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WO1992002237A1 - Analogs of endothelin - Google Patents

Analogs of endothelin Download PDF

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Publication number
WO1992002237A1
WO1992002237A1 PCT/US1991/005056 US9105056W WO9202237A1 WO 1992002237 A1 WO1992002237 A1 WO 1992002237A1 US 9105056 W US9105056 W US 9105056W WO 9202237 A1 WO9202237 A1 WO 9202237A1
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WIPO (PCT)
Prior art keywords
ala
amino acid
compound
cys
formula
Prior art date
Application number
PCT/US1991/005056
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French (fr)
Inventor
James P. Tam
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The Rockefeller University
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Publication of WO1992002237A1 publication Critical patent/WO1992002237A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Endothelin is a recently discovered 21-amino acid residue, bicyclic polypeptide. It is one of the most potent vasoconstrictors known. This peptide hormone is produced by endothelial cells and constricts a variety of blood vessels at concentrations lower than most other currently known vasoconstrictors.
  • ET is shown in Fig. 1. It will be seen that it is a bicyclic molecule bridged through disulfide linkages.
  • ET agonists are useful and are also within the scope of this invention. Such compounds, while they may compete with natural ET for binding sites, will not cause a lowering of blood pressure since they will be selected to have the same muscle contractile and binding activities as ET.
  • Such products are useful in treatment of accident victims who may be in shock from loss of blood.
  • Compounds within the scope of this invention may be either agonists or antagonists.
  • the invention provides methods for modulating blood pressure in patients in need of such therapy by the administration of a selected ET analog of the invention.
  • Novel analogs of ET have now been discovered in which one or more, e.g. up to about ten, of the amino acid residues of the ET polypeptide residues are replaced with the corresponding D-amino acid residue or with an L-alanyl residue provided that there is always at least one disulfide bridge.
  • the compounds therefore, are either monocyclic or bicyclic.
  • the resulting compounds when they bind at least as well as ET and have at least the same muscle contractile activity, are useful as agonists.
  • Those that bind at least as well as ET and have up to about 1% of the contractile activity of ET are useful as antagonists.
  • the compounds of the invention are useful as blood pressure regulators.
  • L-alanine residue or a monocyclic analogue of such compound of the formula A or B with one disulphide bridge.
  • the preferred compounds within the scope of the invention are those having at least the same binding activity as ET and at least the same muscle contractile activity or up to about 1% of the muscle contractile activity of endothelin.
  • amino acid is meant one of the common, naturally occurring amino acids found in proteins comprising Gly, Ala, Val, Leu, lie, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gin, Cys, Met, Phe, Tyr, Pro, Trp and His.
  • amino aci residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.
  • the products of this invention may be synthesized by known solid phase techniques. See, for example, Barany and Merrifield (1979) in The Peptides, eds. Gross and Meienhofer (Academic Press, New York) Vol. 2A, pages 1 to 284.
  • the products can be prepared by manual methods or, for example, on a peptide synthesizer such as the Applied Biosystems 430 unit.
  • Te.rt-Buto.xycarbonyl (Boc) protected amino acids were from Peninsula Laboratories, (San Carlos, CA.) and Boc-Trp(For)-4- oxymethyl-phenylaceta idomethyl copoly (styrene -1% divinyl benzene) was prepared as described by Mitchell et al, J. Org. Chem. 43, 2845 (1978).
  • Boc-Trp(CHO) -OCH_-Pam resin (0.3 g, 0.70 mmol/g, Applied Biosystems, Inc.) was placed in a silanized reaction vessel and carried manually through 20 synthetic cycles in a mechanical shaker. All amino acids were protected with N - tert-butyloxycarbonyl (Boc) . Side chain protecting groups were: Asp(OBzl) , Cys(4-MeBzl) , Glu(OBzl), His(Dnp), Lys(2-C.Z), Ser(Bzl) and Tyr(BrZ) .
  • Each synthetic cycle consisted of (i) 2 l in prewashes with 50% TFA/CH 2 C1 2 (containing 0.05% ethanedithiol and 0.05% dimethylsulfide) , (ii) a 20 min deprotection with 50% TFA/CH_C1 2 , (iii) neutralization with 5% diisopropylethyla ine in DMF/CH-Cl- (1:1), and (iv) double coupling with DCC (3 equiv. of th amino acid) for 1 h each in CH-C1,.
  • the mixtur was equilibrated to 0° by stirring in an ice bath. After 2 h, the HF and DMS were removed in vacuo. In the high-HF, the reaction vessel was recharged at -78° with 14 ml of fresh liquid HF to give total volume of 15 ml of HF-p-cresol-p-thiocresol. The reaction wa carried out at 0°C for 1 h. After evaporation of HF at 0°C and washing with t-butyl methyl ether-mercaptoethanol (98:2, v/v. 100 ml) to remove p-cresol and p-thiocresol, the crude peptide was extracted with 50 ml 8 M urea in 0.1 M Tris. HC1 buffer (containin 0.2 g dithiothreitol, pH 8.4). Dnp groups are removed by 3 treatments with 1M thiophenol in DMF for 8 hours before the low-high HF cleavage procedure.
  • the 8 M urea solution containing the crude peptide was dialyzed against 8 M and 4M urea in 0.1 M Tris. HC1 buffer at pH 8.4 overnight, and then diluted to 2 M urea with 0.1 M Tris. HC1 (pH 8.4) for disulfide formation by air oxidation and by mixed disulfide method (1 mM of equal ratio of reduced and oxidized glutathione)
  • the oxidation reaction monitored by Cl-o Q reverse-phase HPLC was usually completed in 8h.
  • the solution was adjusted to pH 4.0 by cone.
  • hVSM Human vascular smooth muscle cells
  • Adherent cells (2-4 x 10
  • binding buffer consisting of
  • DMEM Dulbecco's modified Eagle's medium
  • BSA bovine serum albumin
  • Specific binding was defined as the total binding after subtraction of the non-specific binding in the presence of 100 nM unlabelled endothelin (a concentration sufficient for maximal competition) . Non-specific binding was always less than 10% of the total binding.
  • vasoconstrictlve activity of peptides were determined in rabbit vena cava. Tissues obtained from male New Zealand rabbits (2.5 - 3 Kg) were spirally cut and mounted in 5 ml organ bath containing Krebs Henseleit solution with indomethacin 1 x 10 M gassed with 95% 0 2 and 5% CO- and thermoregulated at 37°C.
  • Ascorbic acid (5 x 10 " M) was added to prevent cathecolamine oxidation. All tissues were loaded with 1 g and contractions were recorded with a Basile isometric transducer connected to a Watanabe recorder.
  • the tissues were allowed to equilibrate for one hour before being contracted with norepinephrine to obtain a cumulative dose-response curve. After one hour of rest, the preparations were checked with a single dose of potassium 50 mM. The intimal surfaces of vessels were gently rubbed and the failure of acetylcholine to induce relaxation of the tone induced with an EC C . concentration of norepinephrine was taken as an indication of endothelium removal.
  • the ET analogs of this inventio should have a purity of at least about 93% and preferably at least 98%. For administration to animals, a much lower degree of purity may be acceptable.
  • products of the invention are amphoteric they may be administered in the form of non-toxic basic and acid addition salts.
  • Such acid addition salts can be derived from a variety of inorganic and organic acids as sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicylic, 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, diethyla ine, triethylamine and dicyclohexylamine.
  • the ET analogs or the non-toxic salts thereof may be administered to animals, including humans, either intravenously, subcutaneously, intramuscularly, percutaneously, e.g., intranasally or even orally.
  • the administration may be employed by a physician or veterinarian to modulate blood pressure where the host being treated requires such therapeutic treatment.
  • the required dosage will vary with the particular condition being treated, with the severity of the condition, with the duration of desired treatment and other factors the determination of which is well within the skill of a physician or veterinarian.
  • compositions containing a therapeutic agent of this invention carrier and other ingredients should be selected by way of identity and amounts so as not to diminish the therapeutic effects of the active agent.
  • Suitable dosage forms for oral use are tablets, dispersible powders, granules, capsules, caplets, syrups and elixirs.
  • parenteral forms are solutions, suspensions, dispersions, emulsions, and the like.
  • the compositions for oral use may contain one or more conventional adjuvants, such as sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a composition of suitable pharmaceutical elegance.
  • Tablets may contain the active ingredient in admixture with conventional pharmaceutically acceptable excipients including inert diluents such as calcium carbonate, sodium carbonate, lactose and talc; granulating and disintegrating agents such as starch and alginic acid; binding agnets such as starch, gelatin and acacia and lubricating agents such as magnesium stearate, stearic acid and talc.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose and talc
  • granulating and disintegrating agents such as starch and alginic acid
  • binding agnets such as starch, gelatin and acacia and lubricating agents such as magnesium stearate, stearic acid and talc.
  • the tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • suspensions, syrups, and elixirs may contain the active ingredients in admixture with any of the conventional excipients utilized for the preparation of such compositions including suspending agents (e.g., methylcellulose, tragacanth, and sodium alginate) , wetting agents (e.g., lecithin, polyoxyethylene stearate) and preservatives such as ethyl p-hydroxybenzoate.
  • suspending agents e.g., methylcellulose, tragacanth, and sodium alginate
  • wetting agents e.g., lecithin, polyoxyethylene stearate
  • preservatives such as ethyl p-hydroxybenzoate.
  • Capsules may contain the the active ingredient alone or admixed with an inert solid diluent suc ⁇ as calcium carbonate, calcium phosphate and kaolin.
  • the injectable composit; i are formulated as known in the art and may contain appropriate dispersing or wetting agents and suspending agents identical or similar to those mentioned above.
  • The may be administered in isotonic compositions with solutes such as salt or glucose together with appropriate buffering agents.
  • The may also be administered as solutions or suspensions in oils such as sesame oil.
  • Si .mi.lar tablets are prepare with [D-Met7JET
  • Pluronic F-68 and lactose Pass through a No. 80 screen. Add the magnesium stearate, mix and encapsulate into the proper size gela*;in capsule.
  • Procedure Place about 75% of the foreseen final volume of W.F.I. previously deareated with nitrogen, into a suitable glass container, then dissolve the prescribed quantity of mannitol. Under stirring and nitrogen bubbling add the [Ala JET. Sterilize as described for RTU solution, and distribute the filtered solution into sterilized glass vials. The vials are then freeze-dried, stoppered and sealed.

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Abstract

Endothelin analogs useful for modulation of blood pressure, compositions containing such analogs and methods for their use.

Description

RELATED APPLICATION
This application is a continuation in part of copending application Serial Number 07/564,743 filed August 8, 1990.
BACKGROUND OF THE INVENTION
Endothelin (ET) is a recently discovered 21-amino acid residue, bicyclic polypeptide. It is one of the most potent vasoconstrictors known. This peptide hormone is produced by endothelial cells and constricts a variety of blood vessels at concentrations lower than most other currently known vasoconstrictors.
The structure of ET is shown in Fig. 1. It will be seen that it is a bicyclic molecule bridged through disulfide linkages.
The recognition of the strong vasoconstrictor activity of ET suggests that it may be a significant factor in the development of hypertension in mammals and has triggered intensive efforts to produce ET antagonists which will compete successfully with ET for binding sites and have little or no vasoconstrictor activity.
ET agonists are useful and are also within the scope of this invention. Such compounds, while they may compete with natural ET for binding sites, will not cause a lowering of blood pressure since they will be selected to have the same muscle contractile and binding activities as ET.
Such products are useful in treatment of accident victims who may be in shock from loss of blood. Compounds within the scope of this invention may be either agonists or antagonists. Thus, the invention provides methods for modulating blood pressure in patients in need of such therapy by the administration of a selected ET analog of the invention.
BRIEF SϋMJΪARY OF THE INVENTION
Novel analogs of ET have now been discovered in which one or more, e.g. up to about ten, of the amino acid residues of the ET polypeptide residues are replaced with the corresponding D-amino acid residue or with an L-alanyl residue provided that there is always at least one disulfide bridge. The compounds, therefore, are either monocyclic or bicyclic. The resulting compounds, when they bind at least as well as ET and have at least the same muscle contractile activity, are useful as agonists. Those that bind at least as well as ET and have up to about 1% of the contractile activity of ET, are useful as antagonists.
As a class, the compounds of the invention are useful as blood pressure regulators.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with this invention, there are provided therapeutically useful compounds and compositions in which the compounds are represented by the formula A:
I , T 1
Cys-Ser-Cys-Ser-Ser-l^u-Met-Asp-Lys-^lu-Cys-Val-Tyr-Phe-<2ys-His-Leu-Asp-Ile-Ile-Trp
1 5 10 15 20
-_bn"whi-ch -a-t - as -one -and -up -to about 10 -of -the amino acid residues at positions 1, 2, 6 to 10, 12, 13, 15 to 21 are replaced with an L-alanine residue or at least one and up to about 10 of the amino acid residues at positions 1 to 20 are replaced by the corresponding D-amino acid residue, or a compound of the formula B:
I . j tys-Ser-Cys-Ser-Ser-Leu-fet-Asp-Lys-Glu-<^
1 5 10 15 2n
B
in which at least one and up to about 10 of the amino acid residues are replaced by the corresponding D-amino acid residue, or with an
L-alanine residue, or a monocyclic analogue of such compound of the formula A or B with one disulphide bridge.
The compo id of Formula A with Cys ' Cys ' bridges is naturally occurring human or porcine endothelin if all of the amino acid residues are in the natural or L-form. Endothelins rom other species differ slightly.
The preferred compounds within the scope of the invention are those having at least the same binding activity as ET and at least the same muscle contractile activity or up to about 1% of the muscle contractile activity of endothelin.
The nomenclature used to define the peptides is that specified by Schroder and Lubke, "The Peptides", Academic Press (1965), wherein, in accordance with conventional representation, the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus to the right. By natural amino acid is meant one of the common, naturally occurring amino acids found in proteins comprising Gly, Ala, Val, Leu, lie, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gin, Cys, Met, Phe, Tyr, Pro, Trp and His. When the amino aci residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.
In this specification and claims, the conventional three lette abbreviations are employed to represent the amino acid residues. I is believed that those skilled in the art are sufficiently familiar with them so that no further explanation is needed.
The products of this invention may be synthesized by known solid phase techniques. See, for example, Barany and Merrifield (1979) in The Peptides, eds. Gross and Meienhofer (Academic Press, New York) Vol. 2A, pages 1 to 284. The products can be prepared by manual methods or, for example, on a peptide synthesizer such as the Applied Biosystems 430 unit.
Analogs with a free C-terminal carboxyl were made on phenylacetamidomethyl-resin supports (PAM resin) . Side chain protection was Arg(Tos), Asp(OBzl), Glu(OBzl) , His(DnP), Lys(ClZ), Ser(Bzl), Thr(Bzl), Trp(For), and Tyr(BrZ) . Double couplings with preformed symmetric anhydrides in dimethylformamide were used routinely for all tert-butyloxycarbonyl-protected amino acids except for tosyl arginine, glutamine, and asparagine, where active esters in dimethylformamide were required [Konig, W. & Geiger, R. Chem. Ber. 103, 788 (1970) ] . The assembled protected peptide-resins were cleaved by the "low/high HF" technique [Tam, J.P., Heath, W.F. & Merrifield, R.B. J. Am. Chem. Soc. 105, 6442 (1983)], which was developed to avoid a number of potential side reactions. After the usual work-up and oxidation, the crude free peptide was purified by preparative low-pressure reverse-phase liquid chromatography on C.0-silica as described [Andreu, D. & Merrifield, R.B. in Peptides: Structure and Function, eds. Deber, CM., Hruby, V.J. & Kopple, K.D. (Pierce Chem. Co., Rockford, IL) , pp. 595-598. The overall yields were between 8 and 45%. Homogeneity was demonstrated by analytical HPLC, and identity was confirmed by amino acid analysis. The amino acid analysis of all compounds prepared agreed with theory within + 5*.
Te.rt-Buto.xycarbonyl (Boc) protected amino acids were from Peninsula Laboratories, (San Carlos, CA.) and Boc-Trp(For)-4- oxymethyl-phenylaceta idomethyl copoly (styrene -1% divinyl benzene) was prepared as described by Mitchell et al, J. Org. Chem. 43, 2845 (1978).
There follows a more specific description of the general procedures for the preparation of the compounds of this invention. The following abbreviations are utilized in the description:
Boc - t-butoxycarbonyl
TFA - trifluoracetic acid
DMF - dimethylformamide
DCC - dicyclohexylcarbodiimide Tos - tosyl
DMS - dimethylsulfide
For - for yl
4MeBzl - 4-methylbenzyl
Bzl - benzyl Dnp - dinitrophenyl
2C1Z - 2-chlorocarbobenzoxy
DIEA - diisopropylethylamine
TFMSA - trifluormethansulfonic acid
HPLC - high pe. formance liquid chromatography BrZ - 2-bromobenzyloxycarbonyl
Solid-phase Peptide Synthesis. Boc-Trp(CHO) -OCH_-Pam resin (0.3 g, 0.70 mmol/g, Applied Biosystems, Inc.) was placed in a silanized reaction vessel and carried manually through 20 synthetic cycles in a mechanical shaker. All amino acids were protected with N - tert-butyloxycarbonyl (Boc) . Side chain protecting groups were: Asp(OBzl) , Cys(4-MeBzl) , Glu(OBzl), His(Dnp), Lys(2-C.Z), Ser(Bzl) and Tyr(BrZ) . Each synthetic cycle consisted of (i) 2 l in prewashes with 50% TFA/CH2C12 (containing 0.05% ethanedithiol and 0.05% dimethylsulfide) , (ii) a 20 min deprotection with 50% TFA/CH_C12, (iii) neutralization with 5% diisopropylethyla ine in DMF/CH-Cl- (1:1), and (iv) double coupling with DCC (3 equiv. of th amino acid) for 1 h each in CH-C1,. The second coupling of Boc- His(Dnp), Boc-Leu, Boc-Tyr(BrZ) and Boc-Val was mediated by the preformed hydroxybenzatriazole active ester for 2 h in DMF. All couplings were monitored by the ninhydrin test. After completion o the chain assembly, the peptide and the protecting groups except fo the Dnp groups were removed by the low-high HF cleavage procedure. In the low-HF, the peptide resin (300 mg) was dried and premixed with 6.5 ml DMS, 0.5 ml p-cresol and 0.5 ml p-thiocresol. Liquid H at -78 was then added to give a final volume of 10 ml. The mixtur was equilibrated to 0° by stirring in an ice bath. After 2 h, the HF and DMS were removed in vacuo. In the high-HF, the reaction vessel was recharged at -78° with 14 ml of fresh liquid HF to give total volume of 15 ml of HF-p-cresol-p-thiocresol. The reaction wa carried out at 0°C for 1 h. After evaporation of HF at 0°C and washing with t-butyl methyl ether-mercaptoethanol (98:2, v/v. 100 ml) to remove p-cresol and p-thiocresol, the crude peptide was extracted with 50 ml 8 M urea in 0.1 M Tris. HC1 buffer (containin 0.2 g dithiothreitol, pH 8.4). Dnp groups are removed by 3 treatments with 1M thiophenol in DMF for 8 hours before the low-high HF cleavage procedure.
Oxidation and Purification. The 8 M urea solution containing the crude peptide was dialyzed against 8 M and 4M urea in 0.1 M Tris. HC1 buffer at pH 8.4 overnight, and then diluted to 2 M urea with 0.1 M Tris. HC1 (pH 8.4) for disulfide formation by air oxidation and by mixed disulfide method (1 mM of equal ratio of reduced and oxidized glutathione) The oxidation reaction monitored by Cl-oQ reverse-phase HPLC was usually completed in 8h. The solution was adjusted to pH 4.0 by cone. HC1 and loaded directly to a preparative Cl,o0 reverse-phase HPLC column (2.5 x 25 cm, 10-17u) and eluted with buffer A: 5% CH3CN/H20 (0.045% TFA) and buffer B: 60% CH3CN/H20 (0.039% TFA) in a linear gradient of 10-70% buffer B in 60 min at a flow rate of 15 ml/min. Major peak eluting at about 49% of buffer B was collected, neutralized to pH 7.0 and lyophilized to give a homogeneous product. The overall yields of endothelin analogs ranged in 8-45%.
Characterization of Synthetic ET and Analogs. Analytical HPLC of a 10 ug sample was performed in a Vydac column (Cl,o_) with a linear gradient of 15-85% buffer B at a flow rate of 1.5ml/min and monitored at 215 n . Buffer A and B were same as above. All synthetic peptides were characterized by Cf-252 fission ionization mass spectrometry.
The procedures described above were employed to produce a number of compounds of the invention. The procedures generally yielded a mixture of 1,4-isomer (Cys ' , Cys ' ) and the 1,3-
1 11 T 1 *5 isomer (Cys ' Cys ' ). The 1,4-isomer was normally present in the larger amount. The significance of the term "1,4-isomer" is that the longest bridge is between the first and the fourth cysteine. In the 1,3-isomer it is between the first and the third cysteine.
Using the above indicated HPLC method, the Retention Times of the prepared compounds of the invention were determined and are indicated between brackets hereinafter:
(standard, 19.66) [D-Ser2]ET (20.18) .la*]ET (19.88) [Cys 1-11 cys3"15, Ala"]ET (19.57)
[Cys1"11, cys3"15, D-Ser ]ET (18.63) [Cysl-ll' cys3"15, D-Ser5]ET (18.5 [D-Ser5] (20.51) [Cys1-11 cys3'15, Ala5]ET (19.45) [Ala5]ET (20.24) [Cysl-ll' cys3"15, D-Leu6]ET (19.3 [D-Leu6]ET (19.57) [Cys1-11 cys3"15, Ala6]ET 18.57) [Ala6]ET (18.76) [Cysl-ll' cys3"15, D-Met7 ET (20.0
[D-Met7]ET (19.63) [D-Asp8]ET (20.35) [Ala7]ET (18.79) [Ala8]ET (20.41)
XCys1"11, Cys3"15, D-Asp8]ET (19.78) ID-Lys9]ET (20.42) [Cys1"11, Cys3"15, Ala8]ET(19.29) [Cys1-11, Cys3"15, Ala9]ET (21. [Ala9]ET (22.68) [Cys1"11, Cys3"15, D-Glu10]ET
(19.69) [D-Glu10]ET (18.98) [Cys1"11, Cys3"15, Ala10]ET (19.
[Ala10]ET (20.60) [Ala12]ET (19.07)
[D-Val12]ET (21.57) [D-Tyr13] (20.40)
[Cys1"11, Cys3"15, Ala12]ET (18.53) [Ala13]ET (18.85) [Cys1-11, Cys3"15, D-Tyr13]ET (19.90) [Cys1"11, Cys3"15, D-Phe1 ]ET
(20.59) [D-Phe14]ET (21.26) [D-His16]ET (19.14)
[Cys1"11, Cys3"15, D-His10]ET (18.10) [Ala16]ET (22.74) [Cys1-11, Cys3~15Ala16]ET (23.40) [D-Leu17]ET (19.81) [Cys1-11, Cys3~15D-Leu17]ET (19.36) [Ala17]ET (17.74) [Cys1-11, Cys3"15, Ala17]ET (17.23) [D-Asp18]ET (19.49) [Cys1-11, Cys3"15, D-Asp18]ET (19.09) [Ala18]ET (20.03) [D-Ile19]ET (20.62) [Ala19]ET (18.42)
[Cys1"11, Cys3"15, Ala19]ET (17.94) [D-Ile20]ET (21.17) [Cys1"11, Cys3"15, D-Ile20]ET (21.10) [Ala20]ET (18.24)
[Cys1"11, Cys3"15, D-Trp21]ET (19.19)
[Cys1"11, Cys2-15, Ala21]ET
(16.92)
[Ala21]ET (17.48)
The following table shows some of the compounds which were produced. The table also shows the relative percent binding and agonistic activity of the ET analogs which were determined by procedures hereinafter described (ET-1=100) . The figures in parenthesis represent the number of replications. TABLE I
Relative
Relative % Agonistic
Formula % Binding Activity
Et-1 100 100
[D-Ser"]ET 10.0 (1) 0.1 (4) [Ala2]ET 100.0 (1) 70.0 (4)
[cys1'11, cys3"15, D-Ser"*JET 20.0 (1) 5.3 (4)
[D-Ser5]ET 52.2 (1) 45.6 (4y [Cys1"11, cys3"15, Ala3JET 5.8 (1) 3.6 (4)
[D-Leu6]ET 126.0 (1) 58.0 (6)
[Cys1"11, cys3"15, D-LeuuJET 27.3 (1) 5.3 (4) [Ala6JET 101.0 (1) 73.0 (3)
[Cys1"11, cys3"11, Ala°]ET 30.0 (1) 12.6 (4) [D-Met7]ET 126.0 (1) 20.4 (4) tcys1"11, cys3'15, D-Met' JET 57.0 (1) 37.7 (3) [Ala7]ET 350.0 (1) 88.5 (3) [D-Asp8JET 2.7 (1) 0.6 (5) [Ala8JET 103.0 (1) 0.8 (3)
[D-Lys^JET 210.0 (2) 418.0 (4) [Ala9JET 504.0 (1) 56.0 (6)
Figure imgf000011_0001
[D-Glu10]ET 154.0 (1) 53.5 (4) tcys1"11, cys3"15, D-Glu10]ET 14.2 (1) 2.7 (4)
[Ala10JET 162.0 (1) 121.0 (4)
30% p.a.
[Cys1"11, Cys3"15, Ala10JET 20.8 (1) 16.8 (3) 29% p.a.
[Ala12JET 16.2 (1) 29.4 (4) [Ala16]ET 339.0 (1) 382.0 (3) [Cys1"11, Cys3"15, Ala16JET 21.7 (1) <0.1 (4) [D-Leu17]ET 12.6 (1) 0.1 (3) [Ala17JET 25.0 (1) 0.9 (4) [Cys1"11, Cys3"15, Ala17JET 1.7 (1) 0.1 (4) [D-Asp18JET 1.5 (1) 1.5 (3) [Ala18]ET 50.0 (1) 14.0 (3) [Ala19JET 406.0 (1) 121.0 (4) [D-Ile20JET 3.2 (1) 5.0 (4)
22% p.a.
[Ala 20JET 22.0 (1) 22.5 (4)
[Ala21]ET 0.8 (1) <0.1
a) Competitive receptor binding assay for endothelin on human vascular smooth muscle cells
Human vascular smooth muscle cells (hVSM) between passages 6 and 15
5 were used for the binding assays. Adherent cells (2-4 x 10
2 . . . . cells/cm ) , were washed 2 times with binding buffer consisting of
Dulbecco's modified Eagle's medium (DMEM) supplemented with 25 mM HEPES buffer and 0.1% bovine serum albumin (BSA) . Cells were incubated in binding buffer for 2 h at 37 C with 30 to 60 pmol/1 of [ 125I] - endothelin (specific acitivity: 200 Ci/mmol) in the absence or presence of increasing concentrations (ranging from 0.01 to 1000 pH) of unlabelled endothelin or of various analogs. After incubation, cells were extensively washed, solubilized in 0.5N NaOH and the cell-bound radioactivity was measured by gamma counting.
Specific binding was defined as the total binding after subtraction of the non-specific binding in the presence of 100 nM unlabelled endothelin (a concentration sufficient for maximal competition) . Non-specific binding was always less than 10% of the total binding.
Relative binding values were reported in Table I (IC5Q τ
IC
50 analog b) Screening on isolated tissues
The vasoconstrictlve activity of peptides were determined in rabbit vena cava. Tissues obtained from male New Zealand rabbits (2.5 - 3 Kg) were spirally cut and mounted in 5 ml organ bath containing Krebs Henseleit solution with indomethacin 1 x 10 M gassed with 95% 02 and 5% CO- and thermoregulated at 37°C.
Ascorbic acid (5 x 10" M) was added to prevent cathecolamine oxidation. All tissues were loaded with 1 g and contractions were recorded with a Basile isometric transducer connected to a Watanabe recorder.
To avoid non specific binding of the peptides, all organ baths were siliconized.
The tissues were allowed to equilibrate for one hour before being contracted with norepinephrine to obtain a cumulative dose-response curve. After one hour of rest, the preparations were checked with a single dose of potassium 50 mM. The intimal surfaces of vessels were gently rubbed and the failure of acetylcholine to induce relaxation of the tone induced with an ECC. concentration of norepinephrine was taken as an indication of endothelium removal.
If the tested compound contracted the vein, thus showing an agonistic acti .vi.ty, a cumulati.ve dose-response curve from 1 x 10-12 to 1 x 10 M was obtained at least in three preparations and EC._n values were calculated and the relative % agonistic activities were reported in Table I (EC,-. (ET-1) Q ) . EC 50 anal°9
For partial agonists (p.a.) the mean of the relative maximum responses (ET-1 = 100) was also given. For administration to humans, the ET analogs of this inventio should have a purity of at least about 93% and preferably at least 98%. For administration to animals, a much lower degree of purity may be acceptable.
Since the products of the invention are amphoteric they may be administered in the form of non-toxic basic and acid addition salts.
Such acid addition salts can be derived from a variety of inorganic and organic acids as sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicylic, 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, diethyla ine, triethylamine and dicyclohexylamine.
The ET analogs or the non-toxic salts thereof, either alone or combined with a pharmaceutically or veterinarily acceptable carrier to form a pharmaceutical composition, may be administered to animals, including humans, either intravenously, subcutaneously, intramuscularly, percutaneously, e.g., intranasally or even orally. The administration may be employed by a physician or veterinarian to modulate blood pressure where the host being treated requires such therapeutic treatment. The required dosage will vary with the particular condition being treated, with the severity of the condition, with the duration of desired treatment and other factors the determination of which is well within the skill of a physician or veterinarian.
With pharmaceutical compositions containing a therapeutic agent of this invention, carrier and other ingredients should be selected by way of identity and amounts so as not to diminish the therapeutic effects of the active agent. Suitable dosage forms for oral use are tablets, dispersible powders, granules, capsules, caplets, syrups and elixirs. Examples of parenteral forms are solutions, suspensions, dispersions, emulsions, and the like. The compositions for oral use may contain one or more conventional adjuvants, such as sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a composition of suitable pharmaceutical elegance. Tablets may contain the active ingredient in admixture with conventional pharmaceutically acceptable excipients including inert diluents such as calcium carbonate, sodium carbonate, lactose and talc; granulating and disintegrating agents such as starch and alginic acid; binding agnets such as starch, gelatin and acacia and lubricating agents such as magnesium stearate, stearic acid and talc. The tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. Similarly, suspensions, syrups, and elixirs may contain the active ingredients in admixture with any of the conventional excipients utilized for the preparation of such compositions including suspending agents (e.g., methylcellulose, tragacanth, and sodium alginate) , wetting agents (e.g., lecithin, polyoxyethylene stearate) and preservatives such as ethyl p-hydroxybenzoate. Capsules may contain the the active ingredient alone or admixed with an inert solid diluent sucπ as calcium carbonate, calcium phosphate and kaolin. The injectable composit; i are formulated as known in the art and may contain appropriate dispersing or wetting agents and suspending agents identical or similar to those mentioned above. The may be administered in isotonic compositions with solutes such as salt or glucose together with appropriate buffering agents. The may also be administered as solutions or suspensions in oils such as sesame oil.
Although the invention has been described with reference to presently preferred embodiments which constitute the best mode presently known to the inventor for the practice thereof, it should be understood that the invention is only limited by the appended claims since many apparent departures from the spirit and scope of the invention may be conceived by the skilled artisan.
E3.AMPLE 1 Tablet Formulation
Formula: Per/tablet
[Ala JET 200.00 ug
Citric acid 1.00 mg
Lactose 25.00 mg
Dicalcium phosphate 25.00 mg
Sodium Lauryl Sulfate 1.00.mg
Polyvmylpyrrolidone 10.00 mg
Carbowax 1500 5.00 mg
3A alcohol 50 ml./lOOO tablets
Corn Starch 20.00 mg
Dry:
Sodium Lauryl Sulfate 1.00 mg
Magnesium stearate 1.00 mg
Procedure. Mix together the [Ala JET, citric acid, Pluronic F-68, sodium lauryl sulfate, lactose and dicalcium phosphate.
Screen through No. 60 mesh screen. Granulate the screened mix with an alcoholic solution containing the polyvmylpyrrolidone, Carbowax 1500. Add additional alcohol, if necessary, to bring powder mix to a pasty mass. Add corn starch and continue mixing until uniform damp granules are formed. Pass the damp granulation through a No. 10 screen and dry in an oven at 100 C for about 4 hours. Screen the dried granulation using a No. 16 screen, add sodium lauryl sulfate and magnesium stearate, mix and compress on a tablet machine to specifications.
Si .mi.lar tablets are prepare with [D-Met7JET
Example 2
Capsule Formulation
Formula: Per/capsule
[D-Lys9JET 100.00 μq
Citric acid 1.00 mg Pluronic F-68 1.00 mg
Lactose 100.00 mg
Magnesium stearate 1.00 mg
9 Procedure. Mix together the [D-Lys JET, citric acid,
Pluronic F-68 and lactose. Pass through a No. 80 screen. Add the magnesium stearate, mix and encapsulate into the proper size gela*;in capsule.
Similar capsules are prepared with [D-Glu J ET
Example 3 Parenteral Formulation
R.T.U. solution
Formula per ampoule:
[Ala10JET 200.00 ng
Sodium chloride, USP 9.00 mg
Water for injection, USP q.s. to 1.00 ml
Procedure. Under stirring and nitrogen bubbling add sodium chloride to about 85% of the prescribed volume of W.F.I, followed by [Ala JET. Bring the solution to volume with W.F.I, and sterilize through a sterilizing membrane filter 0.22 um porosity, collecting the filtrate in sterile area. Fill the filtered solution into sterilized type I glass ampoules and seal the ampoules by flame.
Freeze-dried vials.
Formula per vial:
[Ala10JET 200.00 ng
Mannitol 20.00 mg
W.F.I.* USP q.s. to 1.00 ml
*W.F.I. is removed during feeze drying process.
Procedure. Place about 75% of the foreseen final volume of W.F.I. previously deareated with nitrogen, into a suitable glass container, then dissolve the prescribed quantity of mannitol. Under stirring and nitrogen bubbling add the [Ala JET. Sterilize as described for RTU solution, and distribute the filtered solution into sterilized glass vials. The vials are then freeze-dried, stoppered and sealed.
Parenteral formulations containing [Ala JET are similarly prepared.

Claims

WHAT IS CLAIMED IS:
1. A compound of the formula A:
Figure imgf000020_0001
1 5 10 15 20
A
in which at least one and up to about 10 of the amino acid residues at positions 1, 2, 6 to 10, 12, 13, 15 to 21 are replaced with an L-alanine residue or at least one and up to about 10 of the amino acid residues at positions 1 to 20 are replaced by the corresponding D-amino acid residue, or a compound of the formula B:
Figure imgf000020_0002
1 5 10 15 20
B
in which at least one and up to about 10 of the amino acid residues are replaced by the corresponding D-amino acid residue or with an
L-alanine residue, or a monocyclic analogue of such compound of the formula A or B with one disulphide bridge.
2. A therapeutic composition for modulating the blood pressure of mammals comprising a compound of the formula A:
Figure imgf000020_0003
1 5 10 15 20 in which at least one and up to about 10 of the amino acid residues at positions 1, 2, 6 to 10, 12, 13, 15 to 21 are replaced with an L-alanine residue or at least onΛ and up to about 10 of the amino acid residues at positions 1 to 20 are replaced by the corresponding D-amino acid residue, or a compound of the formula B:
Figure imgf000021_0001
1 5 10 15 20
in which at least one and up to about 10 of the amino acid residues are replaced by the corresponding D-amino acid residue or with an
L-alanine residue, or a monocyclic analogue of such compound of the formula A or B with one disulphide bridge together with a pharmaceutically acceptable carrier.
3. A method of modulating the blood pressure of mammals in need of such modulation which comprises administration to such mammal of a blood pressure modulating amount of a compound of the formula A:
Figure imgf000021_0002
1 5 10 15 20
A
in which at least one and up to about 10 of the amino acid residues at positions 1, 2, 6 to 10, 12, 13, 15 to 21 are replaced with an L-alanine residue or at least one and up to about 10 of the amino acid residues at positions 1 to 20 are replaced by the corresponding D-amino acid residue, or a compound of the formula B:
Figure imgf000022_0001
1 5 10 15 20
B
in which at least one and up to about 10 of the amino acid residues are replaced by the corresponding D-amino acid residue or with an
L-alanine residue, or a monocyclic analogue of such compound of the formula A or B with one disulphide bridge.
4. A compound as in claim 1 wherein the compound is selected from
6 7 q the group consisting of [Ala ]ET, [D-Met ]ET, [D-Lys ]ET, [D-Glu10]ET, [Ala10]ET and [Ala16]ET.
5. A composition as in claim 2 wherein the compound is selected from the group consisting of [Ala 6]ET, [D-Met7]ET, [D-Lys -)]ET,
[D-Glu10]ET, [Ala10]ET and [Ala16]ET.
6. A method as in Claim 3 wherein the compound is selected from the group consisting of [Ala ]ET, [D-Met ]ET, [D-Lys ]ET,
[D-Glu10]ET, [Ala10]ET and [Ala16]ET.
7. A compound of claim 1 having the same binding activity as endothelin and at least the same muscle contractile activity or up to 1% of the muscle contractile activity of endothelin.
8. A composition as in claim 2 wherein the compound has the same binding activity as endothelin and at least the same muscle contractile activity or up to 1% of the muscle contractile activity of endothelin.
9. A method as in claim 3 wherein the compound has the same binding activity as endothelin and at least the same muscle contractile activity or up to 1% of the muscle contractile activity of endothelin.
PCT/US1991/005056 1990-08-08 1991-07-16 Analogs of endothelin WO1992002237A1 (en)

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Cited By (2)

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US5439887A (en) * 1992-02-12 1995-08-08 Roussel-Uclaf Peptide having antihypertensive activity free of vascoconstrictor activity
EP0487410B1 (en) * 1990-11-21 1999-08-11 Hoechst Marion Roussel Endothelin antagonists

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Publication number Priority date Publication date Assignee Title
US4981950A (en) * 1987-10-09 1991-01-01 Takeda Chemical Ind., Ltd. Vasoconstrictor peptide

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Publication number Priority date Publication date Assignee Title
US4981950A (en) * 1987-10-09 1991-01-01 Takeda Chemical Ind., Ltd. Vasoconstrictor peptide

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Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 156, Number 3, issued 15 November 1988, S. KIMURA et al., "Structure-Activity Relationships of Endothelin: Importance of C-Terminal Moiety", pages 1182-1186. *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 163, Number 1, issued 30 August 1989, K. NAKAJIMA et al., "Structure-Activity Relationship of Endothelin: Importance of changed Groups", pages 424-429. *
BIOCHEMICAL PHARMACOLOGY, Volume 38, Number 12, issued 1989, M. YANAGISAWA et al., "Pharmacological Activities, Regulation, and Possible Roles in Cardiovascular control", pages 1877-1883. *
BRITISH JOURNAL OF PHARMACOLOGY, Volume 98, issued 1989, M.D. RANDALL et al., "Vascular activities of endothelin-1 and some alanyl substituted analogues in resistance beds of the rat", pages 685-699. *
BRITISH JOURNAL OF PHARMACOLOGY, Volume 98, issued 1989, S. TOPOUZIS et al., "Effects of calcium entry blockers on contractions evoked by endothelin-1, (Ala 3,11)-endothelin-1 and (Ala 1,15)-endothelin-1 in rat isolated aorta", pages 669-677. *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0487410B1 (en) * 1990-11-21 1999-08-11 Hoechst Marion Roussel Endothelin antagonists
US5439887A (en) * 1992-02-12 1995-08-08 Roussel-Uclaf Peptide having antihypertensive activity free of vascoconstrictor activity

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