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Justia Patents US Patent for Oligopeptides with specific inhibiting properties of collagen induced aggregation, process for preparing the same and pharmaceutical compositions containing them Patent (Patent # 4,474,761)
Oligopeptides with specific inhibiting properties of collagen induced aggregation, process for preparing the same and pharmaceutical compositions containing them
Dec 26, 1981 - Choay S.A.
Oligopeptides having no more than 10 amino acids containing the sequence--(N-ter) A--X, --Gly--Y--Gly--X.sub.2 --A (C-ter)having the capacity of interacting with blood platelets and an ability to inhibit the aggregation of platelets induced by a collogen containing substance.
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The invention relates to new peptides having biological properties, particularly a capacity of interacting with blood platelets and an ability to inhibit the aggregation of platelets induced by collagen or collagen containing substances.
It also relates to processes for preparing said peptides and lastly to pharmaceutical compositions containing them.
It is known that the vessel wall plays an important role in the maintenance of balance between haemmorrhage and thrombosis. The endothelial lining is thrombo-resistant, while the subendothelium is thrombogenic.
If the endothelium is pathologically or experimentally damaged, adhesion of the blood platelets to the subendothelial surfaces becomes possible.
The subendothelium comprises different constituents among which can be cited collagen, microfibrils, elastin, etc.
Studies have shown that notably subendothelial collagens and microfibrils are potent inducers of platelet adhesion, which adhesion is responsible for cellular activation with the release of numerous intraplatelet constituents, such as serotonin, and the involvement of many complex biochemical phenomena, which ultimately lead to the aggregation of the platelets.
All these mechanisms are so far but little known.
It is not known in particular which portion of subendothelial collagen, preferably of type III collagen is responsible for platelet adhesion and for the resulting subsequent platelet aggregation.
Recent studies have shown that a 149 aminoacid peptide (hereafter called .alpha..sub.1 (III)CB4) obtained by enzymatic cleavage of type III collagen, seems responsible for platelet adhesion, whereas the other derived peptidic fragments, resulting from the same enzymatic cleavage, are inactive as regards platelet adhesion (FAUVEL et al. Thrombosis Research, vol. 12, no. 5, p. 841-850).
Further studies and experiments on .alpha..sub.1 (III)CB4 peptide have been carried out .alpha..sub.1 (III)CB4 peptide was cleaved by chymotrypsin, hydroxylamin and trypsin to give three fragments, respectively designated as C2 (located between the 63rd and 149th aminoacyl residue of .alpha..sub.1 (III)CB4), and HA4 (located between the 76th and 149th aminoacyl residue of .alpha..sub.1 (III)CB.sub.4), and T6 (located between the 52nd and 84th aminoacyl residue of .alpha..sub.1 (III)CB.sub.4) (FAUVEL et al., Thrombosis Research, 16, p. 269-273).
The three peptidic fragments have been found to be active as regards platelet adhesion, and the attendant intracellular modification of the metabolism as well as release of intraplatelet constituents, particularly serotonin. A non isolated sequence:
--Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys--
common to the abovesaid peptide fragments C2, HA4 and T6, was considered as possibly representing the "adhesive site" of collagen towards platelets.
It was however found subsequently that the above adhesion active fragments inhibit the aggregation of platelets induced by the type III collagen molecule (FAUVEL et al. Thrombosis Research, 17, p. 285-287) thereby making any prevision on any possible relationship between adhesion and aggregation virtually impossible.
Applicants have now synthesized much smaller peptides, including a nonapeptide including the nine aminoacyl residues defined hereabove in the same order, which have unexpectedly been found to selectively inhibit the aggregation of platelets induced by collagen, yet while being free of the major phenomena that are consequent to adhesion, that is cellular activation and the attendant liberation of platelet constituents.
These small peptides are the first compounds that have been found to inhibit the aggregation of blood platelets as induced by collagen, yet without modifying the natural intracellular metabolism of the platelets, without causing cellular activation and liberation of intraplatelet constituents.
In addition, these peptides specifically inhibiting the interaction between platelet and collagen, are specific inhibitors of platelet aggregation induced by collagen with, suprisingly, no activity against the aggregation induced by other platelet aggregation inducers.
An object of the invention is to provide peptides enabling the minimum sequence of collagen which specifically inhibits the collagen interaction with platelets to be identified.
Another object of the invention is to provide peptides enabling to understand and regulate the platelet aggregation phenomena, with respect to collagen III, in the absence of modification of the natural intracellular metabolism of the platelets and without liberation of intraplatelet constituents, such as serotonin.
A further object of the invention is also to provide peptide reagents, as a means of identification of the minimum sequence of the microfibrils, of any kind of collagen such as those of type I, IV and V, and of any collagen-containing substances which specifically inhibit the collagen interaction with platelet.
Still another object of the invention is to provide new peptides for the production of pharmaceutical drug compositions, effective as platelet aggregation inhibitors useful in particular for the prevention of thrombosis and microthrombosis caused by the injury of the vessel wall.
Particularly it is an object of the invention to provide an active principle for drug compositions, effective as a specific inhibitor of blood platelet aggregation induced by collagen, yet without substantially interfering with the cellular metabolism of said platelets, particularly without causing release of intraplatelet constituents, particularly serotonin or other platelet constituents liable of possessing themselves undesirable side effects.
Thus the invention aims at providing a method for the control of platelet aggregation, more simple to bring into practice in as much as it does not simultaneously involve the control of the side effects caused by the concommittent effects of other drugs used as platelet aggregation inhibitors on the platelet intracellular metabolism.
The invention is relative, in its broadest aspect to peptides containing no more than 10 amino acids, preferably levorotatory amino-acids, and containing the following sequence:
--(N--ter)A--X.sub.1 --Gly--Y--Gly--X.sub.2 --A(C--ter)--
in which:
X.sub.1 and X.sub.2 represent, independently from each other, either a hydroxyprolyl or a prolyl residue;
Y represents either a --Z--X.sub.3 -- or a --X.sub.3 --X-- residue wherein X.sub.3 is a hydroxyprolyl or prolyl residue and Z is a spacer aminoacyl residue and
A is a polyamino-acyl residue, particularly selected from among arginyl, ornithyl or cystyl, preferably however lysyl residues.
As indicated hereabove Z appears essentially to act, together with the X.sub.3 aminoacyl residue, as a "spacer" between the --A--X.sub.1 --Gly-- and the --Gly--X.sub.2 --A peptidyl moieties. Advantageously Z is selected from among dicarboxylic monoamino-acids or monocarboxylic-mono-amino-acids.
Examples of such monocarboxylic-mono-aminoacyl residues are alanyl, leucyl, isoleucyl, norleucyl, valyl, glycyl, prolyl. Preferably however Z is selected from among glutaminyl, glutamyl, aspartyl and asparaginyl.
In a preferred class of compounds of the invention, one at least of the A terminal residue is engaged in a peptitic bond with a glycyl residue.
In another preferred class of compounds of the invention, the N-terminal residue A is a lysyl residue, possibly itself engaged in a peptidic bond with a glycyl residue.
In another preferred class of compounds of the invention, the C-terminal residue is a lysyl residue, possibly itself engaged in a peptitic bond with a glycyl residue.
A particular preferred class of compounds of the invention contains a sequence having the following formula:
--(N--ter)A--X.sub.1 --Gly--Z--X.sub.3 --Gly--X.sub.2 --A(C--ter)--
A represents a residue chosen from the following: lysyl, arginyl,
X.sub.1, X.sub.2 and X.sub.3 represent independently from each other either the hydroxyprolyl residue, or the prolyl residue;
Z is selected from among dicarboxylic monoaminoacids or monocarboxylic monoaminoacids, and is preferably chosen among glutaminyl, glutamyl, aspartyl and asparaginyl.
Another preferred class of compounds according to the invention, contains a peptidic sequence having the following formula:
--(N--ter)A--X.sub.1 --Gly--X.sub.3 --Z--Gly--X.sub.2 --A(C--ter)--
A, X.sub.1, X.sub.2, X.sub.3 and Z have the above meanings.
In preferred compounds of the invention, Z is a glutamyl residue, the formulae of the corresponding peptides then containing either of the following sequences:
--(N--ter)A--X.sub.1 --Gly--Glu--X.sub.3 --Gly--X.sub.2 --A(C--ter)--
--(N--ter)A--X.sub.1 --Gly--X.sub.3 --Glu--Gly--X.sub.2 --A(C--ter)--
A, X.sub.1, X.sub.2 and X.sub.3 have the above mentioned meanings.
In further preferred compounds of the invention, A represents the lysyl residue, the formulae of the corresponding peptides then containing either of the following sequences:
--(N--ter)Lys--X.sub.1 --Gly--Glu--X.sub.3 --Gly--X.sub.2 --Lys(C--ter)--
--(N--ter)Lys--X.sub.1 --Gly--X.sub.3 --Glu--Gly--X.sub.2 --Lys(C--ter)--
X.sub.1, X.sub.2 and X.sub.3 represent independently from each other either the hydroxyprolyl residue, or the prolyl residue.
In orther preferred compounds of the invention, X.sub.1, X.sub.2 and X.sub.3 simultaneously represent the prolyl residue.
In other preferred compounds of the invention, X.sub.1, X.sub.2 and X.sub.3 simultaneously represent the hydroxyprolyl residue.
In other preferred compounds of the invention, X.sub.1 represents the hydroxyprolyl residue and X.sub.2 the prolyl residue or vice-versa.
In other preferred compounds of the invention, X.sub.1 and X.sub.3 simultaneously represent the hydroxyprolyl residue and X.sub.2 represent the prolyl residue or vice-versa.
Preferred peptides of the invention have the following formula:
Lys--Pro--Gly--Glu--Pro--Gly--Pro--Lys
Lys--Hyp--Gly--Glu--Pro--Gly--Hyp--Lys
Lys--Pro--Gly--Glu--Pro--Gly--Hyp--Lys
Lys--Hyp--Gly--Glu--Pro--Gly--Pro--Lys
Lys--Pro--Gly--Glu--Hyp--Gly--Pro--Lys
Lys--Hyp--Gly--Glu--Hyp--Gly--Hyp--Lys
Lys--Pro--Gly--Glu--Hyp--Gly--Hyp--Lys
Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys
Gly--Lys--Pro--Gly--Glu--Pro--Gly--Pro--Lys (1)
Gly--Lys--Hyp--Gly--Glu--Pro--Gly--Hyp--Lys
Gly--Lys--Pro--Gly--Glu--Pro--Gly--Hyp--Lys
Gly--Lys--Hyp--Gly--Glu--Pro--Gly--Pro--Lys
Gly--Lys--Pro--Gly--Glu--Hyp--Gly--Pro--Lys
Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Hyp--Lys
Gly--Lys--Pro--Gly--Glu--Hyp--Gly--Hyp--Lys
Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys (4)
Lys--Pro--Gly--Glu--Pro--Gly--Pro--Lys--Gly
Lys--Hyp--Gly--Glu--Pro--Gly--Hyp--Lys--Gly
Lys--Pro--Gly--Glu--Pro--Gly--Hyp--Lys--Gly
Lys--Hyp--Gly--Glu--Pro--Gly--Pro--Lys--Gly
Lys--Pro--Gly--Glu--Hyp--Gly--Pro--Lys--Gly
Lys--Hyp--Gly--Glu--Hyp--Gly--Hyp--Lys--Gly
Lys--Pro--Gly--Glu--Hyp--Gly--Hyp--Lys--Gly
Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys--Gly (2)
Tyr--Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys (3)
Lys--Pro--Gly--Pro--Glu--Gly--Pro--Lys
Lys--Hyp--Gly--Pro--Glu--Gly--Hyp--Lys
Lys--Pro--Gly--Pro--Glu--Gly--Hyp--Lys
Lys--Hyp--Gly--Pro--Glu--Gly--Pro--Lys
Lys--Pro--Gly--Hyp--Glu--Gly--Pro--Lys
Lys--Hyp--Gly--Hyp--Glu--Gly--Hyp--Lys
Lys--Pro--Gly--Hyp--Glu--Gly--Hyp--Lys
Lys--Hyp--Gly--Hyp--Glu--Gly--Pro--Lys
Gly--Lys--Pro--Gly--Pro--Glu--Gly--Pro--Lys
Gly--Lys--Hyp--Gly--Pro--Glu--Gly--Hyp--Lys
Gly--Lys--Pro--Gly--Pro--Glu--Gly--Hyp--Lys
Gly--Lys--Hyp--Gly--Pro--Glu--Gly--Pro--Lys
Gly--Lys--Pro--Gly--Hyp--Glu--Gly--Pro--Lys
Gly--Lys--Hyp--Gly--Hyp--Glu--Gly--Hyp--Lys
Gly--Lys--Pro--Gly--Hyp--Glu--Gly--Hyp--Lys
Gly--Lys--Hyp--Gly--Hyp--Glu--Gly--Pro--Lys
Lys--Pro--Gly--Pro--Glu--Gly--Pro--Lys--Gly
Lys--Hyp--Gly--Pro--Glu--Gly--Hyp--Lys--Gly
Lys--Pro--Gly--Pro--Glu--Gly--Hyp--Lys--Gly
Lys--Hyp--Gly--Pro--Glu--Gly--Pro--Lys--Gly
Lys--Pro--Gly--Hyp--Glu--Gly--Pro--Lys--Gly
Lys--Hyp--Gly--Hyp--Glu--Gly--Hyp--Lys--Gly
Lys--Pro--Gly--Hyp--Glu--Gly--Hyp--Lys--Gly
Lys--Hyp--Gly--Hyp--Glu--Gly--Pro--Lys--Gly
Tyr--Gly--Lys--Hyp--Gly--Hyp--Glu--Gly--Pro--Lys
In an advantageous embodiment of the invention, the peptides have the following formula:
Lys--Pro--Gly--Glu--Pro--Gly--Pro--Lys (1)
Other features and advantages of the invention will be apparent for those skilled in the art from the following description and the preferred embodiments of the invention.
SYNTHESIS OF PEPTIDES ACCORDING TO THE INVENTION
One may resort to processes conventional per se, for peptide synthesis for producing the peptides according to the invention. They are recalled hereafter briefly.
The synthesis of peptides in homogeneous solution and by solid phase peptide synthesis are well documented (HOUBEN-WEYL "Methoden der Organischen Chemie" E. Wunsch Eds, vol. 15-I et II THIEME, Stuttgart 1974).
Two different approaches are possible in peptide synthesis, that of fragment condensation and stepwise synthesis. Both strategies require that one aminoacid or peptide fragment have its amino group protected and its carboxyl group activated and that the second aminoacid or peptide fragment have its amino group free and its carboxyl group protected; when the aminoacid involved has two amino functions, such as lysine, or two acid functions, such as glutamic acid, it is necessary to protect either the amino function or the acid function not involved in the peptidic synthesis by protecting groups, preferably carbobenzoxy or t-butyloxycarbonyl groups for the amino function, for instance a lysyl residue and t-butylester groups for the acid functions, for instance of a glutamyl residue.
As regards the stepwise synthesis the aminoacids are added one after each other, starting with the C-terminal aminoacid.
As regards the fragment condensation strategy, all the combinations can be theoretically contemplated, provided they do not involve the condensation of a fragment whose C-terminal residue is either lysine or glutamic acid (because of a possible racemisation).
This way is quick, since several fragments can be prepared at the same time, then linked to each other.
Final purifying is made easier because of the difference in the molecular weight between the fragments and the complete peptide chain.
More particularly, in an advantageous embodiment of the invention, the synthesis of the nine aminoacid peptides of the formula (4) according to the invention, is carried out by resorting to fragment condensation, with the synthesis of the three following fragments:
Gly--Lys--Hyp
Gly--Glu--Hyp
Gly--Pro--Lys
which enables to have fragments to be coupled whose C-terminal residue is not racemisable.
Convention of designation of the individual aminoacyl residues in different peptide fragments considered hereafter:
Numbers have been specifically assigned to the different aminoacyl residues by reference to the position which they occupy, when contained in the decapeptide of formula:
Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys--Gly
Thus any of said numbers, when used to designate the aminoacyl residues contained in a smaller peptide fragment merely refers back to the corresponding aminoacyl residue and is not intended to specify the position of the latter with respect to N-terminal residue of said smaller fragment. ##STR1##
In a more preferred embodiment of the invention, the process for preparing the peptidic sequence of formula (4) is carried out as reported below.
In the course of the specification, the abbreviations which are used have the following meaning:
Z                carbobenzoxy-
BOC              t-butyloxycarbonyl-
OMe              methylester
OBu.sup.t        t-butylester
OSu              succinimid ester
Hyp              hydroxyprolin
Glu              glutamic acid
Pro              prolin
Gly              glycin
Lys              lysin.
Chromatographies on thin layer of silica gel have been carried out, in which the solvent mixtures which have been used (v/v) are the following ones:
chloroform-aceton: 5-2 (A)
chloroform-methanol: 8-1 (B), 50-15 (C), 9-1 (D), 6-1 (E)
chloroform-methanol-benzen: 8-1-1 (F)
ethyl acetate-hexane: 9-1 (G)
ethyl acetate-pyridin-acetic acid-water: 6-2-0, 6-1 (H), 12-2-0, 6-1 (I), 5-5-1-3 (J).
The identifications have been carried out with ninhydrine and chlore-o-tolidine reactive.
For the purification, Silica Gel 60 (trademark of resin commercialized by MERCK-Darmstadt, Germany) was used.
The nonapeptide of the following formula (4):
Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys
has been synthesized by means of fragment condensation in which the three following fragments:
Gly--Lys--Hyp--Gly (1-4 fragment),
Glu--Hyp (5-6 fragment),
Gly--Pro--Lys (7-9 fragment),
have been prepared.
SYNTHESIS OF 1-4 FRAGMENT Z--Gly--Lys--(Z)--Hyp--Gly--OH BOC--Lys--(Z)--Hyp--OMe (I)
1.43 g (3 mM) of BOC--Lys--(Z)--OSu (P. Hartter, Hoppe Seyler's. Physiol. Chem. 357 (1976), 1 683) dissolved in 5 ml of dimethylformamid are added to a solution, cooled to 0.degree. C., of 363 mg (2 mM) of Hyp--OMe, HCl (E. L. Smith and M. Bergmann, j. Biol. Chem. 153 (1944), 627) and 0.22 ml (2 mM) of N-methylmorpholine in 5 ml of dimethylformamid. The reaction mixture is stirred for a night at ambient temperature then treated with 0.30 ml (3 mM) of dimethylaminopropylamine for one hour. The dimethylformamid is concentrated to dryness and the residue is taken up in 50 ml of ethyl acetate. This organic phase is successively washed with KH SO.sub.4 5%, in water, with NaHCO.sub.3 M, in water then dried over Mg SO.sub.4, and concentrated to dryness. The oil which is obtained (860 mg) is chromatographed on a column (28.times.3 cm) of silica 60 (89 g), eluted with the solvent mixture of chloroform-aceton 5-2 (v/v).
The fractions are gathered and concentrated to dryness into a residue which is taken up in aceton, ultrafiltrated, concentrated and precipited in ether-hexan: 675 mg (corresponding to a yield of 66.5%) of compound (I) are obtained.
MP=112.degree. C.
[.alpha.].sub.D.sup.20 =-53.8.degree. (c=0.5, chloroform)
Rf.=0.28 (A)
Rf.=0.27 (G)
The elementary analysis of the product is:
C.sub.25 H.sub.37 N.sub.3 O.sub.8 (507.60)
C %        H %    N %
calculated     59.2       7.3    8.3
found          58.6       7.3    8.0
Lys--(Z)--Hyp-OMe, HCl (II)
597 mg (1.17 mM) of compound (I) are treated with 3.6 ml of HCl solution (1N) in acetic acid, for 30 minutes. After concentration, 560 mg of residue are obtained and dried (corresponding to a yield of 100%).
Rf.=0.38 (H)
Z--Gly--Lys--(Z)--Hyp--OMe (III)
460 mg (1.5 mM) of Z--Gly--OSu (G. W. Anderson, J. E. Zimmermann and F. M. Callahan, J. AM. Chem. Soc. 86 (1964) 1 839) dissolved in 5 ml of dimethylformamid are added to a solution which has been cooled to 0.degree. C., of 560 mg (1.1 mM) of compound (II) and 0.16 ml (1.1 mM) of triethylamin in 5 ml of dimethylformamid. The compound (III) is prepared as it has been described for compound (I). The oily residue obtained (742 mg) is chromatographed on column (30.times.3 cm) of silica 60 (100 g) eluted with a solvent mixture of chloroform methanol 1-1 (v/v). The fractions obtained (533 mg corresponding to a yield of 79%) are treated as usual:
Rf.=0.58 (B)
Rf.=0.39 (E)
Z--Gly--Lys--(Z)--Hyp--OH (IV)
520 mg (0.87 mM) of compound (III) are solubilized in 8.5 ml of absolute methanol and treated for 4 hours with 1 ml of 2M NaOH solution. After addition of 10 ml of water, the methanol is evaporated and the aqueous phase is extracted with ethyl acetate, then acidified with citric acid (pH 3). This aqueous phase is extracted by means of ethyl acetate, then, it is washed with an aqueous solution saturated in NaCl, and it is further dried over MgSO.sub.4 and concentrated to dryness: 390 mg of compound (IV) are obtained, which correspond to a yield of 76.8%.
Rf.=0.18 (I)
Z--Gly--Lys--(Z)--Hyp--Gly--OBu.sup.t (V)
117 mg (0.2 mM) of compound (IV) are dissolved in 2 ml of dimethylformamid. 22 .mu.l (0.2 mM) of Gly--OBu.sup.t, HCl (C. H. Li, B. Gorup, D. Chung, and J. Ramachandran, J. Org. Chem. 28 (1963) 178) and 24 .mu.l (0.22 mM) of N-methylmorpholin in 2 ml of dimethylformamid are successively added to this solution cooled to -15.degree. C. After 4 hours, at -15.degree. C., 0.2 ml of a 2.5 MKHCO.sub.3 solution are added to the reaction mixture, then 30 minutes after, 20 ml of ethyl acetate are added. The organic phase is washed with NaHCO.sub.3 M, in water, with KHSO.sub.4 5%, in aqueous solution saturated in NaCl, then it is dried over MgSO.sub.4 and concentrated to dryness (114 mg corresponding to a yield of 103%).
Z--Gly--Lys--(Z)--Hyp--Gly--OH (VI)
114 mg (0.2 mM) of compound (V) are treated with 0.2 ml of trifluoroacetic acid for 30 minutes. After concentration to dryness, the residue (150 mg) is purified on a column (22.times.1.5 cm) of silica 60 (20 g), eluted with the solvent mixture: ethyl acetate-pyridin-acetic acid-water 4.5-2-0.6-1 (v/v).
The fractions obtained (112 mg corresponding to a yield of 87.3%) are treated as usual.
SYNTHESIS OF 5-6 FRAGMENT Z--Glu--(OBu.sup.t)--Hyp--OH Z--Glu--(OBu.sup.t)--Hyp--OMe (VII)
1.3 g (3 mM) of Z--Glu--(OBu.sup.t)--OSu (A. Eberle, J. L. Franchere, G. I. Tesser and R. Schwyzer, Helv. Chim. Acta. 58 (1975) 2 106) dissolved in 5 ml dimethylformamid, are added to a solution cooled to 0.degree. C. of 363 mg (2 mM) of Hyp--OMe, HCl (E. L. Smith and M. Bergmann, J. Biol. Chem. 153 (1944), 627) and 0.22 ml (2 mM) of N-methylmorpholin. Compound (VIII) is prepared as it has been described for compound (I). The oily residue which is obtained (627 mg corresponding to a yield of 68%) is only crystallized in ethyl acetate petroleum ether.
MP=105.degree.-106.degree.
[.alpha.].sub.D.sup.20 =-58.5.degree. (c=1, glacial acetic acid)
C.sub.23 H.sub.32 O.sub.2 N.sub.8 (464.52)
Z--Glu--(OBu.sup.t)--Hyp--OH (VIII)
495 mg (1.06 mM) of compound (VII) are solubilized in 10 ml of absolute methanol and treated for 4 hours with 1 ml of 2M KOH. Compound (VIII) is then prepared as it has been described for compound (IV). The oily residue which has been obtained (533 mg) is chromatographed on a column (28.times.3 cm) of silica 60 (51 g) eluted with a solvent mixture of ethyl acetate pyridine-acetic acid-water 9-2-0.6-1 (v/v). The fractions are treated as usual. The product is crystallized in acetone-petroleum ether; (309.6 mg which correspond to a yield of 65% of compound (VII) are thus obtained.
MP (decomposition)=55.degree.-68.degree.
SYNTHESIS OF 7-9 FRAGMENT Gly--Pro--Lys--(BOC)--OBu.sup.t, acetate Z--Gly--Pro (IX)
1.9 g (6.2 mM) of Z--Gly--OSu (G. W. Anderson, J. E. Zimmermann and F. M. Callahan, J. Am. Chem. Soc. 86 (1964) 1 839) dissolved in 5 ml of tetrahydrofuran are added to a solution cooled to 0.degree. C., of 691 mg (6 mM) of prolin and 505 mg (6 mM) of NaHCO.sub.3 in 10 ml of tetrahydrofuran and 5 ml of water. Compound (IX) is prepared as it has been described for compound (I). The oily residue is crystallized without any other purification in ethyl acetate. 1.07 g which correspond to a yield of 58.2% of compound (IX) are obtained.
[.alpha.].sub.D.sup.20 =-66.4.degree. (c=1 absolute ethanol)
(Litt. MP=150.degree.-151.degree.)
[.alpha.].sub.D.sup.20 =-60.degree. (absolute ethanol)
Z--Gly--Pro--Lys--(BOC)--OBu.sup.t (X)
1.053 g (3.44 mM) of compound (IX) are dissolved in 10 ml of dimethylformamid. 0.38 ml (3.54 mM) of N-methylmorpholin and 0.45 ml (3.45 mM) of isobutyl chlorocarbonate are added to this solution cooled to -15.degree. C., 3 minutes after, a cooled solution of 1.25 g (3.44 mM) of Lys--(BOC)--OBu.sup.t, acetate and 0.38 ml (3.45 mM) of N-methylmorpholin in 10 ml of dimethylformamid is added. Compound (X) is prepared as it has been described for compound (V). The residue which is obtained (2 g) is purified on a silica column (designated under Chromatospack, manufactured by Jobin and Yvon, France) eluted with a solvent mixture chloroform-aceton 50-15 (v/v). The fractions are treated as usual. The product is crystallized in aceton-ether-hexan. 1.435 g of compound (X) (which correspond to a yield of 70.7%) are thus obtained.
Rf.=0.33 (C)
Gly--Pro--Lys--(BOC)--OBu.sup.t, acetate (XI)
1.435 g (2.43 mM) of compound (X) are solubilized in 20 ml of glacial acetic acid and hydrogenated for 4 hours with palladium 5% on coal. After filtration on the catalyst, the filtrate is concentrated to dryness and the residue is dried. 1.4 g of compound (XI) (which correspond to a yield of 100%) are thus obtained.
SYNTHESIS OF 5-9 FRAGMENT Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--OBu.sup.t Z--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--OBu.sup.t (XII)
288 mg (0.64 mM) of compound (VIII) are dissolved into 3 ml of dimethylformamid. 70 .mu.l (0.64 mM) of N-methylmorpholin and 83 .mu.l (0.64 mM) of isobutyl chlorocarbonate are successively added to this solution cooled to -15.degree. C. 3 minutes after, a cooled solution of 440 mg (0.85 mM) of compound (XI) and 94 .mu.l (0.85 mM) of N-methylmorpholin in 5 ml of dimethylformamid are added. Compound (XII) is prepared as it has been described for compound (V). The residue which is obtained (545 mg) is purified on a column (30.times.3 cm) of silica 60 (100 g), eluted with the solvent mixture: chloroform-methanol 9-1 (v/v). The fractions which are obtained (450 mg corresponding to a yield of 54%) are treated as usual.
Rf.=0.40 (D)
Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--OBu.sup.t, acetate (XIII)
450 mg (0.5 mM) of compound (XII) are solubilized in 40 ml of acetic acid and hydrogenated for 4 hours with palladium 5% on coal. After filtration of the catalyst, the filtrate is concentrated to dryness. 408 mg of compound (XIII) (which correspond to a yield of 100%) are thus obtained.
SYNTHESIS OF 1-9 FRAGMENT Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys Z--Gly--Lys--(Z)--Hyp--Gly--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--OB u.sup.t (XIV)
112 mg (0.17 mM) of compound (VI) are dissolved in 2 ml of dimethylformamid. 19 .mu.l (0.17 mM) of N-methylmorpholin and 22 .mu.l (0.17 mM) of isobutyl chlorocarbonate are successively added to this solution cooled to -15.degree. C. 3 minutes after, a cooled solution of 169 mg (0.2 mM) of compound (XIII) and 22 .mu.l (0.2 mM) of N-methylmorpholin in 5 ml of dimethylformamid are added. Compound (XIV) is prepared as it has been described for compound (V). The residue which is obtained (224 mg) is solubilized in chloroform and chromatographied on a column (22.times.1.5 cm) of silica 60 (20 g), eluted with a solvent mixture, chloroform-methanol 6-1 (v/v). The fractions obtained (53 mg) are treated as usual. The non pure fractions obtained (135 mg) are gathered and concentrated. This raw product is purified on a column (15.times.1.5 cm) of silica 60 (13 g), eluted successively with the following solvent mixtures: chloroform-methanol 10-1, 8-1, 7-1 and 6-1 (v/v). The fractions obtained (66 mg) are treated as usual.
Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys (XV) (4)
53 mg (38.4 mM) of compound (XIV) are treated for 30 minutes with 1 ml of trifluoroacetic acid. After concentration to dryness, the residue is dissolved in 5 ml of acetic acid and hydrogenated for 2 hours with palladium 5% on coal. After filtration of the catalyst, the filtrate is concentrated to dryness. The residue is taken up in water, ultrafiltrated, then lyophilized. 39.6 mg of compound (4) (corresponding to a yield of 91%) are thus obtained.
.vertline..alpha..vertline..sub.D.sup.20 =-73.6.degree. (H.sub.2 O)
Rf.=0.08 (J)
The nonapeptide was obtained in the form of its trifluoroacetic salt in association with water molecules.
C.sub.38 H.sub.63 N.sub.11 O.sub.14 ; 2.5 CF.sub.3 COOH; 1.5 H.sub.2 O (1
210,05)
calculated 42.7         5.7    12.7
found      42.10        5.6    12.5
The nonapeptide can be obtained without water molecules, according to classical methods.
The elementary analysis of the nonapeptide free from water is the following one:
C.sub.38 H.sub.63 N.sub.11 O.sub.14 ; 2.5 CF.sub.3 COOH
calculated 43.65        5.58   13.02
found      43.2         5.6    12.09
The aminoacid analysis after total acid hydrolysis gives the following results:
(HCl 6N - 110.degree.
Gly (3), 3.0-Lys (2) 2.0
Pro (1), 1.1-Glu (1), 1.0-Hyp (2) 2.0
The nonapeptide can be obtained free from anion according to conventional methods of chromatography on ion exchange resins.
The nonapeptide can also be obtained in the form of any of its mineral or organic salts such as in the form of chlorhydrate, acetate, picrate, paratoluenesulfonate, according to classical methods of ion exchange.
The preferred salts of the nonapeptide are those which are physiologically acceptable such as the acetate salt.
The nonapeptide is obtained in the form of its acetate by chromatography of compound (XV) on a resin such as the one known under the designation "Amberlite IRA 45" commercialized by BIORAD Laboratories.
The fragment condensation (in the choice of the peptidic fragments which are first synthesized and then condensed) which has been carried out to obtain peptide of formula (4) can be explained as follows.
PREPARATION OF 5-9 FRAGMENT
Document of the prior art (E. Adams, Synthesis and Derivatives of 3-and 4-hydroxyproline, Int. J. Peptide Protein Res. 8 (1976) 503), relates to some of the difficulties in the preparation of peptides of sequence:
Glu--Hyp.
Accordingly, no result being obtained in the condensation of:
BOC--Glu--(Obu.sup.t)--OSu with H--Hyp--OH (corresponding to 5-6 fragment)
the synthesis of:
has been carried out as follows: ##STR2## (VIII) has been linked with the 7-9 fragment, which has been previously synthesized and whose protective groups have been imposed, as to their type, by the one of 5-6 fragment: ##STR3## 5-9 fragment was thus obtained: ##STR4## (VIII) could also have been readily linked after elimination of Z group, with Z--Gly--OSu, to obtain Z--Gly--Glu--(OBu.sup.t)--Hyp--OMe.
4-6 fragment could have been increased either on the C-terminal side (after saponification) either on the N-terminal side (after eliminating the Z group).
The following dipeptide:
(BOC)--Lys--(Z)--Hyp--OMe (I)
has firstly been prepared, then after eliminating the BOC group, has been condensed to:
Z--Gly--OSu
which gave: ##STR5##
This (IV) fragment could have been linked to 4-6 fragment. But it has preferably been linked to glycine as follows: ##STR6##
The OBu.sup.t ester has been advantageously used (rather than OMe ester) because the saponification of the latter is rather difficult.
The nonapeptide was obtained as follows: ##STR7##
One may add that the fact to protect, as regards 1-4 fragment, the amino functions by hydrogenolysable groups (Z) rather than acidolysable (BOC), takes into account the obtention of the:
Z--Gly--Lys--(Z)--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys
partially deprotected, and which can be easily purified because of its intermediary polarity as regards nonapeptide derivatives (XIV) and (XV).
It must be pointed out that the linking of the three contemplated fragments could have been carried out in an other order without any problem. The tactic choices could have possibly been different. ##STR8##
In order to obtain said octapeptide, recourse is had to fragment condensation as follows:
synthesis of 2-4 fragment,
condensing 2-4 fragment with 5-9 fragment.
SYNTHESIS OF Lys--Hyp--Gly (2-4 fragment)
This synthesis is carried out according to the following reaction diagram: ##STR9##
BOC--Lys--(Z)--Hyp--OH is prepared from BOC--Lys--(Z)--Hyp--OMe according to the same reaction as the one involved for the synthesis of compound (IV) obtained from compound (III) (preparation of 1-4 fragment of nonapeptide of formula (XV) (4).
BOC--Lys--(Z)--Hyp--Gly--OBu.sup.5 is prepared from BOC--Lys--(Z)--Hyp--OH according to the same reaction as the one involved for the synthesis of compound (V) obtained from compound (IV) (preparation of 1-4 fragment of nonapeptide of formula (XV) (4).
BOC--Lys--(Z)--Hyp--Gly--OH is prepared from BOC--Lys--(Z)--Hyp--Gly--OBu.sup.t according to the same reaction as the one involved for the synthesis of compound (VI) obtained from compound (V) (preparation of 1-4 fragment of nonapeptide of formula (XV) (4).
SYNTHESIS OF Glu--Hyp--Gly--Pro--Lys (5-9 fragment)
The preparation of this fragment is carried out as it has been described in the preparation of nonapeptide of formula (XV) (4).
SYNTHESIS OF Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys (2-9 fragment)
The octapeptide 2-9 is obtained as follows: (involving the same reactions as the one described for the synthesis of nonapeptide of formula (XV) (4): ##STR10##
Recourse is had to the fragment condensation as follows:
synthesis of 7-10 fragment,
condensing 5-6 fragment with 7-10 fragment,
condensing 2-4 fragment with 5-10 fragment.
This synthesis is carried out as follows: ##STR11##
This synthesis of 2-4 fragment is carried out as it has been described for the preparation of the same 2-4 fragment in the synthesis of octapeptide Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys.
SYNTHESIS OF Glu--Hyp--Gly--Pro--Lys--Gly (5-10 fragment)
This synthesis is carried out in two stages:
1. Synthesis of 5-6 fragment:
Z--Glu--(OBu.sup.t)--Hyp--OH is obtained as it has been described in the preparation of the nonapeptide of formula (XV) (4).
2. Synthesis of 7-10 fragment:
The 7-10 fragment is obtained as follows: ##STR12##
Z--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t is obtained from Z--Gly--Pro and H--Lys--(BOC)--Gly--OBu.sup.t according to the same reaction as the one involved of the preparation of compound (X) (synthesis of nonapeptide of formula (XV) (4) in which Lys--(BOC)--OBu.sup.t is replaced by Lys--(BOC)--Gly--OBu.sup.t.
H--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t is obtained from Z--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t by hydrogenation.
The synthesis of 5-10 fragment is carried out as follows:
Z--Glu--(OBu.sup.t)--Hyp--OH+H--Gly--Pro--Lys--(BOC)Gly--OBu.sup.t .fwdarw.Z--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t .fwdarw.H--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t
Z--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t is obtained by condensing Z--Glu--(OBu.sup.t)--Hyp--OH on H--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t in the same way as it has been described for the preparation of compound (XII) (in the synthesis of nonapeptide of formula (XV) (4).
H--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--Gly--OBu.sup.t is obtained by hydrogenation in the same way as for the preparation of compound (XIII) obtained from compound (XII).
The nonapeptide is obtained as follows:
BOC--Lys--(Z)--Hyp--Gly--OH+H--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)- -Gly--OBu.sup.t .fwdarw.BOC--Lys--(Z)--Hyp--Gly--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BO C)--Gly--OBu.sup.t .fwdarw.Lys--Hyp--Gly--Glu--Hyp--Pro--Lys--Gly.
BOC--Lys--(Z)--Hyp--Gly--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--Gly-- OBu.sup.t is obtained by condensing 2-4 fragment on 5-10 fragment in the same way as it has been described for the preparation of compound (XIV) (in the synthesis of the nonapeptide of formula (XV) (4).
Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys--Gly is obtained from BOC--Lys--(Z)--Hyp--Gly--Glu--(OBu.sup.t)--Hyp--Gly--Pro--Lys--(BOC)--Gly- -OBu.sup.t as it has been described in the preparation of the nonapeptide (XV) (4).
It must be pointed out that linking of the three contemplated fragments could have been carried out in other order without any problem.
In the synthesis of all the peptides, according to the invention, the hydroxy prolyl residue can be replaced by the prolyl residue, and the prolyl residue can be replaced by the hydroxy prolyl residue without modifying the synthesis.
By resorting to the preparation method described for the nonapeptide of formula (4), other peptides have been prepared among which the three following ones, hereunder identified, as examples:
Gly-Lys-Pro-Gly-Glu-Pro-Gly-Pro-Lys (1)
.vertline..alpha..vertline..sub.D.sup.20 = -115.degree. (c = 0,5 H.sub.2
O)
Elementary analysis: C.sub.38 H.sub.63 N.sub.11 O.sub.12, 1,5 CH.sub.3
COOH, 3H.sub.2 O (1010)
found      48.7         7.2    15.3
Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys-Gly (2)
.vertline..alpha..vertline..sub.D.sup.20 = -98.3.degree. (c = 0.6 H.sub.2
Elementary analysis: C.sub.38 H.sub.63 N.sub.11 O.sub.12, 1,25 CH.sub.3
COOH, 4H.sub.2 O
(1 045.14)
Tyr-Gly-Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys (3)
.vertline..alpha..vertline..sub.D.sup.20 = -67.2.degree. (c = 0.85
H.sub.2 O)
Elementary analysis: C.sub.47 H.sub.70 N.sub.12 O.sub.16, 1.25 CH.sub.3
The peptides according to the invention, have been subjected to the following experiments, in the field of platelet aggregation, induced by type III collagen. The experiments which are described hereunder relate to the nonapeptides of formula (1), (2) and (4) and to the decapeptide of formula (3) of the invention.
STUDY OF PLATELET AGGREGATION INHIBITION Method
The study of platelet aggregation is carried out under continuous stirring in an aggregometer known under the commercial designation LABINTEC, according to Born's method.
0.4 ml of citrated platelet (3.8%) rich plasma (PRP) are placed in the cuvette of the aggregometer. 0% of transmission is regulated with the PRP.
100% of transmission is regulated with a platelet poor plasma (PPP).
After the addition of 2.5 .mu.g of type III polymerized collagen, the aggregation curve is drafted in which the optical transmission percentage value is plotted against the time. There is a phase corresponding to the time duration between the addition of collagen and the beginning of aggregation.
Two parameters are then deduced from the curve:
1. velocity (expressed in %, 30 seconds after the addition of the peptide to be tested):
it is calculated from the curve by measuring the distance on the "optical transmission percentage value" axis (in cm) between 0% transmission and 100% transmission (distance=x) and the distance (in cm) on the "time" axis between 0% transmission and the optical transmission value, 30 seconds after the beginning of aggregation (distance=y)
velocity=(x-y)/x.multidot.100
2. intensity of the reaction (expressed in %):
it is calculated as for velocity, but in this case, y represents the value of optical transmission percentage, 2 minutes after the beginning of aggregation.
The percentage of inhibition of velocity and of inhibition of intensity are calculated as respect to the control test incubation of PRP in 10 .mu.l of buffer solution, in which only type III collagen has been introduced.
EXPERIMENT I
400 .mu.l of citrated (3.8%) platelet rich plasma pH 7.4 are incubated for five minutes, at 33.degree. C., under constant stirring, respectively:
with 10 .mu.l of 0.1M, Tris HCl buffer pH 7.4 (control experiment);
with 500 .mu.g of peptide .alpha..sub.1 (III)CB4 in 10 .mu.l of the above mentioned buffer solution;
with increasing amounts of the nonapeptide of formula (4), in 10 .mu.l of the above mentioned buffer solution (the increasing amounts are the following ones, expressed in n moles:
No aggregation of the platelets was noticed.
A minimal quantity (2.5 .mu.g) of type III polymerized collagen inducing aggregation is added and the subsequent aggregation was recorded. The results relative to velocity (percent), velocity inhibition (percent), intensity, intensity inhibition (percent) are gathered in the following table I.
Preincubations
Inducer    0.4 ml of PRP
% of inhibition
Insensity
of aggregation
+       %    of velocity
%    of intensity
Type III collagen
10 .mu.l of buffer
15.2         62
2.5 .mu.g  10 .mu.l GB4
9.8  35.5    37   40.5
nonapeptide
2.9  81      13.5 78
(840 n moles)
1    93      4    93
0    100     0    100
2.1  86      6.5  88
5.3  65      21   65
(77 n moles)
Another experiment has been carried out with the nonapeptide of formula (4), in the same conditions as for experiment I.
In this case, two different amounts of collagen have been used respectively for each amount of nonapeptide.
The results are gathered in the following table II.
ACTIVITY OF THE NONAPEPTIDE OF FORMULA (4) AS
REGARDS PLATELET AGGREGATION INDUCED BY TYPE III COLLAGEN
type III (.mu.g)
n moles
%    of velocity
%    of intesity
1.25    0     32           90.9
2.5           34.3         78.6
1.25    25    0    100     0    100
2.5           untested
2.5           35   0       78.6 0
1.25   105    0    100     0    100
1.25   210    2.1  90.9    2.9  96.8
2.5           15.9 53.6    73   7.1
2.5           4.2  88      16.6 78.8
1.25   840    untested
2.5           2.7  92      6.8  91
FIGS. 1 and 2 have been drafted accordingly, showing respectively:
the percentage of velocity plotted against the amount of nonapeptide (in n moles) for two respective amounts of collagen (2.5 .mu.g and 1.25 .mu.g);
the percentage of intensity plotted against the amount of nonapeptide (in n moles) for two respective amounts of collagen (2.5 .mu.g and 1.25 .mu.g).
The results show that the peptide of formula (4) presents a strong inhibitory effect on the aggregation of platelets induced by type III collagen. They also show that the concentration requested for inhibiting the aggregation induced by different amounts of collagen is dose-dependant on the nonapeptide.
One may also point out, from the table, that the nonapeptide of formula (4), in the concentration of 420 n moles is responsible for a total inhibition in platelet aggregation. A highly significant inhibition (65%) is observed for a concentration of 77 n moles.
The comparison between the results obtained with the three peptides (C2, HA4, T6), with respect to inhibition of platelet aggregation, shows that the small peptides according to the invention, are in our experiments as efficient as the bigger fragments (C2, HA4 and T6) in their ability to inhibit the platelet aggregation induced by type III collagen.
The peptides according to the invention inhibit aggregation induced only by collagen and collagen containing substances such as type III collagen.
Their specificity as regards the aggregation inducer has been experimented using platelet aggregation inducer such as:
arachidonic acid,
ionophore of the A 23 187 type, commercialized by Calbiochem. Behring.
thrombine in the amount of 0.1.mu. and 0.15.mu.
The results obtained for the nonapeptide of formula (4) are reported below in table III.
ACTIVITY OF THE NONAPEPTIDE OF FORMULA (4) AS REGARDS
PLATELET AGGREGATION INDUCED BY
ARACHIDONIC ACID IONOPHORE AND THROMBINE
Inducer    n moles %    of velocity
Arachidonic acid
0      59.9         81
0.6 nM     830     62.3 0       79.7 0
Ionophore   0      50           81.2
25 .mu.M   830     51.6 0       81.6 0
ADP        20 .mu.l of buffer
10-6 M     600     35.2 0
Thrombine   0      19.6
0.1.mu.    840     18.5
0.15.mu.    0      30.4
840     27.4
From the results, it appears that the peptides according to the invention, do not inhibit aggregation induced by arachidonic acid, ionophore, ADP and thrombine.
EXPERIMENT III
Another experiment has been carried out with the nonapeptides of formula (1), (2) and (4) and the decapeptide of formula (3), in the same conditions as for experiment I.
A pentapeptide of formula:
Gly--Glu--Hyp--Gly--Pro
has also been tested.
In this case, two different amounts of type III collagen (2 and 5 .mu.g) have been used for different amounts of the above mentioned peptides, and the test with the pentapeptide has been carried out with 3 .mu.g of type III collagen.
The results gathered in the following table IV, show that the peptides of formula (1), (2) and (3) inhibit platelet aggregation in the same way as the nonapeptide of formula (4). However, the pentapeptide tested has no activity with respect to aggregation of platelets induced by type III collagen.
EFFECT OF THE DIFFERENT PEPTIDES (1), (2), (3) AND (4)
ON PLATELET AGGREGATION
Nonapeptides  Velocity
Inducer (n moles)     %    of velocity
Collagen 2 .mu.g
0             12.85
--      57.85
decapeptide (3)
840           0    100     0    100
600           4.9  64      21.8 64.3
420           7    48      33.8 44.6
nonapeptide (4)
7.8  42.5    42.8 30
0             6.25 --      22.6 --
6.95 0       24.7 0
(pH 7.4)
0             11   --      40.7 --
5.39 51      22   46
STUDY OF PLATELET ACTIVATION INHIBITION
The peptides according to the invention inhibit aggregation of platelet induced by collagen without modifying the intracellular natural metabolism of the platelets, without involving cellular activation with the liberation of intraplatelet constituents.
A test has been carried out to study the inhibiting effect of the peptides according to the invention with respect to platelet activation induced by type III collagen.
The test has been carried out with the nonapeptide of formula (4) in which the release of endogenous serotonin contained in the platelet granules is studied.
The test consists of a pre-incubation in vitro of a platelet suspension in a physiological buffer (Tris: 0.35M, Mg.sup.+ : 1 mM; K.sup.+ : 2.5 mM; Ca.sup.++ : 2.5 mM; NaCl: 100 mM; glucose: 0.9%/ml; albumine: 3.5 mg/ml (reference: PATSCHEKE and WOMER, "Platelet activation detected by turbidometric shape change analysis. Differential influence of cytocholagin B and prostaglandin E.sub.1 ". Thrombosis Research, vol. 12, 1978, p. 485).
The amount of platelets is of 1.2.times.108 platelets for 0.4 ml of buffer.
The pre-incubation is carried out in the presence of different nonapeptide concentrations, for 5 minutes, at a temperature of 37.degree. C. The pre-incubation is followed by the addition of type III collagen, at the rate of 5 and 7 gammas. The incubation is carried out for 90 seconds and the behaviour of platelets with respect to aggregation is observed at the same time. The platelets are then centrifuged for 15 seconds in an EPPENDORF micro-centrifuge. The supernatants are sampled and the release of endogenous serotonin is measured by the fluorometric method described in Thrombosis Research, vol. 10, 1977, p. 791-RAO, FRIEDLAND, GERRARD and WHITE "The influence of metaboliism on the assay of platelets", in a spectrophotometer commercialized by JOBIN & YVON under the designation JY3.
The results gathered in the following table V show the increasing inhibition of the release of serotonin induced by collagen corresponding to the increasing amounts of nonapeptide.
The inhibition is complete for a concentration of nonapeptides of 850 n moles.
The nonapeptide itself does not induce serotonin release.
The results relative to the study of aggregation which has been carried out in parallel assays, with the study of serotonin release, are also reported in table V.
The inhibition of serotonin release parallels the inhibition of aggregation, as measured by the velocity (%) after one minute.
TABLE V
INHIBITING EFFECT OF NONAPEPTIDE (4)
ON SERONTONIN RELEASE
% of    Re-  % of
Coll-
Nonapetides
Velocity %
agen (n moles)  after 1 mn
of velocity
%    of release
0.gamma.
840        0         --      1.3  --
0         17.4      --      49.8 --
105        11.4      34      32.6 34
210        8         54      27   46
420        6.3       63.7    15.8 68
840        0         100     3.4  93
0         11        --      40   --
210        17        0       34   15
420        0         100     15.2 62
STUDY OF THE NATURE OF THE FIXATION OF THE PEPTIDES OF THE INVENTION TO PLATELETS OR TO PLATELET MEMBRANES
The study of the nature of the interaction between type III collagen and platelets is carried out in the presence of tritiated (labelled) nonapeptide of formula:
This study is carried out with complete platelets or membranes of the platelets.
1. Methods for tritiating the platelets or the platelet membranes
The complete platelets or the platelet membranes solubilized in triton (0.15%) are incubated in a physiological buffer of the type described above in the serotonin release test (in a total volume of 0.2 ml) at a temperature of 37.degree. C. for 15 minutes. The incubation is carried out in presence of tritiated nonapeptide the specific activity of which is of 1 to 2 curie/n mole.
The nonapeptide fixed or linked to the platelet or to the membranes is separated from the free nonapeptide by filtration of the incubation medium through multiporous membranes having porosities of 0.8.mu. and 0.45.mu. respectively. The radio activity is directly determined on the filters and the percentage of linkages determined according to the following formula: ##EQU1##
Different characteristics of the linkage between the platelets or the platelet membranes and the peptides of the invention have been studied as explained below.
A specific linkage of a ligand to its specific receptor is substantially characterized by four properties:
the linkage percentage varies according to the protein concentration (of tables VI, X);
the linkage is liable to reach saturation (tables VII and XI);
the linkage is specific (table VIII);
the linkage is reversible (table IX) as evidenced by a dissociation of labelled ligand from the receptor as a function of time and in the presence of an excess of non labelled ligand.
(a) Tables VI and X respectively relate to the linkage (%) of the nonapeptide as a function of the protein concentration respectively for the platelet membranes and for the platelets.
It results from the study of tables VI and X that similar conclusions can be reached for the platelet membranes and for the complete platelets. In other words, the linkage percentage increases when the protein concentration increases. The linkage percentage is an increasing function of the number of receptors which are present in the incubation medium.
TABLE VI
LINKAGE (%) OF
LABELLED (TRITIATED) NONAPEPTIDE (4) AS A
FUNCTION OF THE PROTEIN CONCENTRATION
(Platelet membranes)
Nonapeptide H.sup.3
Proteic concentration
(ng)    20 .mu.g
40 .mu.g
100 .mu.g
3      2.1%
--  --  --
30      3.5%
13% 13.8%
TABLE X
LINKAGE (%) OF THE NONAPEPTIDE AS A
FUNCTION OF THE PLATELET CONCENTRATION
(complete platelets)
Nona-   Platelet concentration
peptide 0.3 .times. 10.sup.8
0.6 .times. 10.sup.8
2.35%    3.85%    5.1%   7.8%   10.4%
(b) Tables VII an XI relate to the saturation and represent the variation of the linkage percentage as a function of the concentration of nonapeptide respectively for the platelet membranes and for the complete platelets. It results from the study of table VII, that the variation of the linkage percentage is linear up to a concentration of 12.5 ng of nonapeptide. Above this value of the concentration, the variation of the linkage percentages deeply decreases and nearly reaches a constant value.
From the study of table XI, it results that the saturation is complete from 1.2 ng of nonapeptide for a platelet concentration of 1.2.times.10.sup.8 platelets in 0.2 ml of physiological buffer.
TABLE VII
LINKAGE (%) AS A FUNCTION OF THE LABELLED
NONAPEPTIDE (4) (Platelet membranes)
(ng)           Linkage (%)
3             1.6
12             5.5
300            10.5
TABLE XI
LINKAGE SATURATION:LINKAGE (%) AS A FUNCTION OF THE
NONAPEPTIDE (4) CONCENTRATION (Complete platelets)
Nonapeptide concentration
1.2 ng
6 ng
60 ng
--  --  --  --  2.35
--  3.1%
--  --  --  --  3.85%
3%  3%  4%
6%  5%  5.1%
--  --  --  --  0.4%
--  8%  8%
(c) Table VIII relates to the linkage specificity. It represents the linkage percentages between the platelet membranes and different peptides of the invention in the presence of various aggregation inducers (15 ng of peptide, 30 .mu.g of platelet membrane).
The specificity of the linkage is assessed by the total displacement of the corresponding radio-activity by an excess of non labelled peptide of the invention. This excess corresponds to an amount of 100 to 1000 times more than the amount of non labelled peptide which is necessary to induce adhesion of the platelets.
Other platelet aggregation inducers interacting with the platelet membranes are not responsible for any displacement.
From table VIII, it is to be noted that the CB4 central fragment of collagen containing the nonapeptide of formula (4) is responsible for the displacement of the linkage of said nonapeptide.
TABLE VIII
SPECIFICITY OF THE LINKAGE
(platelet membranes 30 .mu.g)
Nonapeptide H.sup.3             Linkage
ng        Agent                 %
15        0                     4
15      .mu.g   0.26
150     .mu.g   0.7
15      .mu.g   0
15      .mu.g   0.4
CB4         100     .mu.g   0.36
ADP         10.sup.-2
M       4.3
ionophore A 23 187
0.5     nM      4.1
Adrenaline  100     .mu.g   5.4
6       nM      4.1
(d) Table IX relates to the study of the reversibility of the linkage of the nonapeptide of formula (4) to the platelets. Said reversibility results from the fact that the peptides according to the invention can be dissociated from the platelets as time goes on.
Said reversibility has been studied in the presence of two concentrations of labelled (nona)peptide which are respectively of 3 and 6 ng, for 30 .mu.g of platelet membranes. After the binding of the labelled nonapeptide to the membranes, the incubation media are diluted to the 80 volumes:
in the absence of non labelled (nona)peptide;
and in the presence of an excess of non labelled (nona)peptide (as indicated under c) for the reversibility test).
The percentage of linkage is then determined as a function of an incubation duration ranging from 0 to 15 minutes.
The reversibility is measured by the dissociation factor and the dissociation time.
The results of table IX show a quick dissociation (measured by the semi-dissociation time). The semi-dissociation time which is of about 5 minutes, when there is no nonapeptide, is decreased to 2 minutes, when there is an excess of non labelled nonapeptide, which corresponds to an increase of the dissociation.
TABLE IX
REVERSIBILITY OF THE LINKAGE
Nona-   Dissociation
Dissociation time
peptide H.sup.3
factor      0'     2'    5'    10'  15'
3 ng    dilution 1/80
3.2%    1.75%
1.55% 1%   0.6%
dilution 1/80 +
3.2%   1%    --    0.5% 0.1%
100 .mu.g of
5.9%   4.8%  3%    2.4% 1.5%
5.9%   3%    --    1.5% 1%
The peptides according to the invention, notably the following peptides:
Tyr--Gly--Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys
Tyr--Gly--Lys--Hyp--Gly--Hyp--Glu--Gly--Pro--Lys (3)
can be used as tracers, particularly if labelled radio-actively.
The peptides according to the invention can be used as identifying means of the particular sites of platelets that are involved in the interaction between the latter and type III collagen.
More particularly, they can be used as a means of discriminating the sites involved in platelet adhesion and for aggregation induced by type III collagen from the other sites possibly involved in platelet adhesion induced by other inducers or more generally of selectively preventing adhesion of platelets liable of being induced by collagen in experimental induction assays in the presence of would-be inducers or mixtures of inducers of platelet adhesion and/or aggregation.
One must point out that the peptides according to the invention, can also be used with any type of collagen such as type I, IV and V, as well as with microfibrils or substances containing any type of collagen.
The peptides according to the invention because of their interesting pharmacological properties of anti-aggregation can also be the active principle, the control of coagulation, particularly for preventing the formation of thrombosis in men whose blood is in hypercoagulable state.
The compositions containing a dose of any of such peptides effective to prevent interaction in vivo between collagen and platelets can be administered by intravenous, intramuscular or subcutaneous route, in the form of a solution pharmaceutically acceptable and in sterile vehicle.
The peptides according to the invention can also be administered by oral, sublingual or nasal route, when associated with solid or liquid pharmaceutically acceptable excipient.
They can also be administered by rectal route when associated to excipients suitable accordingly. They can also be administered in the form of liposomes.
As is self-evident and as emerges besides already from the foregoing, the invention is in no way limited to the embodiments which have been more particularly envisaged; it encompasses, on the contrary:
1. A peptide of the formula:
D is a member selected from the group consisting of H, Gly and Tyr--Gly;
E is a member selected from the group consisting of OH and Gly;
X.sub.1 and X.sub.2 are the same or different and each is an aminoacyl residue selected from the group consisting of hydroxyprolyl and prolyl;
Y is a residue selected from the group consisting of --Z--X.sub.3 -- and --X.sub.3 --Z-- wherein X.sub.3 is hydroxyprolyl or prolyl, and Z is a spacer aminoacyl residue selected from among glutaminyl, glutamyl, aspartyl, asparginyl and alanyl residues, and A is selected from among arginyl, ornithyl, lysyl or hydroxylysyl residues.
2. A peptide comprising amino acids, which, except glycyl, are all levorotatory, of the formula:
Y represents either a --Z--X.sub.3 -- or a --X.sub.3 --Z-- residue, wherein X.sub.3 is a hydroxyprolyl or prolyl residue, and Z is a spacer aminoacyl residue selected from among glutaminyl, glutamyl, aspartyl, asparaginyl and alanyl residues; and A is selected from among arginyl, ornithyl or lysyl residues.
3. Peptide according to claim 2, characterized by the fact that A is lysyl.
4. The peptides of the following formula:
Gly--Lys--Pro--Gly--Glu--Pro--Gly--Pro--Lys
Lys--Hyp--Gly--Glu--Hyp--Gly--Pro--Lys--Gly
Tyr--Gly--Lys--Hyp--Gly--Hyp--Glu--Gly--Pro--Lys.
5. Peptide according to claim 2, of the formula:
A represents a residue chosed from the following: lysyl, arginyl;
X.sub.1, X.sub.2 and X.sub.3 represent independently from each other either the hydroxyprolyl residue, or the prolyl residue; Z is selected from among glutaminyl, glutamyl, aspartyl, asparaginyl and alanyl residues.
A represents a residue chosen from the following: lysyl, arginyl;
Z is selected from among glutaminyl, glutamyl, aspartyl, asparaginyl and alanyl residues.
7. Peptide according to claim 2, wherein Z represents the glutamyl residue, A represents a residue chosen from the following: lysyl, arginyl, the formulae of the corresponding peptides then comprising either of the following:
8. Peptide according to claim 2, wherein A represents the lysyl residue, and Z represents a glutamyl residue, the formulae of the corresponding peptides then comprising:
9. A pharmaceutical composition, particularly for preventing platelet aggregation, comprising in association with a pharmaceutical vehicle, a dose of a peptide according to any of claims 1, 2, 3, 5, 6, 7 or 8 effective to prevent interaction in vivo between collagen and platelets.
10. A pharmaceutical composition, particularly for preventing platelet aggregation, comprising in association with a pharmaceutical vehicle, an effective dose of at least one of the peptides of the following formula:
11. A method of preventing platelet aggregation in a host, which comprises administering to said host an effective amount of a peptide of any one of claims 1, 2, 3, 5, 6, 7, or 8 and a pharmaceutically acceptable vehicle.
5046656 August 1973 JPX
J. M. Seyer, et al., Biochemistry 16, No. 6, (1977), 1158-1164. Chem. Abstr. vol. 92, 1980, 55858r. Chem. Abstr. vol. 92, 1980, 144153z. The Journal of Biological Chemistry 250, No. 13, 5076-5081, 1975. Biochemistry 1981, 20, 2621-2627. Biochem. & Biophys. Research Commun., 1579-1585, 96, (1980). The Journal of Biological Chem. 250, 1975, 7428-7434. Biochemistry 10, (1971), 2076-2081. C. R. Acad. Sc. Paris 4290, (Apr. 28, 1980), Serie D, 1115-1118.
Date of Patent: Oct 2, 1984
Assignee: Choay S.A. (Paris)
Inventors: Jacques Caen (Paris), Yves Legrand (Paris), Pierre Lefrancier (Gif sur Yvette)
Primary Examiner: Delbert R. Phillips
Law Firm: Weiser & Stapler
Current U.S. Class: 424/177; 260/1125R
International Classification: A61K 3700; C07C10352;


text:
Filter: The Parish
Techset Party
Friday March 9, 2012 8:00pm - 11:00pm @ The Parish (214 E Sixth St Austin, TX)
For the 4th consecutive year SXSW Interactive opens with your party…that's right, The Techset will once again kick-off SXSW Interactive with an evening that brings together the new influencers (that's you) to celebrate everything we've done and everything we will do together.
Type Party, Interactive
Tags 21+
Website http://schedule.sxsw.com/2012/events/event_OE01147
PBS Party celebrating @MasterpiecePBS "Sherlock"
Sunday March 11, 2012 8:00pm - 1:30am @ The Parish (214 E Sixth St Austin, TX)
Meet part of the team behind @MasterpiecePBS "Sherlock": Emmy-nominated creator Steven Moffat (of "Doctor Who" fame) and his wife, series producer Sue Vertue. Music, food, pictures, autographs, t-shirts, fun & suds.
Type Party, Film
Tuesday March 13, 2012 8:00pm - 8:40pm @ The Parish (214 E Sixth St Austin, TX)
Eric Mikulak, also known as Click-Clack, is a 21 year old self produced hip-hop and r&b vocalist. Growing up in Austin, Texas, Eric was exposed to and submerged in all different styles of music from an early age. As years passed listening became an obsession and eventually was no longer satisfactory. At the age of 12 he began experimenting with beat production and vocal sampling. Soon after, the age of Macintosh arose, and garage band swiftly became the apple of his eye. As producing beats and recording ate in to his study time, it quickly transitioned from a hobby into an infatuation. At 17, Click-Clack attended Mediatech Institute of Recording Arts and began working with professional producing/recording tools. Presently, he uses a combination of Reason, Recycle and Protools and is entirely self sufficient is his creation process. Eric's driving beats provide the canvas for his emotional, opinionated lyrics. As the front man for several local bands, he has performed at many of Austin's most renown venues, including Stubb's, The Mohawk, and The Parish. Now hitting the stage solo, his energy and stage presence combine to create a intense experience for viewers and listeners. GET YOU SUM.
Eric Mikulak, also known as Click-Clack, is a 21 year old self...
Tags Hip-Hop-Rap, Music
Schmillion
Schmillion formed in the summer of 2009. Back then we were 13-15 years old. But most of us drive now and Natalie has advanced to higher ed. Our sound is built on crunchy guitar riffs and belting vocals. We all have different musical influences and mash it up to make what we think of as garage-punk.
Schmillion formed in the summer of 2009. Back then we were 13-15...
Hashtag #sxsw #Schmillion
Marmalakes
Tuesday March 13, 2012 10:00pm - 10:40pm @ The Parish (214 E Sixth St Austin, TX)
Doug Freeman, The Austin Chronicle:
Amid the nascent pop scene emerging in Austin over the past few years, Marmalakes has found its music at an ideal intersection, balancing exuberant live shows and contemplative, intelligent songwriting. The local trio of Chase Weinacht, Josh Halpern, and Max Colonna, all in their early 20s, delivers folk pop with both a youthful restlessness and eye for detail.
"We're certainly surrounded by an energetic and enthusiastic group of creative folks," offers Weinacht. "As far as impressions, I honestly don't believe that anyone in our community of bands would say their No. 1 goal is 'I want to be famous.' I think the general feeling of all these folks is 'Let's make the best music we know how to make.'
"These bands – Mother Falcon, Little Lo, Hello Wheels, the Sour Notes – we're all playing with pop structures unabashedly. We're all working hard. As far as how Marmalakes fit it into it musically, I think we're wordier than most."
That focus on the live experience and investment in nurturing a scene has helped build an impressive community of both musical collaboration and fans, capped lastmonth at the Mohawk with Marmalakes' split 7-inch release with fellow locals the Sour Notes. With an eye toward releasing its third EP in April, Marmalakes continues to hone its sound, building on 2010's impressive debut Wonder Winds and last year's more adventurous arrangements of Even Clothed.
"The reason our second EP is a bit more exploratory is because we had a better idea of what it means to put together a record," Weinacht surmises. "Since we'd been a live-oriented band for a good while, with Wonder Winds we wanted to try to present our live energy while at the same time keeping it interesting enough for some replay value.
"With Even Clothed, we were able to play around a bit more. I think we felt more adapted to the studio environment altogether, and due to the opportunities we've been given to play lots of different kinds of shows, we've made a handful of different arrangements of each song, and with the Even Clothed version we felt we had to be definitive."
MARMALAKES is a rock band from Austin, TX. Dynamic and lyrically-attentive...
Hashtag #sxsw #Marmalakes
Lex Land
• When you think "singer songwriter," fierce is not a word that often comes to mind. But there is something slightly fierce about the opening seconds of Lex Land's new album "Were My Sweetheart To Go". It's the track "Oh My!" and it just reaches out and grabs you, then descends into a sweet sing song chorus complete with a chipper hand clap beat. Music Director Jason Bentley is a big fan, saying he likes that you can hear her operatic training, her focus before pursuing a life of pop. "She's a great songwriter. She makes herself very vulnerable and her lyrics are revealing about relationships and all those universal themes we love." "She really separates herself from the pack with her demeanor and her lyrics. She's got attitude, she's cool."
- KCRW – Artist You Should Know
• When you think "singer songwriter," fierce is not a word...
Tags 21+, Singer-Songwriter
El Tule
Wednesday March 14, 2012 12:00am - 12:40am @ The Parish (214 E Sixth St Austin, TX)
Simple, El Tule is for those who love to dance, feel good and enjoy the positive expression of music.
For the last decade El Tule (el TOO-lay) has been honing its...
Hashtag #sxsw #ElTule
Tags Latin Rock, Music
Wednesday March 14, 2012 1:00am - 1:50am @ The Parish (214 E Sixth St Austin, TX)
Nakia, known for his larger-than-life, soulful vocal performances and ability to connect with an audience, is a true Austin success story. Since his arrival at "The Live Music Capital of the World" in 2002, he has quickly formed alliances with Austin's best musicians.
Nakia, known for his larger-than-life, soulful vocal performances...
Hashtag #sxsw #Nakia
Tags R&B, Music
The Good Danny's + Navigational Society Party
Wednesday March 14, 2012 2:00pm - 2:30pm @ The Parish (214 E Sixth St Austin, TX)
Free entry with an RSVP via the link below.
Le Castle Vania
Very few can burst on stage and through speakers with boundless...
Hashtag #sxsw #LeCastleVania
Tags 21+, Dance
Wednesday March 14, 2012 9:15pm - 10:00pm @ The Parish (214 E Sixth St Austin, TX)
Everyone can now make music. The increasing access to music related technology and the liquid flow of music and information is a blessing to aspiring musicians, but a curse to those who have to sift through the terrible mash-ups and tunes in order to find quality music. Based out of New York City's Lower East Side, The Knocks are taking a stand and reclaiming good music for all.
http://heavyrocmusic.com/
Hashtag #sxsw #TheKnocks
Wednesday March 14, 2012 10:00pm - 11:00pm @ The Parish (214 E Sixth St Austin, TX)
Whether Kissy Sell Out is DJing to a packed out crowd, mixing avant garde new sounds on his weekly Radio One radio show, producing his own material or remixing for others, the results are always the same: Exhilarating. In the talent rich genre of electro, one name has always stood out as more forward thinking than the rest. Colchester-native Kissy Sell Out first exploded on to the scene with a series of technically ambitious remixes in 2006. His rework of Mark Ronson's take on The Smith's 'Stop Me', gained him particular kudos and was included in numerous compilations. It was enough to bring him to the attention of radio bosses, who offered the twenty-something design student a weekly slot on Radio One. Now over one hundred shows in, he has used it as an opportunity to showcase his own technical abilities, presenting a new Kissy Klub mix each week, a section of the show made entirely of his exclusive remixes made especially for that week. Along with his fresh attitude to mixing records, his amiable persona continues to draw thousands upon thousands of new listeners to his late night slot week on week. Fans not only want to hear what at Kissy has made, but also what he has found; he is a champion of new talent, and regularly plays tracks sent to him by bedroom producers along with tracks from more established artists and record labels.
Whether Kissy Sell Out is DJing to a packed out crowd, mixing...
Wednesday March 14, 2012 11:00pm - 12:00am @ The Parish (214 E Sixth St Austin, TX)
Thursday March 15, 2012 12:00am - 1:00am @ The Parish (214 E Sixth St Austin, TX)
"My music sounds like commercial American hip-hop and death metal being played through various farm animals."
– Borgore, from an interview with Spinner.com
Hailing from Israel via London, dubstep badass BORGORE—the...
Hashtag #sxsw #Borgore
Tags 21+, DJ
Thursday March 15, 2012 1:00am - 1:50am @ The Parish (214 E Sixth St Austin, TX)
SAVOY is a live electronic music hybrid. Consisting of DJs Ben Eberdt, Gray Smith and drummer Mike Kelly; the SAVOY experience can be described as a high energy dance party and laser spectacular that has been referred to as "mind blowing" on multiple occasions. With the combination of a inimitable live show and original repertoire, SAVOY has climbed their way up to national notoriety as well as established themselves as not only purveyor of one of the best parties around, but as world class song writers and producers.
Club hits, blog dominating remixes, and arena rock-ing swagger; SAVOY is bridging the gap from the dance world to the mainstream. Think Daft Punk meets Metallica and give it a bottle of gin and you got SAVOY...
In 2011, SAVOY will continue to rock mainstream festival, live venues, nightclubs and arenas around the world while they produce with and for many high profile artists...
SAVOY is a live electronic music hybrid. Consisting of DJs...
Hashtag #sxsw #Savoy
Shandy Mandies
Thursday March 15, 2012 8:00pm - 8:40pm @ The Parish (214 E Sixth St Austin, TX)
If you believe that music is humankind's universal gift to itself and that it has been passed on over thousands of years from generation to generation in all of its various mind expanding shapes and that it is holy and belongs to us all; if you believe that Elvis opened a door and that the Beatles held it wide open and that the Pistols kicked it down and spat and pissed all over it; if you believe that yesterday is as relevant as today and tomorrow and time and space and love and spirituality and wisdom and beauty all rolled together; if you believe this, then you have to believe in SHANDY MANDIES.
If you believe that music is humankind's universal gift to...
Hashtag #sxsw #ShandyMandies
Bass Drum of Death
Bass Drum of Death is a band from Mississippi. John plays guitar and sings and Colin plays the drums. Together they make blown-out, blazing songs. John was Fat Possum's worst employee 3 years ago. He wrote and recorded the whole record himself using just a drumset, a guitar, a usb microphone, a computer, and a shitload of talent. The songs are about drugs, trying to make it with religious girls, panic attacks, stealing stuff, mild to severe depression, Elvis appearing in your dreams and giving you advice, gravity bongs and the devil living inside your brain. This record is the soundtrack playing in your head when you're fucked up and walking home in the middle of the night.
BASS DRUM OF DEATH It's been a little more than a year since...
Hashtag #sxsw #BassDrumofDeath
Thursday March 15, 2012 10:00pm - 10:40pm @ The Parish (214 E Sixth St Austin, TX)
After a two and a half year break, the San Francisco guitar-drum duo TWO GALLANTS began touring again which took them across North America, through Europe and even to East Asia for their first ever shows in South Korea and China. The well needed time off came after 6 years of incessant touring and the release of 3 full length studio albums and an acoustic EP. The band consists of childhood friends Tyson Vogel and Adam Stephens who met on their elementary school playground at the age of 5. Despite an initial disagreement and ensuing scuffle over the supremacy of White Lion vs. White Snake, the two have been best friends ever since. They have been playing music together in some capacity since 1994, when their parents made the mistake of giving them both electric guitars for Christmas. Around 2002, the two college dropouts found themselves with some time on their hands and silence to kill; and so with an acoustic guitar and a trap set they formed TWO GALLANTS in the basement of the Vogel residence. In an ongoing unspoken competition to out do one another, the intervening years have seen the two-piece increase their volume and gear ten-fold. The bands' sound is somewhat of a syncretism of American old-timey roots music with the grunge and east bay punk of Tyson and Adam's formative years. In the two years since, both have released solo material under The Devotionals and Adam Haworth Stephens, respectively. Now with a new batch of songs in hand TWO GALLANTS are getting back to the haranguing strident noise that fuelled them across the world and a new album is in the works.
After a two and a half year break, the San Francisco guitar-drum...
Hashtag #sxsw #TwoGallants
SLOW CLUB | Paradise Like all good clichés, there's more than...
Hashtag #sxsw #SlowClub
Friday March 16, 2012 12:00am - 12:40am @ The Parish (214 E Sixth St Austin, TX)
Sensual, provocatively percussive pop comes naturally to Friends. But you may be surprised by "I'm His Girl" – the second single from the Brooklyn newcomers – and just how cool and pop it is. As rapturous as Blondie's best, and as stylish as Neneh Cherry's tip-top pop gem "Buffalo Stance," it's the finest fusion of lo-fi indie cool and soulful urban textures of 2011.
Sensual, provocatively percussive pop comes naturally to Friends...
Hashtag #sxsw #Friends
Friday March 16, 2012 1:00am - 1:50am @ The Parish (214 E Sixth St Austin, TX)
Hashtag #sxsw #Chairlift
OurStage
Friday March 16, 2012 11:00am - 3:00pm @ The Parish (214 E Sixth St Austin, TX)
Join us for the taping of the season finale of The OurStage Panel, featuring Bronze Radio Return, Eclectic Approach, Civil Sound & The Reel.
Type Party, Music
Tags 21+, Hip-Hop-Rap
Website http://schedule.sxsw.com/2012/events/event_HB2080
Willy Mason
Friday March 16, 2012 7:45pm - 8:25pm @ The Parish (214 E Sixth St Austin, TX)
Willy Mason was just 19 when his first album was released, a record that propelled him to critical acclaim and showed a young man with a tremendous, broad-oaked voice, a great songwriting talent, and a peculiar wisdom beyond his years. Three years later, in 2007, he returned with If the Ocean Gets Rough, an album that only underlined his ability to capture the spirit of a time and a place and a generation. His as yet untitled third studio album, produced by Dan Carey, has just been completed and is set for release in 2012.
Willy Mason was just 19 when his first album was released, a...
Hashtag #sxsw #WillyMason
Tags 21+, Alt Country
Quantic & Alice Russell
Producer, musician, DJ and composer Quantic and singer/songwriter Alice Russell are two of the UK's best loved independent artists. Through their respective solo projects and various acclaimed collaborations over the last decade, they have become leading lights of the international funk, soul and alternative dance music scene.
Producer, musician, DJ and composer Quantic and singer/songwriter...
Tags 21+, Funk
Friday March 16, 2012 9:45pm - 10:25pm @ The Parish (214 E Sixth St Austin, TX)
Clock Opera, I remember thinking, have this wonderful tendency to sound like their music combines real memories and made up memories, strong memories and shredded memories, and some sense of the mental and physical reality of a classic surprising pop song. They begin a piece of music with exquisite care and end it, just like that, with battle commencing in between, and a dynamic round the bend sense of time. Sound like a group I never remember hearing who decided to imagine what would happen if they sounded like an original pop group whose members consisted of Steve Reich, Scott Walker, Peter Gabriel, Flo and Eddie and To Rococo Rot. That good.
Clock Opera, I remember thinking, have this wonderful tendency...
Hashtag #sxsw #ClockOpera
Friday March 16, 2012 10:45pm - 11:30pm @ The Parish (214 E Sixth St Austin, TX)
Hashtag #sxsw #CrystalFighters
Saturday March 17, 2012 12:00am - 1:30am @ The Parish (214 E Sixth St Austin, TX)
Melbourne born, London based, The Temper Trap return to the live front in wake of their self titled sophomore album, due for worldwide release this June. The bands 2009 breakout release "Conditions" has now scanned over 800,000 albums worldwide with hit singles Sweet disposition and Fader amassing over 2.5 million sales. Anticipation for the bands latest offering is sky high and in the bands words, its a diverse, fully realized body of work. Expect to be blown away by this super talented outfit
Melbourne born, London based, The Temper Trap return to the live...
Hashtag #sxsw #TheTemperTrap
The Jameses
Saturday March 17, 2012 7:00pm - 7:40pm @ The Parish (214 E Sixth St Austin, TX)
Hashtag #sxsw #TheJameses
DIVE is the nom-de-plume of Z. Cole Smith, musical provocateur and front-man of an atmospheric and autumnally-charged new Brooklyn four-piece.
DIVE is the nom-de-plume of Z. Cole Smith, musical provocateur...
Hashtag #sxsw #DIVE
Tags 21+, Avant-Experimental
Evolution is a tricky word when discussing current trends in...
Hashtag #sxsw #Blouse
Saturday March 17, 2012 10:00pm - 10:40pm @ The Parish (214 E Sixth St Austin, TX)
Soft Metals is a multi-disciplinary electronic duo from Portland, Oregon now residing in Los Angeles, California. Its members Ian Hicks and Patricia Hall were brought together through a common love of 1970s and 80s synthesizer music and began writing and recording songs together in the spring of 2009. Inspiration came to them by way of experimental electronic sounds, film soundtracks, early industrial music, minimal synth, house, techno, synth pop, krautrock, psychedelic rock, and shoegaze. Ian and Patricia share songwriting duties and compose the music together before writing lyrics and adding vocals. Their songs are built from moody, improvised sessions together using exclusively electronic instruments. The meaning of the raw music they make is explored and interpreted afterwards with lyrical themes ranging from life experience, films, literature, history, science, love, conflict, and death. Soft Metals prefer to express themselves freely rather than adhere to a particular genre. This freedom gives them a diverse sound somewhere between dance music, austere synthetic pop, and experimental electronic composition. Their two best known songs, "The Cold World Melts" and "Psychic Driving" are demonstrations of this varied sound. "The Cold World Melts" is driving, assertive, and passionate. "Psychic Driving" is delicate, introverted, and vulnerable. Some of their songs remain in their unpolished improvised form to pay homage to authenticity, spontaneity, the joy of sound, and experimentation while others are studio based exercises, fusing new and old production technologies.
Soft Metals is a multi-disciplinary electronic duo from Portland...
Hashtag #sxsw #SoftMetals
Widowspeak
Widowspeak is an American band comprised of Molly Hamilton and...
Hashtag #sxsw #Widowspeak
Craft Spells
Sunday March 18, 2012 12:00am - 12:40am @ The Parish (214 E Sixth St Austin, TX)
In the cold and dreary winter of 2009, alone in his bedroom and hidden from the darkness of the world outside Justin Paul Vallesteros had begun working on something that would change his life forever. What began as a couple notes played in experimentation soon transformed into vibrant melodies that soon shook Vallesteros' musical foundation to the core. Guitar chords laced over pulse sating synth melodies and drum rhythms was a departure from Justin's previous work; he began to create the dreamy nostalgic pop music that is Craft Spells. Joined by Jack Smith (bass), Javier Suarez (lead guitar) and Peter Michel (drums), they recently returned from a European tour and plan on releasing a new ep February 2012.
In the cold and dreary winter of 2009, alone in his bedroom and...
Hashtag #sxsw #CraftSpells
Beach Fossils
Sunday March 18, 2012 1:00am - 1:50am @ The Parish (214 E Sixth St Austin, TX)
Beach Fossils is a Brooklyn-based group, formed in early 2009 by Dustin Payseur. After signing to Captured Tracks and quickly pulling together a live band, they took off playing countless shows across the U.S. gathering a slew of devoted fans in their wake. In May of the same year, their debut LP Beach Fossils met with favorable reviews and became known for its jangly, single note guitar style, dusted with summer-fueled romance.
freak outhttp://beachfossils.com
Hashtag #sxsw #BeachFossils

redpajama_set_name: RedPajamaCommonCrawl


text:
I wanna dance with somebody
Bindi Irwin, daughter of the late "Crocodile Hunter," has already been announced as a cast member for the 21st season of "Dancing With the Stars," which returns Sept. 14. Find out whom she'll be hoofing against when "Good Morning America" reveals the entire cast. 7 a.m. KSTP, Ch. 5
For the Season 8 premiere of "Million Dollar Listing Los Angeles," our real estate team has more than brown lawns to handle. Record low prices for housing have the agents scrambling to make deals, all while grappling with personal issues that range from the death of loved ones to upcoming nuptials. 9 p.m. Bravo
Off the road again
Kenny Alphin, half of country-rock duo Big & Rich, tries to prove he's one of us — at least when he's not on tour — in "Big Kenny and Family," a new special that gives viewers a chance to see what it's like to live large. 9 p.m. TLC
Neal Justin
Nation Expect more worrisome variants after omicron, scientists warn
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On a trip to the United Arab Emirates, the president of South Korea on Sunday reportedly reached a preliminary multibillion-dollar deal to sell Seoul's surface-to-air missiles to Abu Dhabi and pledged deeper cooperation with the Gulf Arab federation.
For 'ice wine' vintners, frigid temps are reason to rejoice
While the vast majority of New Yorkers are hunkering down to ride out frigid temperatures and snow blasts from the latest winter storm, others are seizing on the opportunity to ... pick grapes?
After a month at no. 1, "Spider-Man: No Way Home" has finally been overtaken at the box office. Paramount Pictures' "Scream" reboot debuted with $30.6 million in ticket sales over the weekend, according to studio estimates Sunday.
From the inaugural platform, President Joe Biden saw American sickness on two fronts — a disease of the national spirit and the one from the rampaging coronavirus — and he saw hope, because leaders always must see that.
Will the real Stephen Colbert stand up? Four ways he'll succeed on CBS' 'Late Show' • TV & Media
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Oprah is coming this weekend. Here are 5 ways she changed the world. • TV & Media
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TSC New (BEVOT) Requirements For All Teachers
TSC has issued new requirements for an upcoming teachers Biometric Enrolment and Validation of Teachers (BEVOT) exercise in March 2022, which will be implemented...


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A Sneakpeak at the RTX 2080 Asus ROG Mothership
Home → Uncategorized → A Sneakpeak at the RTX 2080 Asus ROG Mothership
Asus just announced a whole bunch of gaming hardware and laptops the one of interest today is the RTX 2080 Asus ROG Mothership. The Mothership has a completely brand new design and concept of what portable computing and gaming could be.
Instead of a traditional clamshell type of design, this one takes the way we open laptops and spins it on its head. The way you open this one is a bit different than what anyone has seen in the past. This Mothership opens in a completely bonkers way. It is similar to an all-in-one. You lay it on its front edge and a kickstand snaps out of the back of the display panel. Next, the keyboard entirely detaches from the device.
The keyboard can be used wirelessly or wired. If it is wireless, it uses 1.4GHz radio frequency, and if its wored it uses a regular USB-C connection.
The keystroke has a lot of travel in it with 2.4 mm. It doesn't feel like a regular mechanical keyboard it's still distinctly a laptop type of keyboard, but it's very a tactile keyboard.
The trackpad is located on the right-hand side of the keyboard. We have seen this before on the Zephyrus devices. You may not love its positioning, but the way it's designed it pretty much required to be set up like this.
The purpose of separating the keyboard in this kind of bizzar all-in-one design style to this portable device is for thermals. This device has the motherboard, CPU, GPU, and all of its components on the back of the screen. By positioning the internals there they are able to vent out the heat directly at the top and the sides of the device. While it's venting it's also drawing in cool air from behind the screen. You end up with a very impressive thermal solution.
This guy is running the RTX 2080 and that the full, fat non-MaxQ version. As of this year, it's the most powerful GPU we've seen from NVIDIA, and it's running on a 17″ 144 Hertz G-sync enabled display.
A product like ROG Mothership obviously isn't for everyone. Not even most gamers are going to want something like this and it's going to be super expensive once they release the final version. Since we only have our hands on a design sample, it looks really well engineered and well-built. As mentioned, it has an NVIDIA RTX 2080, an i9-8950HK and it weighs a bit over 10-pounds. With these specs, it's supposed to rival the performance of most gaming desktops.
At 10+ pounds it's not super portable, and it's definitely heavier than most gaming laptops, but it's incredibly powerful and it's portable. You can obviously carry this device around if you wanted to.
It's well-built, it looks good, the thermal solutions are seemingly perfect, it rivals a desktop rig, and that's the Asus ROG Mothership. Stay tuned as we learn more about this product after launch. It may be a good time to sell laptop that's getting outdated and buy a new one. Not necessarily the Mothership though.
On January 16, 2019 / Uncategorized
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Roofed Bazaar of Nishapur (Persian:  بازار سرپوش نیشابور)  is a historical roofed Bazaar dating back to the Safavid Persia in Nishapur, Razavi Khorasan province of Iran. Many of its parts have been unfortunately destroyed and only 750 meters of it remain. There is a current effort made by the MCHT to reconstruct and repair this bazaar. This bazaar is located on the Imam Khomeini street of Nishapur. Its reconstruction was started in the year 1998. This Bazaar is now part of the national heritage list of Iran with the registration number of 6739.

Gallery

See also

 Greater Khorasan
 Nishapur

References

Buildings and structures in Nishapur
Tourist attractions in Razavi Khorasan Province
Infobox mapframe without OSM relation ID on Wikidata
Nishapur
Khorasan
Bazaars

redpajama_set_name: RedPajamaWikipedia


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The Battle of Maida, an Epic Poem – epos batalistyczny angielskiego oficera i poety Richarda Scotta, opublikowany w Londynie w 1808 nakładem oficyny W. Bulmer and Co. Utwór opowiada o epizodzie w wojen napoleońskich, mianowicie o bitwie pod Maidą, która dla Brytyjczyków był prestiżowym sukcesem militarnym, ponieważ była jedynym lądowym starciem, w którym oddziały angielskie pokonały armię francuską, a właściwie francusko-polską. Bitwa rozegrała się 4 lipca 1806. Maida jest niewielkim miastem położonym w Kalabrii w południowych Włoszech. Poemat został napisany parzyście rymowanym pentametrem jambicznym (heroic couplet).

Zobacz też: Wellington; or, The Mission of Napoleon, an Epic Poem, Wojny napoleońskie

Przypisy

Bibliografia

Angielskie eposy
Utwory literackie z 1808
Utwory literackie o wojnach napoleońskich


LobbyTorque is TorquePoint's flagship experiential learning programme. It is a full-day immersion in the art of effective political lobbying. Our experiential learning programme will guide you, as a member of a team, through a range of real life simulations relating to lobbying at a variety of levels.
After a day of LobbyTorque you will know who to contact, when and where, how to go about it and what you should be talking about. Your head will be spinning with ideas from simulations designed especially for your business, organisation or industry. If you're looking for a quiet day out of the office, LobbyTorque probably isn't for you – this is a day packed with interactive challenge, learning and fun. Anyone can lobby – only a few lobby effectively – and we will show you how.
What is Unique about LobbyTorque?
Our principal point of difference is that, in the great Kiwi DIY tradition, we teach you how to do your own lobbying.
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We are specialists in the simulation-based learning. We consult and teach using experiential learning methods because we believe that this is the most effective way for adults to achieve positive results. It's intense but fun. The entire focus of LobbyTorque is to transfer the capability to lobby into the hands of the individuals and organisations that seek our assistance. The most important thing is to get your case heard, supported and acted on.
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Meet with a politician and their staff in order to win their support for your cause.
Plan a written submission and also make an oral submission to a Select Committee or local body consultation.
Examine the basic principles of successful media engagement and write a media release.
Attend a media conference and ask questions of the Minister, Mayor or other official who is making an announcement.
Observe and assess a media interview at 'close-up and personal' range.
See how to use informal lobbying opportunities to best advantage.
How often is LobbyTorque run?
Public courses are run 3-4 times per year.
Usually Wellington and Auckland for public courses but if there's enough interest, we will run them anywhere.
Can you run an in-house version?
Absolutely! This gives us the opportunity to fine tune the simulation to your organisation's needs.
Public courses are $895 (excl) per person. In- house courses are quoted separately. Give us a call!
Hon Heather Roy and Dr Simon Ewing-Jarvie. Resumes are on this website or see LinkedIn for more detail. We also employ a well known and highly experienced media facilitator for those aspects of the programme. This person varies from city to city.


Check out these amazing stock vector vintage cameras created by freepik for creative nerds. There are a total of 80 camera vectors that can be used as icons or for other graphic design purposes. Don't miss this fabulous collection.


Slice zucchini and squash into quarters (lengthwise) and then slice the quarters 1″ thick. Boil 10 min. And drain.
In a medium bowl, melt butter and add stuffing mix. Place half of mix into a greased 2-1/2 qt. casserole.
Add drained squash. Combine soup, sour cream, carrot, onion and cheese. Pour soup mixture over squash. Top with remaining stuffing. Cover and bake at 350′ for 50 min.
For the casserole last night, I doubled the squash and used a 9 x 13 x 3″ disposable pan.
Everything else remained the same.