(19)
(11)EP 1 300 416 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
11.07.2012 Bulletin 2012/28

(21)Application number: 01949207.3

(22)Date of filing:  28.05.2001
(51)Int. Cl.: 
C07K 5/113  (2006.01)
A61K 38/07  (2006.01)
A61P 25/28  (2006.01)
C07K 7/06  (2006.01)
A61K 38/08  (2006.01)
(86)International application number:
PCT/CN2001/000875
(87)International publication number:
WO 2002/002592 (10.01.2002 Gazette  2002/02)

(54)

MEMORY-ENHANCING PEPTIDES AND THE USE THEREOF

GEDÄCHTNISVERSTÄRKENDE PEPTIDE UND DEREN VERWENDUNG

PEPTIDE D'AUGMENTATION DE LA MEMOIRE ET SES APPLICATIONS


(84)Designated Contracting States:
AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

(30)Priority: 30.06.2000 CN 00116876

(43)Date of publication of application:
09.04.2003 Bulletin 2003/15

(73)Proprietor: Shanghai Cas Intide Biotech Co., Ltd.
Shanghai 201203 (CN)

(72)Inventors:
  • DU, Yucang
    Shanghai 200031 (CN)
  • SHEN, Jinhuan
    Shanghai 200031 (CN)

(74)Representative: Grosse, Rainer et al
Gleiss Grosse Schrell & Partner Patentanwälte Rechtsanwälte Leitzstrasse 45
70469 Stuttgart
70469 Stuttgart (DE)


(56)References cited: : 
EP-A- 0 106 404
EP-A- 0 620 230
CN-C- 1 036 010
EP-A- 0 393 934
EP-A2- 0 354 820
  
  • HIRATE KENJI ET AL: "No. 302, a newly synthesized (pGlu-4,Cyt-6)AVP(4-9) analogue, prevents the disruption of avoidance behavior" BEHAVIOURAL BRAIN RESEARCH, vol. 83, no. 1-2, 1997, pages 205-208, XP002442027 ISSN: 0166-4328
 
Remarks:
The file contains technical information submitted after the application was filed and not included in this specification
 
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Field of the Invention



[0001] This invention relates to a synthesis of peptide and its analogs. Particularly, it focuses on the synthesis and application.

Background of the Invention



[0002] It has been reported that arginine-vasopressin (AVP) have effect on the memory of animals but not show obvious influence on the learning process. Since AVP is a kind of hormone in vivo and its main function is in anti-diuretic process and to enhance blood pressure, it's not appropriate for the clinical therapy in enhancing memory. EP0354820 discloses peptide derivatives as antidementia agents. Therefore, on the basis of patent "memory-enhancing peptide and its application" (Chinese Patent No. ZL 93112493.X), we go on with work about the structural improvement and perform a series of pharmacological experiments, aiming to modify the defect in the existing patent and search for new memory-enhancing peptides which have simpler structure, stronger pharmacological activity, easy to produce, and have clinically curative effect on promoting the intelligence development in adolescent, ameliorating mental impediment in adults, accelerating the memory.

Summary of the Invention



[0003] The purpose of the invention is to provide for a kind of memory-enhancing peptides which can ameliorate mental impediment in adults and promote the intelligence development in adolescent, even can be a clinical medicine for the patient of Alzheimer's disease.

[0004] Depending on the results of biological activity, we have designed a series of new chemicals with memory- and learning- enhancing function. Their structure has the following general formula:

X represents Ala; Leu; Ile; Cys; Val;

R1 represents PyroGlu; H;

R2 represents S-S-C-(CH3)3; S-C(CH3)3; R2 is a kind of hydrophobic side-chain to stabilize spatial conformation of the whole molecule.

If R1 is PyroGlu, R2 is S-S-C(CH3)3 or S-C(CH3)3

If R1 is H, R2 must be S-S-C(CH3)3 or S-C(CH3)3

X represents Ala; Leu; Ile; Cys; Val.



[0005] Not being part of the invention, X can also be Phe or Tyr and R2 can be other groups which have hydrophobic side-chain to stabilize the spatial conformation of the whole molecule.

[0006] The peptides in this invention can be obtained through solid or liquid peptide synthesis method step by step. The synthesize protocol is as following:





[0007] 2. Synthesis of peptide Asn-Cys(But)-Pro-Arg:



[0008] The pharmacological tests of memory-enhancing peptide in this invention have been performed as following: the animals have been divided into three groups: drug administration group, control group and model group, with each group administrated with memory-enhancing peptide, saline and scopolamine respectively. In the test, the groups except control group were injected with scopolamine after the administration of medicine about 50 min., 10 min. later, the training began. First, the animals will be put into reaction box for 3 min. before each trails to familiarize them with the environment. Then, the reaction box are charged with 36V alternating current. When the animal subjects to electrical shock, it will jump up to the central platform to avoid harmful stimulation as a normal response. Most of animals may be return to the copper curdles twice or even more times, but they will jump back to the platform swiftly as the results of electrical shock. Each test trail will be performed for 5 min. and two following indices will be used to represent the learning results: (I) the number of total error (EN) during the whole training period; (2) the reaction time (RT) for the first returning to the copper curdle in the trail. The trails will repeat again 24 hrs later as memory test. The results as shown in Table 1 demonstrated that memory-enhancing peptide can remarkably ameliorate the memory and learning impediment induced by scopolamine.

[0009] Compared with model group, the memory-enhancing peptide at the dose of 0.5 µg and 1 µglkg can obviously improve the memory and learning impediment induced by scopolamine, shown as longer incubation period and lesser error numbers which have marked difference from those of model group(P<0.01). In addition, memory-enhancing peptide(0.5 µg /kg and 1 µg / kg) have better effects than Huperzine A (100 µg /kg )in ameliorate the memory and learning impediment induced by scopolamine.
Table 1. The effect of memory-enhancing peptide(MEP531) on the learning and memory process of mouse
GroupDrug dosage µg/kgScopolamine mg/kgLearning RT(s)Results ENMemory RT(s)Results EN
Control group Saline - 131.4±98.3 0.9±0.87 285.4±22.6 0.4±0.51
Model group Saline 1 27.5±29A** 2.3±1.4* 140±117** 1.9±1.2**
HuperzineA 100 1 120.5±114.9* 13±23** 2319±78** 0.9±0.7**
MEP-S31 0.25 1 78.8±118.7 0.3:1:0.48** 197.6±75.1 0.9±0.56**
MEP-S31 0.5 1 241.6±51.9** 0.2±0.4** 245.5±62.9** 0.5±0.5**
MEP-531 1 1 267.6±31.4** 0.3±0.5** 282.8±33.4** 0.3±0.5**
◆<0.05, ◆◆<0.01,compared with control group;
*<0.05, * *<0.01, compared with the model group;

Detailed Description of the Invention


Implement case 1


Chemical synthesis of Asn-Cys(But)-Pro-Arg(MEP422):



[0010] Boc-system step-by-step solid phase synthesis method; Started from 2g Merrifield resin.

[0011] First,





[0012] The crude product described above was separated and purified through Silica gel column (Silicagel 60) initially and then subjected to C18 HPLC for further purification. The elution time in HPLC is 19.1 min and solution system is TFA / acetonitrile system.

[0013] The results shown in the following table demonstrated the product has reasonable amino acid component.
Experimental value Asn:1.05 Arg:0.96 Pro:1.08 Cys:not anlysis
Theory value Asn:1.00 Arg:1.00 Pro:1.00  
MS analysis: MW+544.3      


[0014] The method described in the upper phase can also be applied to the synthesis of other analogs as X represents Ala; Leu; Ile; Phe; Tyr; Val respectively.

Implement case 2


Chemical synthesis of PyoGlu-Asn-Cys(S-But)-Pro-Arg(MEP531):



[0015] Boc-system step-by-step solid phase synthesis method; Started from 2g Merrifield resin.

[0016] First,





[0017] The upper crude penta-peptide preparation was separated and purified through Silica gel column initially and then subjected to C18 HPLC for further purification. The elution time in HPLC is 19.5 min and solution system is TFA / acetonitrile system. The results shown in the following table demonstrated the products has reasonable amino acid component.
Experimental value Asn:1.01 Flu:1.03 Arg:0.96 Pro:0.95 Cys:not anlysis
Theory value Asn:1.00 Glu:1.00 Arg:1.00 Pro:1.00  
MS analysis: MW+687      


[0018] The method described in the upper phase also can be applied to the synthesis of other analogs as X represents Ala; Leu; Ile; Phe; Tyr; Val respectively.


Claims

1. A memory-enhancing peptide, characterized by its chemical structure formula which is shown as following:

X represents Ala; Leu; Ile; Cys; Val;

R1 represents PyroGlu; H;

R2 represents S-S-C(CH3)3; S-C(CH3)3·


 
2. A memory enhancing peptide according to claim 1, characterized in that:

If R1 is PyroGlu; R2 is S-S-C(CH3)3 or S-C(CH3)3;

If R1 is H; R2 is S-S-C(CH3)3 or S-C(CH3)3;

X represents Ala; Leu; Ile; Cys; Val.


 
3. A memory-enhancing peptide according to claim 1, characterized in that they are obtained through solid or liquid peptide synthesis method step by step.
 
4. A memory enhancing peptides according to claim 1 for use to ameliorate memory impediment and improve intelligence development on juvenile.
 


Ansprüche

1. Gedächtnis-verbesserndes Peptid, gekennzeichnet durch seine chemische Strukturformel, die nachfolgend dargestellt ist:

wobei

X Ala, Leu, Ile, Cys, Val darstellt,

R1 PyroGlu, H darstellt,

R2 S-S-C(CH3)3, S-C(CH3)3 darstellt.


 
2. Gedächtnis-verbesserndes Peptid nach Anspruch 1, dadurch gekennzeichnet, dass:

wenn R1 PyroGlu ist, R2 S-S-C(CH3)3 oder S-C(CH3)3 ist,

wenn R1 H ist, R2 S-S-C(CH3)3 oder S-C(CH3)3 ist,

X Ala, Leu, Ile, Cys, Val darstellt.


 
3. Gedächtnis-verbesserndes Peptid nach Anspruch 1, dadurch gekennzeichnet, dass es durch Festphasen- oder Flüssig-Peptidsyntheseverfahren Schritt für Schritt erhalten wird.
 
4. Gedächtnis-verbesserndes Peptid nach Anspruch 1 zur Verwendung zur Linderung von Gedächtnisstörungen und zur Verbesserung der Intelligenzentwicklung bei Jugendlichen.
 


Revendications

1. Peptide d'amélioration de mémoire, caractérisé par sa formule structurelle chimique qui est illustrée comme suit :

X représente Ala ; Leu ; Ile ; Cys ; Val ;

R1 représente PyroGlu ; H ;

R2 représente S-S-C(CH3)3 ; S-C(CH3)3.


 
2. Peptide d'amélioration de mémoire selon la revendication 1, caractérisé en ce que :

si R1 est PyroGlu ; R2 est S-S-C(CH3)3 ou S-C(CH3)3 ;

si R1 est H ; R2 est S-S-C(CH3)3 ou S-C(CH3)3 ;

X représente Ala ; Leu ; Ile ; Cys ; Val.


 
3. Peptide d'amélioration de mémoire selon la revendication 1, caractérisé en ce qu'il est obtenu par un procédé de synthèse peptidique solide ou liquide étape par étape.
 
4. Peptide d'amélioration de mémoire selon la revendication 1 destiné à être utilisé pour améliorer un ralentissement de mémoire et améliorer le développement de l'intelligence chez les jeunes.
 




REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description