WO2011004620A1 - 細胞保護剤 - Google Patents
細胞保護剤 Download PDFInfo
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- WO2011004620A1 WO2011004620A1 PCT/JP2010/051711 JP2010051711W WO2011004620A1 WO 2011004620 A1 WO2011004620 A1 WO 2011004620A1 JP 2010051711 W JP2010051711 W JP 2010051711W WO 2011004620 A1 WO2011004620 A1 WO 2011004620A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/04—Ortho-condensed systems
Definitions
- the present invention relates to a cytoprotective agent.
- Ischemic injury is understood as a pathological condition that includes all symptoms caused by restricted blood flow in any part of the body, and as a result of the death of cells due to energy depletion in the ischemic region, the region becomes dysfunctional. It will fall.
- Development of thrombolytic drugs and thrombolytic therapies using the thrombolytic drugs has been promoted as a treatment method when the cause of ischemia is a thrombus in order to dissolve the thrombus and restore the blood supply to the ischemic region. .
- t-PA plasminogen activator
- rt-PA tissue type plasminogen activator
- SMTP Stachybotrys microspora triprenyl phenol
- SMTP Stachybotrys microspora triprenyl phenol
- the SMTP compound leads to a conformational change of plasminogen.
- the sensitivity of plasminogen to t-PA and plasminogen It has been suggested that the mechanism of action increases the binding of thrombi to thrombi, etc., and promotes thrombolysis.
- ischemia reperfusion injury becomes a problem.
- cerebral edema increases or intracranial bleeding occurs. Therefore, it is important to develop a drug having a cytoprotective action that reduces the risk of various cell damages caused by ischemia, particularly the risk of ischemia-reperfusion injury.
- the present invention has been made in view of the above, and an object thereof is to provide a novel use of a cytoprotective agent excellent in the effect of suppressing dysfunction caused by ischemia and a triprenylphenol compound as a medicine.
- the present invention is a cytoprotective agent used for ischemic injury, comprising a triprenyl phenol compound represented by the following general formula (I) as an active ingredient.
- X is —CHY—C (CH 3 ) 2 Z, and Y and Z are each independently —H or —OH, or together form a single bond.
- R represents a hydrogen atom or a substituent having a molecular weight of 1000 or less.
- triprenyl phenol compound represented by the general formula (I) specific examples of the case where R is a substituent having a molecular weight of 1000 or less include, for example, the following general formula (II) or (III) A triprenyl phenol compound is mentioned.
- X 1 , X 2 and X 3 are each independently —CHY—C (CH 3 ) 2 Z, and Y and Z are each independently —H or —OH. Or together form a single bond.
- R 1 represents any one of the following (A) to (D).
- R 2 is a natural amino acid having two amino groups, a D form of a natural amino acid having two amino groups, a natural amino acid having two amino groups, and a D form of a natural amino acid having two amino groups
- H 2 N—CH (COOH) — (CH 2 ) m —S p — (CH 2 ) q —CH (COOH) —NH 2 (m, p and q are each independently an integer of 0 to 9)
- the ischemic disorder is preferably thrombosis (including thromboembolism; hereinafter the same).
- the ischemic disorder is more preferably cerebral infarction.
- cytoprotective agent excellent in the effect of suppressing dysfunction caused by ischemia and a triprenylphenol compound as a medicine.
- a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
- the cytoprotective agent of the present invention contains a triprenyl phenol compound represented by the above general formula (I) as an active ingredient and is used for ischemic injury.
- triprenylphenol compound according to the present invention has an unknown detailed mechanism of action. Protects cells from being damaged in blood disorders and suppresses dysfunction caused by ischemia.
- ischemic disorder is generally understood as a pathological condition including all symptoms caused by restriction of blood flow in any part of the body. That is, it is understood that in an ischemic injury, cell damage due to energy depletion or cell injury after recovery of blood flow (ischemia reperfusion injury) occurs in the ischemic region, and the region becomes dysfunctional. .
- the triprenyl phenol compound is considered to effectively suppress ischemia-reperfusion injury. In this respect, it is a different action from promoting the dissolution of the thrombus to resume the blood flow to the ischemic region, to restore the energy supply, and as a result, to suppress cell damage.
- “dysfunction due to ischemia” includes all symptoms that occur due to restriction of blood flow in any part of the body.
- “Suppression of dysfunction” is a concept that also includes “improvement of dysfunction”, and its effect can be evaluated by the size of the damaged area and the degree of symptoms of each disease.
- the effect can be evaluated by the size of the brain damage area observed by CT, MRI, cerebral angiography, the size of edema, etc., and the neurological symptoms that appear as symptoms, disturbances in daily life movement The effect can be evaluated with indicators such as motor paralysis.
- interleukin-1 ⁇ (IL-1 ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin-6 (IL-6), etc. whose expression increases with tissue inflammation are effective. Can be evaluated.
- thrombosis is generally understood as a blood clot in a blood vessel.
- Specific conditions or diseases include, for example, transient cerebral ischemic attack, disseminated intravascular coagulation syndrome, thrombotic microangiopathy, thrombophlebitis, deep vein thrombosis, idiopathic thrombosis, cerebral infarction ( Brain thrombosis, cerebral embolism), myocardial infarction, pulmonary thromboembolism and the like.
- the cytoprotective agent of the present invention is particularly preferably used for cerebral infarction.
- the triprenyl phenol compound contained as an active ingredient in the cytoprotective agent of the present invention is a triprenyl phenol compound represented by the general formula (I).
- the cytoprotective agent of the present invention contains at least one triprenyl phenol compound according to the present invention as an active ingredient.
- X is —CHY—C (CH 3 ) 2 Z, and Y and Z are each independently —H or —OH, or together form a single bond.
- R represents a hydrogen atom or a substituent having a molecular weight of 1000 or less.
- the substituent having a molecular weight of 1000 or less is preferably a substituent having a molecular weight of 800 or less, more preferably a substituent having a molecular weight of 700 or less, and still more preferably a substituent having a molecular weight of 600 or less, from the viewpoint of the effect as a cytoprotective agent.
- the triprenylphenol compound according to the present invention may be obtained by chemical synthesis or may be obtained by purification from a culture of a filamentous fungus, for example, Stachybotrys microspora.
- a method of obtaining the triprenylphenol compound according to the present invention by purification from a filamentous fungus culture for example, a desired addition from a culture obtained by adding a predetermined added organic amino compound to a culture solution of Stachybotrys microspora And a method comprising purifying the compound.
- the triprenylphenol compound according to the present invention may be an enantiomer, a diastereomer, and a mixture of enantiomers or diastereomers.
- Enantiomers, diastereomers, and mixtures of enantiomers or diastereomers may be obtained by chemical synthesis or may be obtained by purification from a filamentous fungal culture. When it is obtained by purification from a filamentous fungus culture, the corresponding isomers can be obtained by using the D-form or L-form of the added organic amino compound added to the filamentous fungus medium.
- triprenylphenol compound according to the present invention in which R is a substituent having a molecular weight of 1000 or less include, for example, triprenylphenol compounds represented by the following general formula (II) or (III). .
- Triprenylphenol compound represented by general formula (II) One specific example of the triprenyl phenol compound according to the present invention is a compound represented by the following general formula (II).
- X 1 is —CHY—C (CH 3 ) 2 Z, and Y and Z are each independently —H or —OH, or together form a single bond.
- R 1 represents any one of the following (A) to (D).
- (A) is an amino compound selected from the group consisting of natural amino acids, natural amino acid D-forms, and compounds obtained by replacing the carboxy group with a hydrogen atom, hydroxy group or hydroxymethyl group in natural amino acids and natural amino acid D-forms To a residue obtained by removing one amino group (excluding — (CH) 2 —OH).
- Natural amino acids are not particularly limited as long as they are naturally occurring amino acids, and examples include ⁇ -amino acids, ⁇ -amino acids, ⁇ -amino acids, and ⁇ -amino acids. Such amino acids may be obtained from natural products or artificially obtained by techniques such as organic synthesis.
- Natural amino acids for example, ⁇ -amino acids such as glycine, alanine, threonine, valine, isoleucine, tyrosine, cysteine, cystine, methionine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, arginine, lysine, hydroxylysine, ornithine, citrulline , Homocysteine, 3,4-dihydroxyphenylalanine, homocystin, diaminopimelic acid, diaminopropionic acid, serine, leucine, phenylalanine, tryptophan and the like
- ⁇ -amino acids include ⁇ -alanine, and ⁇ -amino acids as Examples include ⁇ -aminobutyric acid and carnitine.
- Examples of ⁇ -amino acids include 5-aminolevulinic acid and 5-aminovaleric acid.
- Examples of the compound in which the carboxy group is replaced with a hydrogen atom, a hydroxy group or a hydroxymethyl group in the natural amino acid or the D form of the natural amino acid include amino alcohols and amines.
- Examples of such amino alcohol include 2-aminoethanol.
- R 1 in the general formula (II) is (A)
- Specific examples of the compound in the case where R 1 in the general formula (II) is (A) include compounds shown in Table 1 below.
- the “added organic amino compound” in the table is used when the triprenyl phenol compound is obtained by purification from a culture obtained when a predetermined added organic amino compound is added to the culture solution of Stachybotrys microspora.
- the added organic amino compounds are shown below (the same applies hereinafter).
- (B) is an aromatic group having at least one selected from the group consisting of a carboxy group, a hydroxyl group, a sulfonic acid group and a second amino group as a substituent or a part of the substituent, or a second amino group And an aromatic group which may contain a nitrogen atom.
- the aromatic group include compounds represented by the following structural formulas.
- (C) is an aromatic amino acid residue represented by the following formula (II-1) (wherein R 3 is a substituent which may or may not be a hydroxyl group, a carboxy group, 5 represents at least one substituent selected from the group consisting of 5 alkyl groups, and n represents an integer of 0 or 1.).
- Examples of the aromatic amino acid residue represented by the formula (II-1) include compounds represented by the following structural formula.
- (D) represents a substituent represented by -L 1 -L 2 -R 4 (wherein L 1 represents a linking group which is a C 1-4 alkylene group having a carboxy group, and L 2 represents A linking group represented by —NH—C ( ⁇ O) — or —NH—C ( ⁇ S) —NH—, wherein R 4 is a 9-fluorenylalkyloxy having an alkyloxy group having 1 to 3 carbon atoms; Or a polyheterocyclic group represented by the following formula (II-2)).
- Triprenylphenol compound represented by general formula (III) One specific example of the triprenylphenol compound according to the present invention is a compound represented by the following general formula (III).
- X 2 and X 3 are each independently —CHY—C (CH 3 ) 2 Z, and Y and Z are each independently —H or —OH, or together.
- R 2 is a natural amino acid having two amino groups, a D form of a natural amino acid having two amino groups, a natural amino acid having two amino groups, and a D form of a natural amino acid having two amino groups
- H 2 N—CH (COOH) — (CH 2 ) n NH 2 (n is an integer of 0 to 9)
- H 2 N—CH (COOH) — (CH 2 ) m —S p — (CH 2 ) q —CH (COOH) —NH 2 (m, p and q are each independently an integer of 0 to 9)
- It represents a residue obtained by removing two amino groups from a selected amino compound.
- N represents an integer of 0 to 9, preferably an integer of 0 to 6, more preferably an integer of 1 to 5, and still more preferably an integer of 1 to 4.
- M represents an integer of 0 to 9, preferably an integer of 0 to 4, more preferably an integer of 1 to 3, and still more preferably 1 or 2.
- P represents an integer of 0 to 9, preferably an integer of 0 to 4, more preferably an integer of 1 to 3, and further preferably 1 or 2.
- Q represents an integer of 0 to 9, preferably an integer of 0 to 4, more preferably an integer of 1 to 3, and further preferably 1 or 2.
- m + q is preferably an integer of 0 to 9, more preferably an integer of 0 to 6, still more preferably an integer of 1 to 5, and particularly preferably an integer of 1 to 4.
- Examples of natural amino acids having two amino groups include hydroxylysine, citrulline, cystine, homocystine, diaminopimelic acid, diaminopropionic acid, lysine and ornithine.
- a carboxy group is replaced with a hydrogen atom, a hydroxy group or a hydroxymethyl group in a D-form of a natural amino acid having two amino groups and a natural amino acid having two amino groups
- triprenyl phenol compound represented by the general formula (II) or (III) As a specific example of the triprenyl phenol compound represented by the general formula (I), the triprenyl phenol shown in Table 6 below is used. Compounds.
- the triprenyl phenol compound according to the present invention can be used in a free form, a pharmaceutically acceptable salt or ester form, or a solvate form.
- Inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or citric acid, formic acid, fumaric acid, malic acid, acetic acid, succinic acid, tartaric acid, methanesulfonic acid, p-toluenesulfonic acid Suitable for the formation of pharmaceutically acceptable salts of the compounds of the present invention.
- alkali metals or alkaline earth metals such as sodium, potassium, calcium, magnesium, basic amines, or basic amino acids are also suitable for the formation of pharmaceutically acceptable salts of the compounds of the present invention.
- alcohols having 1 to 10 carbon atoms or carboxylic acids preferably methyl alcohol, ethyl alcohol, acetic acid, or propionic acid, are suitable for forming a pharmaceutically acceptable ester of the compound of the present invention.
- Water or the like is also suitable for forming a pharmaceutically acceptable solvate of the compound of the present invention.
- the type of carrier and formulation additive used for preparing the cytoprotective agent of the present invention is not particularly limited.
- the cytoprotective agent of the present invention uses the triprenyl phenol compound according to the present invention and a pharmaceutically acceptable solid carrier (for example, gelatin, lactose) or a liquid carrier (for example, water, physiological saline, aqueous glucose solution). To be formulated.
- the cytoprotective agent of the present invention depends on the type of triprenylphenol compound used as an active ingredient, the severity of ischemic injury, and which part of the body the affected part is, the effective amount per adult is 0.01 Administration of ⁇ 100 mg / kg is preferred, and administration of 0.1 to 30 mg / kg is more preferred.
- Administration of ⁇ 100 mg / kg is preferred, and administration of 0.1 to 30 mg / kg is more preferred.
- count of administration It may be used by single administration, may be used by repeated administration, and may be used by continuous administration.
- the administration interval and administration period can be selected by those skilled in the art according to clinical findings, imaging findings, blood findings, comorbid diseases, past medical history, and the like.
- the cytoprotective agent of the present invention When used in repeated administration, it is preferably administered immediately after the onset and 12 hours after the onset from the viewpoint that the affected area continuously contacts the cytoprotective agent of the present invention. Also preferred is a mode of continuous administration.
- the method of administration to an adult is not particularly limited, and various administration routes such as intravenous administration, subcutaneous administration, intramuscular administration, and oral administration can be selected.
- intravenous administration specifically intravenous injection or infusion is preferable from the viewpoint of promptly and reliably administering a desired dose to a patient.
- a person skilled in the art can adopt a method in which 10% of a single dose is rapidly administered intravenously, and 90% is infused over 30 minutes to 1 hour.
- the cytoprotective agent of the present invention is used without particular limitation as long as the possibility of cell damage due to ischemia is increased.
- Examples of the “time when the possibility of cell injury increases” include a time when suffering from the various thrombosis mentioned above. Further, it may be mentioned during or after treatment of thrombosis using anticoagulant, antiplatelet agent, thrombolytic agent and the like. Or the period after recovering from these thrombosis can be mentioned, In this period, it can also use prophylactically. In addition, if there is a possibility of cell damage due to ischemia, it may be used without being limited to the above-mentioned period.
- the cytoprotective agent of the present invention may be used alone or in combination with at least one thrombolytic agent.
- a thrombolytic agent By combining the cytoprotective agent of the present invention and a thrombolytic agent, dysfunction due to ischemia-reperfusion injury caused by thrombolysis by the thrombolytic agent can be effectively suppressed, and the therapeutic effect can be enhanced. Can be expected.
- the cytoprotective agent of the present invention can be used simultaneously with the thrombolytic agent or at different times. Examples of thrombolytic agents that can be used in combination include alteplase, urokinase, desmoteplase, and monteplase.
- the cytoprotective agent of the present invention is effective to use for a patient group in which thrombolytic drugs are not indicated because time has elapsed since onset. In the case of cerebral infarction, it is effective to use the cytoprotective agent of the present invention for a patient who has passed 3 hours or more after the onset and cannot administer alteplase, for example.
- the onset time is defined as the last non-onset time (the time when the patient was finally confirmed asymptomatic).
- the cytoprotective agent of the present invention When the cytoprotective agent of the present invention is used for thrombosis (including thromboembolism), it is effective to use it for patients with contraindicated thrombolytic drugs. Alternatively, it is also effective to use the cytoprotective agent of the present invention for a patient who has stopped administration of a thrombolytic drug due to signs or symptoms that should be contraindicated during the administration of the thrombolytic drug. Contraindications include predisposition to bleeding, bleeding, hypertension, and abnormal blood sugar. For example, the cytoprotective agent of the present invention may be used for patients who cannot be administered a thrombolytic drug due to an increased risk of intracranial hemorrhage in cerebral infarction.
- the cytoprotective agent of the present invention when used for thrombosis (including thromboembolism), it is effective to use it for patients who are not usually used for treatment among thrombosis.
- Such patients include, for example, patients with transient cerebral ischemic attacks, disseminated intravascular coagulation syndrome, thrombotic microangiopathy, thrombophlebitis, deep vein thrombosis, idiopathic thrombosis.
- cytoprotective agent of the present invention may be used without being limited to human use.
- domestic animals such as a cow, a horse, and a sheep
- pets such as a dog, a cat, and a monkey.
- the present invention includes a method for treating ischemic injury comprising administering a drug containing the triprenyl phenol compound according to the present invention to a patient who has developed ischemic injury.
- the “treatment” may be any improvement in symptoms, and this term also includes suppression of severity and reduction or alleviation of symptoms.
- the severity of ischemic injury can be suppressed, or symptoms can be reduced or alleviated.
- the dosage, administration interval, administration period, administration method and the like of the drug containing the triprenylphenol compound according to the present invention in the method for treating ischemic injury of the present invention are the same as those of the above-described cytoprotective agent of the present invention.
- the ischemic disorder treatment method of the present invention is preferably applied to thrombosis (including thromboembolism) as an ischemic disorder, and more preferably applied to cerebral infarction.
- One aspect of the method for treating ischemic injury of the present invention comprises administering a drug containing the triprenyl phenol compound according to the present invention to a patient at a time when the possibility of cell injury caused by ischemia increases. How to treat a disorder.
- the “time when the possibility of cell injury increases” is as described above.
- the treatment method is preferably applied to thrombosis (including thromboembolism) as an ischemic disorder, and more preferably applied to cerebral infarction.
- One aspect of the method for treating ischemic injury of the present invention is a method for treating ischemic injury comprising using a drug containing the triprenylphenol compound according to the present invention together with at least one thrombolytic agent.
- the treatment method is preferably applied to thrombosis (including thromboembolism) as an ischemic disorder, and more preferably applied to cerebral infarction.
- the thrombolytic agent that can be used together with the drug containing the triprenyl phenol compound according to the present invention include alteplase, urokinase, desmoteplase, and monteplase.
- the drug containing the triprenyl phenol compound according to the present invention can be administered to a patient simultaneously with the thrombolytic drug or at different times.
- One embodiment of the method for treating ischemic injury of the present invention is a method for treating ischemic injury comprising administering a drug containing the triprenyl phenol compound according to the present invention to a patient who has passed 3 hours or more after onset.
- the treatment method is preferably applied to thrombosis (including thromboembolism) as an ischemic disorder, and more preferably applied to cerebral infarction.
- cerebral infarction it is effective to use a drug containing the triprenylphenol compound according to the present invention for a patient who has not been able to administer alteplase after 3 hours or more after onset.
- the onset time is defined as the last non-onset time (the time when the patient was finally confirmed asymptomatic).
- One aspect of the method for treating an ischemic disorder of the present invention is a method for treating an ischemic disorder comprising administering a drug containing the triprenylphenol compound according to the present invention to a patient having a contraindicated thrombolytic drug.
- the treatment method is preferably applied to thrombosis (including thromboembolism) as an ischemic disorder, and more preferably applied to cerebral infarction.
- Contraindications include predisposition to bleeding, bleeding, hypertension, and abnormal blood sugar.
- a drug containing the triprenyl phenol compound according to the present invention can be administered to a patient who cannot administer a thrombolytic drug due to an increased risk of intracranial hemorrhage.
- One aspect of the method for treating ischemic injury of the present invention is a method for treating ischemic injury comprising administering a drug containing the triprenylphenol compound according to the present invention to a patient for which a thrombolytic drug is not normally used for treatment. It is.
- the treatment method is preferably applied to thrombosis (including thromboembolism) as an ischemic disorder, and more preferably applied to cerebral infarction.
- Patients who usually do not use thrombolytic drugs for treatment include, for example, transient cerebral ischemic attacks, disseminated intravascular coagulation syndrome, thrombotic microangiopathy, thrombophlebitis, deep vein thrombosis, idiopathic Examples include patients with thrombosis.
- One aspect of the method for treating ischemic injury of the present invention comprises administering a drug containing the triprenyl phenol compound according to the present invention to a patient who has or is predicted to have ischemia reperfusion injury. It is a method for treating blood disorders.
- the treatment method is preferably applied to thrombosis (including thromboembolism) as an ischemic disorder, and more preferably applied to cerebral infarction.
- ischemia-reperfusion injury can be prevented, the severity thereof can be suppressed, or the symptoms can be reduced or alleviated.
- Example 1 ⁇ Production of cerebral infarction model using acetic acid> [Gerbil cerebral infarction model] Anesthesia was induced by inhalation of male gerbils (weight 55-65 g) by inhalation of 5% isoflurane (trade name Escaine, Mylan Pharmaceutical Co., Ltd.), and anesthesia was maintained with 1-1.5% isoflurane. After fixing to the dorsal position and removing the hair from the neck, it was disinfected with 70% ethanol. A midline incision was made in the neck, the right common carotid artery was peeled off, blood flow was temporarily blocked with a clamp, and a thrombus was created by applying 100% acetic acid to the right common carotid artery 30 times.
- isoflurane trade name Escaine, Mylan Pharmaceutical Co., Ltd.
- the blood vessel was resumed and placed on the blood stream to send a thrombus into the brain, resulting in ischemia and cerebral infarction.
- the surgical field was closed with instant adhesive (trade name Aron Alpha, Toagosei Co., Ltd.) and disinfected with 70% ethanol.
- neurological symptoms 24 hours after the start of surgery, neurological symptoms were observed as an index of cerebral infarction. Many kinds of peculiar neurological symptoms such as extension of the hind limb and one-way rotation have been reported due to cerebral infarction, and the method proposed by Longa et al. (Stroke 1989; 20: 84-91) was used as the evaluation method. Asymptomatic; 0 points, leg extension; 1 point, unidirectional turning; 2 points, tilt posture; 3 points, peristalsis; and 4 points, postoperative neurological symptoms were determined.
- mice were decapitated under isoflurane anesthesia. The skin and the skull were peeled off and the head was opened. After the optic nerve was cut, the brain was removed, and the attached hair was removed by immersion in physiological saline. The extracted brain was placed in a brain matrix (RBM-1000C, ASI Instruments), and coronal cuts were made from the frontal pole by 2 mm thickness. Sections were soaked in 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) and incubated for 30 minutes in a 37 ° C. CO 2 incubator for staining.
- TTC 2,3,5-triphenyl-2H-tetrazolium chloride
- TTC colorless TTC is reduced by hydrogen released by the action of dehydrogenase contained in normal cells to become 1,3,5-triphenylformazan (TPF) insoluble in dark red water, and normal cells are stained. .
- the unstained region was defined as the infarct region.
- the corpus callosum the traffic fiber that connects the left and right cerebral hemispheres, is white because it is not dyed.
- image processing was performed with the corpus callosum removed.
- brain slice images were obtained under a stereomicroscope using a digital camera.
- Image analysis software Image J (Version 1.4) was used for the analysis of the infarct area ratio.
- the corpus callosum was removed and the entire image was subjected to monochrome processing. From this grayscale image, the horizontal axis represents the luminance (0-255), the vertical axis represents the frequency of occurrence (or frequency) of each luminance, and a histogram with the entire brain as the target region was obtained.
- Infarct area ratio (%) total area of infarct area excluding corpus callosum ⁇ total area of entire brain ⁇ 100
- Example 2 ⁇ Comparison of effects of various drugs in gerbil cerebral infarction model> Using the gerbil cerebral infarction model, the improvement effect of SMTP-7, alteplase and edaravone (brain protective agent) on infarct area ratio and neurological symptoms was compared. At the same time, the degree of improvement by dose was also examined.
- SMTP-7 was purified from the culture obtained when L-ornithine was added as an organic amino compound to the Stachybotrys microspora IFO30018 medium using the method described in Japanese Patent Application Laid-Open No. 2004-224738. I got it.
- 0.3N NaOH and physiological saline (0.9% NaCl) were added to prepare a 50 mg / ml solution. Thereafter, it was adjusted to 10 mg / ml and pH to be weakly alkaline using 0.3N HCl and physiological saline, sterilized by filtration, and subdivided and stored frozen at ⁇ 30 ° C.
- SMTP-7 was diluted with physiological saline as needed.
- SMTP-7 was frozen and stored at 1 mg / ml in physiological saline immediately before the test.
- Alteplase (trade name: Activacin, Kyowa Hakko Kirin Co., Ltd.) was dissolved in 1.03 mg / ml with the lysis solution for Activacin.
- Edaravone (trade name Radicut, Mitsubishi Tanabe Pharma Corporation) used a 1.5 mg / kg stock solution. These drugs were diluted with saline as needed.
- the dosage of SMTP-7 is 0.1 mg / kg, 1 mg / kg and 10 mg / kg, the dosage of alteplase is 0.01 mg / kg, 0.1 mg / kg and 10 mg / kg, and the dosage of edaravone is 1 mg / kg, 3 mg / kg and 10 mg / kg.
- SMTP-7 and alteplase were started 1 hour, 3 hours or 6 hours after the start of ischemia in the gerbil cerebral infarction model.
- Edaravone started administration immediately after the onset of ischemia (0 hour), 1 hour or 3 hours later.
- anesthesia was introduced by inhalation of 5% isoflurane so that administration could be started after the predetermined time had elapsed from the start of ischemia, and then anesthesia was maintained at 1 to 1.5%.
- a 27G needle-attached polyethylene catheter was inserted into the left femoral vein. From this catheter, SMTP-7 and alteplase were administered at a rapid rate of 10% of the dose, and the remaining dose was continuously administered for 30 minutes. Edaravone was administered continuously for 30 minutes. There were 6 gerbils per condition.
- SMTP-7 When SMTP-7 was administered 1 hour after the onset of ischemia, the infarct area ratio and neurological symptoms improved in a dose-dependent manner. In the 10 mg / kg administration group, both the infarct area ratio and neurological symptoms were significantly improved compared to the control group.
- the ratio of infarct area in the SMTP-7 (10 mg / kg) administration group was controlled even if the administration start time after the start of ischemia was delayed (administration started 1 hour, 3 hours and 6 hours after the start of ischemia) There was a statistically significant improvement compared to the group.
- the improvement rate of the group that started administration 1 hour after the start of ischemia was 65.7%, which was almost the same as the improvement rate of 78.9% when alteplase was administered.
- the neurological symptom score in the SMTP-7 (10 mg / kg) administration group was not significantly improved in the group started administration 1 hour and 3 hours after ischemia compared to the alteplase (10 mg / kg) administration group. A statistically significant improvement was observed compared to the control group. In the SMTP-7 (10 mg / kg) administration group, which was started 6 hours after the start of ischemia, no statistically significant improvement was observed, but an improvement trend was observed.
- the infarct area ratio and neurological symptoms improved in a dose-dependent manner.
- both the infarct area ratio and neurological symptoms were significantly improved compared to the control group.
- the ratio of infarct area in the alteplase (10 mg / kg) administration group was significantly improved by administration 1 hour after the start of ischemia compared to the control group. Although an improvement trend was observed in the administration 3 hours after the start of ischemia, no significant improvement was observed compared to the control group, and there was no statistically significant difference in the administration 6 hours after the start of ischemia compared to the control group. I could't see it.
- the neurosymptom score of the alteplase (10 mg / kg) administration group was significantly improved compared to the control group when administered 1 hour after the start of ischemia and after 3 hours. However, no statistically significant difference was observed in the administration 6 hours after the start of ischemia compared to the control group.
- Example 3 ⁇ Comparison of effects of SMTP-7 and alteplase in mouse cerebral infarction model> Using a mouse cerebral infarction model, the improvement effects of SMTP-7 and alteplase on the infarct area ratio, neurological symptoms, and edema rate were compared. At the same time, the effect of improvement by the dose of SMTP-7 was also examined.
- SMTP-7 and alteplase were prepared in the same manner as in Example 2. 10 mg / kg was administered rapidly at 10% from the femoral vein 1 hour or 3 hours after the start of ischemia in the mouse cerebral infarction model, and the remaining was continuously administered for 30 minutes. Further, 0.1 mg / kg and 1 mg / kg of SMTP-7 were rapidly administered 10% from the femoral vein 1 hour after the onset of ischemia in the mouse cerebral infarction model, and the rest were continuously administered for 30 minutes. Six mice were used for each condition.
- Edema rate (%) (volume of ischemic hemisphere ⁇ volume of contralateral hemisphere) ⁇ volume of contralateral hemisphere ⁇ 100
- Table 8 shows the evaluation results of SMTP-7 and alteplase at a dose of 10 mg / kg.
- FIG. 3 shows the infarct area ratio
- FIG. 4 shows the neurological symptom score
- FIG. 5 shows the edema rate (*; P ⁇ 0.05, **; P ⁇ 0.01, #; P ⁇ 0.05, ##; P ⁇ 0.01).
- Table 9 shows the evaluation results when the dose of SMTP-7 was changed.
- infarct area ratio of the control group was significantly improved in the group in which administration was started 1 hour after the start of ischemia compared to the control group. There was no significant improvement in the group that started administration after 3 hours.
- the percentage of infarct area in the SMTP-7 (10 mg / kg) administration group was significantly improved compared to the control group in both the group started administration after 1 hour and the group started administration after 3 hours.
- a significant increase in the neurological symptom score of the control group was observed compared to the Sham group.
- the neurosymptom score of the alteplase administration group was significantly improved in the group that started administration after 1 hour. There was no significant improvement in the group that started administration after 3 hours.
- the neurological symptom score of the SMTP-7 (10 mg / kg) administration group showed a statistically significant improvement compared to the control group in both the group started administration after 1 hour and the group started administration after 3 hours.
- the infarct area ratio, neurological symptom score, and edema rate were significantly improved in the SMTP-7 (10 mg / kg) administration group and the SMTP-7 (1 mg / kg) administration group as compared to the control group.
- Example 4 ⁇ Comparison of the effects of SMTP-7 and alteplase on cerebral blood flow> Using a mouse cerebral infarction model, the improvement effect of SMTP-7 and alteplase on cerebral blood flow was compared.
- SMTP-7 and alteplase were prepared in the same manner as in Example 2. SMTP-7 and alteplase were administered at 10 mg / kg rapidly after 1 hour from the start of ischemia in the mouse cerebral infarction model, 10% from the femoral vein, and the rest for 30 minutes. Three mice were used for each condition.
- Cerebral blood flow was measured at 6 points before the start of ischemia, immediately after the start of ischemia, 0 hours after the end of drug administration (immediately after the end of drug administration), 1 hour, 3 hours, and 24 hours.
- the cerebral blood flow was measured by incising the skin of the mouse head to expose the skull, and measuring the blood flow over the entire brain surface using a laser Doppler device (moorFLPI, moor instruments Ltd, UK). The analysis was performed using moorFLPI (Version 2.1), and the ratio (%) of each group with respect to that before the start of ischemia was evaluated. The results are shown in Table 10.
- SMTP-6 was obtained in the same manner as SMTP-7 except that L-tryptophan was used as the added organic amino compound.
- SMTP-22 was obtained in the same manner as SMTP-7 except that 4-aminosalicylic acid was used as the added organic amino compound.
- SMTP-25 was obtained in the same manner as SMTP-7 except that 3-aminosalicylic acid was used as the added organic amino compound.
- SMTP-43 was obtained in the same manner as SMTP-7 except that L-phenylglycine was used as the added organic amino compound.
- SMTP-44D was obtained in the same manner as SMTP-7 except that D-4-hydroxyphenylglycine was used as the added organic amino compound.
- triprenyl phenol compounds were prepared to a 50 mg / ml solution by adding 0.3 N NaOH and physiological saline (0.9% NaCl). Thereafter, it was adjusted to 10 mg / ml and pH to be weakly alkaline using 0.3 N HCl and physiological saline, sterilized by filtration, and subdivided and stored frozen at ⁇ 30 ° C.
- the above triprenylphenol compound was frozen and stored at 1 mg / ml in physiological saline immediately before the test. 10 mg / kg each was rapidly administered 10% from the femoral vein 1 hour after the start of ischemia in the mouse cerebral infarction model, and the remaining was continuously administered for 30 minutes. Six mice were used for each condition.
- Example 3 24 hours after the start of surgery, the infarct area ratio, neurological symptoms, and edema rate of each mouse were evaluated in the same manner as in Example 3. The evaluation results are shown in Table 11. Table 11 also shows the evaluation results of the group in which administration of SMTP-7 (10 mg / kg) in Example 3 was started after 1 hour.
- Example 6 ⁇ Examination of parameter variation by real-time RT-PCR method> Using a mouse cerebral infarction model, real-time RT in a triprenyl phenol compound (10 mg / kg) administration group, alteplase (10 mg / kg) administration group, and alteplase (10 mg / kg) and aspirin (10 mg / kg) administration group -Fluctuations in parameters involved in inflammation were assessed by PCR.
- the parameters were IL-1 ⁇ , TNF- ⁇ and IL-6, which are typical parameters involved in inflammation.
- SMTP-7, SMTP-6, SMTP-22, SMTP-25, SMTP-43 and SMTP-44D were used as the triprenylphenol compound.
- the triprenylphenol compound and alteplase were prepared in the same manner as in Example 2 and Example 5.
- Aspirin was dissolved in physiological saline to 1 mg / ml.
- Administration of SMTP-7 (10 mg / kg) and alteplase (10 mg / kg) was started 1 hour or 3 hours after the onset of ischemia.
- the administration method was the same as in Example 3.
- Alteplase (10 mg / kg) and aspirin (10 mg / kg) were administered together 3 hours after the start of ischemia.
- Alteplase was administered in the same manner as in Example 3, and aspirin was rapidly injected intravenously from the femoral vein.
- Administration of SMTP-6, SMTP-22, SMTP-25, SMTP-43 and SMTP-44D was started 1 hour after the onset of ischemia.
- the administration method was the same as in Example 5. Six mice were used for each condition.
- Krebs-HEPES buffer was perfused from the heart, and the brain was removed.
- the fourth slice was divided into the left brain and the right brain, and homogenized with 1 mL of TRIZOL (registered trademark) Reagent (Invitrogen). After incubating at room temperature for 5 minutes, chloroform (0.2 mL) was added and mixed for 15 seconds, followed by incubation at room temperature for 3 minutes. The sample was centrifuged at 12,000 ⁇ g for 15 minutes at 4 ° C.
- RNA moves to the aqueous layer, the aqueous layer was collected, 0.5 mL of isopropyl alcohol was added, incubated at room temperature for 10 minutes, and then centrifuged at 12,000 ⁇ g for 10 minutes at 4 ° C. After centrifugation, since the pellet was formed in the tube, the supernatant was removed. To wash the pellet, 1 mL of 75% ethanol was added and stirred, and centrifuged at 7,500 ⁇ g for 5 minutes at 4 ° C. . The supernatant was again removed and air-dried for 8 minutes, and the pellet containing RNA was dissolved in 100 ⁇ L of RNase-free water.
- RNA was reverse transcribed by Thermal Cycler 2720 (Applied Biosystems) to prepare cDNA. That is, 4 ⁇ L of 5 ⁇ VILO TM ReactionMix, 2 ⁇ L of 10 ⁇ SuperScript (registered trademark) Enzyme® Mix, and 1 ⁇ g of each RNA were mixed and made 20 ⁇ L using RNase-free water.
- reverse transcription was performed under the following conditions. The reaction was carried out at 25 ° C. for 10 minutes, 42 ° C. for 60 minutes, and 85 ° C. for 5 minutes, and then stored at 4 ° C.
- Real-time RT-PCR was performed using SYBR (registered trademark) GreenERTM (qPCR) SuperMix (for ABI PRISM (registered trademark) (Invitrogen) with ABI PRISM (7000) using the cDNA as a template.
- Real-time RT-PCR was performed using the above sample under the following conditions. After reacting at 50 ° C. for 2 minutes and at 95 ° C. for 15 minutes, 40 cycles of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds were repeated. Each parameter variation was calculated by the calibration curve method. ⁇ -actin was measured as an endogenous control, and each parameter was calculated as a relative value to ⁇ -actin. The results are shown in Table 12. In addition, the numerical value of Table 12 is a numerical value on a disease side (right brain).
- ⁇ -actin forward 5′-CCTCTCCTTCTGGGTATGGAATC-3 ′ (SEQ ID NO: 1)
- ⁇ -actin reverse 5′-TGCTAGGAGCCAGCAGTAATC-3 ′ (SEQ ID NO: 2)
- IL-1 ⁇ Qiagen, QT01048355
- TNF- ⁇ Qiagen
- IL-6 Qiagen, QT00098875)
- SMTP-22, SMTP-25 administration group, the SMTP-43 administration group, and the SMTP-44D administration group no significant increase in IL-1 ⁇ , TNF- ⁇ , and IL-6 was observed, and SMTP-22, SMTP-25, SMTP-43, and SMTP-44D suppressed the increase of these parameters.
- Example 7 ⁇ Examination of free radical scavenging activity of triprenylphenol compounds>
- H-ORAC hydrophilic oxygen radical absorbance capacity
- mH-ORAC modified H-ORAC
- the SMTP compounds evaluated by H-ORAC were each used as an aqueous sodium salt solution, and the compounds evaluated by mH-ORAC were each used as an acetone solution.
- the test compound was appropriately diluted with a buffer (75 mM phosphate buffer, pH 7.4) and used for measurement.
- mH-ORAC the test compound was appropriately diluted with a 50% (v / v) acetone solution obtained by diluting an acetone solution with water to prepare a solution 40 times the final concentration. This solution was diluted 10 times with a buffer and used for measurement (acetone final concentration 1.25%).
- Trolox 500 ⁇ M / buffer was appropriately diluted with buffer.
- Example 8 ⁇ Examination of plasmin activity by fibrinogen zymography> The amount of plasmin ⁇ ⁇ 2 -antiplasmin in plasma was measured by fibrinogen zymography, and the plasmin activity when a triprenylphenol compound was administered to a mouse cerebral infarction model was evaluated.
- a triprenyl phenol compound SMTP-7, SMTP-6, SMTP-22, SMTP-25, SMTP-43, and SMTP-44D were used.
- the triprenyl phenol compound was prepared in the same manner as in Example 2 and Example 5.
- the administration of a triprenyl phenol compound at 10 mg / kg was started 1 hour after the start of ischemia.
- the administration method was the same as in Example 3. Six mice were used for each condition.
- sample buffer 125 mM Tris-HCl (pH 6.8), 4% (w / v) SDS, 0.04% (w / v) bromophenol blue, 20% (w / v) Sucrose
- sample buffer 125 mM Tris-HCl (pH 6.8), 4% (w / v) SDS, 0.04% (w / v) bromophenol blue, 20% (w / v) Sucrose
- a gel for electrophoresis As a gel for electrophoresis, a gel in which a stacking gel was overlaid on a 7.5% running gel containing 2 mg / ml fibrinogen (manufactured by SIGMA) was prepared. Based on the quantified amount of protein, 3-15 ⁇ l of sample was applied so that the same amount of protein was applied. After electrophoresis at 10 mA / plate for 3 hours, the stacking gel was removed and washed twice with a washing solution (2.5% triton X-100) of about 100 ml / gel for 30 minutes.
- a washing solution (2.5% triton X-100
- incubation buffer 0.1 M glycine-50 mM Tris-HCl, pH 8.3 at 37 ° C.
- the image was taken from the gel with an imaging analyzer (printed by ATTO, Printgraph (AE-6933FXCF)), and the band intensity was evaluated. The results are shown as a ratio of 0 hour after the end of administration in the control group (immediately after the end). The results are shown in Table 14.
- Example 9 ⁇ Examination of therapeutic effect of SMTP-7 in cynomolgus monkey cerebral infarction model> The effect of SMTP-7 on reducing the cerebral infarct size and improving neurological symptoms in a cynomolgus monkey cerebral infarction model was examined by the following test method.
- Test substance administration ⁇ Test substance administration route: Intravenous administration ⁇ Test substance dosage: 10 mg / kg Test substance administration method: The administration volume was 10 mL / kg. A surflow indwelling needle was placed in the left saphenous vein, and 1 mg / kg (1 mL / kg) was rapidly administered for 5 seconds after 1 hour of ischemia. Thereafter, 9 mg / kg (9 mL / kg) was 30 by using a syringe pump. Administration was continued for a minute. -Preparation of administration solution: SMTP-7 was dissolved in physiological saline to 1 mg / mL.
- SMTP-7 sodium salt including 63.1 mg of SMTP-7
- a magnetic stirrer stir and dissolve. Sonication was performed as needed. After dissolution, the solution was sterilized by filtration with a sterile filter (0.22 ⁇ m, made of cellulose acetate). The dosing solution was prepared at the time of use. After the preparation was completed, it was placed in a 37 ° C. hot water bath until just before use, and the administration was terminated within 4 hours after the preparation.
- the fundus bone outside the optic nerve exit was removed with a dental drill to expose the dura mater.
- the dura mater was carefully detached to confirm the middle cerebral artery.
- the origin of the middle cerebral artery was separated from the arachnoid membrane, and a light irradiation probe for preparing a thrombus was fixed on the middle cerebral artery. Further, a probe of a pulse Doppler blood flow meter (Crystal Bio, PDV-20) was placed at the distal portion of the probe.
- Rose Bengal (Wako Pure Chemical Industries, Ltd.) (20 mg / kg) was started intravenously in 6 minutes, and green light (1.4 million LUX) with a wavelength of 540 nm was irradiated for 20 minutes to clot the middle cerebral artery. Obstructed.
- the start of ischemia was defined as the start of administration of light irradiation and rose bengal.
- the wound was closed. A series of operations were all performed under a surgical microscope to minimize intraoperative bleeding as much as possible.
- the rectal temperature of the animals during the operation was monitored, and the animals were kept warm with a heating pad so that the body temperature of the animals was in the physiological range (37.0-38.5 ° C.).
- Infection prevention measures are carried out by intramuscular administration of penicillin G (Meiji Seika Co., Ltd.) (100,000 Unit / head / day), and buprenorphine hydrochloride (Repetan, Otsuka Pharmaceutical Co., Ltd.) 0.02 mg / Analgesic treatment with head administration was performed.
- Time to Occlusion and Total Occlusion Time are shown in Table 15 below. There was no significant difference in Time to Occlusion and Total Occlusion Time between the vehicle group and the SMTP-7 administration group. Since the administration of the vehicle and SMTP-7 was started 60 minutes after ischemia, the total occlusion time was divided into the occlusion times from 60 minutes to 120 minutes after the onset of ischemia. As shown in FIG. 16, no significant occlusion time shortening effect was observed.
- SMTP-7 (10 mg / kg) was administered 1 hour after ischemia, and as a result, the infarct size was significantly reduced 24 hours after ischemia. It became clear. As a result, it was considered that the neurosymptom was significantly improved and the hemorrhagic infarction was significantly reduced. Although the cerebral infarction focus reducing action of SMTP-7 is thought to be based on the thrombolytic action of this drug, the results of this study did not affect the occlusion time of the middle cerebral artery. This may be because the blood flow measurement time after administration of SMTP-7 was short.
- SMTP-7 significantly reduced infarct size of the basal ganglia centered on the striatum.
- the striatum is the most vulnerable part to ischemia, and is a region that receives blood flow control from the penetrating artery from the origin of the middle cerebral artery.
- SMTP-7 gradually dissolved the thrombus from the origin side of the middle cerebral artery that was obstructed thromboticly, resulting in a marked infarction of the basal ganglia It is thought that the size was reduced.
- Examples 1 to 9 are summarized as follows. As is clear from the results of Examples 2 and 3, it was confirmed that SMTP-7 exhibits an action of suppressing the infarct area ratio and the expression of neurological symptoms in cerebral infarction model animals. As is clear from the results of Example 4, it was confirmed that SMTP-7 gradually recovered cerebral blood flow after infarction in cerebral infarction model animals. From this, it is presumed that SMTP-7 has a low risk of causing ischemia-reperfusion injury accompanying rapid blood flow recovery. As is clear from the results of Example 6, it was confirmed that SMTP-7 exhibits an action of suppressing the increase in inflammation parameters IL-1 ⁇ , TNF- ⁇ and IL-6 in cerebral infarction model animals.
- SMTP-7 was confirmed to exhibit a strong scavenging action against free radicals, which are one of the damaging factors in cell injury caused by ischemia, and has antioxidant activity. It was confirmed. As is clear from the results of Example 8, it was confirmed that SMTP-7 exhibits an action of increasing plasmin activity in blood. As is clear from the results of Example 9, in the cerebral infarction model animal, SMTP-7 exhibits a significant reduction action of infarct size, a significant improvement action of neurological symptoms, and a significant reduction action of hemorrhagic infarction. Was confirmed. From the above, the composition containing SMTP-7 can be used as a cytoprotective agent having an effect of suppressing dysfunction caused by ischemia.
- Example 5 it was confirmed that SMTP-22 and SMTP-43 have an action of suppressing the infarct area ratio and the expression of neurological symptoms in cerebral infarction model animals.
- Example 6 SMTP-22, SMTP-25, SMTP-43 and SMTP-44D increased IL-1 ⁇ , TNF- ⁇ and IL-6, which are inflammatory parameters, in cerebral infarction model animals. It was confirmed that the action which suppresses was shown.
- SMTP-6, SMTP-22, SMTP-25, SMTP-43, and SMTP-44D are against free radicals that are one of the damaging factors in cytotoxicity caused by ischemia. It was confirmed to have a strong erasing action and to have an antioxidant activity.
- Example 8 it was confirmed that SMTP-22, SMTP-25, and SMTP-43 have an action of increasing plasmin activity in blood.
- SMTP-6, SMTP-25, and SMTP-44D it was not confirmed in Example 5 that they have the action of suppressing the infarct area ratio and the expression of neurological symptoms.
- SMTP-6 has free radical scavenging activity.
- SMTP-25 suppresses an increase in inflammatory parameters, has a free radical scavenging activity, and increases plasmin activity in blood.
- SMTP-44D was confirmed to suppress the increase of inflammatory parameters and to have free radical scavenging activity. It is speculated that SMTP-6, SMTP-25, and SMTP-44D have an effect of suppressing the infarct area ratio and the expression of neurological symptoms if the dose to the model animal is appropriately adjusted.
- composition containing any one selected from SMTP-6, SMTP-22, SMTP-25, SMTP-43 and SMTP-44D has an effect of suppressing dysfunction caused by ischemia. It can be used as a cytoprotective agent.
- SMTP-0, SMTP-1, SMTP-4, SMTP-5D, SMTP-8, SMTP-11-14, SMTP-18-21, SMTP-23, SMTP-24, SMTP-26 to 29, SMTP-36, SMTP-37, SMTP-42, SMTP-43D, SMTP-44, SMTP-46 and SMTP-47 are one of the damaging factors in cytotoxicity caused by ischemia. It was confirmed that the free radical exhibits an erasing action equivalent to or stronger than Trolox and has an antioxidant activity.
- the composition containing the triprenyl phenol compound according to the present invention can be used as a cytoprotective agent having an effect of suppressing dysfunction caused by ischemia. Moreover, the composition containing the triprenyl phenol compound according to the present invention can be used in a method for treating ischemic injury comprising administering the composition to a patient who has developed ischemic injury.
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Abstract
Description
R1は、下記(A)から(D)のいずれか1つを表す。
(A)天然アミノ酸、天然アミノ酸のD体、並びに天然アミノ酸及び天然アミノ酸のD体においてカルボキシ基を水素原子、ヒドロキシ基又はヒドロキシメチル基に置き換えた化合物からなる群より選択されるアミノ化合物から、1個のアミノ基を除いた残基(ただし、-(CH)2-OHは除く)、
(B)カルボキシ基、水酸基、スルホン酸基及び第二アミノ基からなる群より選択される少なくとも1つを置換基として若しくは置換基の一部として有する芳香族基、又は第二アミノ基を含み且つ窒素原子を含んでいてもよい芳香族基、
(C)下記式(II-1)で表される芳香族アミノ酸残基(式中R3はあってもなくてもよい置換基であって、水酸基、カルボキシ基及び炭素数1~5のアルキル基からなる群より選択される少なくとも1つの置換基を表し、nは0又は1の整数を表す。)、
「機能不全の抑制」とは、「機能不全の改善」をも含む概念で、その効果は、損傷領域の大きさ、各病気の症状の程度によって評価することができる。例えば、脳の機能不全に関しては、CT、MRI、脳血管造影などで認められる脳損傷領域の大きさ、浮腫の大きさ等で効果を評価でき、また、症状として現れる神経症状、日常生活動作障害、運動麻痺などの指標で効果を評価できる。さらには、組織の炎症に伴って発現が増強するインターロイキン-1β(IL-1β)、腫瘍壊死因子-α(TNF-α)、インターロイキン-6(IL-6)等の上昇度で効果を評価できる。
分子量1000以下の置換基としては、細胞保護剤としての効果の観点から、分子量800以下の置換基が好ましく、分子量700以下の置換基がより好ましく、分子量600以下の置換基が更に好ましい。
本発明に係るトリプレニルフェノール化合物の具体例の一つは、下記一般式(II)で表される化合物である。
R1は、下記(A)から(D)のいずれか1つを表す。
(A)天然アミノ酸、天然アミノ酸のD体、並びに天然アミノ酸及び天然アミノ酸のD体においてカルボキシ基を水素原子、ヒドロキシ基又はヒドロキシメチル基に置き換えた化合物からなる群より選択されるアミノ化合物から、1個のアミノ基を除いた残基(ただし、-(CH)2-OHは除く)、
(B)カルボキシ基、水酸基、スルホン酸基及び第二アミノ基からなる群より選択される少なくとも1つを置換基として若しくは置換基の一部として有する芳香族基、又は第二アミノ基を含み且つ窒素原子を含んでいてもよい芳香族基、
(C)下記式(II-1)で表される芳香族アミノ酸残基(式中R3はあってもなくてもよい置換基であって、水酸基、カルボキシ基及び炭素数1~5のアルキル基からなる群より選択される少なくとも1つの置換基を表し、nは0又は1の整数を表す。)、
前記(A)は、天然アミノ酸、天然アミノ酸のD体、並びに天然アミノ酸及び天然アミノ酸のD体においてカルボキシ基を水素原子、ヒドロキシ基又はヒドロキシメチル基に置き換えた化合物からなる群より選択されるアミノ化合物から、1個のアミノ基を除いた残基(ただし、-(CH)2-OHは除く)である。
天然アミノ酸として、例えば、α-アミノ酸として、グリシン、アラニン、スレオニン、バリン、イソロイシン、チロシン、システイン、シスチン、メチオニン、ヒスチジン、アスパラギン酸、グルタミン酸、アスパラギン、グルタミン、アルギニン、リシン、ヒドロキシリシン、オルニチン、シトルリン、ホモシステイン、3,4-ジヒドロキシフェニルアラニン、ホモシスチン、ジアミノピメリン酸、ジアミノプロピオン酸、セリン、ロイシン、フェニルアラニン及びトリプトファンなどが挙げられ、β-アミノ酸として、β-アラニンなどが挙げられ、γ-アミノ酸として、γ-アミノ酪酸及びカルニチンなどが挙げられ、δ-アミノ酸として、5-アミノレブリン酸及び5-アミノ吉草酸などが挙げられる。
前記(B)は、カルボキシ基、水酸基、スルホン酸基及び第二アミノ基からなる群より選択される少なくとも1つを置換基として若しくは置換基の一部として有する芳香族基、又は第二アミノ基を含み且つ窒素原子を含んでいてもよい芳香族基である。
前記芳香族基としては、例えば、下記構造式で表される化合物が挙げられる。
前記(C)は、下記式(II-1)で表される芳香族アミノ酸残基(式中R3はあってもなくてもよい置換基であって、水酸基、カルボキシ基及び炭素数1~5のアルキル基からなる群より選択される少なくとも1つの置換基を表し、nは0又は1の整数を表す。)である。
前記(D)は、-L1-L2-R4で表される置換基(式中、L1はカルボキシ基を有する炭素数1~4のアルキレン基である連結基を表し、L2は-NH-C(=O)-又は-NH-C(=S)-NH-で示される連結基を表し、R4は炭素数1~3のアルキルオキシ基を有する9-フルオレニルアルキルオキシ基又は下記式(II-2)で表される多複素環基を表す。)である。
本発明に係るトリプレニルフェノール化合物の具体例の一つは、下記一般式(III)で表される化合物である。
前記mは、0~9の整数を表し、好ましくは0~4の整数、より好ましくは1~3の整数、更に好ましくは1又は2である。
前記pは、0~9の整数を表し、好ましくは0~4の整数、より好ましくは1~3の整数、更に好ましくは1又は2である。
前記qは、0~9の整数を表し、好ましくは0~4の整数、より好ましくは1~3の整数、更に好ましくは1又は2である。
前記pが0の場合、m+qとしては、0~9の整数が好ましく、より好ましくは0~6の整数、更に好ましくは1~5の整数、特に好ましくは1~4の整数である。
2個のアミノ基を有する天然アミノ酸及び2個のアミノ基を有する天然アミノ酸のD体においてカルボキシ基を水素原子、ヒドロキシ基又はヒドロキシメチル基に置き換えた化合物として、H2N-(CH2)k-NH2(kは1~10の整数、好ましくは1~6の整数、より好ましくは1~4の整数である)が挙げられる。
なお、虚血に起因する細胞傷害の可能性があれば、上述した時期に制限されず使用してよい。
本発明の細胞保護剤と血栓溶解薬とを併用することにより、血栓溶解薬による血栓溶解に起因した虚血再灌流障害による機能不全を効果的に抑制することができ、治療効果を増強することが期待できる。この場合、本発明の細胞保護剤は、血栓溶解薬と同時に又は時間をかえて、用いることができる。
組み合わせて使用可能な血栓溶解薬としては、アルテプラーゼ、ウロキナーゼ、デスモテプラーゼ、モンテプラーゼ、などが挙げられる。
本発明の虚血性障害の治療方法により、虚血性障害の重症化の抑制、又は、症状の軽減若しくは緩和をすることができる。
本発明の虚血性障害の治療方法は、虚血性障害として血栓症(血栓塞栓症をも含む)に対する適用が好適であり、脳梗塞に対する適用がより好適である。
本発明に係るトリプレニルフェノール化合物を含む薬剤とともに用いうる血栓溶解薬としては、アルテプラーゼ、ウロキナーゼ、デスモテプラーゼ、モンテプラーゼ、などが挙げられる。本発明に係るトリプレニルフェノール化合物を含む薬剤は、血栓溶解薬と同時に又は時間をかえて、患者に投与することができる。
例えば脳梗塞の場合、発症後3時間以上が経過し、アルテプラーゼを投与することができない患者に、本発明に係るトリプレニルフェノール化合物を含む薬剤を用いることが有効である。なお、発症時刻が不明な場合は、最終未発症時刻(患者が無症状であることが最後に確認された時刻)をもって発症時刻とする。
禁忌としては、出血素因、出血、高血圧症、血糖異常などである。例えば、脳梗塞において、頭蓋内出血の危険性が増大するために血栓溶解薬を投与できない患者に、本発明に係るトリプレニルフェノール化合物を含む薬剤を投与することができる。
通常、血栓溶解薬が治療に用いられない患者としては、例えば、一過性脳虚血発作、播種性血管内凝固症候群、血栓性微小血管障害、血栓症静脈炎、深部静脈血栓症、特発性血栓症の患者が挙げられる。
上記の虚血性障害の治療方法により、虚血再灌流障害の予防、重症化の抑制、又は、症状の軽減若しくは緩和をすることができる。
<酢酸を用いた脳梗塞モデルの作製>
〔スナネズミ脳梗塞モデル〕
雄性スナネズミ(体重55~65g)を5%イソフルラン(商品名エスカイン、マイラン製薬(株))の吸入により麻酔導入後、1~1.5%イソフルランで麻酔を維持した。背位に固定し、頸部を除毛後、70%エタノールで消毒した。頸部を正中切開し、右総頸動脈を剥離後、クレンメで一時的に血流を遮断し、右総頸動脈に100%酢酸を30往復塗布することによって血栓を作製した。血流遮断から10分後、血管を再開通させ、血流にのせて血栓を脳内へ送り込むことによって虚血とし、脳梗塞を惹起した。瞬間接着剤(商品名アロンアルフア、東亞合成(株))で手術野を閉じ、70%エタノールで消毒した。
手術開始から24時間後に、脳梗塞の指標として神経症状を観察した。脳梗塞により後肢の伸展や一方向旋回等の特有な神経症状が多種類報告されているが、その評価法としてLongaらが提唱した方法 (Stroke 1989;20:84-91) の変法を用い、無症状;0点、足の伸展;1点、一方向旋回;2点、傾斜姿勢;3点、寡動;4点と評価することで、術後の神経症状の判定を行った。
上記の方法で神経症状を評価した後、イソフルラン麻酔下にて断頭した。皮膚と頭蓋骨を剥離して開頭し、視神経切断後に脳を摘出し、生理食塩水に浸して付着した体毛等を除去した。摘出した脳をブレインマトリックス(RBM-1000C、ASI Instruments)に入れ、前頭極から厚さ2mmずつ冠状切断を行った。切片を2%2,3,5-トリフェニル-2H-テトラゾリウムクロリド(TTC)に浸して、37℃のCO2インキュベーター内で30分間インキュベーションを行い染色した。無色のTTCは正常細胞に含まれる脱水素酵素の働きで遊離した水素で還元されて、暗赤色の水に不溶な1,3,5-トリフェニルホルマザン(TPF)となり、正常細胞が染色される。染色されていない部位(白色の部位)を梗塞領域とした。ただし、左右の大脳半球をつなぐ交通繊維である脳梁は染色されないため白色であるので、以下の解析では、脳梁を除いて画像処理をした。
梗塞領域割合(%)=脳梁を除いた梗塞領域の合計面積÷脳全体の合計面積×100
雄性ddY系マウス(体重35~45g) を用いて、上記のスナネズミ脳梗塞モデルの作製方法および評価方法と同様の方法で、脳梗塞モデルを作製し、神経症状と梗塞領域割合を評価した。
<スナネズミ脳梗塞モデルにおける各種薬剤の効果の比較>
スナネズミ脳梗塞モデルを用い、梗塞領域割合および神経症状に対する、SMTP-7、アルテプラーゼ及びエダラボン(脳保護薬)の改善効果を比較した。同時に投与量による改善の程度も検討した。
SMTP-7(10mg/kg)投与群の梗塞領域割合は、虚血開始後の投与開始時間が遅くなっても(虚血開始1時間後、3時間後、及び6時間後に投与開始)、対照群に比べ統計学的に有意な改善が認められた。虚血開始1時間後に投与開始した群の改善率は65.7%であり、アルテプラーゼを投与した場合の改善率78.9%とほぼ同等の効果を示した。
SMTP-7(10mg/kg)投与群の神経症状のスコアは、虚血開始1時間後、及び3時間後に投与開始した群では、アルテプラーゼ(10mg/kg)投与群に比べ顕著な改善ではないものの、対照群に比べ統計学的に有意な改善が認められた。虚血開始6時間後に投与開始したSMTP-7(10mg/kg)投与群では、対照群に比べ統計学的に有意な改善は認められないものの、改善傾向は認められた。
アルテプラーゼ(10mg/kg)投与群の梗塞領域割合は、虚血開始1時間後の投与では対照群に比べ有意な改善が認められた。虚血開始3時間後の投与では改善傾向は認められたが、対照群に比べ有意な改善は認められず、虚血開始6時間後の投与では対照群と比べ統計学的に有意な差は見られなかった。
アルテプラーゼ(10mg/kg)投与群の神経症状のスコアは、虚血開始1時間後、及び3時間後の投与では対照群に比べ有意な改善が認められた。しかし、虚血開始6時間後の投与では、対照群と比べ統計学的に有意な差は見られなかった。
エダラボン(3mg/kg)投与群の梗塞領域割合は、虚血開始0時間後と1時間後の投与では、改善傾向が認められた。虚血開始3時間後の投与では改善が認められなかった。
エダラボン(3mg/kg)投与群の神経症状のスコアは、虚血開始0時間後と1時間後の投与では、有意な改善が認められた。虚血開始3時間後の投与では、有意な改善は認められなかった。
<マウス脳梗塞モデルにおけるSMTP-7とアルテプラーゼの効果の比較>
マウス脳梗塞モデルを用い、梗塞領域割合、神経症状、及び浮腫率に対する、SMTP-7とアルテプラーゼの改善効果を比較した。同時にSMTP-7の投与量による改善の効果も検討した。
浮腫率(%)=(虚血を起こした大脳半球の体積-反対側の大脳半球の体積)÷反対側の大脳半球の体積×100
アルテプラーゼ投与群の梗塞領域割合は、虚血開始1時間後に投与開始した群では、対照群に比し有意な改善が認められた。3時間後に投与開始した群では、有意な改善は認められなかった。
SMTP-7(10mg/kg)投与群の梗塞領域割合は、1時間後に投与開始した群、3時間後に投与開始した群ともに、対照群に比べ有意な改善が認められた。
アルテプラーゼ投与群の神経症状のスコアは、1時間後に投与開始した群では、有意な改善が認められた。3時間後に投与開始した群では、有意な改善は認められなかった。
SMTP-7(10mg/kg)投与群の神経症状のスコアは、1時間後に投与開始した群、3時間後に投与開始した群ともに、対照群に比べ統計学的に有意な改善が認められた。
SMTP-7(10mg/kg)投与群では、1時間後に投与開始した群、3時間後に投与開始した群ともに、対照群に比べ有意な改善が認められた。
<脳血流量に対するSMTP-7とアルテプラーゼの効果の比較>
マウス脳梗塞モデルを用い、脳血流量に対するSMTP-7とアルテプラーゼの改善効果を比較した。
また、脳血流量の測定は、マウスの頭部の皮膚を切開して頭蓋骨を露出させ、脳表面全体の血流をレーザードップラー装置( moorFLPI, moor instruments Ltd, UK)を用いて測定した。解析は moorFLPI(Version 2.1)を用いて行い、各群の虚血開始前に対する割合(%)で評価した。結果を表10に示す。
<マウス脳梗塞モデルにおけるトリプレニルフェノール化合物の効果の検討>
トリプレニルフェノール化合物であるSMTP-6、SMTP-22、SMTP-25、SMTP-43及びSMTP-44Dを用い、マウス脳梗塞モデルに対する改善効果を検討した。
<リアルタイムRT-PCR法によるパラメータの変動の検討>
マウス脳梗塞モデルを用い、トリプレニルフェノール化合物(10mg/kg)投与群、アルテプラーゼ(10mg/kg)投与群、及びアルテプラーゼ(10mg/kg)とアスピリン(10mg/kg)の併用投与群において、リアルタイムRT-PCR法により、炎症に関与するパラメータの変動を評価した。パラメータは、炎症に関与する代表的なパラメータであるIL-1β、TNF-α及びIL-6とした。
トリプレニルフェノール化合物及びアルテプラーゼは、実施例2及び実施例5と同様にして調製した。アスピリンは、生理食塩水で1mg/mlに溶解した。
SMTP-7(10mg/kg)及びアルテプラーゼ(10mg/kg)は、虚血開始から1時間後または3時間後に投与開始した。投与方法は実施例3と同様であった。
アルテプラーゼ(10mg/kg)とアスピリン(10mg/kg)の併用投与は、虚血開始から3時間後に投与開始した。アルテプラーゼは実施例3と同様に投与し、アスピリンは大腿静脈より急速静注した。
SMTP-6、SMTP-22、SMTP-25、SMTP-43及びSMTP-44Dは、虚血開始から1時間後に投与開始した。投与方法は実施例5と同様であった。
用いたマウスは条件ごとに6匹ずつであった。
脳スライスを作製後、4枚目のスライスを左脳および右脳に切り分け、TRIZOL(登録商標) Reagent(Invitrogen)1mLでそれぞれホモジナイズを行った。室温で5分間インキュベーションし、クロロホルム(0.2mL) を加え、15秒間混和させた後、室温で3分間インキュベーションを行った。そのサンプルを4℃で15分間、12,000×gにて遠心した。
<トリプレニルフェノール化合物のフリーラジカル消去活性の検討>
トリプレニルフェノール化合物のフリーラジカルの消去活性を検討するため、以下のH-ORAC( hydrophilic oxygen radical absorbance capacity )試験、及びmodified H-ORAC(mH-ORAC)試験を行った。
H-ORACにおいては、試験化合物を緩衝液(75mMリン酸緩衝液、pH7.4)で適宜稀釈し、測定に用いた。mH-ORACにおいては、試験化合物を、アセトン溶液を水で稀釈した50%(v/v)アセトン溶液で適宜稀釈し、終濃度の40倍溶液を調製した。この溶液を緩衝液で10倍稀釈し、測定に用いた(アセトン終濃度1.25%)。標準物質トロロックスは、500μM/緩衝液を緩衝液で適宜稀釈した。
96穴マイクロプレート(透明、平底、ブラックウォール)に試験化合物溶液またはトロロックス溶液50μLを添加し、さらに蛍光物質フルオレセイン140nM/緩衝液(終濃度70nM)100μLを添加して、37℃で10分間インキュベーションした。
フリーラジカル発生剤2,2’-アゾビスアミジノプロパン二塩酸塩48mM/緩衝液(終濃度12mM)50μLを加え、励起波長485nm、蛍光波長535nmで蛍光強度を2分おきに90分間測定した。検量線はトロロックス終濃度5~15μMで作成した。測定は濃度を振って、n=3で行い、横軸に時間、縦軸に蛍光強度をとり、試料の曲線下面積からブランクの曲線下面積を差し引いた値を用いて評価した。結果はトロロックス等量で示した。結果を表13に示す。表13中、TEはトロロックス等量を表す。
<フィブリノーゲンザイモグラフィー法によるプラスミン活性の検討>
フィブリノーゲンザイモグラフィー法により、血漿中のプラスミン・α2-アンチプラスミン量を測定し、マウス脳梗塞モデルにトリプレニルフェノール化合物を投与した場合のプラスミン活性を評価した。トリプレニルフェノール化合物は、SMTP-7、SMTP-6、SMTP-22、SMTP-25、SMTP-43、及びSMTP-44Dを用いた。
一部の血漿をとりおき、電気泳動用ゲルにアプライするタンパク質量をそろえるため、0.1N NaOHで300倍稀釈し、Bradford法によりタンパク質を定量した。
10mA/枚、3時間で電気泳動後、スタッキングゲルを取り外し、洗浄液(2.5% triton X-100)100ml/ゲル程度で30分、2回洗浄した。洗浄後、インキュベーションバッファー(0.1Mグリシン-50mM Tris-HCl、37℃におけるpH8.3)100ml/枚程度で、37℃で24~60時間ゆるやかに揺らしながらインキュベーションした。
続いて、染色液(0.075% CBB R250 、22.5%メタノール、2.25%スルホサリチル酸二水和物、7.5%トリクロロ酢酸)で、室温下で15~30分間ゆるやかに揺らしながら染色した。染色液を取り除き、脱色液(メタノール:酢酸:水=1:1:6)で脱色し、バンドの出方を見ながら適当なところで水に置換にした。
イメージングアナライザ(ATTO製、Printgraph(AE-6933FXCF))でゲルからイメージを取り込み、バンド強度を評価した。結果は対照群の投与終了から0時間後(終了直後)との比で示した。結果を表14に示す。
<カニクイザル脳梗塞モデルにおけるSMTP-7の治療効果の検討>
SMTP-7のカニクイザル脳梗塞モデルにおける脳梗塞サイズの縮小効果および神経症状改善効果を、以下の試験方法により検討した。
・媒体群:生理食塩液、2~3歳齢の雄のカニクイザル6例
・SMTP-7投与群:10mg/kg、2~3歳齢の雄のカニクイザル6例
・ケージ:ステンレス製ケージ、W×D×H=600×600×800(mm)
・ケージ床面:0.36m2
・温度(許容範囲):20~26℃
・湿度(許容範囲):40~70%
・照明時間(設定):12時間/日(7:00~19:00)
・収容条件:個別飼育
〔飼料〕
・種類:固型飼料LabDiet(PMI社製)
・給餌方法:1日100g
〔飲料水〕
・種類:水道水
・給水方法:500mL給水瓶にて自由摂取させた。
・被験物質投与経路:静脈内投与
・被験物質投与量:10mg/kg
・被験物質投与法:投与容量は10mL/kgとした。左伏在静脈にサーフロー留置針を留置し、虚血1時間後に1mg/kg(1mL/kg)を5秒間急速投与し、その後、シリンジポンプを用いて、9mg/kg(9mL/kg)を30分間持続投与した。
・投与液の調製:SMTP-7を生理食塩液で1mg/mLに溶解した。すなわち、SMTP-7ナトリウム塩の70.0mg(63.1mgのSMTP-7を含む)に対し、生理的食塩液63.1mLを加え、湯浴にて加温下(37℃)、マグネティックスターラーにより撹拌し、溶解させた。必要に応じて、超音波処理を行った。溶解後、無菌フィルター(0.22μm、セルロースアセテート製)によりろ過滅菌を行った。投与液は用時調製した。調製完了後は使用直前まで37℃湯浴中に置き、調製後4時間以内に投与を終了させた。
標準実験手順書(SPHPR400-3A)に従って、動物麻酔を実施した。すなわち、ケタミン(第一三共プロファーマ(株))(10mg/kg)+アトロピン(田辺三菱製薬(株))(0.05mg/kg)を筋肉内投与することにより、導入麻酔を実施した。その後、気管挿管した後、イソフルラン(Abbott)の吸入麻酔下で手術台に固定した。
脳梗塞処置は、標準実験手順書(SPHPR710-15A)に従った。すなわち、右眼球を摘出した後、視神経出口外側の眼底骨をデンタルドリルで除去し、硬膜を露出させた。慎重に硬膜を剥離し、中大脳動脈を確認した。中大脳動脈起始部をクモ膜から分離し、中大脳動脈上に血栓作製用光照射プローブを固定した。また、プローブの遠位部にパルスドップラー血流計(Crystal Bio、PDV-20)のプローブを設置した。ローズベンガル(和光純薬工業(株))(20mg/kg)を6分間で静脈内投与開始するとともに、波長540nmの緑色光(140万LUX)を20分間照射することにより、中大脳動脈を血栓性に閉塞した。光照射とローズベンガルの投与開始時を虚血開始時とした。パルスドップラー血流計により、中大脳動脈血流を光照射開始から2時間連続的に測定した後、創を閉じた。一連の操作は、全て手術用顕微鏡下で行い、術中の出血を極力最小限に止めた。また、手術中の動物の直腸温をモニターし、動物の体温が生理的範囲(37.0~38.5℃)となるようヒーティングパッドで保温した。
ペニシリンG(明治製菓(株))(100,000 Unit/head/day)の筋肉内投与を実施することによる感染防止対策を行うとともに、塩酸ブプレノルフィン(レペタン、大塚製薬(株))0.02mg/head投与による鎮痛処置を実施した。
虚血24時間後にJ.Neurosci.Meth.2001;105:45-53.に従い、以下に記す神経症状の観察を行った。
神経症状のスコア化;
(1)意識障害(Consciousness)
・正常動物と同様、活発に行動(0)
・覚醒状態、攻撃的(4)
・覚醒状態、逃避可能(6)
・覚醒状態であるが反応が鈍い(8)
・傾眠(軽度)、刺激に対して反応(10)
・傾眠(重度)、強い刺激により開眼(16)
・知覚消失、持続的な刺激により反応(20)
・昏睡(軽度)、反射運動のみ(24)
・昏睡(重度)、動かない(28)
(2)知覚系(Sensory system)
顔面の感覚(障害側/非障害側)
・顔面への刺激に対して正常に反応(0/0)
・顔面への刺激に対して正常に反応しない(3/3)
耳介の反応(障害側/非障害側)
・耳を引っ張ると反応する(0/0)
・耳を引っ張っても反応しない(3/3)
痛み刺激(下肢、障害側/非障害側)
・足指をつかむとすばやく引っ込める(0/0)
・足指をつかむとゆっくりと引っ込める(3/3)
・足指をつかんでも引っ込めない(5/5)
(3)運動系(Motor system)
手(握れる/動く、障害側/非障害側)
・正常(0/0)
・握る力の低下/動作の不調和(2/2)
・麻痺、握ることができない(4/4)
脚(握れる/動く、障害側/非障害側)
・正常(0/0)
・膝を曲げて持ち上げる(2/2)
・動かすことはできるが、持ち上げられない(4/4)
・麻痺、動かすことができない(6/6)
上腕の筋緊張(障害側/非障害側)
・正常(0/0)
・明らかな筋の弛緩(痙攣性)(3/3)
下肢の筋緊張(障害側/非障害側)
・正常(0/0)
・明らかな筋の弛緩(痙攣性)(3/3)
(4)統御系(Skeletal muscle coordination)
・正常、歩行可能(0)
・運動失調(軽度)、歩行障害有り(4)
・運動失調、止まり木に登ることができない(6)
・自発的に立つことが可能、歩行困難(10)
・床で座位、刺激により回転運動をする(12)
・床で横臥位(16)
・動かない(18)
術後24時間後の神経症状観察後に、ペントバルビタールの大量投与による安楽死処置を施し、脳を摘出した。6mm厚の冠状切片を作製し、出血性梗塞の定量のため写真撮影した。その後。2%TTC液中で梗塞巣の染色を行い、梗塞サイズ定量用の写真撮影を行った。出血性梗塞および梗塞巣の評価には、各切片の後頭葉側を用いた。
個体別写真は、TIFイメージに変換した後、Photoshop 7.0(Adobe)にて出血性梗塞巣および各梗塞巣のマーキングを行い、Scion Image 0.4.0.3(Scion Corporation)にて面積を測定した。
出血性梗塞巣は、各断面の面積の総和とした。
梗塞巣は、各断面毎に、大脳皮質、白質、基底核に分けて測定し、その面の厚み(6mm)を乗じることにより、梗塞体積(mm3)を算出した。
実験結果の数値はMicrosoft Excel(Version 2003、Microsoft Inc.)を用いて集計及び作図し、平均値±標準偏差(S.D.)で表示した。
統計解析は、Microsoft Excel(Version 2003、Microsoft Inc.)を用いて行い、媒体群とSMTP-7投与群との間で対応のないt検定(unpaired t-test)(等分散)を行い、P<0.05の場合、有意差ありと判断した。
Time to Occlusion及びTotal Occlusion Timeを以下の表15に示す。媒体群とSMTP-7投与群との間に、Time to Occlusion及びTotal Occlusion Timeに有意な差は認められなかった。
媒体およびSMTP-7は、虚血60分後に投与開始を行ったため、Total Occlusion Timeを虚血開始から60分後までと60分後から120分後までのOcclusion Timeに分けて解析したが、表16に示す通り、有意な閉塞時間の短縮作用は認められなかった。
虚血24時間後の神経症状観察結果を以下の表17に記す。SMTP-7投与群においては、知覚系および筋統合系の有意な神経症状改善作用(P<0.05)が認められた。また、トータルスコアにおいても、有意な神経症状改善作用(P<0.05)が認められた。
虚血24時間後の出血性梗塞面積および脳梗塞サイズの測定結果を以下の表18及び表19に記す。出血性梗塞に関しては、SMTP-7投与群において有意な軽減(P<0.05)が認められた。また、SMTP-7投与により、基底核の梗塞サイズの有意な縮小(P<0.05)と全梗塞サイズの有意な縮小(P<0.05)が認められた。
SMTP-7の脳梗塞巣縮小作用は、本剤の血栓溶解作用に基づくと考えられるが、今回の検討結果では、中大脳動脈の閉塞時間には影響を与えなかった。この原因として、SMTP-7投与後の血流測定時間が短かったことが考えられる。SMTP-7は、線条体を中心とする基底核の梗塞サイズを有意に縮小した。線条体は最も虚血に脆弱な部位であり、中大脳動脈起始部からの穿通枝動脈からの血流支配を受けている領域である。一方、血流測定部位は中大脳動脈遠位部であることから、SMTP-7は血栓性に閉塞した中大脳動脈の起始部側から徐々に血栓を溶解した結果、顕著な基底核の梗塞サイズを縮小したと考えられる。
実施例2及び3の結果から明らかな通り、SMTP-7は、脳梗塞モデル動物において、梗塞領域割合及び神経症状の発現を抑制する作用を示すことが確認された。
実施例4の結果から明らかな通り、SMTP-7は、脳梗塞モデル動物において、梗塞後の脳血流量を徐々に回復させることが確認された。このことから、SMTP-7は、急激な血流回復に伴なう虚血再灌流障害を起こす危険性が少ないことが推測される。
実施例6の結果から明らかな通り、SMTP-7は、脳梗塞モデル動物において、炎症パラメータであるIL-1β、TNF-α及びIL-6の上昇を抑制する作用を示すことが確認された。
実施例7の結果から明らかな通り、SMTP-7は、虚血に起因する細胞傷害における傷害因子の一つであるフリーラジカルに対して強い消去作用を示すことが確認され、抗酸化活性を有することが確認された。
実施例8の結果から明らかな通り、SMTP-7は、血中のプラスミン活性を上昇させる作用を示すことが確認された。
実施例9の結果から明らかな通り、脳梗塞モデル動物において、SMTP-7は、梗塞サイズの有意な縮小作用を示し、神経症状の有意な改善作用と出血性梗塞の有意な軽減作用を示すことが確認された。
以上のことから、SMTP-7を含む組成物を、虚血に起因する機能不全を抑制する効果を有する細胞保護剤として用いることができる。
実施例6の結果から明らかな通り、SMTP-22、SMTP-25、SMTP-43及びSMTP-44Dは、脳梗塞モデル動物において、炎症パラメータであるIL-1β、TNF-α及びIL-6の上昇を抑制する作用を示すことが確認された。
実施例7の結果から明らかな通り、SMTP-6、SMTP-22、SMTP-25、SMTP-43及びSMTP-44Dは、虚血に起因する細胞傷害における傷害因子の一つであるフリーラジカルに対して強い消去作用を示すことが確認され、抗酸化活性を有することが確認された。
実施例8の結果から明らかな通り、SMTP-22、SMTP-25及びSMTP-43は、血中のプラスミン活性を上昇させる作用を示すことが確認された。
SMTP-6、SMTP-25及びSMTP-44Dは、モデル動物への投与量を適切に調節すれば、梗塞領域割合及び神経症状の発現を抑制する作用が認められるものと推測される。
また、本発明に係るトリプレニルフェノール化合物を含む組成物は、虚血性障害を発症した患者に該組成物を投与することを含む虚血性障害の治療方法に用いることができる。
本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。
Claims (4)
- 前記一般式(I)で表されるトリプレニルフェノール化合物が、下記一般式(II)又は(III)で表されるトリプレニルフェノール化合物である請求項1に記載の細胞保護剤。
[式中X1、X2及びX3は、それぞれ独立に-CHY-C(CH3)2Zであり、Y及びZは、それぞれ独立に-H若しくは-OHであるか、又は一緒になって単結合を形成する。
R1は、下記(A)から(D)のいずれか1つを表す。
(A)天然アミノ酸、天然アミノ酸のD体、並びに天然アミノ酸及び天然アミノ酸のD体においてカルボキシ基を水素原子、ヒドロキシ基又はヒドロキシメチル基に置き換えた化合物からなる群より選択されるアミノ化合物から、1個のアミノ基を除いた残基(ただし、-(CH)2-OHは除く)、
(B)カルボキシ基、水酸基、スルホン酸基及び第二アミノ基からなる群より選択される少なくとも1つを置換基として若しくは置換基の一部として有する芳香族基、又は第二アミノ基を含み且つ窒素原子を含んでいてもよい芳香族基、
(C)下記式(II-1)で表される芳香族アミノ酸残基(式中R3はあってもなくてもよい置換基であって、水酸基、カルボキシ基及び炭素数1~5のアルキル基からなる群より選択される少なくとも1つの置換基を表し、nは0又は1の整数を表す。)、
(D)-L1-L2-R4で表される置換基(式中、L1はカルボキシ基を有する炭素数1~4のアルキレン基である連結基を表し、L2は-NH-C(=O)-又は-NH-C(=S)-NH-で示される連結基を表し、R4は炭素数1~3のアルキルオキシ基を有する9-フルオレニルアルキルオキシ基又は下記式(II-2)で表される多複素環基を表す。)。
R2は、2個のアミノ基を有する天然アミノ酸、2個のアミノ基を有する天然アミノ酸のD体、2個のアミノ基を有する天然アミノ酸及び2個のアミノ基を有する天然アミノ酸のD体においてカルボキシ基を水素原子、ヒドロキシ基又はヒドロキシメチル基に置き換えた化合物、H2N-CH(COOH)-(CH2)n-NH2(nは0~9の整数)、並びにH2N-CH(COOH)-(CH2)m-Sp-(CH2)q-CH(COOH)-NH2(m、p及びqはそれぞれ独立に0~9の整数)で示される化合物からなる群より選択されるアミノ化合物から、2個のアミノ基を除いた残基を表す。] - 前記虚血性障害が血栓症である請求項1に記載の細胞保護剤。
- 前記虚血性障害が脳梗塞である請求項1に記載の細胞保護剤。
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WO2012115209A1 (ja) * | 2011-02-24 | 2012-08-30 | 国立大学法人東京農工大学 | 可溶性エポキシドハイドロラーゼ阻害剤 |
WO2013129661A1 (ja) | 2012-03-02 | 2013-09-06 | 株式会社ティムス | クロマン誘導体 |
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JP2020070280A (ja) * | 2018-11-02 | 2020-05-07 | 学校法人昭和大学 | Smtp一群又はsmtp−7の製造中間体及びその化学的製造方法 |
US11440920B2 (en) | 2018-11-02 | 2022-09-13 | Showa University | Chemical method of producing SMTP groups or SMTP-7 and intermediates used in the method |
WO2021100031A1 (ja) * | 2019-11-20 | 2021-05-27 | 株式会社ティムス | 脳出血を治療又は予防するための薬剤及び該薬剤を用いて脳出血を治療又は予防する方法 |
JP7482618B2 (ja) | 2019-11-20 | 2024-05-15 | バイオジェン・エムエイ・インコーポレイテッド | 脳出血を治療又は予防するための薬剤及び該薬剤を用いて脳出血を治療又は予防する方法 |
US11850232B2 (en) | 2019-11-20 | 2023-12-26 | Biogen Ma Inc. | Drug for treating or preventing cerebral hemorrhage, and method for treating or preventing cerebral hemorrhage using the drug |
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WO2022226013A1 (en) | 2021-04-20 | 2022-10-27 | Biogen Ma Inc. | Pharmaceutical composition comprising the compound smtp-7 |
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Also Published As
Publication number | Publication date |
---|---|
JPWO2011004620A1 (ja) | 2012-12-20 |
ES2476041T3 (es) | 2014-07-11 |
EP2452679B1 (en) | 2014-05-28 |
CN102470124A (zh) | 2012-05-23 |
CN102470124B (zh) | 2014-11-05 |
US20120135996A1 (en) | 2012-05-31 |
JP5605659B2 (ja) | 2014-10-15 |
EP2452679A4 (en) | 2013-01-23 |
USRE47684E1 (en) | 2019-11-05 |
USRE49351E1 (en) | 2023-01-03 |
US9078880B2 (en) | 2015-07-14 |
EP2452679A1 (en) | 2012-05-16 |
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