Compounds and pharmaceutical combinations for the treatment of neurodegenerative and ischemic brain diseases

Abstract

The present invention describes peptides comprising phycocyanobilin (PCB), as well as the medical use of said peptides and that of PCB, due to the neuroprotector and/or neuroregenerative effects identified for them. Furthermore, pharmaceutical combinations of said peptides and of PCB with proteins or other peptides with synergic effect justify their use for ischemic or neurodegenerative CNS disease treatment.

Claims

1-16. (canceled)

17. A method for the prophylaxis or the treatment of diseases of the central nervous system (CNS) that progress with ischemic, inflammatory or neurodegenerative damage wherein said method comprises administering a compound selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5 and phycocyanobilin to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0057] FIGS. 1A-D. Characterization by mass spectrometry of PCB obtained by metanolic treatment (Figure A) and the chromogenic peptides obtained by trypsin digestion, defined together as PCB-aa, Figure B: SEQ ID NO. 1; Figure C: SEQ ID NO. 2; Figure D: SEQ ID NO. 3-5.

[0058] FIGS. 2A-B. In vitro study of the neuroprotector effect of C-Phycocyanin (C-Phyco) (Figure A) and of PCB and PCB-aa (Figure B) against the Sodium glutamate (50 mM) induced damage in the neuronal line PC12. The symbols indicate the presence (+), absence () or concentrations of the respective compounds in the culture medium. Different letters indicate statistically significant difference, according to ANOVA followed by the Newman-Keuls multiple comparison test, p<0.05. The values presented in the graphs are the meansstandard error of the mean (MSE).

[0059] FIG. 3. PCB-aa (peptides 1 to 5) therapeutic treatment effect on the brain infarct volume, 24 h after the transient occlusion (10 min) of the common carotid arteries (CCA) in Mongolian gerbils. Different letters indicate statistically significant difference, according ANOVA followed by the Newman-Keuls multiple comparisons test, p<0.05. The values are presented in the graphs as the meansMSE.

[0060] FIG. 4. Effect of therapeutic treatment with PCB, PCB-aa (peptide 1), IFN-a and IFN-b and the PCB/IFN-a, PCB/IFN-b, PCB-aa/IFN-a and PCB-aa/IFN-b combinations on the brain infarct volume, 24 h after the transient (10 min) occlusion of the CCA in Mongolian gerbils. Different letters indicate statistically significant difference regarding the I/R group+saline, p<0.05. The values are presented in the graphs as the meansMSE.

[0061] FIG. 5. Effect of the PCB-aa (peptide 1)/IFN-b, PCB/IFN-b combinations and their independent active principles on the clinical course of EAE in C57BL6 mice. The values presented in the graphs are the means of the clinical index of each group.

[0062] FIG. 6. Effect of the PCB-aa (peptide 1)/IFN-b combination administered by different routes: intraperitoneally, nasal, oral y rectal on the clinical index of sick EAE C57BL6 mice. The values presented in the graphs are the means of the clinical index of each group.

[0063] FIG. 7. Therapeutic effect with PCB, PCB-aa (peptide 2), IL-2, or the combinations PCB/IL-2 and PCB-aa (peptide 2)/IL-2, on the volume of the brain infarct, 24 h after the transient (10 min) occlusion of the CCA in Mongolian gerbils. Different letters indicate significant differences with respect to the I/R+saline group, *p<0.05. The values presented in graphs are meansMSE

[0064] FIGS. 8A-B. Morphometric evaluation of the therapeutic effect with PCB-aa (peptide 3), the GHRP-6 peptide, or its combination in Mongolian gerbils submitted to transient (10 min) occlusion of CCA. Figure A: representative images (4magnification) of the left hippocampus of animals that underwent fake surgery (sham), or treated with saline solution, or with PCB-aa (peptide 3) and GHRP-6 (6,25 g/kg, intraperitoneal route), or with PCB-aa (peptide 3)/GHRP-6 (maintaining the corresponding doses and administration routes) for 30 min, 3, 6 and 12 h after the ischemic event. Figure B: bilateral cell count performed in the C2, CA3 and CA4 regions of both hippocampi for each experimental group. Different letters indicate significant differences with respect to the I/R+saline group, *p<0.05. The values presented in the graphs are meansMSE

[0065] FIG. 9. Effect of therapeutic treatment with PCB, PCB-aa (peptide 4), EPO, asyaloEPO, or their respective combinations PCB/EPO, PCB/asyaloEPO, PCB-aa (peptide 4)/EPO and PCB-aa (peptide 5)/asyaloEPO on the brain infarct volume, 24 h after transient occlusion (10 min) of CCA in Mongolian gerbils. Different letters indicate significant differences with respect to the I/R+saline group, p<0.05. The values presented in graphs are meansMSE

DETAILED EXPOSITION OF THE REALIZATION MODES/REALIZATION EXAMPLES

EXAMPLE 1

Mass Spectrometry of Phycocyanobilin (PCB) Obtained by Metanolic Treatment and of the Chromogenic Peptides (PCB-aa)

[0066] FIG. 1A shows the m/z signal 587.26 corresponding to the chromophore PCB, obtained by differential ultrafiltration of the C-Phyco metanolic extract.

[0067] FIGS. 1B, C and D show the mass spectrometry pattern of PCB-aa obtained by trypsin digestion of C-Phyco.

EXAMPLE 2

Neuroprotector Effect of C-Phyco, PCB and PCB-aa Against the Sodium Glutamate Induced Damage in the PC12 Cell Line

[0068] PC12 cells (1,510.sup.4 cells/well) were pretreated with C-Phyco (25, 50 M) or PCB (0,5; 1; 5 M) or PCB-aa (0,25; 1; 2 M), during 24 h, and then submitted to co-incubation with 50 M Sodium glutamate together with the corresponding product (different doses) for 4 h. The cell viability was measured by the (3-(4,5-Dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide method (MTT) and the percentage with respect to the control was reported, as shown in FIG. 2. It can be observed that to attain similar cell viability to that of C-Phyco lower concentrations of PCB and PCB-aa were needed. The PCB-aa (peptide 1) concentrations that achieved a similar effect to PCB and C-Phyco were even lower in respect to the said compounds.

EXAMPLE 3

Demonstration of Neuroprotector and/or Neuroregenerator Properties of PCB-aa Peptides, by Means of the Infarct Volume Reduction, in the Mongolian Gerbil I/R Model

[0069] The animals were treated with saline solution or with an accumulative dose of the PCB-aa peptides (3.375 mg/kg of each one), by intraperitoneal route for 30 min, 3, 6 and 12 h after the ischemic event.

[0070] The effectiveness percentage of each treatment was calculated according to the following formula: effectiveness %=(1Vi/VI/R)100. Vi: infarct volume of the ischemic group treated with the corresponding product; VI/R: volume of the infarct of the ischemic group treated with saline solution.

[0071] As may be observed in FIG. 3, the animals treated with the chromogenic peptides (PCB-aa) consisting in the SEQ ID NO: 1 to the SEQ ID NO: 5, showed a significant reduction of the infarct volume with respect to the ischemic group treated with saline solution.

EXAMPLE 4 cl Effect of the Combinations PCB/IFN-a, PCB-aa (Peptide 1)/IFN-a, PCB/IFN-b and PCB-aa (peptide 1)/IFN-b in the Bilateral Ischemia-Reperfusion Model in Gerbils

[0072] The animals were treated with saline solution (by intraperitoneal route) or with the individual compounds at a dose of PCB (750 g/Kg, intraperitoneal route), or PCB-aa (peptide 1) (3.375 mg/kg), IFN-a and IFN-b (500 ng/Kg, subcutaneous route) or with the combinations PCB/IFN-a, PCB/IFN-b, PCB-aa (peptide 1)/IFN-a and PCB-aa (peptide 1)/IFN-b according to the doses indicated in FIG. 4 by 30 min, 3, 6 and 12 h after the ischemic event. The percentage of effectiveness was calculated as described in Example 3.

[0073] The reduction of the brain infarct volume, by groups, evidences the effectiveness of the evaluated treatments (FIG. 4), observing a reduction of the infarct index in the group treated with PCB with an effectiveness of 43.1%; with PCB-aa (peptide 1) of 49.2%; in the group treated with IFN-a of 35.4%; with IFN-b of 37.0%; in the group treated with PCB/IFN-a of 83.3%; PCB-aa (peptide 1)/IFN-a of 89.3%; PCB/IFN-b of 87.0%; and PCB-aa (peptide 1)/IFN-b of 93.6%; evidencing a synergic effect of both active principles in the animals treated with the combination.

EXAMPLE 5

Demonstration of the Pharmacological Effect of the PCB/IFN-a and PCB-aa (peptide 1)/IFN-a Combinations with Respect to the Active Principles Independently, Referred to the Clinical Signs in the EAE Model

[0074] On the other hand, the evaluation of the combinations PCB/IFN-a and PCB-aa (peptide 1)/IFN-a was performed in the prophylactic schedule, in the EAE model (Table 1), where a synergic effect of the said combination regarding the prevention of the development of the disease at the doses declared formerly, was demonstrated.

TABLE-US-00002 TABLE 1 Evaluation of the PCB-aa (peptide 1)/IFN-a and PCB/IFN-a combinations and their active principles independently in the prophylactic schedule in the EAE model. Clinical Index Clinical Incidence Starting Day score Days of the Groups (%) (Mean SD) (Mean SD) disease Control 0 0 0 0 PCB-aa (peptide 1) 75 9.5 0.2 1.23 1.1 7.2 0.3 (3.375 mg/Kg) PCB 70 10.5 0.5 1.37 1.7 8.5 0.1 (750 g/Kg) IFN-a 60 10.7 0.3 1.5 1.6 7.3 0.7 (500 ng/Kg) PCB-aa (peptide 1) 0 0 0 0 (3.375 mg/Kg) + IFN-a (500 ng/Kg) PCB-aa (peptide 1) 12 11.3 0.2 0.5 0.1 4.5 0.5 (0.9 mg/Kg) + IFN-a (5000 ng/Kg) PCB (750 g/Kg) + 10 12.4 0.2 0.9 0.1 5.4 0.6 IFN-a (500 ng/Kg) PCB (300 g/Kg) + 15 12.8 0.3 1.12 0.5 6.6 0.2 IFN-a (5000 ng/Kg) EAE 100 10.2 0.7 2.7 0.4 15.2 1.1

[0075] As shown in Table 1, the combinations with different doses of PCB/IFN-a and PCB-aa (peptide 1)/IFN-a provide protection of between 85% and 100% of the animals induced to develop EAE, respectively.

EXAMPLE 6

Demonstration of the Pharmacological Effect of the Combinations PCB/IFN-b and PCB-aa (Peptide 1)/IFN-b in Relation to the Active Principles Independently, Referred to the Clinical Signs in the EAE Model

[0076] To demonstrate that the pharmacological effect of the combinations PCB/IFNb and PCB-aa (peptide 1)/IFN-b, in the prophylactic (Table 2) as well as in the therapeutic schedule (FIG. 5), in relation to the reduction of the clinical signs in C57BL6 mice the following individual compounds were used: PCB (750 g/kg, intraperitoneal route), PCB-aa (3.375 mg/kg, intraperitoneal route) administered daily for 15 days and IFNb (500 ng/Kg, subcutaneous route) 6 doses, 3 times a week, or their combinations according to the doses indicated in Table 2 were used.

[0077] The percentage of effectiveness of each treatment was calculated according to the following formula: % of effectiveness=(1AC.sub.i/AC.sub.EAE)100. AC.sub.i: area under the curve of the group treated with the corresponding product; AC.sub.EAE: area under the curve of the EAE group.

[0078] In the prophylactic schedule the treatment was performed 15 days before EAE induction and in the therapeutic schedule starting from the beginning of the clinical signs. The control group corresponds to healthy animals that did not receive any treatment.

[0079] Table 2 shows the results obtained in the prophylactic schedule, where the combinations PCB/IFN-b and PCB-aa (peptide 1)/IFN-b protected from 90% to 100% of the animals from EAE development respectively. Hence, a synergic effect was observed regarding the independent active principles.

TABLE-US-00003 TABLE 2 Evaluation of the combinations PCB-aa (peptide 1)/IFN-b and PCB/IFN-b, and their active principles independently, in the EAE prophylactic model. Clinical index Clinical Days Incidence Starting day score of the Groups (%) (Mean DS) (Mean SD) disease Control 0 0 0 0 PCB-aa (peptide 1) 70 9.5 0.2 1.23 1.1 7.2 0.3 (3.375 mg/Kg) PCB 60 12.3 0.1 1.55 0.3 7.3 0.4 (750 g/Kg) IFN-b 70 12.6 0.5 1.4 0.2 7.1 0.3 (500 ng/Kg) PCB-aa (peptide 1) 0 0 0 0 (3.375 mg/Kg) + IFN-b (500 ng/Kg) PCB-aa (peptide 1) 10 12.1 0.1 0.4 0.1 4.2 0.2 (0.9 mg/Kg) + IFN-b (5000 ng/Kg) PCB (750 g/Kg) + 5 12.5 0.1 1.1 0.1 4.4 0.3 IFN-b (500 ng/Kg) PCB (300 g/Kg) + 10 12.5 0.2 1.21 0.4 6.2 0.1 IFN-b (5000 ng/Kg) EAE 100 10.1 0.3 2.3 0.6 1.2 1.1

[0080] FIG. 5 shows a reduction of the clinical signs that is greater than the reduction obtained with the active principles independently, PCB (750 g/kg, intraperitoneal route), PCB-aa (3.375 mg/kg, intraperitoneal route), IFN-b (500 ng/Kg, subcutaneous route), evidencing also in the therapeutic schedule a synergic effect in the group treated with the combinations in the EAE model, PCB (750 g/kg)/IFN-b (500 ng/Kg) or PCB-aa (3.375 mg/kg)/IFN-b (500 ng/Kg), with 87.7% effectiveness for the combination PCB/IFN-b; 94.5% for the combination PCB-aa/IFN-b; 46.1% for PCB; 53.9% for PCB-aa and 34.8% for IFN beta.

EXAMPLE 7

Demonstration of the Therapeutical Effect of the Combination PCB-aa (Peptide 1)/IFNb by Different Routes in the EAE Model

[0081] The animals of the EAE group received a daily administration of saline solution by the intraperitoneal route. The mice treated with the combination PCB-aa (peptide 1)/IFN-b (PCB-aa 3.375 mg/kg+500 ng de IFN-b/Kg) were divided into different groups, according the administration route: intraperitoneal, oral, nasal and rectal. The therapeutic schedule was followed for 15 consecutive days, from day 9 and up to day 24 after immunization. The control group corresponds to the healthy mice that did not receive any treatment. The clinical evaluation was performed day 27 after immunization.

[0082] As evidenced in FIG. 6, statistically significant differences were not detected between the different routes evaluated (intraperitoneal, nasal, oral and rectal), which indicates that they can be used with equal effectiveness.

EXAMPLE 8

Neuroprotector and/or Neuroregenerator Effects of the Combination PCB/IL-2 and PCB-aa (Peptide 2)/IL-2 in the Bilateral I/R Model in Gerbils

[0083] The animals were treated with saline solution (by intraperitoneal route) or with an accumulative dose of PCB (750 g/Kg, by intraperitoneal route), PCB-aa (peptide 2) (3.375 mg/kg), IL-2 (100 ng/Kg, subcutaneous route) or with PCB/IL-2 and PCB-aa (peptide 2)/IL-2 (maintaining the corresponding dose and administration route) for 30 min, 3, 6 and 12 h after the ischemic event. In FIG. 7 a reduction of the infarct volume by group can be observed.

[0084] The percentage of effectiveness of each treatment was calculated as described in Example 3, which was 43.1% for PCB; 49.2% for PCB-aa (peptide 2); 25.8% for IL-2; 74.5% for the PCB/IL-2 combination; and 84.3% for PCB-aa (peptide 2)/IL-2), which evidence a synergic effect of both active principles in the animals treated with the combination.

EXAMPLE 9

Therapeutic Effect of the PCB/GHRP-6 Combination and its Active Principles Independently, in the Bilateral Ischemia-Reperfusion Model in Gerbils

[0085] The morphometric evaluations of the therapeutic treatment with PCB-aa (peptide 3), the GHRP-6 peptide or its combinations were tested in Mongolian gerbils submitted to transient (10 min) CCAs occlusion. Accumulative doses of PCB-aa (peptide 3) (750 g/Kg, by intraperitoneal route), GHRP-6 (6.25 g/kg, intraperitoneal route), or with PCB-aa (peptide 3)/GHRP-6 (maintaining the corresponding dose and administration route) for 30 min, 3, 6 and 12 h after the ischemic event were evaluated. The bilateral cell count was performed in the C2, CA3 and CA4 regions for each experimental group and it was expressed in percentage with respect to the sham (negative or fake surgery) group. The results showed that in the animals of the I/R group, there was an almost complete loss of the cell line which encompasses practically all hippocampus zones (CA2, CA3, CA4).

[0086] A synergic effect was observed in the group treated with the PCB-aa combination (peptide 3)/GHRP-6 (85% effectiveness), in relation to the independent active principles with an effectiveness of 35.8% for the PCB-aa (peptide 3) and 36.1% for GHRP-6 (FIG. 8).

EXAMPLE 10

Therapeutic Effect of the PCB/EPO, PCB-aa (Peptide 4)/EPO, PCB/asyaloEPO and PCB-aa (Peptide 5)/asyaloEPO Combinations and Their Independent Active Principles in the Bilateral I/R Model in Gerbils

[0087] The animals were treated with a saline solution (through the intraperitoneal route) or with an accumulative dose of PCB (750 g/Kg, by the intraperitoneal route), PCB-aa (peptide 4) 3.375 mg/kg, EPO (500 U/Kg, through the intraperitoneal route), asyaloEPO (200 U/Kg, through the nasal route) or with PCB/EPO or PCB-aa (peptide 4)/EPO, PCB/asyaloEPO and PCB-aa (peptide 5)/asyaloEPO (maintaining the dose and the corresponding administration route) for 30 min, 3, 6 and 12 h after the ischemic event.

[0088] The evaluation of the therapeutic effect of the combinations and their independent components was carried out. The percentage of effectiveness of each treatment was calculated as described in Example 3.

[0089] A decrease of the brain infarct volume per group was observed, which evidenced the effectiveness of the treatments evaluated (FIG. 9), with a reduction of the infarct volume in the group treated with PCB of 43.1% effectiveness; PCB-aa (peptide 4) of 49.2%; EPO of 36.9%; asyaloEPO of 39.4%; and even greater in the groups treated with PCB/EPO (87.7% effectiveness), PCB/asyaloEPO (90.5% effectiveness), PCB-aa (peptide 4)/EPO (91.7%) and PCB-aa (peptide 5)/asyaloEPO (94.5%), which shows a synergic effect of the active principles in the combinations.

SUMMARY

[0090] The present invention describes peptides comprising phycocyanobilin (PCB), as well as the medical use of said peptides and that of PCB, due to the neuroprotector and/or neuroregenerative effects identified for them. Furthermore, pharmaceutical combinations of said peptides and of PCB with proteins or other peptides with synergic effect justify their use for ischemic or neurodegenerative CNS disease treatment.