WO2015159963A1 - ヒトcsfの毒性を測定する方法 - Google Patents
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Definitions
- the present invention relates to a method for measuring toxicity of human cerebrospinal fluid (hereinafter referred to as CSF), a method for determining Alzheimer's disease, a method for measuring the efficacy as a prophylactic and / or therapeutic agent for Alzheimer's disease, and a subject
- CSF human cerebrospinal fluid
- the present invention relates to a method for determining the susceptibility of an Alzheimer's disease to a preventive and / or therapeutic agent, a method for screening a preventive and / or therapeutic agent for Alzheimer's disease, and a method for producing an Alzheimer's disease-like model animal.
- AD Alzheimer's disease
- a ⁇ amyloid ⁇
- a ⁇ oligomers in CSF are important as clinical biomarkers for measuring the efficacy of drugs targeting A ⁇ such as anti-A ⁇ antibodies, they actually cause pathology in AD patients.
- the detailed properties (structure, size, origin, etc.) of the A ⁇ oligomers are unknown (Non-patent Document 2). Therefore, there is a demand for a method for measuring A ⁇ oligomers causing a disease state.
- bioassays include in vitro tests and in vivo tests described below.
- Non-patent Document 3 it has been reported that cell death, synaptic degeneration, and the like occur when a high concentration of artificial A ⁇ oligomer of nM order is allowed to act on cultured neurons.
- Non-patent Documents 4 and 5 it has been reported that when a CSF collected from an AD patient (hereinafter referred to as an AD patient CSF) is allowed to act on a neuronal cell line, the neuron is damaged (Non-patent Documents 4 and 5).
- LTP Long-Term Potentation
- Non-patent Document 7 In an in vivo test, it is reported that LTP is suppressed in the rat brain when an electrical stimulation is applied after administering AD patient CSF into the rat ventricle (Non-patent Document 7).
- Non-patent Documents 8 and 9 there are reports that their learning behavior is impaired. However, there are no reports of administering AD patient CSF to rodent animals and measuring the presence or absence of impairment in their learning behavior.
- an object of the present invention is to provide a method capable of efficiently evaluating toxicity in human CSF in a short time using a small amount of human CSF.
- Another object of the present invention is to provide a method for efficiently determining Alzheimer's disease in a short time using a small amount of human CSF.
- Another object of the present invention is to provide a method for efficiently measuring the efficacy as a prophylactic and / or therapeutic drug for Alzheimer's disease in a short time using a small amount of human CSF.
- Another object of the present invention is to provide a method for efficiently measuring a subject's sensitivity to Alzheimer's disease prophylactic and / or therapeutic agents in a short time using a small amount of human CSF.
- Another object of the present invention is to provide a method for efficiently screening a prophylactic and / or therapeutic drug for Alzheimer's disease in a short time using a small amount of human CSF.
- Another object of the present invention is to provide a method for efficiently producing an Alzheimer's disease model animal in a short time using a small amount of human CSF.
- the present inventors solved the above problem by administering human CSF into the ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique.
- the present invention has been completed by finding out what can be done.
- Human CSF comprising the step of administering human cerebrospinal fluid (hereinafter referred to as CSF) into a ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique. To measure the toxicity of sucrose. 2. 2. The method according to 1 above, wherein the evaluation of the cognitive function is to evaluate short-term memory of the rodent animal. 3. 3. The method according to 1 or 2 above, wherein the behavioral pharmacological technique is a Y-shaped maze test. 4). 4. The method according to any one of 1 to 3 above, wherein the dose of human CSF into the ventricle of the rodent is 5 ⁇ l or more. 5. 5. 5.
- a method for determining Alzheimer's disease comprising a step of administering human CSF into a ventricle of a rodent animal and evaluating the cognitive function of the rodent animal by a behavioral pharmacological technique. 7). 7. The method according to 6, wherein the evaluation of the cognitive function is to evaluate short-term memory of the rodent animal. 8). 8. The method according to 6 or 7 above, wherein the behavioral pharmacological technique is a Y-shaped maze test. 9. 9. The method according to any one of 6 to 8, wherein the dose of human CSF into the ventricle of the rodent is 5 ⁇ l or more. 10. 10.
- the method according to any one of 6 to 9 above comprising the step of comparing the cognitive function of the rodent animal to which the control is administered intraventricularly and the cognitive function of the rodent animal to which human CSF is administered intraventricularly. . 11. 11. The method according to any one of 6 to 10, wherein the rodent animal is a mouse. 12 It has a medicinal effect as a prophylactic and / or therapeutic drug for Alzheimer's disease, including the step of administering human CSF into the ventricle of a rodent animal and evaluating the cognitive function of the rodent animal by a behavioral pharmacological technique. How to measure. 13 13. The method according to 12 above, wherein the evaluation of the cognitive function is to evaluate short-term memory of the rodent animal. 14 14.
- the method according to 12 or 13 above, wherein the behavioral pharmacological technique is a Y-shaped maze test. 15.
- the method according to any one of 12 to 14, wherein the dose of human CSF into the ventricle of the rodent is 5 ⁇ l or more. 16.
- the method according to any one of 12 to 15, comprising the following steps (1) and (2). (1) Steps of collecting human CSF before and after administration of the drug for subjects who have started administration or continuous administration of prophylactic and / or therapeutic drugs for Alzheimer's disease (2) Before and after administration of the drug collected in step (1) A step of administering each human CSF into the ventricle of a rodent and comparing the cognitive function of the rodent. 17.
- a ⁇ anti-amyloid ⁇
- the preventive agent and / or therapeutic agent for Alzheimer's disease is one antibody selected from the following (a) and (b).
- CDR Complementarity determining region
- VH Heavy chain variable region
- VL light chain variable region
- SEQ ID NOs: 6, 7 and 8 amino acid sequences represented by SEQ ID NOs: 6, 7 and 8, respectively.
- B a monoclonal antibody comprising the amino acid sequence represented by SEQ ID NO: 1 and VL comprising the amino acid sequence represented by SEQ ID NO: 2; 19. The method according to any one of 12 to 18, wherein the rodent animal is a mouse.
- human CSF is administered into the cerebral ventricles of rodents, and the cognitive function of the rodents is evaluated by behavioral pharmacological techniques, whereby toxicity in human CSF is reduced with a small amount of humans.
- a method that can be efficiently evaluated in a short time using CSF a method that efficiently determines Alzheimer's disease in a short time using a small amount of human CSF, and a medicinal effect as a prophylactic and / or therapeutic drug for Alzheimer's disease.
- a method capable of efficiently measuring in a short time using human CSF a method capable of efficiently predicting a subject's sensitivity to a preventive and / or therapeutic drug for Alzheimer's disease in a short time using a small amount of human CSF, and prevention of Alzheimer's disease
- Method for efficiently screening drug and / or therapeutic agent in a short time using a small amount of human CSF and using a small amount of human CSF It is possible to provide a method for producing efficiently Alzheimer's disease-like model animal to time.
- FIG. 2 is a diagram in which the effect of a plurality of AD patient CSFs on mouse cognitive function is examined. Spontaneous alternation rate (%) as an index of cognitive function is shown on the vertical axis.
- FIG. 3 is a diagram in which the effect of a plurality of healthy human CSFs on the mouse cognitive function is examined. Spontaneous alternation rate (%) as an index of cognitive function is shown on the vertical axis.
- FIG. 4 is a diagram showing the effect of intravenous administration of an anti-A ⁇ oligomer humanized antibody (6E4HV0LV0) on cognitive dysfunction caused by AD patient CSF.
- shaft shows the spontaneous alternation action rate (spontaneous alternation) (%) used as the parameter
- FIG. 5 is a diagram in which the effect of pretreatment with an anti-A ⁇ oligomer antibody (6E4HV0LV0) on cognitive dysfunction caused by AD patient CSF is examined.
- FIG. 6A and FIG. 6B are diagrams for explaining the Y-shaped maze testing apparatus.
- the method includes the steps of administering human CSF into the ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique.
- the human CSF is not particularly limited as long as it is a CSF collected from a human.
- it may be a commercially available human CSF or a human CSF collected from a subject by a doctor.
- CSF immediately after being collected from a human CSF obtained by freezing and storing CSF collected from a human, and thawed when necessary may be used.
- mice examples include mice, rats, hamsters and the like, with mice being preferred.
- the administration of human CSF to the ventricle may be in the left ventricle, the right ventricle, or both.
- a method for administering human CSF into the ventricle a conventionally known method for administering a drug or the like into the ventricle can be used (see Scientific Reports, 2014, vol. 4, 6777).
- the dose of human CSF to the ventricle is preferably 5 ⁇ l or more, more preferably 10 ⁇ l.
- the dose of human CSF to the ventricle can be set according to the method described in Example 1 of the present application.
- behavioral pharmacological techniques can be used to evaluate the cognitive function of rodent animals.
- Cognitive functions include functions such as short-term memory, motor memory, associative learning, fear learning, latent learning, visual cognitive memory, long-term memory, spatial work, reference memory, spatial learning, spatial memory, and working memory.
- methods for behavioral pharmacological evaluation of rodent cognitive functions include, for example, the well-known Y-shaped maze test, T-shaped maze test, rotarod test, fear conditioning contextual learning test, water exploration test, novel Examples include a substance search test, a passive avoidance test, a radial maze test, a Morris water maze test, a delayed sample matching / non-sample matching test, and the like.
- the Y-shaped maze test is a method described in Alkam et al. [Behavior Brain Research, Volume 180, page 139 (2007)].
- a Y-shaped maze testing apparatus shown in FIGS. 6 (a) and 6 (b) is prepared.
- the Y-shaped maze testing apparatus is an apparatus in which, for example, three arms A, B, and C made of black acrylic walls are connected at an angle of 120 degrees. Rodents can move freely in the three arms A, B and C.
- the rate of spontaneous alternation behavior in a certain period of time is lower in rodents having abnormal cognitive functions than in normal rodents.
- spontaneous alternation behavior rate (Spontaneous Alternation) (%)
- spontaneous alternation behavior rate (Spontaneous Alternation) (%)
- toxicity of human CSF that is, the presence / absence of toxicity of human CSF can be determined.
- Equation (1) is a calculation formula for calculating the spontaneous alternation action rate (%).
- Equation (2) shows a method of counting the number of spontaneous alternation actions.
- the following shows the results of the mouse Y-shaped maze test and the spontaneous alternation action rate (%) calculated using the above formula (1).
- the results of a mouse Y-shaped maze test using the apparatus shown in FIGS. 6 (a) and 6 (b) are shown below.
- spontaneous alternation behavior rate (%) of rodent animals administered human CSF into the ventricle and controls such as physiological saline are applied to the brain. Comparing the rate of spontaneous alternation behavior (%) of rodent animals administered indoors, if the rate of voluntary alternation behavior (%) is lower than the control, it is determined that the human CSF has toxicity can do.
- Examples of the degree of decrease in the spontaneous alternation behavior rate (%) include a statistically significant decrease. “Statistically significant” means, for example, rodent animals administered with a control such as physiological saline in the ventricle and rodents administered human CSF into the ventricle in Example 1 of the present application. The difference of the spontaneous alternation action rate (%) obtained according to the method is p ⁇ 0.05 in the Dunnett test.
- the degree of decrease in the spontaneous alternation action rate (%) may be 3% or more, 5% or more, 7% or more, 10% or more, or 20% or more.
- the method includes the steps of administering human CSF into the ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique.
- the human CSF is not particularly limited as long as it is a CSF collected from a human as described above.
- rodent animals include mice, rats, and hamsters as described above, and mice are preferred.
- the method for administering human CSF to the ventricle is the same as described above.
- the dose of human CSF to the ventricle is, for example, 5 ⁇ l or more, preferably 10 ⁇ l in the case of mice.
- the dose of human CSF to the ventricle can be set according to the method described in Example 1 of the present application.
- the cognitive function of rodent animals is evaluated by behavioral pharmacological techniques, and examples of the techniques include various known tests similar to those described above.
- the method for determining Alzheimer's disease of the present invention it is preferable to evaluate the short-term memory of a rodent animal, and among these, it is preferable to employ a Y-shaped maze test for evaluating the short-term memory of a rodent animal. .
- the spontaneous alternation behavior rate (%) of a rodent animal to which a control for example, physiological saline
- the spontaneous alternation of a rodent animal to which human CSF is administered intracerebroventricularly is preferable to include a step of comparing the action rate (%).
- the spontaneous alternation behavior rate (%) of a rodent animal administered with human CSF into the ventricle was higher than that of a rodent animal administered with control (eg, physiological saline) in the ventricle.
- a rodent animal administered with control eg, physiological saline
- the spontaneous alternation behavior rate (%) decreases, it can be determined that there is a high possibility that a patient who has collected human CSF has Alzheimer's disease.
- Examples of the degree of decrease in the spontaneous alternation behavior rate (%) include a statistically significant decrease. “Statistically significant” means, for example, rodent animals administered with a control such as physiological saline in the ventricle and rodents administered human CSF into the ventricle in Example 1 of the present application. The difference of the spontaneous alternation action rate (%) obtained according to the method is p ⁇ 0.05 in the Dunnett test.
- the degree of decrease in the spontaneous alternation action rate (%) may be 3% or more, 5% or more, 7% or more, 10% or more, or 20% or more.
- the determination method of the present invention can also be used for determination of progenitor Alzheimer's disease.
- the method includes the steps of administering human CSF into the ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique.
- the human CSF is not particularly limited as long as it is a CSF collected from a human as described above.
- rodent animals include mice, rats, and hamsters as described above, and mice are preferred.
- the method for administering human CSF to the ventricle is the same as described above.
- the dose of human CSF to the ventricle is preferably 5 ⁇ l or more, more preferably 10 ⁇ l.
- the dose of human CSF to the ventricle can be set according to the description in Example 1 of the present application.
- the cognitive function of rodents is evaluated by a behavioral pharmacological technique, and the technique is known in the same manner as described above. The following tests can be exemplified.
- the method for measuring the efficacy of the present invention as a preventive and / or therapeutic agent for Alzheimer's disease it is preferable to evaluate short-term memory of rodents, and among these, short-term memory of rodents is evaluated. It is preferable to employ a Y-shaped maze test.
- the determination of the efficacy includes the following steps (1) and (2).
- Steps of collecting human CSF before and after administration of the drug for subjects who have started administration or continuous administration of prophylactic and / or therapeutic drugs for Alzheimer's disease (2) Before and after administration of the drug collected in step (1) Each of the human CSF is administered into the ventricle of a rodent animal, and the cognitive function of the rodent animal is compared.
- the spontaneous alternation behavior rate (%) (A) of a rodent animal into which the human CSF before the drug administration was administered into the ventricle and the drug administration The spontaneous alternation behavior rate (%) (B) of rodent animals to which human CSF is administered intraventricularly is compared.
- the term “after drug administration” may refer to a case in which human CSF is collected before the drug administration and then administered once or after multiple administrations.
- the drug has a medicinal effect can be confirmed by the fact that the spontaneous alternation behavior rate (%) (B) is higher than the spontaneous alternation behavior rate (%) (A). It can be done.
- the degree of increase in the spontaneous alternation behavior rate (%) is, for example, statistically significant. “Statistically significant” means, for example, that the difference between the two groups is p ⁇ 0.05 by Dunnett's test.
- the fact that the drug has a medicinal effect means that, for example, in the case of a mouse, the improvement rate (%) of the spontaneous alternation behavior rate calculated according to Equation 3 described in Example 6 of the present application is 40% or more. , 60% or more, 80% or more, or 100%.
- the preventive and / or therapeutic agent for Alzheimer's disease includes all drugs that are inhibitors of Alzheimer's disease, from prevention to improvement of Alzheimer's disease.
- Examples include known acetylcholinesterase inhibitors, NMDA receptor antagonists, anti-A ⁇ oligomer antibodies, amyloid ⁇ vaccines, etc. Among them, anti-A ⁇ oligomer antibodies are preferred as the preventive and / or therapeutic agents for Alzheimer's disease.
- the anti-A ⁇ oligomer antibody is more preferably one antibody selected from the following (a) and (b).
- (A) Complementarity determining region (hereinafter referred to as CDR) 1, CDR2, and CDR3 of the heavy chain variable region (hereinafter referred to as VH) of the antibody are represented by SEQ ID NOs: 3, 4, respectively.
- CDR3, CDR2, and CDR3 of the light chain variable region (hereinafter referred to as VL) of the antibody are amino acid sequences represented by SEQ ID NOs: 6, 7 and 8, respectively.
- the medicinal effect of the drug may represent the therapeutic effect of the subject at the time of collecting the CSF after the administration of the drug, or represents the therapeutic effect that the subject is expected to obtain in the future. May be.
- the present invention can also measure the efficacy of the drug over time with respect to a subject who has continuously administered the prophylactic and / or therapeutic drug for Alzheimer's disease.
- the method of the present invention can also be used as a method for measuring the efficacy as a prophylactic and / or therapeutic agent for prodromal Alzheimer's disease.
- the present invention also relates to a method for determining sensitivity to Alzheimer's disease prophylactic and / or therapeutic agents for Alzheimer's disease patients as subjects.
- the method includes the steps of administering human CSF into the ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique.
- the subject is preferably an Alzheimer's disease patient who has never received the drug.
- the human CSF is not particularly limited as long as it is a CSF collected from a human as described above.
- rodent animals include mice, rats, and hamsters as described above, and mice are preferred.
- the method for administering human CSF to the ventricle is the same as described above.
- the dose of human CSF to the ventricle is preferably 5 ⁇ l or more, more preferably 10 ⁇ l.
- the dose of human CSF to the ventricle can be set according to the description in Example 1 of the present application.
- the cognitive function of a rodent animal is evaluated by a behavioral pharmacological technique.
- Known tests can be exemplified.
- the method for determining the subject's sensitivity to the preventive and / or therapeutic agent for Alzheimer's disease of the present invention it is preferable to evaluate the short-term memory of the rodent, and the short-term memory of the rodent can be evaluated. It is preferable to employ a Y-shaped maze test.
- the sensitivity to the drug is determined by comparing the spontaneous alternation behavior rate of the rodent when the drug and AD patient CSF are administered to the rodent, and the control and AD patient CSF by the rodent. This can be confirmed by comparing with the rate of spontaneous alternation behavior of the rodent animals when administered to the animal.
- the drug and AD patient CSF or control and AD patient CSF may be premixed or may be administered separately. There is no particular limitation on the administration interval and the administration order when separately administered.
- the administration site or administration method of the test drug is not particularly limited, and examples thereof include intravenous administration and intraventricular administration. The presence or absence of sensitivity to the drug in Alzheimer's disease patients can be confirmed, for example, according to the methods described in Examples 4 and 5 of the present application.
- Susceptibility to the drug means that the spontaneous alternation behavior rate of the rodent when the drug and AD patient CSF are administered to a rodent is the control and AD patient CSF is administered to the rodent It means that it is higher than the spontaneous alternation behavior rate of the rodent at the time.
- the drug i. v. -Spontaneous alternation behavior rate of AD CSF administration group is Human IgG4 i. v. -It means that it is higher than the spontaneous alternation behavior rate in the AD CSF administration group.
- Example 5 of the present application confirm that the spontaneous alternation action rate of the drug-AD CSF administration group is higher than the spontaneous alternation behavior rate of the Vehicle-AD CSF administration group. Can do.
- the degree of increase in the spontaneous alternation behavior rate (%) is statistically significant. “Statistically significant” means, for example, that the difference between the two groups is p ⁇ 0.05 by Dunnett's test.
- the sensitivity to the drug indicates that the improvement rate (%) of the spontaneous alternation behavior rate calculated according to the formula (3) described in Example 6 of the present application is 40% or more and 60%. As mentioned above, it may be 80% or more or 100%.
- the invention relating to the preventive and / or therapeutic drug for Alzheimer's disease is the same as described above.
- the method of the present invention can also be used as a method for measuring the sensitivity of a subject to a prophylactic and / or therapeutic drug for prodromal Alzheimer's disease.
- the present invention also relates to a method for producing an Alzheimer's disease-like model animal.
- the method includes the steps of administering human CSF into the ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique.
- the human CSF is not particularly limited as long as it is a CSF collected from a human as described above.
- rodent animals include mice, rats, and hamsters as described above, and mice are preferred.
- the method for administering human CSF to the ventricle is the same as described above.
- the dose of human CSF to the ventricle is preferably 5 ⁇ l or more, more preferably 10 ⁇ l.
- the dose of human CSF to the ventricle can be set according to the description in Example 1 of the present application.
- the cognitive function of a rodent is evaluated by a behavioral pharmacological technique, and examples of the technique include various known tests similar to the above. .
- the method for producing an Alzheimer's disease-like model animal of the present invention it is preferable to evaluate the short-term memory of a rodent animal, and among these, a Y-shaped maze test for evaluating the short-term memory of a rodent animal is adopted. It is preferable to do this.
- spontaneous alternation behavior rate (%) of a rodent animal administered with human CSF into the ventricle and control such as physiological saline administered into the ventricle If the spontaneous alternation behavior rate (%) of the rodent animal is compared with that of the control and the spontaneous alternation behavior rate (%) is lower than the control, the rodent animal to which the human CSF is administered intracerebroventricularly is an Alzheimer's disease-like model. It can be said that it is an animal.
- Examples of the degree of decrease in the spontaneous alternation behavior rate (%) include a statistically significant decrease. “Statistically significant” means, for example, rodent animals administered with a control such as physiological saline in the ventricle and rodents administered human CSF into the ventricle in Example 1 of the present application. The difference of the spontaneous alternation action rate (%) obtained according to the method is p ⁇ 0.05 in the Dunnett test.
- the degree of decrease in the spontaneous alternation action rate (%) may be 3% or more, 5% or more, 7% or more, 10% or more, or 20% or more.
- the present invention also relates to a method for screening a preventive and / or therapeutic agent for Alzheimer's disease.
- the method includes the steps of administering human CSF into the ventricle of a rodent and evaluating the cognitive function of the rodent by a behavioral pharmacological technique.
- the model animal obtained by the above-described method for producing an Alzheimer's disease-like model animal may be used.
- the human CSF is not particularly limited as long as it is a CSF collected from a human as described above.
- rodent animals include mice, rats, and hamsters as described above, and mice are preferred.
- the method for administering human CSF to the ventricle is the same as described above.
- the dose of human CSF to the ventricle is preferably 5 ⁇ l or more, more preferably 10 ⁇ l.
- the dose of human CSF to the ventricle can be set according to the description in Example 1 of the present application.
- the cognitive function of rodents is evaluated by behavioral pharmacological techniques, and as such techniques, various known tests similar to the above are performed. It can be illustrated.
- the method for screening a prophylactic and / or therapeutic agent for Alzheimer's disease of the present invention it is preferable to evaluate short-term memory of rodent animals, and among these, Y-shape for evaluating short-term memory of rodent animals It is preferred to employ a type maze test.
- the spontaneous alternation behavior rate of the animal is the control and AD patient CSF.
- the test drug is selected as a candidate for a prophylactic and / or therapeutic drug for Alzheimer's disease.
- the test drug and AD patient CSF or the control and AD patient CSF may be administered in a premixed manner or separately. There is no particular limitation on the administration interval and the administration order when separately administered.
- the administration site or administration method of the test drug is not particularly limited, and examples thereof include intravenous administration and intraventricular administration.
- the test drug i. v. -Spontaneous alternation behavior rate of AD CSF administration group is Human IgG4 i. v. -Selection of a test drug that is higher than the spontaneous alternation behavior rate in the AD CSF administration group as a candidate for a prophylactic and / or therapeutic drug for Alzheimer's disease.
- a test drug in which the voluntary alternation behavior rate of the test drug-AD CSF administration group is higher than the voluntary alternation behavior rate of the Vehicle-AD CSF administration group is Alzheimer's. It may be selected as a preventive and / or therapeutic drug for diseases.
- the degree of increase in the spontaneous alternation behavior rate (%) is statistically significant. “Statistically significant” means, for example, that the difference between the two groups is p ⁇ 0.05 by Dunnett's test.
- a method for screening the preventive and / or therapeutic agent for Alzheimer's disease of the present invention for example, in the case of a mouse, improvement of the spontaneous alternation behavior rate calculated according to the formula (3) described in Example 6 of the present application.
- Examples include selecting a test drug having a rate (%) of 40% or more, 60% or more, 80% or more, or 100% as a prophylactic and / or therapeutic drug for Alzheimer's disease.
- test drug in the present invention examples include, but are not particularly limited to, low molecules, proteins including antibodies, peptides and the like.
- the method of the present invention can also be used as a method for screening a prophylactic and / or therapeutic drug for precursor Alzheimer's disease.
- Example 1 Measurement of cognitive dysfunction in mice by AD patient CSF
- the cognitive function of mice administered AD patient CSF was measured using a Y-shaped maze test.
- the Y-shaped maze test was carried out in accordance with the method described in Alkam et al. [Behavior Brain Research, 180, 139 (2007)].
- ICR mice male, SLC Japan
- AD patient CSF PrecisionMed
- a maximum volume of 10 ⁇ l of physiological saline (hereinafter referred to as Sham) (Otsuka Pharmaceutical Factory) was administered into the cerebral ventricles of mice as described above.
- the mouse was placed in a Y-shaped maze test apparatus [three arms A, B, C (black acrylic walls A, B, C, respectively) as shown in FIGS. 6 (a) and 6 (b). Having a length of 25 cm, a width of 5 cm, and a height of 20 cm] was placed at the tip of any of the arms connected to each other at an angle of 120 degrees, and the maze was freely searched for 7 minutes.
- the spontaneous alternation behavior rate in the AD patient CSF administration group decreased with an increase in the dose of the AD patient CSF.
- the spontaneous alternation behavior rate of the 10 ⁇ l AD patient CSF administration group was significantly lower than the spontaneous alternation behavior rate of the Sham administration group.
- Example 2 Measurement of mouse cognitive dysfunction with different AD patient CSFs
- the cognitive function of a mouse administered with an AD patient CSF different from that in Example 1 was measured using a Y-shaped maze test.
- AD patient CSF a sample (Cureline) obtained by mixing two AD patient-derived CSFs (both PrecisionMed) different from Example 1 and three patient-derived CSFs indicated as “pool” was used.
- Example 2 From the results of Example 1 and Example 2, it was revealed that when 10 ⁇ l of AD patient CSF was administered into the ventricle of a mouse, the cognitive function of the mouse was significantly reduced compared to the Sham group.
- Example 3 Measurement of the effect of healthy human CSF on mouse cognitive function
- the cognitive function of mice administered with healthy human CSF was measured using a Y-shaped maze test.
- Three healthy person-derived CSFs (PrecisionMed) were used as healthy persons CSF, physiological saline (hereinafter referred to as Sham) as a negative control, and AD patient CSF (PrecisionMed) as a positive control.
- each of the above samples was intraventricularly administered under isoflurane anesthesia to the ICR mouse, and a Y-shaped maze test was performed by the method described in Example 1 to determine the spontaneous alternation action rate (%).
- the obtained results are shown in FIG.
- Each of the above samples was administered in an amount of 5 ⁇ l each in the left ventricle and right ventricle of the mouse.
- Example 4 Measurement of the effect of intravenous administration of anti-A ⁇ antibody on cognitive dysfunction in mice caused by AD patient CSF
- mice administered intravenously with anti-A ⁇ oligomer antibody the cognitive function of the mouse by administration of AD patient CSF was measured by a Y-shaped maze test.
- an anti-A ⁇ oligomer antibody an anti-A ⁇ oligomer humanized antibody 6E4HV0LV0 was prepared according to the method disclosed in International Publication No. 2011/016567 and a known method, and used for the experiment.
- amino acid sequences of VH and VL of 6E4HV0LV0 are shown in SEQ ID NOs: 1 and 2, respectively.
- amino acid sequences of heavy chain CDR1, CDR2, and CDR3 of 6E4HV0LV0 are described in SEQ ID NOs: 3, 4, and 5, respectively, and the amino acid sequences of light chain CDR1, CDR2, and CDR3 are described in SEQ ID NOs: 6, 7, and 8, respectively. .
- ICR mice were intravenously administered with 3 mg / kg human IgG4 (Sigma-Aldrich) or 0.3 mg / kg, 1 mg / kg or 3 mg / kg 6E4HV0LV0.
- mice using human IgG4 for intravenous administration and Sham for intraventricular administration will be referred to as Human IgG4 i. v. -Notated as a Sham administration group, the same notation was applied to mice administered with other samples.
- 6E4HV0LV0 i. v. -In the AD CSF administration group the spontaneous alternation behavior rate increased as the dose of 6E4HV0LV0 increased. Furthermore, 3 mg / kg 6E4HV0LV0 i. v. -In the AD CSF administration group, Human IgG4 i. v. -Spontaneous alternation behavior rate significantly increased compared to AD CSF administration group, Human IgG4 i. v. -It recovered to the same level as the spontaneous alternation rate in the Sham administration group.
- Example 5 Measurement of effect of anti-A ⁇ antibody pretreatment on cognitive dysfunction in mice by AD patient CSF
- Example 4 the effect of intravenous administration of 6E4HV0LV0 was examined.
- this measurement system to the clinical efficacy of drugs that target Alzheimer's disease prophylactic and / or therapeutic drugs, such as A ⁇ oligomers, before and after administering the drug from the same patient.
- CSF is collected, and the cognitive function of the mice administered with each CSF is measured. That is, in the patient-derived CSF after the administration of the drug, the drug is present in the ventricle of the mouse.
- the cognitive function of a mouse administered with AD patient CSF pretreated with 6E4HV0LV0 was measured using a Y-shaped maze test.
- 6E4HV0LV0 was added to AD patient CSF (PrecisionMed) to a final concentration of 1 ng / ml, 10 ng / ml or 100 ng / ml, and incubated at room temperature for about 1 hour.
- artificial cerebrospinal fluid artificial cerebrospinal fluid (artificial cerebrospinal fluid (Otsuka Pharmaceutical Factory) supplemented with 0.02 w / v% human serum albumin (Sigma-Aldrich)] (hereinafter referred to as Vehicle) CSF or physiological It was added to a saline solution (hereinafter referred to as Sham) and incubated at room temperature. The prepared sample was then stored at ⁇ 80 ° C. and thawed at room temperature on the day of the Y-shaped maze test and used for the experiment.
- Vehicle artificial cerebrospinal fluid
- Sham saline solution
- Vehicle-AD CSF a sample in which Vehicle is added to AD patient CSF
- Vehicle-AD CSF a sample in which Vehicle is added to AD patient CSF
- the spontaneous alternation behavior rate increased compared to the Vehicle-AD CSF administration group.
- the spontaneous alternation behavior rate of the 6E4HV0LV0-AD CSF administration group with a final concentration of 100 ng / ml recovered to the same level as that of the Vehicle-Sham administration group.
- Example 6 Measurement of cognitive dysfunction in mice with blinded CSF samples, and measurement of changes in cognitive function of the mice with 6E4HV0LV0 pretreatment of mice administered multiple blinded healthy CSF or AD patient CSF Cognitive function was measured by the same Y-shaped maze test as in Example 1. At the same time, the effect of 6E4HV0LV0 pretreatment was also examined. Distributing healthy CSF or AD patient CSF, preparing and blinding AD patient CSF pre-treated with 6E4HV0LV0, and administering the sample to mice to perform a Y-shaped maze test The experiment was conducted.
- a blind number was assigned to the healthy CSF (PrecisionMed) and AD patient CSF (PrecisionMed) distributed by the person in charge (A) at random.
- A person in charge
- 6E4HV0LV0 was added to the dispensed CSF to a final concentration of 10 ng / ml, and a sample incubated at room temperature for about 1 hour was also prepared. All CSF samples dispensed and prepared were stored at ⁇ 80 ° C. until the day of the Y-shaped maze test.
- a sample blinded by the person in charge (B) was received from the person in charge (A) and thawed at room temperature. Thereafter, 10 ⁇ l of each sample or 10 ⁇ l of physiological saline (hereinafter referred to as Sham) was intraventricularly administered to the ICR mice under isoflurane anesthesia, and the Y-shaped maze test was performed according to the method described in Example 1. The voluntary alternation action rate (%) was obtained.
- Each of the above samples and physiological saline was administered in an amount of 5 ⁇ l each in the left ventricle and right ventricle of the mouse.
- Improvement rate of spontaneous alternation behavior rate of mice by 6E4HV0LV0 pretreatment ⁇ (Spontaneous alternation behavior rate of mice administered CSF pretreated with 6E4HV0LV0) ⁇ (same AD patient not pretreated with 6E4HV0LV0) Spontaneous alternation behavior rate of mice administered CSF) ⁇ / ⁇ (Spontaneous alternation behavior rate of Sham administration group) ⁇ (Spontaneous alternation behavior rate of mice administered the same AD patient CSF not pretreated with 6E4HV0LV0) ⁇ ⁇ 100 ...
- mice administered with AD patient CSF that was not pretreated with 6E4HV0LV0 the cognitive function of the mice was impaired in all 15 cases.
- mouth which administered healthy person CSF the cognitive function of the mouse
- mice administered with healthy human CSF cognitive function of the mice was impaired in 1 out of 11 cases.
- mouth by 6E4HV0LV0 pretreatment was measured similarly to said AD patient CSF.
- the cognitive impairment that occurred in the mice was not improved by 6E4HV0LV0 pretreatment.
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Abstract
Description
1.ヒト脳脊髄液(cerebrospinal fluid、以下CSFと記載する)をげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む、ヒトCSFの毒性を測定する方法。
2.前記認知機能の評価が、前記げっ歯類動物の短期記憶を評価するものである前記1に記載の方法。
3.前記行動薬理学的な手法が、Y字型迷路試験である前記1または2に記載の方法。
4.前記げっ歯類動物の脳室内へのヒトCSFの投与量が、5μl以上である前記1~3のいずれか1に記載の方法。
5.前記げっ歯類動物が、マウスである前記1~4のいずれか1に記載の方法。
6.ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む、アルツハイマー病の判定方法。
7.前記認知機能の評価が、前記げっ歯類動物の短期記憶を評価するものである前記6に記載の方法。
8.前記行動薬理学的な手法が、Y字型迷路試験である前記6または7に記載の方法。
9.前記げっ歯類動物の脳室内へのヒトCSFの投与量が、5μl以上である前記6~8のいずれか1に記載の方法。
10.コントロールを脳室内投与した前記げっ歯類動物の認知機能と、ヒトCSFを脳室内投与した前記げっ歯類動物の認知機能とを比較する工程を含む前記6~9のいずれか1に記載の方法。
11.前記げっ歯類動物が、マウスである前記6~10のいずれか1に記載の方法。
12.ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む、アルツハイマー病の予防薬及び/または治療薬としての薬効を測定する方法。
13.前記認知機能の評価が、前記げっ歯類動物の短期記憶を評価するものである前記12に記載の方法。
14.前記行動薬理学的な手法が、Y字型迷路試験である前記12または13に記載の方法。
15.前記げっ歯類動物の脳室内へのヒトCSFの投与量が、5μl以上である前記12~14のいずれか1に記載の方法。
16.下記工程(1)及び(2)を含む、前記12~15のいずれか1に記載の方法。
(1)アルツハイマー病の予防薬及び/または治療薬を投与開始または連投中の被験者について、ヒトCSFを該薬剤の投与前後に採取する工程
(2)工程(1)で採取した該薬剤の投与前後のヒトCSFを、それぞれげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を比較する工程
17.前記アルツハイマー病の予防薬及び/または治療薬が、抗アミロイドβ(amyloid beta、以下Aβと記載する)オリゴマー抗体である前記12~16のいずれか1に記載の方法。
18.前記アルツハイマー病の予防薬及び/または治療薬が、下記(a)及び(b)から選ばれる一つの抗体である前記12~17のいずれか1に記載の方法。
(a)抗体の重鎖可変領域(heavy chain variable region、以下VHと記載する)の相補性決定領域(complementarity determining region、以下CDRと記載する)1、CDR2及びCDR3が、それぞれ配列番号3、4及び5で表わされるアミノ酸配列を含み、かつ抗体の軽鎖可変領域(light chain variable region、以下VLと記載する)のCDR1、CDR2及びCDR3が、それぞれ配列番号6、7及び8で表わされるアミノ酸配列を含むモノクローナル抗体
(b)抗体のVHが配列番号1で表わされるアミノ酸配列を含み、かつVLが配列番号2で表わされるアミノ酸配列を含むモノクローナル抗体
19.前記げっ歯類動物が、マウスである前記12~18のいずれか1に記載の方法。
まず、ヒトCSFの毒性を測定する方法について説明する。該方法は、ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む。
次に、本発明のアルツハイマー病の判定方法について説明する。該方法は、ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む。
次に、本発明のアルツハイマー病の予防薬及び/または治療薬としての薬効を測定する方法について説明する。該方法は、ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む。
(1)アルツハイマー病の予防薬及び/または治療薬を投与開始または連投中の被験者について、ヒトCSFを該薬剤の投与前後に採取する工程
(2)工程(1)で採取した該薬剤の投与前後のヒトCSFを、それぞれげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を比較する工程
(a)抗体の重鎖可変領域(Heavy chain variable region、以下VHと表記する)の相補性決定領域(Complementarity determining region,以下CDRと表記する)1、CDR2及びCDR3が、それぞれ配列番号3、4及び5で表わされるアミノ酸配列を含み、かつ抗体の軽鎖可変領域(Light chain variable region、以下VLと表記する)のCDR1、CDR2及びCDR3が、それぞれ配列番号6、7及び8で表わされるアミノ酸配列を含むモノクローナル抗体
(b)抗体のVHが配列番号1で表わされるアミノ酸配列を含み、かつVLが配列番号2で表わされるアミノ酸配列を含むモノクローナル抗体
また本発明は、被験者であるアルツハイマー病患者について、アルツハイマー病の予防薬及び/または治療薬に対する感受性を判定する方法に関する。該方法は、ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む。
また、本発明はアルツハイマー病様モデル動物を作製する方法に関する。該方法は、ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む。
また、本発明は、アルツハイマー病の予防薬及び/または治療薬をスクリーニングする方法に関する。該方法は、ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む。当該方法は、上述したアルツハイマー病様モデル動物を作製する方法により得られた当該モデル動物を用いてもよい。
AD患者CSFを投与したマウスの認知機能を、Y字型迷路試験を用いて測定した。Y字型迷路試験はAlkamらの文献[ビヘイビオラル・ブレイン・リサーチ(Behav Brain Res)、180巻、139ページ(2007年)]に記載の方法に準じて行った。
次に、実施例1とは異なるAD患者CSFを投与したマウスの認知機能を、Y字型迷路試験を用いて測定した。AD患者CSFとして、実施例1とは異なる2名のAD患者由来CSF(いずれもPrecisionMed社)及びpoolと表記する3名の患者由来のCSFを混ぜ合わせたサンプル(Cureline社)を用いた。
次に、健常人CSFを投与したマウスの認知機能を、Y字型迷路試験を用いて測定した。健常人CSFとして3名の健常人由来CSF(PrecisionMed社)、陰性対照として生理食塩液(以下Shamと表記する)及び陽性対照としてAD患者CSF(PrecisionMed社)を用いた。
次に、抗Aβオリゴマー抗体を静脈内投与したマウスについて、AD患者CSF投与による当該マウスの認知機能の変化を、Y字型迷路試験により測定した。抗Aβオリゴマー抗体として、抗Aβオリゴマーヒト化抗体6E4HV0LV0を国際公開公報第2011/016567号に開示された方法及び既知の方法に準じて作製し、実験に使用した。
実施例4では、6E4HV0LV0の静脈内投与での作用を検討した。しかし、本測定系を臨床でアルツハイマー病の予防薬及び/または治療薬、例えばAβオリゴマーを標的とする薬剤の薬効の測定に応用することを考えた場合、同一の患者から該薬剤を投与する前後のCSFを採取し、それぞれのCSFを投与したマウスの認知機能を測定することになる。つまり、該薬剤投与後の患者由来CSF中には、該薬剤が存在した状態でマウスの脳室内に投与することになる。
盲検化した複数の健常人CSFまたはAD患者CSFを投与したマウスの認知機能を、実施例1と同様のY字型迷路試験によって測定した。同時に、6E4HV0LV0前処置の作用についても検討した。健常人CSFまたはAD患者CSFの分注、6E4HV0LV0で前処置したAD患者CSFの調製及び盲検化を行う担当者と、該サンプルをマウスに投与してY字型迷路試験を行う担当者を別にして実験を行った。
認知機能障害惹起の程度の判定方法を以下に記す。
-:Sham投与群と比較して、自発交替行動率が0~3%低下した(認知機能障害惹起なし)
+:Sham投与群と比較して、自発交替行動率が3~7%低下した(弱い認知機能障害惹起)
++:Sham投与群と比較して、自発交替行動率が7~10%低下した(強い認知機能障害惹起)
配列番号2-人工配列の説明;6E4HV0LV0の軽鎖可変領域のアミノ酸配列
配列番号3-人工配列の説明;6E4HV0LV0の重鎖CDR1のアミノ酸配列
配列番号4-人工配列の説明;6E4HV0LV0の重鎖CDR2のアミノ酸配列
配列番号5-人工配列の説明;6E4HV0LV0の重鎖CDR3のアミノ酸配列
配列番号6-人工配列の説明;6E4HV0LV0の軽鎖CDR1のアミノ酸配列
配列番号7-人工配列の説明;6E4HV0LV0の軽鎖CDR2のアミノ酸配列
配列番号8-人工配列の説明;6E4HV0LV0の軽鎖CDR3のアミノ酸配列
Claims (19)
- ヒト脳脊髄液(cerebrospinal fluid、以下CSFと記載する)をげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む、ヒトCSFの毒性を測定する方法。
- 前記認知機能の評価が、前記げっ歯類動物の短期記憶を評価するものである請求項1に記載の方法。
- 前記行動薬理学的な手法が、Y字型迷路試験である請求項1または2に記載の方法。
- 前記げっ歯類動物の脳室内へのヒトCSFの投与量が、5μl以上である請求項1~3のいずれか1項に記載の方法。
- 前記げっ歯類動物が、マウスである請求項1~4のいずれか1項に記載の方法。
- ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む、アルツハイマー病の判定方法。
- 前記認知機能の評価が、前記げっ歯類動物の短期記憶を評価するものである請求項6に記載の方法。
- 前記行動薬理学的な手法が、Y字型迷路試験である請求項6または7に記載の方法。
- 前記げっ歯類動物の脳室内へのヒトCSFの投与量が、5μl以上である請求項6~8のいずれか1項に記載の方法。
- コントロールを脳室内投与した前記げっ歯類動物の認知機能と、ヒトCSFを脳室内投与した前記げっ歯類動物の認知機能とを比較する工程を含む請求項6~9のいずれか1項に記載の方法。
- 前記げっ歯類動物が、マウスである請求項6~10のいずれか1項に記載の方法。
- ヒトCSFをげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を行動薬理学的な手法により評価する工程を含む、アルツハイマー病の予防薬および/または治療薬としての薬効を測定する方法。
- 前記認知機能の評価が、前記げっ歯類動物の短期記憶を評価するものである請求項12に記載の方法。
- 前記行動薬理学的な手法が、Y字型迷路試験である請求項12または13に記載の方法。
- 前記げっ歯類動物の脳室内へのヒトCSFの投与量が、5μl以上である請求項12~14のいずれか1項に記載の方法。
- 下記工程(1)および(2)を含む、請求項12~15のいずれか1項に記載の方法。
(1)アルツハイマー病の予防薬および/または治療薬を投与開始または連投中の被験者について、ヒトCSFを該薬剤の投与前後に採取する工程
(2)工程(1)で採取した該薬剤の投与前後のヒトCSFを、それぞれげっ歯類動物の脳室内に投与し、該げっ歯類動物の認知機能を比較する工程 - 前記アルツハイマー病の予防薬および/または治療薬が、抗アミロイドβ(amyloid beta、以下Aβと記載する)オリゴマー抗体である請求項12~16のいずれか1項に記載の方法。
- 前記アルツハイマー病の予防薬および/または治療薬が、下記(a)および(b)から選ばれる一つの抗体である請求項12~17のいずれか1項に記載の方法。
(a)抗体の重鎖可変領域(heavy chain variable region、以下VHと記載する)の相補性決定領域(complementarity determining region、以下CDRと記載する)1、CDR2およびCDR3が、それぞれ配列番号3、4および5で表わされるアミノ酸配列を含み、かつ抗体の軽鎖可変領域(light chain variable region、以下VLと記載する)のCDR1、CDR2およびCDR3が、それぞれ配列番号6、7および8で表わされるアミノ酸配列を含むモノクローナル抗体
(b)抗体のVHが配列番号1で表わされるアミノ酸配列を含み、かつVLが配列番号2で表わされるアミノ酸配列を含むモノクローナル抗体 - 前記げっ歯類動物が、マウスである請求項12~18のいずれか1項に記載の方法。
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