Disclosed is a cytoprotective agent for use with respect to ischemic damage, including as an active ingredient a triprenyl phenol compound represented by the following general formula (I), wherein X is —CHY—C(CH3)2Z, Y and Z are each independently —H or —OH, or jointly form a single bond, and R represents a hydrogen atom or a substituent with a molecular weight of 1000 or less.
##STR00001##
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0. 5. A method for treating cerebral thrombosis in a human subject in need thereof, comprising administering a therapeutically effective amount of a compound represented by formula (III) to the human subject suffering from cerebral thrombosis, wherein use of a thrombolytic drug is contraindicated in the human subject:
##STR00070##
wherein X2 and X3 are each —CHY—C(CH3)2Z; Y and Z jointly form a bond, and R2 and the two nitrogens to which it is attached are derived from compounds having the formula H2N—CH(COOH)—(CH2)n—NH2 wherein n is 3.
0. 6. A method for treating cerebral embolism in a human subject in need thereof, comprising administering: to a subject experiencing a cerebral embolism and having experienced at least one symptom of said cerebral embolism for three or more hours, a therapeutically effective amount of a compound represented by formula (III) to the human subject:
##STR00071##
wherein X2 and X3 are each —CHY—C(CH3)2Z; Y and Z jointly form a bond, and R2 and the two nitrogens to which it is attached are derived from compounds having the formula H2N—CH(COOH)—(CH2)n—NH2 wherein n is 3.
0. 7. A method for treating cerebral embolism in a human subject in need thereof, comprising administering: to a subject experiencing a cerebral embolism and having a contraindication for use of a thrombolytic drug, a therapeutically effective amount of a triprenyl phenol of formula (III) to the human subject:
##STR00072##
wherein X2 and X3 are each independently —CHY—C(CH3)2Z; Y and Z are each independently —H or —OH, or jointly form a bond, and
R2 and the two nitrogens to which it is attached are derived from compounds having the formula H2N—CH(COOH)—(CH2)n—NH2 wherein n is an integer from 0 to 9.
0. 9. A method for treating cerebral thrombosis in a human subject in need thereof, comprising administering, to the subject suffering from cerebral thrombosis and having experienced at least one symptom of said cerebral thrombosis for three or more hours, a therapeutically effective amount of a triprenyl phenol of formula (III) to the human subject:
##STR00074##
wherein X2 and X3 are each independently —CHY—C(CH3)2Z; Y and Z are each independently —H or —OH, or jointly form a bond, and
R2 and the two nitrogens to which it is attached are derived from compounds having the formula H2N—CH(COOH)—(CH2)n—NH2 wherein n is an integer from 0 to 9.
0. 8. A method of reducing ischemia reperfusion damage in a human subject, wherein the human subject is suffering from cerebral thrombosis or cerebral embolism and having experienced at least one symptom of said cerebral thrombosis or cerebral embolism for three or more hours, said method comprising administering a therapeutically effective amount of a triprenyl phenol of formula (III) to the human subject:
##STR00073##
wherein X2 and X3 are each independently —CHY—C(CH3)2Z; Y and Z are each independently —H or —OH, or jointly form a bond, and
R2 and the two nitrogens to which it is attached are derived from compounds having the formula H2N—CH(COOH)—(CH2)n—NH2 wherein n is an integer from 0 to 9.
0. 1. A method of treatment of cerebral hemorrhagic infarction, comprising the step of administering a drug containing a triprenyl phenol of formula (II) or formula (III) to a patient affected by cerebral hemorrhagic infarction:
##STR00068##
wherein X1, X2 and X3 are each independently —CHY—C(CH3)2Z; Y and Z are each independently —H or —OH, or jointly form a bond, and R1 is selected from among any one of the following (A) through (C):
(A) a residue of an amino compound selected from the group consisting of a natural amino acid, a D-isomer of a natural amino acid, and a compound derived by replacing a carboxy group in a natural amino acid, or a D-isomer of a natural amino acid, with a hydrogen atom, a hydroxy group, or a hydroxymethyl group, from which one amino group has been removed, with the proviso that R1 is not hydroxymethyl;
(B) an aromatic group having at least one selected from the group consisting of a carboxy group, a hydroxy group, a sulfonic group and a secondary amino group as a substituent or a part of a substituent, or an aromatic group that contains a secondary amino group and may contain a nitrogen atom; and
(C) an aromatic amino acid residue represented by the following formula (II-1)
##STR00069##
wherein, in formula (II-1), n is 0 or 1, and R3 is at least one substituent selected from the group consisting of a hydroxy group, a carboxy group, and a C1 to C5 alkyl group, wherein R3 may be present or absent; and
wherein, in formula III, R2 and the two nitrogens to which it is attached are derived from a diamine selected from the group consisting of: a natural amino acid with two amino groups; a D-isomer of a natural amino acid with two amino groups; a compound derived from a natural amino acid with two amino groups by replacing a carboxy group with a hydrogen atom, a hydroxy group, or a hydroxymethyl group; a compound derived from a D-isomer of a natural amino acid with two amino groups by replacing a carboxy group with a hydrogen atom, a hydroxy group, or a hydroxymethyl group; compounds having the formula H2N—CH(COOH)—(CH2)n—NH2 wherein n is an integer from 0 to 9; and compounds having the formula H2N—CH(COOH)—(CH2)m—Sp—(CH2)q—CH(COOH)—NH2 wherein m, p and q are each independently an integer from 0 to 9.
0. 2. The method of treatment of cerebral hemorrhagic infarction according to
0. 3. The method of treatment of cerebral hemorrhagic infarction according to
0. 4. The method of treatment of cerebral hemorrhagic infarction according to
0. 10. The method of claim 9, wherein said administering is carried out within 12 hours of onset of at least one symptom of cerebral thrombosis.
0. 11. The method of claim 9, wherein said subject has discontinued thrombolytic drug treatment for cerebral thrombosis.
0. 12. The method of claim 9, wherein said administering is carried out after the administration of a thrombolytic drug.
0. 13. The method of claim 9, wherein no additional thrombolytic drug is administered to the subject in combination with the compound of formula (III).
0. 14. The method of claim 7, wherein said triprenyl phenol of formula (III) has Y and Z jointly forming a bond and n is 3.
0. 15. The method of claim 8, wherein said triprenyl phenol of formula (III) has Y and Z jointly forming a bond and n is 3.
0. 16. The method of claim 9, wherein said triprenyl phenol of formula (III) has Y and Z jointly forming a bond and n is 3.
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This application
[Mouse Cerebral Infarction Model]
Using male ddY line mouse (body weight 35 to 45 g), a cerebral infarction model was produced and evaluated about neurological symptoms and the infarction area percentage according to methods similar to the production method and the evaluation method for the Mongolian gerbil cerebral infarction model.
The improvement effectiveness of SMTP-7, alteplase and edaravone (cerebroprotective drug) on the infarction area percentage and the neurological symptom was compared using the Mongolian gerbil cerebral infarction model. At the same time the dose dependence of the degree of improvement was investigated.
SMTP-7 was produced according to a process described in Japanese Patent Laid-Open No. 2004-224738 by purifying a culture obtained by adding L-ornithine as an additive organic amino compound to a culture medium of Stachybotrys microspora, strain IFO30018. To SMTP-7 produced through purification and exsiccated, 0.3 N NaOH and a physiological saline solution (0.9% NaCl) were added to prepare a 50 mg/mL solution. Thereafter, the solution was adjusted to 10 mg/mL, and weak alkaline pH with 0.3 N HCl and a physiological saline solution, subjected to filtration sterilization, divided into small fractions, and cryopreserved at −30° C. The SMTP-7 was used after dilution according to need with a physiological saline solution.
The SMTP-7 cryopreserved as above was dissolved at 1 mg/mL in a physiological saline solution just before a test. Alteplase (trade name Activacin, Kyowa Hakko Kirin Co., Ltd.) was dissolved in a solvent for Activacin at 1.03 mg/mL. As for Edaravone (trade name Radicut, Mitsubishi Tanabe Pharma Corp.), a stock solution of 1.5 mg/kg was used. The above medicaments were used after dilution according to need with a physiological saline solution.
The doses of SMTP-7 were set at 0.1 mg/kg, 1 mg/kg and 10 mg/kg; the doses of alteplase at 0.01 mg/kg, 0.1 mg/kg and 10 mg/kg; and the doses of edaravone at 1 mg/kg, 3 mg/kg and 10 mg/kg.
Administrations of SMTP-7 and alteplase were started 1 hour, 3 hours, or 6 hours after the initiation of ischemia of a Mongolian gerbil cerebral infarction model. An administration of edaravone was started immediately after (0 hour), 1 hour after, or 3 hours after the initiation of ischemia.
Anesthesia was induced to a Mongolian gerbil cerebral infarction model by inhalation of 5% isoflurane and thereafter maintained at 1 to 1.5%, so that an administration should be able to start as soon as the above defined time elapsed from the initiation of ischemia. The Mongolian gerbil was fixed in a dorsal position, and a polyethylene catheter with a 27 G needle was inserted into the left femoral vein. SMTP-7 and alteplase were administered through the catheter, wherein 10% of the dose by a bolus administration and the rest by a continuous administration over 30 min. Edaravone was administered continuously over 30 min. Six Mongolian gerbils were used for each condition.
Twenty four hours after the initiation of the operation neurological symptoms of the respective Mongolian gerbils were examined to rate the neurological symptom. Then the brain of each Mongolian gerbil was extirpated and the infarction area was measured and the infarction area percentage was determined. The infarction area percentage and scores of the neurological symptom are shown in Table 7. Further, for SMTP-7 and alteplase, with respect to groups of 10 mg/kg administration, the infarction area percentage is shown in
TABLE 7
Infarction area [%]
Neurological scores
Sham
2.9 ± 0.2
0 ± 0
Control
14.9 ± 3.08
3.33 ± 0.67
Initiation time of administration
Initiation time of administration
0 hr
1 hr
3 hr
6 hr
0 hr
1 hr
3 hr
6 hr
SMTP-7
—
11.4 ± 2.69
—
—
—
2.67 ± 0.33
—
(1 mg/kg)
SMTP-7
—
7.01 ± 0.56**
5.90 ± 0.36**
7.93 ± 1.64**
—
1.50 ± 0.67*
1.67 ± 0.61*
2.00 ± 0.45
(10 mg/kg)
alteplase
—
11.2 ± 2.74
—
—
—
2.67 ± 0.56
—
—
(0.01 mg/kg)
alteplase
—
9.67 ± 2.35
—
—
—
2.00 ± 0.77
—
—
(0.1 mg/kg)
alteplase
—
5.43 ± 1.33##
11.0 ± 3.61
13.8 ± 2.40
—
0.17 ± 0.17##
1.33 ± 0.67##
3.17 ± 0.48
(10 mg/kg)
edaravone
—
12.8 ± 2.87
—
—
—
3.17 ± 0.17
—
—
(1 mg/kg)
edaravone
9.95 ± 3.22
8.29 ± 1.61*
12.3 ± 5.59
—
1.50 ± 0.67*
1.50 ± 0.67*
2.50 ± 0.56
—
(3 mg/kg)
edaravone
—
9.29 ± 2.17
—
—
—
1.83 ± 0.65
—
—
(10 mg/kg)
argatroban
10.0 ± 2.66
12.1 ± 2.46
—
—
2.33 ± 0.61
2.50 ± 0.50
—
—
(1 mg/kg)
argatroban
—
11.5 ± 2.57
—
—
—
2.50 ± 0.56
—
—
(10 mg/kg)
ticlopidine
8.83 ± 1.73
13.1 ± 3.89
—
—
2.33 ± 0.42
2.67 ± 0.56
—
—
HCl
(1 mg/kg)
ticlopidine
—
9.84 ± 1.81
—
—
—
2.33 ± 0.42
—
—
HCl
(10 mg/kg)
Numerical expression represents: mean ± SE (n = 6)
Sham: Group of sham treatment for generating cerebral infarction
*P < 0.05;
**P < 0.01 (The values of the control and the values of the groups of SMTP-7 (10 mg/kg) administration, and the values of the control and the values of the groups of edaravone (3 mg/kg) administration were compared by ANOVA and thereafter by Bonferroni test.)
#P < 0.05;
##P < 0.01 (The values of the control and the values of the groups of alteplase (10 mg/kg) administration were compared by ANOVA and thereafter by Bonferroni test.)
When SMTP-7 was administered 1 hour after the initiation of ischemia, dose dependent improvement of the infarction area percentage and the neurological symptom was recognized. In the group of 10 mg/kg administration, for both of the infarction area percentage and the neurological symptom, significant improvement compared to the control group was recognized.
With respect to the infarction area percentage in the SMTP-7 (10 mg/kg) administration group, there was statistically significant improvement compared to the control group, even if the administration initiation time was delayed after the initiation of ischemia (administrations were initiated 1 hour, 3 hours, and 6 hours after the initiation of ischemia). The improvement rate of the group, for which the administration was initiated 1 hour after the initiation of ischemia, was 65.7% showing effectiveness nearly equivalent to the case of administration of alteplase, whose improvement rate was 78.9%.
With respect to the neurological symptom score of the SMTP-7 (10 mg/kg) administration groups, for which the administration was initiated 1 hour after or 3 hours after the initiation of ischemia, there was not remarkable improvement compared to the alteplase (10 mg/kg) administration group, but statistically significant improvement compared to the control group was recognized. In the SMTP-7 (10 mg/kg) administration group, for which the administrations was initiated 6 hours after the initiation of ischemia, there was no statistically significant improvement recognized, but some improving tendency was recognized.
When alteplase was administered 1 hour after the initiation of ischemia, dose dependent improvement of the infarction area percentage and the neurological symptom was recognized. In the group of 10 mg/kg administration, for both of the infarction area percentage and the neurological symptom, significant differences compared to the control group were recognized.
Among the alteplase (10 mg/kg) administration groups, significant improvement in the infarction area percentage compared to the control group was recognized in the case of the administration 1 hour after the initiation of ischemia. In the case of the administration 3 hours after the initiation of ischemia, some improving tendency was recognized, but there was no significant improvement compared to the control group recognized. In the case of the administration 6 hours after the initiation of ischemia, there was no statistically significant difference compared to the control group.
With respect to the neurological symptom score of the alteplase (10 mg/kg) administration groups, for which the administration was initiated 1 hour after or 3 hours after the initiation of ischemia, there was significant improvement compared to the control group recognized. In the case of the administration 6 hours after the initiation of ischemia, however, there was no statistically significant difference compared to the control group.
When edaravone was administered 1 hour after the initiation of ischemia, dose dependent improvement of the infarction area percentage and the neurological symptom was recognized. In the group of 3 mg/kg administration, for both of the infarction area percentage and the neurological symptom, significant improvement compared to the control group was recognized. By increasing the dose to 10 mg/kg, no further improvement of the effectiveness was recognized and the same level was maintained.
Among the edaravone (3 mg/kg) administration groups, improving tendency was recognized in the case of the administration 0 hour or 1 hour after the initiation of ischemia. No improvement was recognized in the case of the administration 3 hours after the initiation of ischemia.
With respect to the neurological symptom score among the edaravone (3 mg/kg) administration groups, significant improvement was recognized in the case of the administration 0 hour or 1 hour after the initiation of ischemia. No significant improvement was recognized in the case of the administration 3 hours after the initiation of ischemia.
The improvement effectiveness of SMTP-7 and alteplase on the infarction area percentage, the neurological symptom and the edema percentage was compared using the mouse cerebral infarction model. At the same time the dose dependence of the degree of improvement of SMTP-7 was investigated.
SMTP-7 and alteplase were prepared as in Example 2. Ten mg/kg each was administered into the femoral vein 1 hour or 3 hours after the initiation of ischemia in a mouse cerebral infarction model, wherein 10% was administered by a bolus administration, and the rest by continuous administration over 30 min. Further, 0.1 mg/kg or 1 mg/kg of SMTP-7 was administered into the femoral vein 1 hour after the initiation of ischemia in a mouse cerebral infarction model, wherein 10% was administered by a bolus administration, and the rest by continuous administration over 30 min. Six mice were used for each condition.
Twenty four hours after the initiation of the operation neurological symptom of each mouse was examined to rate the neurological symptom. Then the brain of each mouse was extirpated and the infarction area was measured and the infarction area percentage was determined. The edema percentage was determined according to the following formula:
Edema percentage (%)=(Volume of the cerebral hemisphere affected by ischemia−Volume of the other side cerebral hemisphere)/Volume of the other side cerebral hemisphere×100
The evaluation results for SMTP-7 and alteplase at the dose of 10 mg/kg are shown in Table 8. The infarction area percentage of the same is shown in
Evaluation results of SMTP-7 at different doses are shown in Table 9.
TABLE 8
Infarction area [%]
Neurological scores
Edema percentage [%]
Sham
2.99 ± 0.27
0 ± 0
0.568 ± 0.796
Control
11.9 ± 2.54
3.17 ± 0.477
13.2 ± 1.76
Initiation time of
Initiation time of
Initiation time of
administration
administration
administration
1 hr
3 hr
1 hr
3 hr
1 hr
3 hr
SMTP-7
4.87 ± 1.06**
5.05 ± 0.576**
1.67 ± 0.422*
1.33 ± 0.558**
5.81 ± 0.967**
6.23 ± 1.07**
alteplase
6.15 ± 1.94#
11.9 ± 2.26
1.17 ± 0.749##
2.50 ± 0.500
3.63 ± 2.67##
12.6 ± 1.84
Numerical expression represents: mean ± SE (n = 6)
Sham: Group of sham treatment for generating cerebral infarction
*P < 0.05;
**P < 0.01 (The values of the control and the values of the groups of SMTP-7 administration were compared by ANOVA and thereafter by Bonferroni test.)
#P < 0.05;
##P < 0.01 (The values of the control and the values of the groups of alteplase administration were compared by ANOVA and thereafter by Bonferroni test.)
Significant increase in the infarction area percentage was recognized in the control group compared to the sham group (Group of sham treatment for generating cerebral infarction).
Among the alteplase administration groups, significant improvement in the infarction area percentage compared to the control group was recognized in the group having initiated the administration 1 hour after the initiation of ischemia. In the group having initiated the administration 3 hours after the initiation of ischemia, no significant improvement compared to the control group was recognized.
Among the SMTP-7 (10 mg/kg) administration groups, significant improvement in the infarction area percentage compared to the control group was recognized in both of the group having initiated the administration after 1 hour, and the group having initiated the administration after 3 hours.
Significant increase in the neurological symptom score was recognized in the control group compared to the sham group (Group of sham treatment for generating cerebral infarction).
Among the alteplase administration groups, significant improvement in the neurological symptom score was recognized in the group having initiated the administration after 1 hour. In the group having initiated the administration after 3 hours, no significant improvement was recognized.
Among the SMTP-7 (10 mg/kg) administration groups, statistically significant improvement in the neurological symptom score compared to the control group was recognized in both of the group having initiated the administration after 1 hour, and the group having initiated the administration after 3 hours.
With respect to the edema percentage, among the alteplase administration groups, significant improvement was recognized in the group having initiated the administration after 1 hour, but no significant improvement was recognized in the group having initiated the administration after 3 hours.
Among the SMTP-7 (10 mg/kg) administration groups, significant improvement compared to the control group was recognized in both of the group having initiated the administration after 1 hour, and the group having initiated the administration after 3 hours.
TABLE 9
Infarction
Neurological
Edema
area [%]
scores
percentage [%]
Sham
2.99 ± 0.27
0 ± 0
0.568 ± 0.796
Control
11.9 ± 2.54
3.17 ± 0.477
13.2 ± 1.76
SMTP-7
0.1
mg/kg
10.08 ± 2.07
2.5 ± 0.5
10.15 ± 1.817
1
mg/kg
6.66 ± 0.71*
2 ± 0.632
8.81 ± 2.292
10
mg/kg
4.87 ± 1.06**
1.67 ± 0.422*
5.81 ± 0.967**
Numerical expression represents: mean ± SE (n = 6)
Sham: Group of sham treatment for generating cerebral infarction
*P < 0.05;
**P < 0.01 (Comparison with the values of the control by ANOVA and thereafter by Bonferroni test.)
Significant improvement in any of the infarction area percentage, the neurological symptom score and the edema percentage was recognized compared to the control group in the SMTP-7 (10 mg/kg) administration group and the SMTP-7 (1 mg/kg) administration group.
The improvement effectiveness of SMTP-7 and alteplase on the cerebral blood flow was compared using the mouse cerebral infarction model.
SMTP-7 and alteplase were prepared as in Example 2. SMTP-7 and alteplase were administered in an amount of 10 mg/kg into the femoral vein 1 hour after the initiation of ischemia in a mouse cerebral infarction model, wherein 10% was administered by a bolus administration, and the rest by continuous administration over 30 min. Three mice were used for each condition.
The cerebral blood flow was measured at 6 time points, namely before the initiation of ischemia, immediately after the initiation of ischemia, and after the completion of the drug administration 0 hour (immediately after the completion), 1 hour, 3 hours, and 24 hours.
The cerebral blood flow measurement was conducted by cutting the head skin of a mouse to expose the cranial bone, and measuring the blood flow of the whole brain surfaces by a laser-Doppler apparatus (moorFLPI, Moor Instruments Ltd, UK). The analysis was conducted using moorFLPI (Version 2.1) by rating percentage (%) relative to the value before the initiation of ischemia of each group. The results are shown in Table 10.
TABLE 10
Before
Immediately after
initiation of
initiation of
After completion of administration
ischemia
ischemia
0 hr
1 hr
3 hr
24 hr
Control
100%
30.61 ± 2.22%
45.08 ± 7.99%
38.52 ± 2.92%
31.97 ± 4.92%
43.37 ± 7.51%
alteplase
100%
27.30 ± 2.35%
56.45 ± 14.70%
73.49 ± 20.33%*
66.83 ± 11.10%*
71.63 ± 6.06%*
SMTP-7
100%
34.17 ± 5.22%
43.74 ± 12.67%
44.48 ± 12.59%
48.90 ± 15.89%
76.01 ± 9.71%*
Numerical expression represents: mean ± SE (n = 3)
*P < 0.05 (Comparison with the values immediately after the initiation of ischemia by ANOVA and thereafter by Bonferroni test.)
In the SMTP-7 administration group the recovery of the cerebral blood flow was slower than the alteplase administration group, but 24 hours after the completion of the administration the cerebral blood flow recovered to the same level as the alteplase administration group. There was statistically significant difference compared to the control group.
Improving effectiveness on the mouse cerebral infarction model was studied using SMTP-6, SMTP-22, SMTP-25, SMTP-43, and SMTP-44D as triprenyl phenol compounds.
The used SMTP-6 was prepared identically as SMTP-7, except that L-tryptophan was used as an additive organic amino compound. The used SMTP-22 was prepared identically as SMTP-7, except that 4-aminosalicylic acid was used as an additive organic amino compound. The used SMTP-25 was prepared identically as SMTP-7, except that 3-aminosalicylic acid was used as an additive organic amino compound. The used SMTP-43 was prepared identically as SMTP-7, except that L-phenylglycine was used as an additive organic amino compound. The used SMTP-44D was prepared identically as SMTP-7, except that D-4-hydroxyphenylglycine was used as an additive organic amino compound. With these triprenyl phenol compounds 50 mg/mL solutions were prepared by adding 0.3 N NaOH and a physiological saline solution (0.9% NaCl) Thereafter, the solutions were adjusted to 10 mg/mL, and weak alkaline pH with 0.3 N HCl and a physiological saline solution, subjected to filtration sterilization, divided into small fractions, and cryopreserved at −30° C.
The cryopreserved triprenyl phenol compounds were dissolved at 1 mg/mL in a physiological saline solution just before a test. Ten mg/kg each was administered into the femoral vein 1 hour after the initiation of ischemia in a mouse cerebral infarction model, wherein 10% was administered by a bolus administration, and the rest by continuous administration over 30 min. Six mice were used for each condition.
Twenty four hours after the initiation of the operation, the infarction area percentage, the neurological symptom, and the edema percentage of each mouse were evaluated. The evaluation results are shown in Table 11. In Table 11, the evaluation results of the group, in which the administration of SMTP-7 (10 mg/kg) was initiated after 1 hour in Example 3, are also shown.
TABLE 11
Infarction
Neurological
Edema
area [%]
scores
percentage [%]
Sham
2.99 ± 0.27
0 ± 0
0.568 ± 0.796
Control
11.9 ± 2.54
3.17 ± 0.477
13.2 ± 1.76
SMTP-7
4.87 ± 1.06**
1.67 ± 0.422*
5.81 ± 0.967**
SMTP-6
9.81 ± 1.43
3 ± 0.447
9.80 ± 2.74
SMTP-22
4.37 ± 0.51**
1.67 ± 0.422*
4.55 ± 0.99**
SMTP-25
10.16 ± 1.04
2.5 ± 0.5
10.95 ± 2.57
SMTP-43
5.70 ± 1.17**
1.5 ± 0.5*
3.28 ± 1.37**
SMTP-44D
7.75 ± 2.71
2.17 ± 0.401
6.86 ± 2.92*
Numerical expression represents: mean ± SE (n = 6)
Sham: Group of sham treatment for generating cerebral infarction
*P < 0.05;
**P < 0.01 (Comparison with the values of the control by ANOVA and thereafter by Bonferroni test.)
In the SMTP-22 administration group and the SMTP-43 administration group significant improvement in the infarction area percentage, the neurological symptom score and the edema percentage was recognized.
Using the Mouse Cerebral Infarction model changes of parameters related to inflammation among the triprenyl phenol compound (10 mg/kg) administration groups, the alteplase (10 mg/kg) administration groups, and the alteplase (10 mg/kg) and aspirin (10 mg/kg) combined administration group were evaluated by a real time RT-PCR method. As the parameters IL-1β, TNF-α nd IL-6 were selected, which were typical parameters related to inflammation.
As triprenyl phenol compounds, SMTP-7, SMTP-6, SMTP-22, SMTP-25, SMTP-43 and SMTP-44D were used.
The triprenyl phenol compounds and alteplase were prepared same as in Example 2 and Example 5. Aspirin was dissolved at 1 mg/mL in a physiological saline solution.
The administrations of SMTP-7 (10 mg/kg) and alteplase (10 mg/kg) were initiated 1 hour or 3 hours after the initiation of ischemia. The administration method was same as Example 3.
The combined administration of alteplase (10 mg/kg) and aspirin (10 mg/kg) was initiated 3 hours after the initiation of ischemia. Alteplase was administered same as in Example 3, and aspirin was administered by a bolus intravenous injection into the femoral vein.
The administrations of SMTP-6, SMTP-22, SMTP-25, SMTP-43 and SMTP-44D were initiated 1 hour after the initiation of ischemia. The administration method was same as Example 5.
Six mice were used for each condition.
After 24 hours of the initiation of the operation, a Krebs-HEPES buffer was perfused from the heart and then the brain was extirpated.
After preparing brain slices, the fourth slice was divided to the left brain and the right brain, and homogenized respectively with 1 mL of TRIZOL (registered trademark) reagent (Invitrogen). After incubating at room temperature for 5 min, adding chloroform (0.2 mL), and mixing for 15 sec, incubation was carried out at room temperature for 3 min. The samples were centrifuged at 4° C. for 15 min at 12,000×g.
Since RNA moved to a water layer, the water layer was collected and, after addition of 0.5 mL of isopropyl alcohol, followed by incubation at room temperature for 10 min, centrifuged at 4° C. for 10 min at 12,000×g. Since a pellet was formed in a tube after the centrifugation, the supernatant was removed, and 1 mL of 75% ethanol was added followed by stirring to wash the pellet. The mixture was then centrifuged at 4° C. for 5 min at 7,500×g. After removing again the supernatant, being left for natural drying for 8 min, the pellet containing the RNA was dissolved in 100 μL of RNase-free water. cDNA was produced by reverse transcription of the RNA using a SuperScript (registered trademark) VILOTM cDNA Synthesis Kit (Invitrogen) by a Thermal Cycler 2720 (Applied Biosystems). More particularly, 4 μL of 5×VILOTM Reaction Mix, 2 μL of 10× SuperScript (registered trademark) Enzyme Mix, and 1 μg of each RNA were mixed and diluted to 20 μL with RNase-free water.
Reverse transcription was conducted using the above samples under the following conditions. After reacting at 25° C. for 10 min, at 42° C. for 60 min, and at 85° C. for 5 min, the product was stored at 4° C. With the cDNA as a template real time RT-PCR was carried out by ABI PRISM 7000 using SYBR (registered trademark) GreenERTM qPCR SuperMix for ABI PRISM (registered trademark; Invitrogen). More particularly, 12.5 μL of SYBR (registered trademark) GreenERTM qPCR SuperMix for ABI PRISM (registered trademark), 0.5 μL of a forward primer, 0.5 μL of reverse primer and 2.5 μL of the 20-fold diluted template were mixed and diluted to 25 μL with RNase-free water.
Real time RT-PCR was conducted using the above samples under the following conditions. After reacting at 50° C. for 2 min and at 95° C. for 15 min, a cycle of 94° C. for 15 sec, 55° C. for 30 sec, and 72° C. for 30 sec was repeated 40 times. Changes of the respective parameters were determined by a calibration curve method. β-actin was measured as an internal control, and the respective parameters were calculated as a relative value based on the β-actin. The results are shown in Table 12. The values in Table 12 are the ipsilesional values (the right brain).
The following primers were used:
β-actin forward:
(SEQ. ID. NO: 1)
5′-CCTTCCTTCTTGGGTATGGAATC-3′,
β-actin reverse:
(SEQ. ID. NO: 2)
5′-TGCTAGGAGCCAGAGCAGTAATC-3′,
IL-1β (Quiagen, QT01048355),
TNF-α (Quiagen, QT00104006),
and
IL-6 (Quiagen, QT00098875).
TABLE 12
Relative value
to β-actin
IL-1β
TNF-α
IL-6
Sham
0.00358 ± 0.000676
0.0102 ± 0.00334
0.00235 ± 0.000457
Control
0.116 ± 0.0428*
0.474 ± 0.191**
0.173 ± 0.0464*
Initiation time of administration
Initiation time of administration
Initiation time of administration
1 hr
3 hr
1 hr
3 hr
1 hr
3 hr
alteplase
0.0408 ± 0.0173
0.347 ± 0.172**,##
0.164 ± 0.0332##
0.381 ± 0.155**
0.0582 ± 0.0254
0.341 ± 0.138**,#
alteplase +
—
0.172 ± 0.0502
—
0.143 ± 0.0330##
—
0.169 ± 0.0532*
Aspirin
SMTP-7
0.0314 ± 0.0171
0.0728 ± 0.0380
0.151 ± 0.0313##
0.147 ± 0.0293##
0.0356 ± 0.0138
0.0651 ± 0.0308
SMTP-6
0.165 ± 0.0930
—
0.261 ± 0.119*
—
0.220 ± 0.0850**
—
SMTP-22
0.040 ± 0.0140
—
0.087 ± 0.0295##
—
0.071 ± 0.0437
—
SMTP-25
0.060 ± 0.0249
—
0.049 ± 0.0158##
—
0.0560 ± 0.0259
—
SMTP-43
0.025 ± 0.0118
—
0.058 ± 0.0147##
—
0.0234 ± 0.0115
—
SMTP-44D
0.070 ± 0.0435
—
0.081 ± 0.0304##
—
0.0562 ± 0.0354
—
Numerical expression represents: mean ± SE (n = 6)
Sham: Group of sham treatment for generating cerebral infarction
*P < 0.05;
**P < 0.01 (Comparison with the values of Sham by ANOVA and thereafter by Bonferroni test.)
#P < 0.05;
##P < 0.01 (Comparison with the values of the control by ANOVA and thereafter by Bonferroni test.)
TNF-α and IL-6 in the control group were increased significantly compared to the Sham group.
In the group, in which the administration of alteplase was initiated after 1 hour from the initiation of ischemia, no parameter was recognized, which showed remarkable increase compared to the Sham group, but in the group, in which the administration was initiated after 3 hours, significant increase in IL-1β, TNF-α and IL-6 compared to the Sham group was recognized. Among others, significant increase in IL-1β and IL-6 relevant to the control group was also recognized.
In the group administered with alteplase and aspirin in combination, the increase of IL-1β and TNF-α, which was obvious when alteplase was administered singly, was not recognized.
Among the SMTP-7 administration groups, not only in the group, in which the administration was initiated after 1 hour, but also in the group, in which the administration was initiated after 3 hours, remarkable increase in IL-1β, TNF-α and IL-6 was not recognized, namely SMTP-7 inhibited increase of such parameters.
In the SMTP-22 administration group, the SMTP-25 administration group, the SMTP-43 administration group, and the SMTP-44D administration group, remarkable increase in IL-1β, TNF-α and IL-6 was not recognized, namely SMTP-22, SMTP-25, SMTP-43, and SMTP-44D inhibited increase of such parameters.
In the SMTP-6 administration group, the increase in IL-1β, TNF-α and IL-6 was not inhibited.
To study the free radical scavenging activity of a triprenyl phenol compound, the following H-ORAC (hydrophilic oxygen radical absorbance capacity) assay, and modified H-ORAC (mH-ORAC) assay were conducted.
SMTP compounds evaluated by H-ORAC were used respectively in a form of a sodium salt aqueous solution, and the compounds evaluated by mH-ORAC were used respectively in a form of an acetone solution.
In H-ORAC a test compound was diluted appropriately with a buffer solution (75 mM phosphate buffer solution, pH 7.4) and used for measurement. In mH-ORAC a test compound was diluted appropriately by a 50% (v/v) acetone solution, prepared by diluting an acetone solution with water, to prepare a solution of 40-fold the final concentration. The solution was diluted 10-fold with a buffer solution for measurement (acetone final concentration 1.25%). As for the reference material Trolox, 500 μM/buffer solution was diluted appropriately with a buffer solution.
To a 96-well microplate (transparent, flat bottom, black wall) 50 μL of a test compound solution or a Trolox solution was placed, and additionally 100 μL of a fluorescein 140 nM/buffer solution as a fluorescent substance was added (final concentration 70 nM), followed by incubation at 37° C. for 10 mM.
As a free radical generator, 50 μL of a 48 mM/buffer solution of 2,2′-azobis(amidinopropane)dihydrochloride was added (final concentration 12 mM), and fluorescence intensity at fluorescence wavelength of 535 nm with excitation wavelength of 485 nm was measured at 2 min intervals for 90 min. The calibration curve was prepared between the Trolox final concentrations of 5 to 15 μM. The measurement was conducted by changing the concentration with n=3, and plotted on a diagram with time on the abscissa and the fluorescence intensity on the ordinate. Therewith evaluation was made using the value obtained by reducing the area under the curve for the blank from the area under the curve for the sample. The result was expressed as an equivalent to Trolox. The results are shown in Table 13, wherein TE stands for a Trolox equivalent.
TABLE 13
Measuring
method
molTE/mol
SMTP-0
mH-ORAC
7.76 ± 0.36
SMTP-1
mH-ORAC
4.35 ± 0.55
SMTP-4
H-ORAC
1.79 ± 0.02
SMTP-5D
H-ORAC
1.71 ± 0.20
SMTP-6
H-ORAC
3.73 ± 0.42
SMTP-7
H-ORAC
2.08 ± 0.13
SMTP-7
mH-ORAC
1.82 ± 0.27
SMTP-8
mH-ORAC
1.69 ± 0.14
SMTP-11
H-ORAC
2.52 ± 0.12
SMTP-12
H-ORAC
3.18 ± 0.13
SMTP-13
H-ORAC
3.07 ± 0.51
SMTP-14
H-ORAC
4.43 ± 0.49
SMTP-18
mH-ORAC
7.34 ± 0.22
SMTP-19
H-ORAC
1.36 ± 0.05
SMTP-20
H-ORAC
1.80 ± 0.46
SMTP-21
H-ORAC
3.57 ± 0.13
SMTP-22
H-ORAC
3.63 ± 0.15
SMTP-23
H-ORAC
6.34 ± 0.37
SMTP-24
H-ORAC
3.95 ± 0.13
SMTP-25
H-ORAC
5.03 ± 0.72
SMTP-26
mH-ORAC
2.05 ± 0.27
SMTP-27
H-ORAC
5.01 ± 0.27
SMTP-28
H-ORAC
6.89 ± 0.03
SMTP-29
H-ORAC
5.60 ± 0.08
SMTP-36
H-ORAC
3.61 ± 0.04
SMTP-37
H-ORAC
4.09 ± 0.07
SMTP-42
H-ORAC
3.28 ± 0.25
SMTP-43
H-ORAC
2.74 ± 0.26
SMTP-43D
H-ORAC
2.32 ± 0.12
SMTP-44
H-ORAC
4.84 ± 0.43
SMTP-44D
H-ORAC
5.56 ± 0.18
SMTP-46
H-ORAC
3.19 ± 0.13
SMTP-47
H-ORAC
2.13 ± 0.22
Meanwhile, just as a reference value, the ORAC value for α-tocopherol measured by an ORAC assay same as the above was 0.50 ± 0.02 by Trolox equivalent (Huang et al., J. Agric. Food Chem., 50, 1815-1821 (2002)).
As obvious from Table 13, all of the SMTP compounds according to the present invention have free radical scavenging activity equivalent to or higher than Trolox. Consequently, it is clear that the SMTP compounds according to the present invention have high antioxidative activity.
The quantity of plasmin/α2-antiplasmin in plasma was measured by a fibrinogen zymography method for evaluating the plasmin activity, when a triprenyl phenol compound was administered to a mouse cerebral infarction model. As triprenyl phenol compounds, SMTP-7, SMTP-6, SMTP-22, SMTP-25, SMTP-43, and SMTP-44D were used.
The triprenyl phenol compounds were prepared same as in Example 2 and Example 5. The administration of 10 mg/kg of a triprenyl phenol compound was initiated 1 hour after the initiation of ischemia. The administration method was same as Example 3. Six mice were used for each condition.
A blood sample was taken according to the following method at 3 time points, namely after the completion of the administration 0 hour (immediately after the completion), 1 hour, and 3 hours. Under anesthesia with 1 to 1.5% isoflurane, the tail was cut in and 90 μL of blood was withdrawn from the abdominal vena cava into a syringe containing 10 μL of sodium citrate (3.8%) (sodium citrate:blood=1:9, final concentration of sodium citrate 0.38%).
After collection the blood was centrifuged (5000 rpm, 15 min, 4° C.) to separate plasma. A part of the plasma was mixed with equal quantity of a sample buffer (125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 0.04% (w/v) bromophenol blue, 20% (w/v) sucrose), which was then divided into small fractions, and cryopreserved.
A part of the plasma was reserved and diluted 300-fold with 0.1 N NaOH and the protein content was determined by the Bradford method in order to even out the protein mass to be applied to an electrophoresis gel.
As the standard sample, a sample was prepared by reacting 10 μL of 120 nM plasmin (by SIGMA) and 10 μL of 600 nM α2-antiplasmin (by Wako Pure Chemical Industries, Ltd.) at 37° C. for 30 min, and mixing the same with 20 μL of the sample buffer.
An electrophoresis gel was prepared by overlaying a stacking gel on a 7.5% running gel containing 2 mg/mL fibrinogen (by SIGMA). Based on the determined protein mass, 3 to 15 μL of the sample was applied, so that the equal amount of protein was applied.
After electrophoresis at 10 mA/sheet, for 3 hours the stacking gel was removed and rinsed for 30 min twice with about 100 mL/gel of a rinse liquid (2.5% Triton X-100). After rinsing, incubation was carried out at 37° C. for 24 to 60 hours under gentle shaking with about 100 mL/sheet of an incubation buffer (0.1 M glycine-50 mM Tris-HCl, pH 8.3 at 37° C.).
Thereafter the gel was stained by a stain solution (0.075% CBB 8250, 22.5% methanol, 2.25% sulfosalicylic acid dihydrate, 7.5% trichloroacetic acid) at room temperature for 15 to 30 min with gentle shaking. Removing the stain solution, the gel was decolored by a decoloring solution (methanol:acetic acid:water=1:1:6), which was replaced with water at an appropriate time point according to appearance of bands observed.
The image on the gel was read in by an imaging analyzer (Printgraph (AE-6933FXCF), by ATTO) and the band intensity was evaluated. The results are expressed as a ratio to the value of the control group at 0 hour after the completion of the administration (immediately after the completion). The results are shown in Table 14.
TABLE 14
After completion of administration
0 hr
1 hr
3 hr
Control
1 ± 0.26
1.01 ± 0.27
0.99 ± 0.23
SMTP-7
1.49 ± 0.13
3.23 ± 0.47**
3.27 ± 0.67**
SMTP-6
1.08 ± 0.12
1.17 ± 0.14
1.68 ± 0.10
SMTP-22
1.79 ± 0.21*
2.08 ± 0.33*
1.99 ± 0.78
SMTP-25
2.44 ± 0.28**
2.68 ± 0.33**
2.98 ± 0.60**
SMTP-43
2.64 ± 0.56**
3.04 ± 0.47**
3.18 ± 0.78**
SMTP-44D
0.53 ± 0.11
0.66 ± 0.10
0.75 ± 0.22
Numerical expression represents: mean ± SE (n = 6)
*P < 0.05;
**P < 0.01 (Comparison with the values of the control of the same administration time by ANOVA and thereafter by Bonferroni test.)
Among the SMTP-7 administration groups, in case of 1 hour after the completion of the administration significant increase of the plasmin activity was recognized and similar activity was maintained in case of 3 hours after the completion of the administration.
Among the SMTP-22 administration groups, the SMTP-25 administration groups, and the SMTP-43 administration groups, even in the case immediately after the completion significant increase of the plasmin activity was recognized and similar activity was maintained in cases of 1 hour and 3 hours after the completion of the administration.
Among the SMTP-6 administration groups, and the SMTP-44D administration groups, no significant increase of the plasmin activity was recognized.
A reducing effect of a cerebral infarction size and an improving effect of a neurological symptom of SMTP-7 in a crab-eating monkey cerebral infarction model were studied according to the following testing method.
[Constitution of Groups]
Medium group: physiological saline solution, 2 to 3 year old male crab-eating monkeys, 6 monkeys
SMTP-7 administration group: 10 mg/kg, 2 to 3 year old male crab-eating monkeys, 6 monkeys
[Environmental Conditions of Animal Room]
Cage: stainless steel cage, W×D×H=600×600×800 (mm)
Cage floor size: 0.36 m2
Temperature (allowable range): 20 to 26° C.
Humidity (allowable range): 40 to 70%
Lighting hours (setup): 12 hours/day (7:00 to 19:00)
Poundage condition: individual housing
[Feed]
Kind: solid feed LabDiet (by PMI Nutrition International)
Feeding method: daily 100 g
[Drinking Water]
Kind: tap water
Water feeding method: free-feeding by a 500 mL water bottle
[Administration of Test Substance]
Administration route of test substance: intravenous administration
Test substance dose: 10 mg/kg
Administration method of test substance: The administration volume was set at 10 mL/kg. A Surflo indwelling needle was indwelled in the left saphenous vein, 1 mg/kg (1 mL/kg) was administered 1 hour after ischemia as a bolus over 5 sec, then 9 mg/kg (9 mL/kg) was administered continuously over 30 min using a syringe pump.
Preparation of administration liquid: SMTP-7 was dissolved in a physiological saline solution at 1 mg/mL. More particularly, to 70.0 mg of a SMTP-7 sodium salt (containing 63.1 mg of SMTP-7) was added 63.1 mL of a physiological saline solution, and the liquid was stirred by a magnetic stirrer under heating in a hot-water bath (37° C.) for dissolution. An ultrasonic treatment was conducted according to need. After dissolution the liquid was subjected to filter sterilization by a sterile filter (0.22 μm, made of cellulose acetate). The administration liquid was prepared before using. After the preparation it was kept in a 37° C. hot-water bath until immediately before the use, and the administration was finished within 4 hours after the preparation.
[Treatment of Cerebral Infarction]
Animal anesthesia was conducted according to the standard experimental protocol (SPHPR400-3A). More particularly, anesthesia was induced by intramuscular administration of ketamine (Daiichi Sankyo Propharma Co. Ltd.) (10 mg/kg)+atropine (Mitsubishi Tanabe Pharma Corp.) (0.05 mg/kg). Thereafter, conducting tracheal intubation, the animal was fixed to an operating table under inhalation anesthesia with isoflurane (Abbott).
The treatment of cerebral infarction was conducted according to the standard experimental protocol (SPHPR710-15A). More particularly, after extirpating the right eyeball, the eyeground bone outside the optic nerve exit was removed by a dental drill to expose the dura mater. The dura mater was carefully detached to identify the middle cerebral artery. The origin part of the middle cerebral artery was separated from the arachnoid mater and a light irradiation probe for generating a thrombus was fixed on the middle cerebral artery. At the distal end of the probe a probe of a pulse Doppler blood flowmeter (Crystal Bio, PDV-20) was placed. An intravenous administration of rose bengal (Wako Pure Chemical Industries, Ltd.) (20 mg/kg) over 6 min was initiated and an irradiation with green light with the wavelength of 540 nm (1.40 million LUX) was conducted for 20 min to block the middle cerebral artery by a thrombus. The initiation time of the light irradiation and the administration of the rose Bengal was defined as the initiation time of ischemia. The middle cerebral artery blood flow was measured by the pulse Doppler blood flowmeter continuously for 2 hours from the initiation time of the light irradiation and then the incision was closed. A series of procedures were carried out completely under an operating microscope to minimize intraoperative hemorrhage to the utmost. Further, the rectal temperature of an animal under operation was monitored, and the animal was kept warmed by a heating pad, so as to maintain the body temperature of the animal in a physiological range (37.0 to 38.5° C.).
[Postoperative Management]
An intramuscular administration of penicillin G (Meiji Seika Pharma, Limited) (100,000 Unit/head/day) was carried out as a preventive measure against infection and an analgesic treatment by means of an administration of 0.02 mg/head of buprenorphine hydrochloride (Lepetan, Otsuka Pharmaceutical Co., Ltd.).
[Evaluation Method of Neurological Symptom]
Neurological symptoms as described below were observed 24 hours after ischemia according to J. Neurosci. Meth., 2001; 105: 45-53.
Rating of a neurological symptom score:
(1) Consciousness
Active movement, same as normal animal (0)
Arousal state, aggressive (4)
Arousal state, able to escape (6)
Arousal state, but slow to react (8)
Drowsiness (mild), reactive to stimulation (10)
Drowsiness (severe), eye is opened by strong stimulation (16)
Loss of sensation, reactive to persistent stimulation (20)
Coma (mild), reflex movement only (24)
Coma (severe), motionless (28)
(2) Sensory system
Sense of face (ipsilesional/contralesional)
Reaction of auricular (ipsilesional/contralesional)
Painful stimulation (inferior limb, ipsilesional/contralesional)
Hand (ability to grip/move, ipsilesional/contralesional)
Leg/foot (ability to grip/move, ipsilesional/contralesional)
Upper arm muscle tone (ipsilesional/contralesional)
Inferior limb muscle tone (ipsilesional/contralesional)
Normal, able to walk (0)
Failure of muscular coordination (mild), disturbance in walking (4)
Failure of muscular coordination, unable to climb to a roost (6)
Able to stand up spontaneously, difficult to walk (10)
Sit down position on floor, circling movement by stimulation (12)
Side-lying position on floor (16)
Motionless (18)
[Collection of Brain Specimen]
After the neurological symptom observation 24 hours after the operation, a euthanasia treatment was conducted by means of a megadose of pentobarbital and the brain was extirpated. Six mm-thick coronal slices were prepared and photographed for quantitative determination of a hemorrhagic infarction. Thereafter, the infarction area was stained in a 2% TTC solution and photographs were taken for quantitative determination of an infarction size. For evaluations of the hemorrhagic infarction and the infarction area, the occipital lobe side of each slice was used.
[Analysis Method]
Photographs of an individual were converted to TIF images and hemorrhagic infarction areas and the respective infarction areas were marked by Photoshop 7.0 (Adobe), which sizes were then measured by Scion Image 0.4.0.3 (Scion Corporation).
As a hemorrhagic infarction area the sum of area sizes of the respective cross-sections was used.
An infarction area was measured for cerebral cortex, white matter, and basal ganglion separately with respect to each cross-section, and the infarction volume (mm3) was calculated by multiplying the thickness (6 mm) of the slice.
The experiment result data were tabulated and graphed by Microsoft Excel (Version 2003, Microsoft Inc.) and expressed in mean±standard deviation (S.D.).
Statistical analysis was conducted using Microsoft Excel (Version 2003, Microsoft Inc.), and an unpaired t-test (equal variances) was carried out between the medium group and the SMTP-7 administration group to judge that significant difference was present if P<0.05.
[Results of Blood Flow Measurement]
Time to Occlusion and Total Occlusion Time are shown in Table 15. With respect to Time to Occlusion and Total Occlusion Time, no significant difference was recognized between the medium group and the SMTP-7 administration group.
Since the administration of the medium and SMTP-7 was initiated 60 min after ischemia, the Total Occlusion Time was divided into the Occlusion Time from the initiation of the ischemia to 60 min after the same, and the Occlusion Time from 60 min after the same to 120 min after the same, and analyzed respectively. As shown in Table 16, no significant reducing action on the occlusion time was recognized.
TABLE 15
Time to
Total Occlusion
Treatment
Occlusion (min)
Time (min)
Medium group
6.9 ± 2.8
103.3 ± 6.5
SMTP-7 administration
6.9 ± 2.5
86.8 ± 18.8
group
TABLE 16
Occlusion Time (min)
Treatment
0~60 min
60~120 min
Medium group
46.3 ± 4.9
57.0 ± 2.3
SMTP-7 administration
45.5 ± 3.8
41.3 ± 18.9
group
[Observation Results of Neurological Symptom]
The observation results of a neurological symptom 24 hours after ischemia are shown in the following Table 17. In the SMTP-7 administration group significant neurological symptom improving action (P<0.05) was recognized in the sensory system and the skeletal muscle coordination system. Further, significant neurological symptom improving action (P<0.05) was recognized also in the total score.
TABLE 17
Skeletal muscle
Treatment
Consciousness
Sensory system
Motor system
coordination system
Total
Medium group
10.3 ± 3.2
9.7 ± 1.0
13.7 ± 2.7
10.0 ± 3.1
43.7 ± 8.1
SMTP-7 administration
7.7 ± 2.0
7.5 ± 1.6*
10.7 ± 2.7
5.3 ± 2.4*
31.2 ± 7.9*
group
*P < 0.05 vs. medium group
[Measurement Results of Infarction Size]
The measurement results of the hemorrhagic infarction area size and the cerebral infarction size 24 hours after ischemia are shown in the following Table 18 and Table 19. In the SMTP-7 administration group, significant reduction (P<0.05) about the hemorrhagic infarction was recognized. Further, by the SMTP-7 administration, significant reduction (P<0.05) in the infarction size of the basal ganglion, and significant reduction (P<0.05) in the whole infarction size were recognized.
TABLE 18
Hemorrhagic infarction area size
Treatment
(mm2)
Medium group
29.0 ± 9.6
SMTP-7 administration
14.1 ± 7.4 (P < 0.05)
group
TABLE 19
Treatment
Total
Cerebral cortex
Basal ganglion
White matter
Medium group
3358.2 ± 1299.3
877.7 ± 1081.3
2291.2 ± 379.7
189.3 ± 105.8
SMTP-7 administration
1745.1 ± 749.5*
237.8 ± 198.8
1419.7 ± 633.8*
87.7 ± 71.8
group
Unit: mm3,
*P < 0.05 vs. medium group
From the above it has become clear that significant reducing action of the infarction size 24 hours after ischemia was exhibited in a crab-eating monkey thrombotic middle cerebral artery occlusion model as the result of the administration of SMTP-7 (10 mg/kg) 1 hour after ischemia. According to this result, a significant improving action of a neurological symptom and a significant reducing action of a hemorrhagic infarction were indicated.
Although the cerebral infarction area reducing action of SMTP-7 is believed to be attributable to the thrombolytic action of the same, there was no influence on the occlusion time of the middle cerebral artery according to the above study results. A reason for this may be attributable to a short measurement time of the blood flow after the SMTP-7 administration. SMTP-7 reduced significantly the infarction size of the basal ganglion including a striate body as a main part. The striate body is the site most vulnerable to ischemia and is a region depending on the blood flow of the perforating artery from the origin part of the middle cerebral artery. While, the blood flow measuring point was at a distal part of the middle cerebral artery, and SMTP-7 should have lysed gradually a thrombus from the origin part of the middle cerebral artery occluded by the thrombus, thus presumably resulting in remarkable reduction in the infarction size of the basal ganglion.
The results of Examples 1 to 9 can be summarized as follows.
As obvious from the results of Examples 2 and 3, it has been confirmed that SMTP-7 exhibits a suppressing action on the infarction area percentage and on the expression of a neurological symptom in a cerebral infarction model animal.
As obvious from the results of Example 4, it has been confirmed that SMTP-7 can gradually recover the cerebral blood flow after infarction in a cerebral infarction model animal. From this fact it can be presumed that with SMTP-7 the risk of causing ischemia reperfusion damage inherent to rapid recovery of the blood flow is limited.
As obvious from the results of Example 6, it has been confirmed that SMTP-7 exhibits an inhibitory action on increase in inflammation parameters of IL-1β, TNF-α, and IL-6 in a cerebral infarction model animal.
As obvious from the results of Example 7, it has been confirmed that SMTP-7 exhibits a strong scavenging action on a free radical, which is one of the damaging factors with respect to cell damaging caused by ischemia, and has an antioxidative activity.
As obvious from the results of Example 8, it has been confirmed that SMTP-7 exhibits an enhancing action on the plasmin activity in the blood.
As obvious from the results of Example 9, it has been confirmed that SMTP-7 exhibits a significant reducing action on the infarction size, and exhibits a significant improving action on a neurological symptom and a significant mitigating action on a hemorrhagic infarction, in a cerebral infarction model animal.
From the above, a composition containing SMTP-7 can be used as a cytoprotective agent having the effectiveness for inhibiting the dysfunction caused by ischemia.
As obvious from the results of Example 5, it has been confirmed that SMTP-22 and SMTP-43 exhibit a suppressing action on the infarction area percentage and on the expression of a neurological symptom in a cerebral infarction model animal.
As obvious from the results of Example 6, it has been confirmed that SMTP-22, SMTP-25, SMTP-43 and SMTP-44D exhibit an inhibitory action on increase in inflammation parameters of IL-1β, TNF-α, and IL-6 in a cerebral infarction model animal.
As obvious from the results of Example 7, it has been confirmed that SMTP-6, SMTP-22, SMTP-25, SMTP-43 and SMTP-44D exhibit a strong scavenging action on a free radical, which is one of the damaging factors with respect to cell damaging caused by ischemia, and have an antioxidative activity.
As obvious from the results of Example 8, it has been confirmed that SMTP-22, SMTP-25 and SMTP-43 exhibit an enhancing action on the plasmin activity in the blood.
With respect to SMTP-6, SMTP-25 and SMTP-44D, however, it has been not confirmed in Example 5 that they exhibit a suppressing action on the infarction area percentage and on the expression of a neurological symptom. On the other hand, it has been confirmed that SMTP-6 has a scavenging activity on a free radical. Further, it has been confirmed that SMTP-25 inhibits increase of an inflammation parameter, has scavenging activity on a free radical, and enhances the plasmin activity in the blood. It has been confirmed that SMTP-44D inhibits increase of an inflammation parameter, and has scavenging activity on a free radical.
It can be presumed that SMTP-6, SMTP-25 and SMTP-44D would exhibit a suppressing action on the infarction area percentage and on the expression of a neurological symptom, if the dose thereof to a model animal should be adjusted appropriately.
From the above, a composition containing any one selected out of SMTP-6, SMTP-22, SMTP-25, SMTP-43 and SMTP-44D can be used as a cytoprotective agent having the effectiveness for inhibiting the dysfunction caused by ischemia.
As obvious from the results of Example 7, it has been confirmed that SMTP-0, SMTP-1, SMTP-4, SMTP-5D, SMTP-8, SMTP-11 to 14, SMTP-18 to 21, SMTP-23, SMTP-24, SMTP-26 to 29, SMTP-36, SMTP-37, SMTP-42, SMTP-43D, SMTP-44, SMTP-46 and SMTP-47 exhibit a scavenging action as strong as, or stronger than Trolox, on a free radical, which is one of the damaging factors with respect to cell damaging caused by ischemia, and have an antioxidative activity.
From the above, a composition containing any one selected out of SMTP-0, SMTP-1, SMTP-4, SMTP-5D, SMTP-8, SMTP-11 to 14, SMTP-18 to 21, SMTP-23, SMTP-24, SMTP-26 to 29, SMTP-36, SMTP-37, SMTP-42, SMTP-43D, SMTP-44, SMTP-46 and SMTP-47 can be used as a cytoprotective agent having the effectiveness for inhibiting the dysfunction caused by ischemia.
Consequently, a composition containing a triprenyl phenol compound according to the present invention can be used as a cytoprotective agent having the effectiveness for inhibiting the dysfunction caused by ischemia.
Further, a composition containing a triprenyl phenol compound according to the present invention can be used for a method of treatment for ischemic damage including the administration of the composition to a patient affected by ischemic damage.
The entire disclosures of Japanese Patent Application No. 2009-160278 applied on Jun. 7, 2009 are hereby incorporated by reference.
All the literature, patent literature, and technical standards cited herein are also herein incorporated to the same extent as provided for specifically and severally with respect to an individual literature, patent literature, and technical standard to the effect that the same should be so incorporated by reference.
Sequence Listing
Honda, Kazuo, Hashimoto, Terumasa, Shibata, Keita, Hasegawa, Keiko, Hasumi, Keiji
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