INHIBITIONS OF ENDOTHELIN FORMATION

Abstract: Peptides of up to 20 amino acids, which are capable of binding to human big-endothelin, contain a sequence of the formula (I): A3-A4-A5-A'4-A'3 wherein A3 and A'3 independently represent Ser, Gly, Asp or Asn residue, and A4 and A'4 independently represent Val, Pro, Gly or Ala residue, and A5 represents Asp or Asn residue. Also provided are enantiomers in which each amino acid is in D-form and pharmaceutically acceptable salts. The peptides are inhibitors of endothelin formation and can, therefore, find application in the therapy of human diseases caused by endothelin.

Description: Inhibitions of endothelin formation The present invention relates to peptides capable of binding to human big-endothelin (bigET) / to their preparation and to pharmaceutical composition containing them. Endothelin (ET) is a mammalian hormone of 21 amino acids which possesses high vasoconstrictor activity. The presence in endothelial cells of mKNA encoding a large precursor of ET indicates that this peptide is produced in a manner similar to that of many biological relevant peptides or hormones, that is through enzymatic proteolyεis of a protein of 212 aminoacids with formation of a fragment of 38 amino acid named big-endothelin (Yanagisawa et al.. Nature 332, p. 411-415, 1988). The present invention relates to peptides having endothelin formation inhibitor activity, useful in the therapy of diseases which are dependent from endothelin effects. The invention provides a peptide of up to 20 amino acids which is capable of binding to human big endothelin and which contains a sequence of the formula I wherein A 3 and A' 3 independently represent Ser, Gly, Asp or Asn residue, A_ a and A'_ » independently represent Val, Pro, Gly or Ala residue, A s represents Asp or Asn residue. The enantiomers of these peptides having all the optically active amino acid residues in the D-form are within the scope of the present invention. Salts of peptides according to the invention with pharmaceutically acceptable acids or bases are also within the scope of the present invention. Such acid addition salts can be derived from a. variety of. inorganic and organic acids such as sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicyclic, gluconic, ascorbic and related acids. Such base addition salts can be derived from a variety of inorganic and organic bases as sodium hydroxide, potassium hydroxide, diethylamine, triethylamine and dicyclohexylamine. The amino acid residues are denoted by the three letter code (Eur.J.Bioche . 138. 9-37, 1984). The sequence of the formula I was derived from the non-coding DNA strain of the 212 amino acid endothelin precursor and from the hydropatic map of big-endothelin according to the indeces of Kyte and Doolittle (J. Mol. Biol. .152, 105-132, 1982). The peptides tipically comprise at least 5 aminoacid residues. The peptides contain no more that 20 aminoacid residues, for example up to 18 aminoacid residues. Preferably the peptides have the formula II: Λ"Ae"Λ 7 "Aβ"A "Aιo*Λ 3ι , "A 2 "A 3 "A ' , "Λ "A < 4 ~A 3 ""A 2 *"A χ~Α ιo"« 9 ™*" B wherein A 3 ,A 4 ,A 5 ,A , ,A' 3 are as defined above, X represents hydrogen atom or a nitrogen protecting group, A a . and A' x independently represent Val, Leu, Cys or lie residue, or a valence bond, A 2 and A' 2 independently represent Gly, Ala or Asp residue, or a valence bond, A 6 and A' 6 independently represent Val or a valence bond, - 7 and A ! -j independently represent Arg, Ala, Gly, Cys, Glu or Lys residue or a valence bond, A 8 and A' β independently represent Asn, Pro, Gly, Ala residue or a valence bond, Ag and A' 9 independently represent Asn, Asp, Val, Leu, Met residue or a valence bond, A αo and A' xo independently represent Val, Leu, Met, His, Glu residue or a valence bond and W represents hydroxy or amino group. More preferably the peptides have the formula III or IV: X-Av-Asn-A 9 -A α o-Aι-A 2 -A 3 -Val-A 5 -A ' 4-A , 3 -Asp-A , α -A , α o -A ' s ,-Α ' β -A -Val-W HI X-Val-A , v -A 1 B -A ' 9 -A ' 3 . o -A ' α -Asp-A , 3 -A , ^-A 5 -Val-A 3 -A 2 -A a .-A 3. 0 -A 9 - Asn-A v -V? IV wherein A α represents Val, Leu, or Cys residue, A' α represents Val or He residue, A 2 represents Gly or Ala residue, A 3 represents Ser or Gly residue, A' 3 represents Asp or Asn residue. A' * represents Pro, Gly or Ala residue, A s represents Asp or Asn residue, A 7 represents Arg, Ala, Gly or Cys residue, '- 7 represents Glu or Lys residue, A'„ represents Pro, Gly or Ala residue, A 9 represents Asn or Asp residue, A' s represents Val, Leu or Met residue, A 10 represents Val, Leu or Met residue, A' αo represents His or Glu residue, X represents hydrogen atom or a nitrogen protecting group and V. represents hydroxy or amino group, or an enantiomer thereof having all the optically active amino acid residues in the D-form. Preferred peptides have the sequences: H-Arg-Asn-Asn-Val-Leu-Gly-Ser-Val-Asp-Pro-Asn-Asp-Val-His-Val- -Ala-Glu-Val-OH (As big 1) and H-Gly-Asn-Asn-Met-Leu-Gly-Ser-Val-Asn-Pro-Asp-Asp-Ile-Glu-Met- -Pro-Lys-Val-OH (As big 2) The synthesis of the peptides of the invention may be accomplished either by classical solution methods or by solid phase on polymeric supports. In the classical solution method the synthesis consists essentially in appropriate successive condensations of protected amino acids or peptides. The condensation is carried out so that the resulting peptides have the desired sequence of 5 to 20 amino acid residues. The amino acids and peptides, which are condensed according to methods known in themselves in polypeptide chemistry, have such of their amino and carboxy groups that are not involved in the formation of the peptide linkage blocked by a suitable protecting group. The hydroxy functions of hydroxy amino acids may be protected by suitable protecting groups (throughout all the synthesis or only during a few steps) or may be kept unprotected. The protecting groups are capable of being removed by acidolysis, saponification or hydrogenolysis. For the protection of the amino groups the following protective groups - A - may for example be employed: benzyloxycarbonyl, t-butoxycarbon- yl, trityl, for yl, trifluoroacetyl, o-nitrophenylsulphenyl, 4- methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl or 3,5- dimethoxy-α,α'- dimethylbenzyloxycarbonyl. For the protection of the carboxy groups the following protective groups may for example be employed: methyl, ethyl, t-butyl, benzyl or p-nitro-benzyl. For the protection of the hydroxy groups the following protecting groups may for example be used: acetyl, t-butoxycar- bonyl, benzyloxycarbonyl, 2-bromobenzyloxycarbonyl, tetrahydro- pyranyl, t-butyl, trityl, benzyl or 2,4-dichlorobenzyl. The condensation between an amino group of one molecule and a carboxyl group of another molecule to form the peptide linkage may be carried out through an activated acyl-derivative such as a mixed anhydride, an azide or an activated ester, or by direct condensation between free amino group and a free carboxyl group, in the presence of a condensing agent such.as dicyclo- hexylcarbodiimide, alone or together with a racemization preventing agent, such as N-hydroxysuccinimide or 1-hydroxyben- zotriazole. Hydrazido or substituted hydrazido derivatives according to the invention are prepared by condensation of the N-protected peptide or amino acid with a suitably substituted hydrazido such as benzylcarbazate, t-butylcarbazate, adamantylcarbazate, phenylhydrazine or adamanthylhydrazine, or reacting the N-protected peptide or amino acid hydrazide with a suitable alkylating agent such as benzylchloroformate, t-butylchloro- formate, di-t-butyldicarbonate or adamantylfluoroformate. The condensation may be carried out in a solvent such as dimethylformamide, pyridine, acetonitrile, tetrahydrofuran or N-methyl-2-pyrrolidone. The reaction temperature may be from -30°C to room temperature. The reaction time is generally from 1 to 120 hours. The scheme of synthesis, the protecting groups and the condensing agents are selected so as to avoid the risk of racemization. D-protecting reactions are carried out according to methods known per se in polypeptide chemistry. In the solid-phase method a polymeric εuport is used. Th polymer is preferably a copolymer of styrene with from 1 to percent by weight of divinyl benzene as a cross-linking agen which causes the polystyrene polymer to be completely insolubl in most organic solvents. The synthesis is commenced from th C-terminal end of the peptide, by attaching the reguired amin acid to a chloromethylated resin, a hydroxymethyl resin, or benzhydrylamine resin. The amino and side chain protectin groups are those described in the classical solution synthesis In the preparation of the compounds of this invention, an amin protected amino acid is coupled to the chloromethylated resi via caesium salt, or to hydroxymethyl or benzhydrylamine resin with the aid of a condensing agent such a dicyclohexylcarbodiimide. After the initial coupling, the amino protecting group i removed by a choice of reagents including trifluoroacetic aci or hydrogen chloride solutions in organic solvents at roo temperature. After removal of the amino protecting group, the remainin protected amino acids are coupled stepwise in the desired orde to obtain the desired peptide. Each protected amino acid i generally reacted in a 3-fold excess using an appropriat carboxy group activator such as dicyclohexylcarbodiimide i solution, in, for example, methylene dichloride; dimethylformamide mixtures. After the desired amino acid sequence has been completed, th peptide is removed from the resin support by treatment with reagent such as hydrogen fluoride, which not only cleaves th peptides from the resin, but also cleaves most of the remainin side-chain protecting groups. When chloromethylated o hydroxymethyl resin is used, hydrogen fluoride treatmen results in the formation of the free peptide acid (W=OH). Whe benzhydrylamine resin is used, hydrogen fluoride treatmen results directly in the free peptide amide (W=NH 2 ). Alternatively the peptide amides can be obtained from th peptide acids by amminolysis. The compounds according to the invention have an interesting pharmacological activity on inhibition of maturation process of endothelin. Several proteolytic enzymes were shown to convert bigEt into ET in-vitro (R. Tkegawa et al. BBRC 167:860-866 1990). More precisely, α-chymotrypsin has been shown to cleave the human 38 residue ET precursor (bigET (1.38) in vitro, at neutral pH (E.G. McMahon Et al. BBRC 161:406-414 1989). The inhibition of bigET cleavage was determined in "in vitro" tests by using α-chymotrypsin as preteolytic enzyme. Under our experimental conditions, within the first minute of reaction, bigET is totally converted into bigET (1-31) which in turn is cleaved at the Trp(21) with formation of ET(1-21). This second fragmentation is greatly retarded by the presence of peptides of the present invention. The sequences of ET (1-21), bigET (1-38) and bigET (1-31) are shown in the following formula: Cyε-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys- 1 5 10 15 His-Leu-Asp-Ile-Ile-Trp-Val-Asn-Thr-Pro-Glu-His-Val-Val-Pro- 20 25 30 Tyr-Gly-Leu-Gly-Ser-Pro-Arg-Ser 35 Biological tests Alpha-chymotrypsin (0.014 mg/ml) was incubated with synthetic bigET (0.2 mg/ml) in glycine buffer (25 mM). The enzyme/bigET molar ratio was 1/500. The proteolysis of bigET was followed as a function of time, at 37 β C, pH 7.1. 10 μl of the initial reaction mixture were injected in a RP-HPLC column (SuperPacPepS, 4x250 mm, Pharmacia) after 0, 45, 60, 100, 150, 180, 255 in of incubation. Elution was carried out in a acetonitrile/water (0.1% TFA) gradient (from 10% to 80% acetonitrile in 30 min). The peptides were assessed by their absorbance at 210 nm. Analogously, the proteolysis of the substrate was followed i the presence of the peptides of the formula I at a bigET peptide molar ratio of 1/1 (Table 1). Table 1: Inhibition of bigET cleavage in the presence o peptides as formula I as a function of time (a). ( a) Substrate (bigET) and inhibitor were mixed at the constant molar ratio of 1 to 1 (23.3 uM) . Incubation with chymotrypsin (pH 7.1, 37°C) was stopped with guanidine hydrochloride (4M) and the mixture applied to a RP-HPLC column (see above). (b) P/S = product (ET)/substrate (bigET 1-31) values were calculated as ratio of the area of the correspondent HPLC peaks. Standard errors on P/S values were estimated leεε than 9%. Proteolysis of bigET was also followed as a function of peptide/bigET ratio at 120 and 180 min of incubation and at the enzyme/bigET molar ratio of 1/1000 (37 β C, pH 7.1) (Table 2). Table 2: Inhibition of bigET cleavage as a function of Asbig2/bigET ratio (a) Asbig2 / bigET P/S (b) (molar ratio) t=120' (a) t=180' (a) 0.0 1.19 1.91 0.2 - 1.22 0.6 0.34 0.47 1.4 0.23 0.34 2.0 0.19 0.28 4.0 0.07 0.07 (a) Substrate and inhibitor were mixed at different inh. to substrate molar ratios and incubated with alpha-chymotrypsin (at pH 7.1 and 37°C) for either 120 or 180 min. The enzyme was denaturated with guanidine hydrochloride (4M) and the mixture applied to a RP-HPLC column (see above) . BigET concentration was kept constant and equal to 23.3 μM. (b) P/S = product (ET) to subεtrate (bigET 1-31) ratio was obtained from the correspondent HPLC areas. Standard errors on P/S values were estimated less than 9%. The P/S value of 40% is reached in 100 min in blank incubation. In the presence of ASbigl (1/1, substrate/peptide, molar ratio), the same P/S ratio is realized in 150 min. In the presence of ASbig2 such a value is not achieved even after 250 min of incubation (Table 1). These data are indicative of a kinetic retardation of the proteolysis of bigET (1-31). In fact, titration experiments (Table 2) at constant incubation times (e.i. 120' and 180') of bigET with Asbig2 show that a constant P/S value is reached when the initial bigET/Asbig2 molar ratio is equal to 1/2 or lower. The peptides of the present invention bind to the internal segment (i.e. 13-30) of bigET (1-38), thus inhibiting ET(1-21) formation. On this respect, then, the initial proteolytic step which cuts the terminal segment bigET(32-38) becomes irrelevant as far as ET formation is concerned. The peptideε of the formula I are inhibitors of endothelin formation and can, therefore, find application in the therapy of human diseases which are caused by endothelin, either directly or in concert with other factors. In addition, these peptides can be used in the management of the clinical symptoms asεociated with these deseases such as hypertension. The compounds of the invention can be administered by the usual routes, for example, parenterally, e.g. by intravenous injection or infusion, or by intramuscular, subcutaenous, intracavity and intranasal administration. The dosage depends on the age, weight and condition of the patient and on the administration route. It can be estimated that the therapeutic doseε in humanε will be in the range 10 ng/kg - 10 mg/kg, once to 6 times daily. The invention also provides pharmaceutical compositions containing a compound of formula (I) as the active substance, in association with one or more pharmaceutically acceptable excipients. The pharmaceutical compositionε of the invention are usually prepared following conventional methods and are administered in a pharmaceutically suitable form. For instance, solutions for intravenous injection or infusion may contain as carrier, for example, sterile water or, preferably, they may be in the form of sterile aqueous isotonic saline solutions. Suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride. The following examples illustrate the invention. High performance liquid chromatography (HPLC) was carried out using a Hewlett-Packard 1084B apparatus equipped with a UV detector operating at 215 nm. The peptides are separated on a 4 x 250 mm (I.D.) nucleosil 300 C 18 5μ column. The following solvents are used: A) 0.25 N H 3 PO_ » adjusted to pH 2.25 with triethyla ine B) 0.25 N HaPO * adjusted to pH 2.25 with triethylamine/CH 3 CN= 4/6 by volume. The elution is programmed with a linear gradient from 15 to 50 B over a period of 35 min. with a flow rate of 1.5 ml/min. The peptides are characterized by their retention time (RT) . Example 1 Preparation of H-Arq-Asn-Asn-Val-Leu-Gly-Ser-Val-Asp-Pro-Asn- -Asp-Val-His-Val-Ala-Glu-Val-OH (As big 1) The peptide was synthesised uεing an adaption of the Merrifield method (Merrifield, JACS 5, 2149-2154, 1963). The peptide was synthesised on a phenylacetanidomethyl resin. The α-amino protecting group on each amino acid was N-tert-butyloxycarbonyl (Boc) . Each coupling cycle was as follows: 1. Wash resin with dichloro ethane - 10 minutes 2. Wash with 5% diisopropylethylamine in DHF/CH 2 C1 2 2 minutes x 3 3. Dichloromethane wash - 1 minute x 2 4. Couple with 3 equivalents of the aminoacid in CH 2 C1 2 with 0.3M diisopropylcarbodiimide - 60 minutes. 5. As 3 6. Deprotect with 50% trifluoroacetic acid in dichloromethane - 20 minutes. 7. Dichloromethane wash - 1 minute x 6. 8. Return to 2. When the coupling cycles were completed the peptide was cleaved off the resin using hydrogen fluoride for 1 hour with an anisole scavenger 10%. The peptide was then ether washed, dried, dissolved in 15% acetic acid and lyophilized. RT = 27.9 min. Example 2 Operating as described in example 1, the following peptides were also prepared: a) H-Gly-Asn-Aεn-Met-Leu-Gly-Ser-Val-Asn-Pro-Asp-Asp-Ile-Glu- Met-Pro-Lys-Val-OH (As big 2) RT= 20.7 min. b) H-Ala-Asn-Aεn-Leu-Val-Ala-Ser-Val-Asn-Gly-Asp-Asp-Val-His- Leu-Pro-Glu-Val-OH RT= 27.8 min c) H-Cys-Gly-Ser-Val-Asn-Pro-Asp-Asp-Ile-Glu-Met-Pro-Lys-Val-OH RT= 21.3 min. d) H-Cys-Gly-Ser-Val-Asn-Pro-Asp-Aεp-OH RT= 15.9 min. e) Fmoc-Ser-Val-Asn-Pro-Asp-Asp-Gly-Lys-OH RT= 18.8 min.

Claims: CLA I MS 1. A peptide of up to 20 amino acids which is capable of binding to human big endothelin and which contains a sequence of the formula I A 3 —A^—As-A <—A 3 wherein A 3 and A' 3 independently represent Ser, Gly, Asp or Asn residue, and A., and A' * independently represent Val, Pro, Gly or Ala residue, -and A s represents Asp or ^n residue j or an enantiomer thereof in which each amino acid is in D-form, or a pharmaceutically acceptable salt thereof. 2. A peptide according to claim 1 having the formula II A_f—A 3 —A 41 "A 3 —A —A a.-A o — A β — g wherein A 3 ,A <* ,A 5 ,A' < ,A' 3 , are as defined in claim 1, X represents hydrogen atom or a nitrogen protecting group, A α and A' α independently represent Val, Leu, Cys or He residue or a valence bond, A 2 and A' 2 independently represent Gly, Ala or Asp residue or a valence bond, A β and A' β independently represent Val or a valence bond, A τ and A'-y independently represent Arg, Ala, Gly, Cys, Glu or Lys residue or a valence bond, A B and A' β independently represent Asn, Pro, Gly, Ala residue or a valence bond, A » and A' » independently represent Asn, Asp, Val, Leu, Met residue or a valence bond, A o and A' αo independently represent Val, Leu, Met, His, Glu •residue, or a valence bond.and W represents hvdroxv or amino group; or an enantiomer thereof in which each amino acid residue is in D-form, or a pharmaceutically acceptable salt thereof. 3. A peptide according to claim 1 or 2 having the formula: H-Arg-Asn-Asn-Val-Leu-Gly-Ser-Val-Asp-Pro-Asn-Aεp-Val-Hiε-Val- Ala-Glu-Val-OH, or a pharmaceutically acceptable salt thereof. 4. A peptide according to claim 1 or 2 having the formula: H-gly-Asn-Asn-Met-Leu-Gly-Ser-Val-Asn-Pro-Asp-Asp-Ile-Glu-Met- Pro-Lys-Val-OH, or a pharmaceutically acceptable salt thereof. 5. A method of preparing a peptide as defined in any of claims l to 4, which method comprises condensing single amino acids and/or preformed peptides of two or more amino acids in the required order and, if desired, converting the terminal carboxy group into amide form and/or, if desired, converting a peptide into a pharmaceutically acceptable salt thereof. 6. A pharmaceutical composition comprising a pharmaceuticall acceptable diluent or carrier and, as an active principle, a peptide as defined in any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof. 7. A peptide as defined in any one of claims 1 to , or a pharmaceutically acceptable salt thereof, for use in a method of treatment of the human or animal body by therapy. 8. A peptide or salt according to claim 7 for use as an inhibitor of endothelin formation. 9. A peptide or salt according to claim 7 or 8 for use in th treatment of hypertension.

Inventors: CAIOLFA VALERIA, ZAMAI MORENO, CAIOLFA, VALERIA, ZAMAI, MORENO
Applicants: ERBA CARLO SPA, FARMITALIA CARLO ERBA S.R.L.
Application year: 1991
Main classification: C07K7
IPC codes: A61K38, A61P5, A61P9, C07K1, C07K7, C07K14