WO1987005029A1 - Peptides affecting blood pressure regulation - Google Patents
Peptides affecting blood pressure regulationInfo
- Publication number
- WO1987005029A1 WO1987005029A1 PCT/US1987/000367 US8700367W WO8705029A1 WO 1987005029 A1 WO1987005029 A1 WO 1987005029A1 US 8700367 W US8700367 W US 8700367W WO 8705029 A1 WO8705029 A1 WO 8705029A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- val
- leu
- tyr
- peptide
- glu
- Prior art date
Links
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Definitions
- the regulation of blood pressure in mammals, especially humans, is an important medical problem.
- Hypertension which is an abnormally high level of blood pressure, is a disease of particular concern, along with hypotension, which is low blood pressure. Therefore, the discovery of substances that can aid in the detection and treatment of blood pressure abnormalities is an important one.
- the present invention encompasses a class of peptides and their antibodies which affect the regulation of blood pressure.
- Angiotensinogen also known as renin substrate
- renin an enzyme that renin I does not have a major hormonal effect on blood pressure.
- angiotensin I is converted to angiotensin II by an enzyme called angiotensin converting enzyme.
- Angiotensin II an octapeptide
- W is a radical derived from an aliphatic ⁇ -aminooxycarboxylic acid, hydroxyacetyl, or ⁇ -hydroxypropyl;
- Z is a radical derived from an aliphatic ⁇ -aminocarboxylic acid, leucyl, isoleucyl, alanyl, or threonyl.
- NH is the ⁇ amino group of amino acid AA 1 ;
- C is the carbonyl group of amino acid AA 7 ;
- Y is -OH, -NH 2 , or -PRO;
- AA 1 is LYS, GLU, GLN, pGLU, BALA, PRO, PRO-OH,
- AA 2 is VAL, LEU, ILE, MET, or AIB
- AA 3 is ASP, TYR, GLU, HIS, or PHE
- AA 4 is VAL, MET, ILE, LEU, or AIB
- AA 5 is TYR, HIS, or GLU
- AA 6 is ALA, PRO, SER, BALA, PRO-OH, or AIB; and AA 7 is VAL, LEU, ILE, or AIB; as well as salts thereof.
- antibodies raised to these peptides along with pharmaceutical compositions comprising a peptide or antibody and a pharmaceutically-acceptable carrier.
- this invention includes diagnostic and therapeutic methods for blood pressure regulation.
- amino acids an L-form is usually preferred, although the D-form or racemic mixtures of the amino acid may be used.
- GLN glutamine
- Tos p-toluenesulfonyl
- GLU glutamic acid Boc: tert-butyloxycarbonyl
- pGLU pyroglutamic acid
- Bzl benzyl LYS: lysine Cl 2 -BZl: 2,6-dichlorobenzyl
- PRO proline Cl-Z: 2-chlorobenzyloxycarbonyl
- PRO-OH hydroxyproline DNA: deoxyribonucleic acid
- VAL valine
- EBV Epstein Barr Virus
- GLY glycine ELISA: enzyme-linked immunosorbent
- PIC picolinic acid
- EIA enzyme immunoassay
- ALA alanine
- DEAE diethylaminoethyl
- BALA beta-alanine Tris-HCI: tris (hydroxymethyl)-
- TYR tyrosine aminomethane hydrochloride
- AIB alpha-amino- BSA: bovine serum albumin isobutyric acid
- DMSO dimethylsulfoxide
- DCM dichloromethane
- BrZ 2-bromobenzyloxycarbonyl HF: hydrogen fluoride
- DICD diisopropylcarbodiimide
- HBr hydrogen bromide
- DIEA diisopropylethylamine
- HOAc acetic acid
- DMF dimethylformamide
- DCC dicylohexylHOBt: N-hydroxybenzotriazole carbodiimide
- AA 1 is LYS, GLU, or GLN;
- AA 2 is VAL or LEU
- AA 3 is ASP, TYR, or GLU
- AA 4 is VAL, MET, ILE, or LEU;
- AA 5 is TYR, HIS, or GLU; AA 6 is ALA, PRO, or SER; and
- AA 7 is VAL or LEU.
- the peptides of the present invention can be prepared by conventional processes for synthesizing peptides; more specifically, using processes as described in Schroder and Lubke, The Peptides, Vol. 1, published by Academic Press, New York (1966), or Izumiya, et al., Synthesis of Peptides, published by Maruzen Publishing Co., Ltd., (1975).
- an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, a DCC process, an active ester process (for example: p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or a DCC/additive process can be used.
- Solid phase and solution phase syntheses are both applicable to the foregoing processes.
- the peptides of the present invention are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid.
- stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid.
- amino groups that are not being used in the coupling reaction must be protected to prevent coupling at an incorrect location.
- the C terminal amino acid is bound to an insoluble carrier or support through its carboxyl group.
- the insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group. Examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, and the like
- An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide.
- This solid-phase approach is generally described by Merrifield, et al. in J. Am. Chem. Soc. 85, 2149-2156 (1963).
- the peptide can be cleaved and the protecting groups removed by either stirring the insoluble carrier or resin in anhydrous, liquid HF at about 0°C for about 20 to 90 minutes, preferrably 60 minutes, or by bubbling HBr continuously through a 1 mg/10 ml suspension of the resin in TFA for 30 to 60 minutes at about room temperature, depending on the protecting groups selected. Other deprotection methods may also be used.
- protective groups for amino groups include: benzyloxycarbonyl, Boc, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, Cl-Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl, and the like.
- protective groups for the imino group of histidine include: Tos, Bzl, benzyloxycarbonyl, trityl, and the like.
- protective groups for the hydroxy group of tyrosine include: Bzl, Cl 2 -BZl, BrZ, benzyloxycarbonyl, acetyl, Tos, and the like.
- protective groups for the amino group of lysine include: benzyloxycarbonyl, Cl-Z, Cl 2 -Bzl, Boc, Tos, and the like.
- Protection for the carboxyl groups of glutamic acid and aspartic acid includes: esterification of the carboxylic acids with benzyl alcohol, methanol, ethanol, tert-butanol, and the like.
- protective groups for the hydroxy of serine include: Bzl, tert-butyl, and the like.
- activated carboxyl groups include: the corresponding acid chlorides, acid anhydrides, mixed acid anhydrides, azides, and active esters (esters with pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboxydiimide, and the like).
- active esters esters with pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboxydiimide, and the like.
- the residues PIC and pGLU may only be used as the amino terminus of the final peptide.
- the residue AIB is often coupled to the growing peptide chain in a solvent mixture which is about one part DMSO to about one part DMF.
- the peptides of this invention can also be prepared through DNA techniques.
- the amino acid sequence of the desired peptide is used to deduce the codon sequence for the single-stranded DNA, synthesized using conventional synthetic techniques, then the double-stranded DNA is prepared and inserted at a suitable site in a cloning vehicle, vector, or plasmid.
- An appropriate organism such as bacteria cells, yeast cells, or mammalian cells, is transformed to obtain expression of the desired peptide.
- the prepared peptides of the present invention can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, countercurrent distribution, column chromatography, high performance liquid chromatography, and the like.
- the peptides of this invention form salts with a variety of inorganic or organic bases.
- the non-toxic, pharmaceutically-acceptable salts are preferred, although other salts are also useful in isolating or purifying the product.
- Such pharmaceutically-acceptable salts include metal salts, such as sodium, potassium, or lithium, alkaline earth metal salts, such as calcium or magnesium, and salts derived from amino acids, such as arginine or lysine.
- the salts are obtained by reacting the acid form of the peptide with an equivalent of the base supplying the desired ion in a medium in which the salt precipitates or in aqueous medium and then lyophilizing.
- the peptides form salts with a variety of inorganic and organic acids.
- non-toxic, pharmaceutically-acceptable salts are preferred, although other salts are also useful in isolating or purifying the product.
- Such pharmaceutically-acceptable salts include those formed with hydrochloric acid, methanesulfonic acid, sulfuric acid, maleic acid, and the like.
- the salts are obtained by reacting the product with an equivalent amount of the acid in a medium in which the salt precipitates.
- Antigens can be prepared by using the peptides or fragments of the peptides of the present invention as haptens and reacting the peptides or fragments with a suitable carrier in the presence of a hapten-carrier binding agent.
- a suitable carrier in the presence of a hapten-carrier binding agent.
- natural and synthetic proteins having a high molecular weight which are conventionally employed in the preparation of antigens, can be employed as carriers to be bound to the haptens.
- Such carriers include: albumins of animal sera, globulins of animal sera, thyroglobulins of animals, hemoglobulins of animals, hemocyanins of animals, such as KLH, proteins extracted from ascaris, polylysine, polyglutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine.
- hapten-carrier binding agents those conventionally employed in the preparation of antigens can be employed.
- these agents include: diazonium compounds for cross linking, aliphatic dialdehydes for cross linking an amino group with an amino group, dimaleimide compounds for cross linking a thiol group with a thiol group, maleimidocarboxyl-N-hydroxysuccinimide esters for cross linking an amino group with a thiol group, and agents used in conventional peptide bond forming reactions in which amide bonds are formed from an amino group and a carboxyl group.
- hapten-carrier binding agent it is also possible to use diazonium aryl carboxylic acids, such as p-diazonium phenylacetic acid, in combination with conventional peptide bond-forming agents, such as the dehydrating and condensing agents described above.
- the coupling reaction for preparing the antigenic forms of the peptides of the present invention is suitably carried out in an aqueous solution or a conventional buffer solution having a pH of 7 to 10, preferably in a buffer solution having a pH of 8 to 9, at temperatures of about 0o to 40°C, preferably around, room temperature.
- the coupling reaction is generally completed within about 1 to about 24 hours, preferably 3 to 5 hours.
- Proportions of the hapten, hapten-carrier binding agent, and carrier can be appropriately determined, but it is preferred that the molar ratio of hapten to carrier be about 1 to about 20, preferably about 10, and the molar ratio of binding agent to hapten be about 1 to about 10, preferably about 2 to about 5.
- the carrier is bound to the hapten via the hapten-carrier binding agent to obtain a desired antigen composed of a peptide-carrier complex.
- the antigen can easily be isolated and purified by means of dialysis, gel filtration, fractionation precipitation, and the like.
- the antibody or antibodies of the present invention which are raised to a peptide or peptides of this invention can be monoclonal or polyclonal, but monoclonal is preferred.
- antibodies may be obtained by injecting the desired immunogen or antigen into a wide variety of vertebrates in accordance with conventional techniques. Suitable vertebrates include mice, rats, sheep, and goats, with mice being preferred. Usually, the animals are bled periodically with the successive bleeds having improved titer and specificity.
- the antigens may be injected intramuscularly, intraperitoneally, subcutaneously, or the like.
- Polyclonal antibodies are prepared by hyperimmunization of the animal with antigen. Then the blood of the animal is collected shortly after the repeated immunizations and the gamma globulin is isolated. Suitable methods for preparing polyclonal antibodies are described in the Handbook of Experimental Immunology, 3d edition, (ed. Weir, 1978).
- spleen cells from the immunized vertebrate demonstrating the desired antibody response are immortalized.
- the manner of immortalization is not critical, but the most common method is fusion with a myeloma fusion partner.
- Other techniques of immortalization include EBV transformation, transformation with bare DNA, such as oncogenes or retroviruses, or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies.
- the general process for obtaining monoclonal antibodies is described by Kohler and Milstein, in Nature, 256 495-497 (1975), which is herein incorporated by reference.
- Human monoclonal antibodies may be obtained by fusion of the spleen cells with an appropriate human fusion partner, such as WI- L2, described in European Application No. 82.301103.6.
- WI- L2 human fusion partner
- a detailed technique for producing mouse x mouse monoclonal antibodies is taught by Oi and Herzenberg, in Selected Methods in Cellular Immunology, 351-372 (eds. Mishell and Shiigi, 1980).
- the resulting hybridomas are screened to isolate individual clones, each of which secretes a single antibody species to the antigen.
- the peptides and/or antibodies may be used without modification or may be modified in a variety of ways, for example, by labeling such as joining, either covalently or non-covalently, a moiety which directly or indirectly provides for a means of detection.
- labeling such as joining, either covalently or non-covalently, a moiety which directly or indirectly provides for a means of detection.
- labels include: radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles and the like.
- the peptides and/or antibodies may be used in assays, which are homogeneous (without a separation step between free reagent and receptor-ligand complex) or heterogeneous (with a separation step between free reagent and receptor-ligand complex).
- assays which are homogeneous (without a separation step between free reagent and receptor-ligand complex) or heterogeneous (with a separation step between free reagent and receptor-ligand complex).
- Various commercial assays exist, such as RIA, ELISA, EIA, and the like.
- the assays detect the presence of a related antibody or antigen in a physiological fluid, such as urine, serum, plasma, and the like.
- unlabeled antibodies can be employed by employing a second antibody, which is labeled and recognizes the antibody to a subject peptide.
- the peptides and/or antibodies may be used to characterize the cell surface receptor.
- the tissue sample may be lysed by conventional methods using bases, detergents, or the like, cellular debris separated by filtration or centrifugation, and the filtrate or supernatant isolated and assayed.
- the reagents are supplied in diagnostic kits, so as to optimize the sensitivity of the assay.
- the protocol, and the label either labeled or unlabeled antibody, or labeled peptide is provided, usually in conjunction with other additives, such as surface-active agents, buffers, stabilizers, materials necessary for signal production, such as substrates for enzymes, and the like.
- the peptides of this invention and their antibodies can be used to reduce or increase blood pressure depending upon their respective antagonistic or agonistic properties. Therefore, hypertension or hypotension can be diagnosed or treated depending upon the peptide or antibody selected and the concentrations utilized.
- the human or other mammal being treated with the peptide or antibody is one that has been diagnosed as having blood pressure regulation needs, hypertension or hypotension. Therefore, the patient to be treated is in need of treatment due to an existing condition.
- compositions employing the peptides of the invention, or antibodies, are also a part of this invention.
- therapeutic compositions comprise effective amounts of the peptides or antibodies in admixture with pharmaceutically- or physiologically-acceptable carriers.
- Pharmaceutical compositions that contain the polypeptides or antibodies as an active ingredient will normally be formulated with an appropriate solid or liquid carrier depending upon the particular mode of administration being used.
- parenteral formulations are usually injectable fluids that use pharmaceutically- or physiologically-acceptable fluids, such as physiological saline, balanced salt solutions, or the like, as a vehicle.
- Other drug delivery systems that may be used include: liposomes, biodegradable or bioerodible polymers, polyethylene glycol, and the like.
- Also a part of this invention are methods of regulating or aiding in the regulation of the blood pressure of a mammalian host which comprises administering to the host an effective amount of a peptide or antibody described above.
- the host being treated is human and this human host is diagnosed as having hypertension, hypotension, or another blood pressure regulation abnormality.
- the peptides or antibodies may be administered to a human or any other mammalian host in various manners, such as orally, topically, intranasally, and parenterally, which includes: intravenously, intramuscularly, intraperitoneally, intradermally and subcutaneously.
- dosages of about 0.1 to 50 mg/kg of body weight per day of active ingredient should be appropriate to alter blood pressure.
- the dosage should be from about 0.1 to about 10 mg/kg.
- the peptide or antibody therapy of the invention may be combined with other treatments and may be combined with or used in association with other chemotherapeutic or chemopreventive agents.
- the peptides or antibodies can be formulated in combination with a beta-adrenergic blocker, a diuretic, or an angiotensin converting enzyme inhibitor, for the treatment of hypertension.
- beta-adrenergic blockers that can be used include: propranolol, practolol, acebutolol, timolol, and the like.
- Exemplary of the diuretics contemplated for use in combination with a peptide of this invention are the thiazide diuretics, as well as ethacrynic acid, ticrynafen, chlorthalidone, furosemide, musolimine, bumetanide, triamterene, amiloride, and spironolactone and salts of such compounds.
- Examples of angiotensin converting enzyme inhibitors include enalapril and captopril.
- the peptides may be used in combination with adjuvants to generate vaccines.
- the immune system of an organism may be used to moderate the biological response of its hormone systems.
- the presentation of antigen may require special modifications to ensure the highest population of antibodies that give the desired biological responses.
- Example 1 is illustrative of the present invention, but are not to be construed as limitations on it.
- Example 1 is illustrative of the present invention, but are not to be construed as limitations on it.
- Example 1 is illustrative of the present invention, but are not to be construed as limitations on it.
- N ⁇ -Boc-L-Val esterified to a benzyl moiety on polystyrene/divinylbenzene copolymer (1% crosslink, 0.6 meq/g) was used as the starting resin.
- Other N ⁇ -Boc protected amino acids with appropriate side-chain functional group protection were also used during the synthesis.
- the reagents were used as received, except DIEA and TFA were distilled before use.
- the completed octapeptide was cleaved from the resin with simultaneous removal of side-chain protecting groups by treatment with anhydrous HF, in the presence of 10% (v/v) anisole for 60 min at 0°C.
- the HF/anisole was removed by water aspiration; and the resin wash was thoroughly dried under vacuum to remove trace amounts of HF.
- the peptide was washed from the resin with 15-20 ml portions of DMF, DMF/H20 (9:1), DMF/H20 (1:9), 10% HOAC/H 2 O, and water. The combined washings were concentrated in vacuo and lyophilized to give a crude peptide powder.
- the crude peptide material was purified by reverse phase high performance liquid chromatography using a preparative column (2.2 ⁇ 50 cm, Whatman Partisil 10 ODS-3); solvent A was 0.05% TFA/distilled water and solvent B was 0.05% TFA/acetonitrile.
- a typical purification chromatogram incorporated gradient development from 10 to 90% solvent B over 80 minutes at a flow rate of 10 ml/min to give purified peptide eluting at 34% solvent B.
- Inhibition of angiotensin II binding to angiotensin II receptors by peptides of this invention was measured through the use of radioactive angiotensin II.
- Rabbit livers were homogenized and centrifuged in order to isolate particles sedimenting between 1,000 and 100,000 ⁇ g.
- a portion of the particles which binds angiotensin II was solubilized with 1% digitonin, followed by ammonium sulfate fractionation between 49 and 65% saturation, followed by DEAE-cellulose chromatography at pH 7.5 using a linear gradient between 0.0 and 0.3 M KC1.
- the partially purified, solubilized receptor preparation bound 17 pmoles of angiotensin II per mg of protein when analyzed by Scatchard analysis, indicating a purity of approximately 0.1%.
- the assay for binding of angiotensin II to the receptor was as follows: The complete system (150 ⁇ l) contained 30 mM Tris-HCl, pH 7.5, 2.5 mM K 2 EDTA, 0.2 mM
- PCMS 0.25 nM [ 125 I] angiotensin II (ca. 100,000 cpm), 100 ⁇ g BSA, 0.25% (v/v) Brij 99 (a polyoxyethylene ether of a fatty alcohol) and 30 ⁇ g of partially purified receptor.
- the reaction was initiated by addition of the receptor and samples were incubated for 60 minutes at 20°C.
- the reaction was terminated with 1 ml of cold 0.5% charcoal/0.05% dextran in 100 mM Tris-HCl, pH 7.5. Tubes were vortexed and then allowed to stand 10 minutes at 4°C, after which they were centrifuged and their supernatants, containing protein-bound angiotensin II, were counted.
- a ID 50 is the concentration of peptide that inhibited binding of radiolabeled angiotensin II by 50%. This experiment demonstrates that peptides of this invention can inhibit the binding of angiotension II to its receptor.
- the peptides of this invention were tested in vitro, using an assay of isolated rabbit aorta strips mounted in a muscle bath.
- the left anterior descending coronary artery was excised from the heart and cut helically into a strip under a dissecting microscope. Strips were mounted vertically on a glass holder in a tissue bath containing 50 ml of PSS. The upper end of each strip was connected to a force transducer. The bathing medium was maintained at 37°C and aerated with a mixture of 95% O 2 - 5% CO 2 .
- the pH of the PSS was 7.2 and the composition, in mmoles/1, was as follows: NaCl 130, KCl 14.7, KH 2 PO 4 1.18, MgSO 4 -7H 2 O 1.17, CaCl 2 -2H 2 O 1.6, NaHCO 2 14.9, dextrose 5.5, and CaNa 2 EDTA 0.03.
- the passive force on each strip was adjusted to 2,000 mg.
- peptides were tested for ability to stimulate contractile response.
- Peptide was added to the bath in a step-wise, cumulative manner.
- Three peptides were tested: angiotensin II, Peptide 2 and Peptide 5.
- Muscle strips from between 2 and 6 rabbits were used to test each peptide. Results are given as average baseline response (Response -2,000 mg (passive force)); and are shown in Tables 2, 3, and 4.
- the muscle strips were bathed in PSS containing All, 10 -9 M. Strips were allowed to contract until a steadystate contraction was reached (-8 minutes), then peptide was added in a step-wise, cumulative manner. The change in muscle strip contraction was measured after each addition of peptide, and more peptide was not added until contraction had stabilized from the previous peptide addition (10-15 minutes). Each peptide was tested in strips from 2 or 4 rabbits. Results are given as percentage (%) change from All contractile response, and are shown in Tables 5 through 9.
- a positive value indicates increased contraction, while a negative value represents a decreased contractile response.
- Peptide 2 of this invention was tested in vivo, using a rat hypertension model.
- Bolus injections of angiotensin II were delivered via the same venous catheter.
- the right carotid artery was cannulated for measurement of blood pressure.
- Injection of angiotensin II (1 ng) caused an increase in mean arterial blood pressure (-50 mm Hg).
- Constant infusion of saralasin (1 mg/min) inhibited the pressor activity of angiotensin II.
- Infusion of Peptide 2 (0.1 to 0.5 mg/min) did not alter baseline blood pressure and did not influence pressor responses to angiotensin II.
- AA' is LYS, GLU, GL pGLU, ⁇ ALA, PRO, PRO-OH, PIC, or AIB
- AA- is VAL, LEU, ILE, MET, or AIB
- AA ⁇ is ASP, TYR, GLU, HIS, PHE
- AA 4 is VAL, MET, ILE, LEU, or AIB
- AA* is TYR, HIS, or GLU:
- AA 6 is ALA, PRO, SER, ⁇ ALA, PRO-OH, AIB: and
- a A 7 is VAL, LEU, ILE, or AIB; and salts thereof are claimed as effective blood pressure regulators.
- pr v ided are antibodies to these peptides, as well as diagnostic and therapeutic methods for blood pressure regulation.
Abstract
Peptides of formula (I), wherein NH is the alpha amino group of amino acid AA1; (II) is the carbonyl group of amino acid AA7; X is H-, (III), GLU-VAL-, or VAL-TYR-HIS-GLU-VAL; Y is -OH, -NH2, or -PRO; AA1 is LYS, GLU, GLN, pGLU, betaALA, PRO, PRO-OH, PIC, or AIB; AA2 is VAL, LEU, ILE, MET, or AIB; AA3 is ASP, TYR, GLU, HIS, or PHE; AA4 is VAL, MET, ILE, LEU, or AIB; AA5 is TYR, HIS, or GLU; AA6 is ALA, PRO, SER, betaALA, PRO-OH, or AIB; and AA7 is VAL, LEU, ILE, or AIB; and salts thereof are claimed as effective blood pressure regulators. Further provided, are antibodies to these peptides, as well as diagnostic and therapeutic methods for blood pressure regulation.
Description
PEPTIDES AFFECTING BLOOD PRESSURE REGULATION
The regulation of blood pressure in mammals, especially humans, is an important medical problem. Hypertension, which is an abnormally high level of blood pressure, is a disease of particular concern, along with hypotension, which is low blood pressure. Therefore, the discovery of substances that can aid in the detection and treatment of blood pressure abnormalities is an important one. The present invention encompasses a class of peptides and their antibodies which affect the regulation of blood pressure.
A physiological pathway believed to affect blood pressure is as follows. Angiotensinogen, also known as renin substrate, is acted upon by an enzyme, called renin, to produce angiotensin I. Angiotensin I is a relatively inactive decapeptide; in other words, angiotensin I does not have a major hormonal effect on blood pressure. However, angiotensin I is converted to angiotensin II by an enzyme called angiotensin converting enzyme. Angiotensin II, an octapeptide, is often at elevated levels in those individuals with hypertension and its action at these elevated levels causes blood vessel
constriction and water retention, both of which are associated with hypertension. By modification or blockage of the peptides or enzymes in this pathway, the level of blood pressure can be controlled.
The pathway is also shown in the following diagram:
Various peptides or angiotensin analogs have been prepared and disclosed. An extensive review of known analogs is found in Khosla, et al., "Structure-Activity Relationship in Angiotensin II Analogs", Angiotensin (Page and Bumpus eds). Handbook of Experimental Pharmacology, Vol. 34, 126-161 (1974). Other analogs are described in U.S. Patents 4,179,433 and 4,209,442, which analogs have the following amino acid sequence:
W-ARG-VAL-TYR-ILE-HIS-PRO-Z wherein:
W is a radical derived from an aliphatic α-aminooxycarboxylic acid, hydroxyacetyl, or α-hydroxypropyl; and
Z is a radical derived from an aliphatic α-aminocarboxylic acid, leucyl, isoleucyl, alanyl, or threonyl.
U.S. Patent 4,302,386 describes the production of antibodies raised against hormones.
Peptides of the following formula are part of the present invention:
NH is the α amino group of amino acid AA1 ; O
''
C is the carbonyl group of amino acid AA7;
VAL-TYR-HIS-GLU-VAL-; Y is -OH, -NH2, or -PRO;
AA1 is LYS, GLU, GLN, pGLU, BALA, PRO, PRO-OH,
PIC or AIB; AA2 is VAL, LEU, ILE, MET, or AIB AA3 is ASP, TYR, GLU, HIS, or PHE; AA4 is VAL, MET, ILE, LEU, or AIB; AA5 is TYR, HIS, or GLU;
AA6 is ALA, PRO, SER, BALA, PRO-OH, or AIB; and AA7 is VAL, LEU, ILE, or AIB;
as well as salts thereof.
Further provided by this invention are antibodies raised to these peptides, along with pharmaceutical compositions comprising a peptide or antibody and a pharmaceutically-acceptable carrier. In addition, this invention includes diagnostic and therapeutic methods for blood pressure regulation.
The following abbreviations are used throughout the specification. In the case of amino acids, an L-form is usually preferred, although the D-form or racemic mixtures of the amino acid may be used.
SER: serine PSS: physiological salt solution
LEU: leucine TFA: trifluoroacetic acid
GLN: glutamine Tos: p-toluenesulfonyl
GLU: glutamic acid Boc: tert-butyloxycarbonyl pGLU: pyroglutamic acid Bzl: benzyl LYS: lysine Cl2-BZl: 2,6-dichlorobenzyl
PRO: proline Cl-Z: 2-chlorobenzyloxycarbonyl
PRO-OH: hydroxyproline DNA: deoxyribonucleic acid
VAL: valine EBV: Epstein Barr Virus
HIS: histidine KLH: Keyhole limpet hemocyanin ASP: aspartic acid RIA: radioimmunoassay
GLY: glycine ELISA: enzyme-linked immunosorbent
ILE: isoleucine assay
PIC: picolinic acid EIA: enzyme immunoassay ALA: alanine DEAE: diethylaminoethyl
BALA: beta-alanine Tris-HCI: tris (hydroxymethyl)-
TYR: tyrosine aminomethane hydrochloride
PHE: phenylalanine EDIA: ethylenediaminetetraacetate
MET: methionine PCMS: para-chloromercurisulfate ARG: arginine BBzl: beta-benzyl
AIB: alpha-amino- BSA: bovine serum albumin
isobutyric acid DMSO: dimethylsulfoxide DCM: dichloromethane BrZ: 2-bromobenzyloxycarbonyl HF: hydrogen fluoride DICD: diisopropylcarbodiimide HBr : hydrogen bromide DIEA: diisopropylethylamine HOAc: acetic acid DMF: dimethylformamide DCC: dicylohexylHOBt: N-hydroxybenzotriazole carbodiimide
Preferred are peptides wherein:
AA1 is LYS, GLU, or GLN;
AA2 is VAL or LEU;
AA3 is ASP, TYR, or GLU;
AA4 is VAL, MET, ILE, or LEU;
AA5 is TYR, HIS, or GLU; AA6 is ALA, PRO, or SER; and
AA7 is VAL or LEU.
In addition to the peptides defined above, the following are preferred: wherein AA 1 is LYS; AA2 is VAL;
AA3 is TYR; AA4 is ILE; AA5 is HIS; AA6 is ALA; and AA7 is
LEU. Also preferred are the following peptides:
Peptide SEQUENCE Number
1 NH2-LYS-GLY-VAL-ASP-VAL-TYR-ALA-VAL-COOH
2 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH
3 NH2-LYS-GLY-VAL-ASP-MET-HIS-ALA-LEU-COOH
4 GLU-VAL-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH 5 VAL-TYR-HIS-GLU-VAL-LYS-GLY-VAL-TYR-ILE-HIS- ALA-LEU-COOH
6 NH2-GLU-GLY-LEU-GLU-LEU-GLU-ALA-LEU-COOH 7 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-CONH2
8 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-PRO O
''
9 CH3CNH-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-CONH2 O
''
10 CH3CNH-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-PRO and
11 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH (all D-form amino acids) 12 NH2-GLN-GLY-VAL-ASP-VAL-HIS-PRO-VAL-COOH
13 NH2-LYS-GLY-VAL-TYR-MET-HIS-SER-LEU-COOH
The peptides of the present invention can be prepared by conventional processes for synthesizing peptides; more specifically, using processes as described in Schroder and Lubke, The Peptides, Vol. 1, published by Academic Press, New York (1966), or Izumiya, et al., Synthesis of Peptides, published by Maruzen Publishing Co., Ltd., (1975). For example, an azide process, an acid chloride process, an acid anhydride process, a mixed anhydride process, a DCC process, an active ester process (for example: p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or a DCC/additive process can be used. Solid phase and solution phase syntheses are both applicable to the foregoing processes.
The peptides of the present invention are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. (Amino groups that are not being used in the coupling reaction must be protected to prevent coupling at an incorrect location.)
In case that a solid phase synthesis is adopted, the C terminal amino acid is bound to an insoluble carrier or support through its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group. Examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, and the like.
An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide. This solid-phase approach is generally described by Merrifield, et al. in J. Am. Chem. Soc. 85, 2149-2156 (1963).
The peptide can be cleaved and the protecting groups removed by either stirring the insoluble carrier or resin in anhydrous, liquid HF at about 0°C for about 20 to 90 minutes, preferrably 60 minutes, or by bubbling HBr continuously through a 1 mg/10 ml suspension of the resin in TFA for 30 to 60 minutes at about room temperature, depending on the protecting groups selected. Other deprotection methods may also be used.
In the foregoing process, it is preferred that respective amino acids of histidine, tyrosine, glutamic acid, lysine, serine, and aspartic acid be protected at the side chain functional groups. These functional groups at the side chain are protected with ordinary protective groups which are split off after completion of the
reaction. The functional groups that take part in the reaction are generally activated.
Examples of protective groups for amino groups include: benzyloxycarbonyl, Boc, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, Cl-Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl, and the like.
Examples of protective groups for the imino group of histidine include: Tos, Bzl, benzyloxycarbonyl, trityl, and the like.
Examples of protective groups for the hydroxy group of tyrosine include: Bzl, Cl2-BZl, BrZ, benzyloxycarbonyl, acetyl, Tos, and the like.
Examples of protective groups for the amino group of lysine include: benzyloxycarbonyl, Cl-Z, Cl2-Bzl, Boc, Tos, and the like.
Protection for the carboxyl groups of glutamic acid and aspartic acid includes: esterification of the carboxylic acids with benzyl alcohol, methanol, ethanol, tert-butanol, and the like.
Examples of protective groups for the hydroxy of serine include: Bzl, tert-butyl, and the like.
Examples of activated carboxyl groups include: the corresponding acid chlorides, acid anhydrides, mixed acid anhydrides, azides, and active esters (esters with pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboxydiimide, and the like).
In the foregoing process, the residues PIC and pGLU may only be used as the amino terminus of the final peptide. Furthermore, the residue AIB is often coupled to the growing peptide chain in a solvent mixture which is about one part DMSO to about one part DMF.
The peptides of this invention can also be prepared through DNA techniques. When the peptide contains only naturally occurring amino acids, the amino acid sequence of the desired peptide is used to deduce the codon sequence for the single-stranded DNA, synthesized using conventional synthetic techniques, then the double-stranded DNA is prepared and inserted at a suitable site in a cloning vehicle, vector, or plasmid. An appropriate organism, such as bacteria cells, yeast cells, or mammalian cells, is transformed to obtain expression of the desired peptide.
The prepared peptides of the present invention can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, countercurrent distribution, column chromatography, high performance liquid chromatography, and the like.
The peptides of this invention form salts with a variety of inorganic or organic bases. The non-toxic, pharmaceutically-acceptable salts are preferred, although other salts are also useful in isolating or purifying the product. Such pharmaceutically-acceptable salts include metal salts, such as sodium, potassium, or lithium, alkaline earth metal salts, such as calcium or magnesium, and salts derived from amino acids, such as arginine or lysine. The salts are obtained by reacting the acid form of the peptide with an equivalent of the base supplying the desired ion in a medium in which the salt precipitates or in aqueous medium and then lyophilizing.
Similarly, the peptides form salts with a variety of inorganic and organic acids. Again, the non-toxic, pharmaceutically-acceptable salts are preferred, although other salts are also useful in isolating or purifying the product. Such pharmaceutically-acceptable salts include those formed with hydrochloric acid, methanesulfonic acid, sulfuric acid, maleic acid, and the like. The salts are obtained by reacting the product with an equivalent amount of the acid in a medium in which the salt precipitates.
Antigens can be prepared by using the peptides or fragments of the peptides of the present invention as haptens and reacting the peptides or fragments with a suitable carrier in the presence of a hapten-carrier binding agent. In this case, natural and synthetic proteins having a high molecular weight, which are conventionally employed in the preparation of antigens, can be employed as carriers to be bound to the haptens. Examples of such carriers include: albumins of animal sera, globulins of animal sera, thyroglobulins of animals, hemoglobulins of animals, hemocyanins of animals, such as KLH, proteins extracted from ascaris, polylysine, polyglutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine.
As hapten-carrier binding agents, those conventionally employed in the preparation of antigens can be employed. Specific examples of these agents include: diazonium compounds for cross linking, aliphatic dialdehydes for cross linking an amino group with an amino group, dimaleimide compounds for cross linking a thiol group with a thiol group, maleimidocarboxyl-N-hydroxysuccinimide esters for cross linking an amino group with a thiol group, and agents used in conventional peptide bond forming reactions in which amide bonds are formed from an amino group and a carboxyl group. Also as
the hapten-carrier binding agent, it is also possible to use diazonium aryl carboxylic acids, such as p-diazonium phenylacetic acid, in combination with conventional peptide bond-forming agents, such as the dehydrating and condensing agents described above.
The coupling reaction for preparing the antigenic forms of the peptides of the present invention is suitably carried out in an aqueous solution or a conventional buffer solution having a pH of 7 to 10, preferably in a buffer solution having a pH of 8 to 9, at temperatures of about 0º to 40°C, preferably around, room temperature.
The coupling reaction is generally completed within about 1 to about 24 hours, preferably 3 to 5 hours.
Representative examples of buffer solutions which can be used in the above process include:
0.2 N sodium hydroxide-0.2 M boric acid-0.2 M potassium chloride buffer solution;
0.2 M sodium carbonate-0.2 M boric acid-0.2 M potassium chloride buffer solution;
0.05 M sodium tetraborate-0.2 M boric acid-0.05 M sodium chloride buffer solution; and 0.1 M dihydrogen potassium phosphate-0.05 M sodium tetraborate buffer solution.
Proportions of the hapten, hapten-carrier binding agent, and carrier can be appropriately determined, but it is preferred that the molar ratio of hapten to carrier be about 1 to about 20, preferably about 10, and the molar ratio of binding agent to hapten be about 1 to about 10, preferably about 2 to about 5. In the coupling reaction, the carrier is bound to the hapten via the hapten-carrier binding agent to obtain a desired antigen composed of a peptide-carrier complex.
After completion of the coupling reaction, the antigen can easily be isolated and purified by means of dialysis, gel filtration, fractionation precipitation, and the like.
The antibody or antibodies of the present invention which are raised to a peptide or peptides of this invention, can be monoclonal or polyclonal, but monoclonal is preferred. In general, antibodies may be obtained by injecting the desired immunogen or antigen into a wide variety of vertebrates in accordance with conventional techniques. Suitable vertebrates include mice, rats, sheep, and goats, with mice being preferred. Usually, the animals are bled periodically with the successive bleeds having improved titer and specificity. The antigens may be injected intramuscularly, intraperitoneally, subcutaneously, or the like.
Polyclonal antibodies are prepared by hyperimmunization of the animal with antigen. Then the blood of the animal is collected shortly after the repeated immunizations and the gamma globulin is isolated. Suitable methods for preparing polyclonal antibodies are described in the Handbook of Experimental Immunology, 3d edition, (ed. Weir, 1978).
To obtain monoclonal antibodies, spleen cells from the immunized vertebrate demonstrating the desired antibody response are immortalized. The manner of immortalization is not critical, but the most common method is fusion with a myeloma fusion partner. Other techniques of immortalization include EBV transformation, transformation with bare DNA, such as oncogenes or retroviruses, or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. The general process for obtaining
monoclonal antibodies is described by Kohler and Milstein, in Nature, 256 495-497 (1975), which is herein incorporated by reference. Human monoclonal antibodies may be obtained by fusion of the spleen cells with an appropriate human fusion partner, such as WI- L2, described in European Application No. 82.301103.6. A detailed technique for producing mouse x mouse monoclonal antibodies is taught by Oi and Herzenberg, in Selected Methods in Cellular Immunology, 351-372 (eds. Mishell and Shiigi, 1980). The resulting hybridomas are screened to isolate individual clones, each of which secretes a single antibody species to the antigen.
The peptides and/or antibodies may be used without modification or may be modified in a variety of ways, for example, by labeling such as joining, either covalently or non-covalently, a moiety which directly or indirectly provides for a means of detection. A wide variety of labels are known and include: radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles and the like.
Many of the techniques for linking the peptides to suitable labels involve the use of activated carboxyl groups, either through the use of carbodiimide or active esters to form peptide bonds: the formation of thioethers by reaction of a mercapto group with an activated halogen, such as chloroacetyl; or activated olefin, such as maleimide, or the like.
The peptides and/or antibodies may be used in assays, which are homogeneous (without a separation step between free reagent and receptor-ligand complex) or heterogeneous (with a separation step between free reagent and receptor-ligand complex). Various commercial assays exist, such as RIA, ELISA, EIA, and the like. Usually,
the assays detect the presence of a related antibody or antigen in a physiological fluid, such as urine, serum, plasma, and the like. For example, unlabeled antibodies can be employed by employing a second antibody, which is labeled and recognizes the antibody to a subject peptide. In a related application, the peptides and/or antibodies may be used to characterize the cell surface receptor.
Where the antigen may not be found in a physiological fluid or if found there is not diagnostic, then cells will have to be isolated and the cells assayed for the presence of the antigen. For detecting the antigen, the tissue sample may be lysed by conventional methods using bases, detergents, or the like, cellular debris separated by filtration or centrifugation, and the filtrate or supernatant isolated and assayed.
Frequently, the reagents are supplied in diagnostic kits, so as to optimize the sensitivity of the assay. For the subject invention, depending upon the nature of the assay, the protocol, and the label; either labeled or unlabeled antibody, or labeled peptide is provided, usually in conjunction with other additives, such as surface-active agents, buffers, stabilizers, materials necessary for signal production, such as substrates for enzymes, and the like.
The peptides of this invention and their antibodies can be used to reduce or increase blood pressure depending upon their respective antagonistic or agonistic properties. Therefore, hypertension or hypotension can be diagnosed or treated depending upon the peptide or antibody selected and the concentrations utilized. Typically, the human or other mammal being treated with the peptide or antibody is one that has been diagnosed as having blood pressure regulation needs, hypertension or
hypotension. Therefore, the patient to be treated is in need of treatment due to an existing condition.
Methods and compositions employing the peptides of the invention, or antibodies, are also a part of this invention. In particular, therapeutic compositions comprise effective amounts of the peptides or antibodies in admixture with pharmaceutically- or physiologically-acceptable carriers. Pharmaceutical compositions that contain the polypeptides or antibodies as an active ingredient will normally be formulated with an appropriate solid or liquid carrier depending upon the particular mode of administration being used. For instance, parenteral formulations are usually injectable fluids that use pharmaceutically- or physiologically-acceptable fluids, such as physiological saline, balanced salt solutions, or the like, as a vehicle. Other drug delivery systems that may be used include: liposomes, biodegradable or bioerodible polymers, polyethylene glycol, and the like.
Also a part of this invention are methods of regulating or aiding in the regulation of the blood pressure of a mammalian host which comprises administering to the host an effective amount of a peptide or antibody described above. Usually, the host being treated is human and this human host is diagnosed as having hypertension, hypotension, or another blood pressure regulation abnormality. In the therapeutic methods of the invention, the peptides or antibodies may be administered to a human or any other mammalian host in various manners, such as orally, topically, intranasally, and parenterally, which includes: intravenously, intramuscularly, intraperitoneally, intradermally and subcutaneously. The particular mode of administration and dosage regimen will be selected by the skilled artisan taking into account the particulars of the patient and the nature of treatment
required. For example, dosages of about 0.1 to 50 mg/kg of body weight per day of active ingredient should be appropriate to alter blood pressure. Preferably, the dosage should be from about 0.1 to about 10 mg/kg.
The peptide or antibody therapy of the invention may be combined with other treatments and may be combined with or used in association with other chemotherapeutic or chemopreventive agents. For example, the peptides or antibodies can be formulated in combination with a beta-adrenergic blocker, a diuretic, or an angiotensin converting enzyme inhibitor, for the treatment of hypertension. Examples of beta-adrenergic blockers that can be used include: propranolol, practolol, acebutolol, timolol, and the like. Exemplary of the diuretics contemplated for use in combination with a peptide of this invention are the thiazide diuretics, as well as ethacrynic acid, ticrynafen, chlorthalidone, furosemide, musolimine, bumetanide, triamterene, amiloride, and spironolactone and salts of such compounds. Examples of angiotensin converting enzyme inhibitors include enalapril and captopril.
In addition, the peptides may be used in combination with adjuvants to generate vaccines. In this way, the immune system of an organism may be used to moderate the biological response of its hormone systems. The presentation of antigen may require special modifications to ensure the highest population of antibodies that give the desired biological responses.
The following examples are illustrative of the present invention, but are not to be construed as limitations on it.
Example 1 :
Peptide Preparation
All peptide molecules were prepared on an automated peptide synthesizer (Beckman 990C) using Boc solid phase synthetic chemistry essentially as described by Merrifield and others [Merrifield, et al., J. Am. Chem. Soc., 85, 2149 (1963). Erickson. et al., Proteins (3rd Ed.) 2, 2156 (1976): and Stewart, et al.. Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, IL (1984)]. The octapeptide, NH2- LYS-GLY-VAL-ASP-VAL-TYR-ALA-VAL-COOH designated as Peptide 1, was synthesized as follows.
Nα-Boc-L-Val esterified to a benzyl moiety on polystyrene/divinylbenzene copolymer (1% crosslink, 0.6 meq/g), was used as the starting resin. Other Nα-Boc protected amino acids with appropriate side-chain functional group protection were also used during the synthesis. The reagents were used as received, except DIEA and TFA were distilled before use.
Beginning with one gram of Boc-L-Val resin in the automated peptide synthesizer, the following double coupling protocol was used.
1. Resin wash, DCM 3 times (×) @ 2 min
2. Boc removal, TFA/DCM/anisole 1 × 1 min
(45:50:5) 1 × 20 min
3. Resin wash, DCM 3 × @ 2 min 4. Neutralization, DIEA/DCM (1:9) 2 × @ 5 min
5. Resin wash, DCM 3 × @ 3 min
6. Add Boc-amino acid/HOBt 0.5 min
(1.25/1.0, 50% DCM/DMF)
7. Add DICD (0.5M/DCM) 0.5 min 8. Mix 30 min
9. Resin wash, DCM 3 × @ 2 min
10. Neutralize, DIEA/DCM (1:9) 2 × @ 5 min
11. Resin wash 3 × @ 2 min
12. Add Boc-amino acid/HOBt 0.5 min
(1.25/1.0, 50% DCM/DMF) 13. Add DICD (0.5M/DCM) 0.5 min
14. Mix 30 min
15. Resin wash, DCM 3 × @ 2 min
16. To step #2, repeat
Subsequent residues were attached following the same procedure in accordance with the desired sequence. For this example the following protected amino acids were used: Nα-Boc-4-bromobenzyloxycarbonyl-L-tyrosine (Nα-Boc- (BrZ)-L-tyrosine), Nα-Boc-L-valine, Nα-Boc-beta-benzyl-L-aspartic acid (Nα-Boc-(βBzl)-L-aspartic acid), Nα-Boc-L-valine, Boc-glycine, and Nα-Boc-N',-2-chlorobenzyloxycarbonyl-L-lysine (Nα-Boc-(Nα-Cl-Z)-L-lysine) to give the Boc protected peptide resin. The final Boc group was removed by washing with DCM then exposing to TFA/anisole/DCM. The finished protected peptide resin was washed thoroughly with DCM (four times) and dried under vacuum for about 16 hours to remove traces of solvent.
The completed octapeptide was cleaved from the resin with simultaneous removal of side-chain protecting groups by treatment with anhydrous HF, in the presence of 10% (v/v) anisole for 60 min at 0°C. The HF/anisole was removed by water aspiration; and the resin wash was thoroughly dried under vacuum to remove trace amounts of HF. The peptide was washed from the resin with 15-20 ml portions of DMF, DMF/H20 (9:1), DMF/H20 (1:9), 10% HOAC/H2O, and water. The combined washings were concentrated in vacuo and lyophilized to give a crude peptide powder.
The crude peptide material was purified by reverse phase high performance liquid chromatography using a preparative column (2.2×50 cm, Whatman Partisil 10 ODS-3); solvent A was 0.05% TFA/distilled water and solvent B was 0.05% TFA/acetonitrile. A typical purification chromatogram incorporated gradient development from 10 to 90% solvent B over 80 minutes at a flow rate of 10 ml/min to give purified peptide eluting at 34% solvent B.
In addition to Peptide 1 , the following peptides of this invention were prepared using the general procedure described above:
Peptide Number SEQUENCE
2 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH
3 NH2-LYS-GLY-VAL-ASP-MET-HIS-ALA-LEU-COOH
4 GLU-VAL-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH
5 VAL-TYR-HIS-GLU-VAL-LYS-GLY-VAL-TYR-ILE-HIS- ALA-LEU-COOH
6 NH2-GLU-GLY-LEU-GLU-LEU-GLU-ALA-LEU-COOH
7 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-CONH2
8 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-PRO
O "
9 CH3CNH-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-CONH2
O
''
10 CH3CNH-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-PRO 11 NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH
(all D-form amino acids)
Example 2:
Effects of Peptides on the Binding of
Radiolabeled Angiotensin II to its Receptor
Inhibition of angiotensin II binding to angiotensin II receptors by peptides of this invention was measured through the use of radioactive angiotensin II. Rabbit livers were homogenized and centrifuged in order to isolate particles sedimenting between 1,000 and 100,000 × g. A portion of the particles which binds angiotensin II was solubilized with 1% digitonin, followed by ammonium sulfate fractionation between 49 and 65% saturation, followed by DEAE-cellulose chromatography at pH 7.5 using a linear gradient between 0.0 and 0.3 M KC1. The partially purified, solubilized receptor preparation bound 17 pmoles of angiotensin II per mg of protein when analyzed by Scatchard analysis, indicating a purity of approximately 0.1%.
The assay for binding of angiotensin II to the receptor was as follows: The complete system (150 μl) contained 30 mM Tris-HCl, pH 7.5, 2.5 mM K2EDTA, 0.2 mM
PCMS, 0.25 nM [125I] angiotensin II (ca. 100,000 cpm), 100 μg BSA, 0.25% (v/v) Brij 99 (a polyoxyethylene ether of a fatty alcohol) and 30 μg of partially purified receptor. The reaction was initiated by addition of the receptor and samples were incubated for 60 minutes at 20°C. The reaction was terminated with 1 ml of cold 0.5% charcoal/0.05% dextran in 100 mM Tris-HCl, pH 7.5. Tubes were vortexed and then allowed to stand 10 minutes at 4°C, after which they were centrifuged and their supernatants, containing protein-bound angiotensin II, were counted.
The complete system under these conditions regularly yields about 10,000 cpm of bound radioactivity. A control, which lacks receptor, yields values of 50-200
cpm, which were subtracted from these data. Values of 50-200 cpm were obtained when 10 μM cold angiotensin II was present in the reaction mixture, indicating that virtually all binding was specific. A sample including 20 nM cold angiotensin II was also run. Residual binding of radioactivity in this control was 35-45%. Certain peptides of this invention were dissolved in water, except for Peptide 5, which was dissolved in 3% DMSO, 0.04 M acetic acid, and 0.05 M HCl in water. Results of these assays are given in Table 1.
Table 1 Inhibition by Peptides on Angiotensin II Binding to Isolated Hepatic Receptor
Peptide ID50 (nM)a
Angiotensin II 12-15 1 4,000 2 8-14 3 5,000 4 490 5 40
7 40-160 8 7-40
9 2,400-3,100
10 300-1,350
11 >10,000
12 5,000 13 6
a ID50 is the concentration of peptide that inhibited binding of radiolabeled angiotensin II by 50%.
This experiment demonstrates that peptides of this invention can inhibit the binding of angiotension II to its receptor.
Example 3:
Effects of Peptides on Arterial Smooth Muscle Contraction
The peptides of this invention were tested in vitro, using an assay of isolated rabbit aorta strips mounted in a muscle bath.
The left anterior descending coronary artery was excised from the heart and cut helically into a strip under a dissecting microscope. Strips were mounted vertically on a glass holder in a tissue bath containing 50 ml of PSS. The upper end of each strip was connected to a force transducer. The bathing medium was maintained at 37°C and aerated with a mixture of 95% O2 - 5% CO2. The pH of the PSS was 7.2 and the composition, in mmoles/1, was as follows: NaCl 130, KCl 14.7, KH2PO4 1.18, MgSO4-7H2O 1.17, CaCl2-2H2O 1.6, NaHCO2 14.9, dextrose 5.5, and CaNa2EDTA 0.03. In all experiments, the passive force on each strip was adjusted to 2,000 mg.
In one set of experiments, peptides were tested for ability to stimulate contractile response. Peptide was added to the bath in a step-wise, cumulative manner. Three peptides were tested: angiotensin II, Peptide 2 and Peptide 5. Muscle strips from between 2 and 6 rabbits were used to test each peptide. Results are given as average baseline response (Response -2,000 mg (passive force)); and are shown in Tables 2, 3, and 4.
Table 2 Contractile Response to Angiotensin II (AII)
[AII] (M) Average Baseline Response (mg)
10-11 0
3×10-11 0
10-10 0
3×10-10 991
10-9 1,726
3×10-9 2,332
10-8 2,562
3×10-8 2,613
Table 3
Contractile Response to Peptide 5
[Pept ide 5] (M) Average Baseline Response (mg)
3×10-10 0
10-9 0
3×10-9 50
10-8 245
3x10-8 540
10-7 1,070
3×10-7 1,230
10-6 1,380
3×10-6 1,380
Table 4
Contractile Response to Peptide 2
[Peptide 2] (M) Average Baseline Response (mg)
3×10-10 0
10-9 0
3x10-9 0
10-8 0
3x10-8 0
10-7 0
3x10-7 0
10-6 0
These experiments demonstrate that Peptide 5 of this invention can stimulate contraction of arterial smooth muscle.
In another set of experiments, the ability of peptides to increase or decrease contraction caused by angiotensin II was measured.
The muscle strips were bathed in PSS containing All, 10 -9M. Strips were allowed to contract until a steadystate contraction was reached (-8 minutes), then peptide was added in a step-wise, cumulative manner. The change in muscle strip contraction was measured after each addition of peptide, and more peptide was not added until contraction had stabilized from the previous peptide addition (10-15 minutes). Each peptide was tested in strips from 2 or 4 rabbits. Results are given as percentage (%) change from All contractile response, and are shown in Tables 5 through 9.
A positive value indicates increased contraction, while a negative value represents a decreased contractile response.
Table 5
Peptide 2 Effect on All-stimulated Contraction
[Peptide 2] (M) % Change from All Response
10-6 0
3x10-6 0 10-5 -18
3x10-5 -73
10-4 -83
Table 6 Peptide 5 Effect on All-stimulated Cont raction
[Peptide 5] (M) % Change from All Response
3x10-8 0
10-7 +4
3x10-7 +24
10-6 +36
3x10-6 +40
10-5 -6
3x10-5 -28
10-4 -38
Table 7 Peptide 8 Effect on All-stimulated Contraction [Peptide 8] (M) % Change from All Response
10-6 0
3x10-6 -8
10-5 -9
3x10-5 -11 10-4 -21
Table 8
Peptide 10 Effect on All-stimulated Contraction
[Peptide 10] (M) % Change from All Response 3x10-6 0
10-5 +5
3x10-5 +34
10-4 +85
Table 9 Peptide 11 Effect on All-stimulated Contraction Peptide 11] (M) % Change from All Response
10-6 0
3x10-6 -3
10-5 -5
3x10-5 -7
10-4 -33
These experiments demonstrate that peptides of this invention can either increase or decrease contraction of arterial smooth muscle caused by angiotensin II.
In a third type of experiment, the ability of saralasin to block All contraction and peptide-stimulated contraction was tested. The muscle strips were bathed in All, 10 -9 M. Once a steady contraction was established, saralasin, 10 -8 M, was added to the bath. The addition of saralasin decreased All contraction. Upon restabilization of the contractile response, Peptide 5, 10-6 M was added to the bath. No change in contractile response was seen.
This experiment demonstrates that contraction caused by peptides of this invention can be inhibited by a competitive antagonist for angiotensin Il-stimulated contraction.
Example 4 :
Effects of Peptides on Venous Smooth Muscle
Relaxation
Peptide 2 of this invention was tested in vitro, using an assay of isolated dog venous strips mounted in a muscle bath. Isolated segments of the dog femoral vein (n=2) were mounted in organ baths for measurement of isometric force generation. The conditions were as described in Example 3. The vascular segments were made to contract in response to prostaglandin F2a (8.5 × 10-7M). After the contraction reached a plateau, angiotensin II (3 × 10-7M) was added to the organ bath and the venous segments relaxed to approximately 50% of the response to prostaglandin F2a. This relaxation response to angiotensin II was inhibited 100% by saralasin (10-6M) and by Peptide 2 (10-5M).
These experiments demonstrate that peptides of this invention can inhibit angiotensin 11-caused relaxation of venous smooth muscles.
Example 5 :
Effects of Peptides on Blood Pressure
Peptide 2 of this invention was tested in vivo, using a rat hypertension model. Adult male rats (n=4) were anesthetized with sodium pentobarbital (-50 mg/kg) and the left jugular vein was cannulated for infusion of drugs. Bolus injections of angiotensin II were delivered via the same venous catheter. The right carotid artery was cannulated for measurement of blood pressure. Injection of angiotensin II (1 ng) caused an increase in mean arterial blood pressure (-50 mm Hg). Constant infusion of saralasin (1 mg/min) inhibited the pressor activity of angiotensin II. Infusion of Peptide 2 (0.1 to 0.5 mg/min) did not alter baseline blood pressure and did not influence pressor responses to angiotensin II.
INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(51) International Patent Classification** : (11) International Publication Number: WO 87/ 05
C07K 7/00, 7/06, 7/08 A3 A61K 37/02 (43) International Publication Date: 27 August 1987 (27.08.
(21 ) International Application Number: PCT/US87/00367 (74) Agent: PARKER, David, L ; Arnold, White & Dur P.O. Box 4433, Houston, TX 77210 (US). , (22) International Filing Date: 19 February 1987 (19.02.87)
*1
(81) Designated States: AT (European patent), AU, BE (
(31) Priority Application Numbers: 830,795 ropean patent), CH (European patent), DE (Eu 002,298 pean patent), FR (European patent), GB (Europ patent), IT (European patent), JP, LU (European 1 (32) Priority Dates: 19 February 1986 (.9.02.86) tent), NL (European patent), SE (European paten 20 January 1987 (20.01.87) I (33) Priority Country: US Published
With international search report.
Before the expiration of the time limit for amending
(71) Applicant: TRITON BIOSCIENCES INC. [US/US]; claims and to be republished in the event of the receipt
1501 Harbor Bay Parkway, Alameda, CA 94501 (US). amendments.
(72) Inventors: BRUNCK, Terence, . : 3353 Washington
Court, Alameda, CA 94501 (US). COLBY, Clarence. (88) Date of publication of the international search report: B., Jr. ; 138 Sea Bridge, Alameda, CA 94501 (US). 19 November 1987 (19.11.
(54) Title: PEPTIDES AFFECTING BLOOD PRESSURE REGULATION
O
II (I)
X-NH-AA1 -GLY-AA2-AA3-AA4-AA5-AA6-AA7-C-Y
(IH)
O σi) o
II II
CH3C-
(57) Abstract
Peptides of formula (I), wherein NH is the α amino group of amino acid AA1 ; (II) is the carbonyl group of ami acid AA"; X is H-, (III), GLU-VAL-, or VAL-TYR-HIS-GLU-VAL: Y is -OH, -NH . or -PRO; AA' is LYS, GLU, GL pGLU, βALA, PRO, PRO-OH, PIC, or AIB; AA- is VAL, LEU, ILE, MET, or AIB; AA^ is ASP, TYR, GLU, HIS, PHE; AA4 is VAL, MET, ILE, LEU, or AIB; AA* is TYR, HIS, or GLU: AA6 is ALA, PRO, SER, βALA, PRO-OH, AIB: and A A7 is VAL, LEU, ILE, or AIB; and salts thereof are claimed as effective blood pressure regulators. Further pr v ided, are antibodies to these peptides, as well as diagnostic and therapeutic methods for blood pressure regulation.
FOR THE PURPOSES OF INFORMAπON ONLY
Codesused to identify States party to the PCTon the frontpages of pamphlets publishingϊnternational applications under the PCT.
AT Austria FR France ML Mali
AU Australia GA Gabon MR Mauritania
BB Barbados GB United Kingdom MW Malawi
BE Belgium HU Hungary NINetherlands
BG Bulgaria II Italy NO Norway
BJ Benin JP Japan RO Romania
BR Brazil KP Democratic People's Republic SD Sudan
CF Central Africa Republic ofKorea SE Sweden
CG Congo KR Republic ofKorea SN Senegal
CH Switzerland LI Liechtenstein SU Soviet Union
CM Cameroon LK Sri Lanka TD Chad
DE Germany, Federal Republic of LU Luxembourg TG Togo
DK Denmark MC Monaco US United States of America π Finland MG Madagascar
Claims
1. A peptide of the formula:
wherein:
VAL-TYR-HIS-GLU-VAL-;
Y is -OH, -NH2, or -PRO;
AA1 is LYS, GLU, GLN, pGLU, BALA, PRO, PRO-OH, PIC, or AIB;
AA2 is VAL, LEU, ILE, MET, or AIB;
AA3 is ASP, TYR, GLU, HIS, or PHE:
AA4 is VAL, MET, ILE, LEU, or AIB;
AA5 is TYR, HIS, or GLU;
AA6 is ALA, PRO, SER, BALA, PRO-OH, or AIB; and
AA7 is VAL, LEU, ILE, or AIB;
or a salt thereof.
2. The peptide of Claim 1 wherein:
AA1 is LYS, GLU, or GLN; AA2 is VAL or LEU; AA3 is ASP, TYR, or GLU; AA4 is VAL, MET, ILE, or LEU; AA5 is TYR, HIS, or GLU;
AA6 is ALA, PRO, or SER; and
AA7 is VAL or LEU .
3. The peptide of Claim 2 wherein:
AA1 is LYS; AA2 is VAL;
AA3 is TYR; AA4 is ILE;
AA5 is HIS; AA6 is ALA; and AA 7 is LEU.
4. The peptide of Claim 1 which is selected from one of the following:
NH2-LYS-GLY-VAL-ASP-VAL-TYR-ALA-VAL-COOH;
NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH;
NH2-LYS-GLY-VAL-ASP-MET-HIS-ALA-LEU-COOH;
GLU-VAL-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH; VAL-TYR-HIS-GLU-VAL-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH;
NH2-GLU-GLY-LEU-GLU-LEU-GLU-ALA-LEU-COOH;
NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-CONH2;
NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-PRO;
5. The peptide of Claim 4 which is:
NH2-LYS-GLY-VAL-TYR-ILE-HIS-ALA-LEU-COOH .
6. An antibody raised to a peptide of Claim 1, 2, 3, 4, or 5.
7. The antibody of Claim 6 which is a monoclonal antibody.
8. A method of regulating or aiding in the regulation of the blood pressure of a mammalian host which comprises administering to the host an effective amount of a peptide of Claim 1, 2, 3, 4, or 5.
9. The method of Claim 8 wherein the mammalian host is human.
10. The method of Claim 9 wherein the human host is diagnosed as having hypertension or hypotension.
11. A method of regulating or aiding in the regulation of the blood pressure of a mammalian host which comprises administering to the host an effective amount of an antibody of Claim 6.
12. The method of Claim 11 wherein the antibody is a monoclonal antibody.
13. The method of Claim 12 wherein the mammalian host is human.
14. The method of Claim 13 wherein the human host is diagnosed as having hypertension or hypotension.
15. A pharmaceutical composition which comprises a peptide of Claim 1, 2, 3, 4, or 5 and a pharmaceutically-acceptable carrier.
16. The composition of Claim 15 which is administered at a daily dosage from about 0.1 to about 50 milligrams of peptide per kilogram of body weight of a mammalian host.
17. The composition of Claim 16 wherein the mammalian host is a human.
18. The composition of Claim 17 wherein the dosage is from about 0.1 to about 10 milligrams.
19. The method of Claim 8 wherein the dosage is administered intranasally, parenterally, orally, or topically.
20. A pharmaceutical composition which comprises an antibody of Claim 6 and a pharmaceutically-acceptable carrier.
21. The composition of Claim 20 wherein the antibody is a monoclonal antibody.
22. The composition of Claim 21 which is administered at a daily dosage from about 0.1 to about 50 milligrams of antibody per kilogram of body weight of a mammalian host.
23. The composition of Claim 22 wherein the mammalian host is a human.
24. The composition of Claim 23 wherein the dosage is from about 0.1 to about 10 milligrams.
25. The method of Claim 11 wherein the antibody is a human x human monoclonal.
26. A pharmaceutical combination which comprises a peptide of Claims 1, 2, 3, 4, or 5 and a chemotherapeutic agent.
27. The combination of Claim 26 wherein the agent is a beta adrenergic blocker compound.
28. The combination of Claim 26 wherein the agent is a diuretic compound.
29. A pharmaceutical combination which comprises an antibody of Claim 6 and a chemotherapeutic agent.
30. The combination of Claim 29 wherein the antibody is a monoclonal antibody.
31. The combination of Claim 30 wherein the agent is a beta adrenergic blocker compound.
32. The combination of Claim 31 wherein the agent is a diuretic compound.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US83079586A | 1986-02-19 | 1986-02-19 | |
US830,795 | 1986-02-19 | ||
US229887A | 1987-01-20 | 1987-01-20 | |
US002,298 | 1987-01-20 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO1987005029A1 true WO1987005029A1 (en) | 1987-08-27 |
WO1987005029A2 WO1987005029A2 (en) | 1987-08-27 |
WO1987005029A3 WO1987005029A3 (en) | 1987-11-19 |
Family
ID=26670201
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1987/000367 WO1987005029A2 (en) | 1986-02-19 | 1987-02-19 | Peptides affecting blood pressure regulation |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0294390A1 (en) |
AU (1) | AU595597B2 (en) |
WO (1) | WO1987005029A2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989011657A1 (en) * | 1988-05-25 | 1989-11-30 | Cambridge Research Biochemicals Limited | Assay for oncogene expression |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5077195A (en) * | 1985-03-01 | 1991-12-31 | Board Of Reagents, The University Of Texas System | Polypeptides complementary to peptides or proteins having an amino acid sequence or nucleotide coding sequence at least partially known and methods of design therefor |
-
1987
- 1987-02-19 WO PCT/US1987/000367 patent/WO1987005029A2/en not_active Application Discontinuation
- 1987-02-19 EP EP19870901916 patent/EP0294390A1/en not_active Withdrawn
- 1987-02-19 AU AU71258/87A patent/AU595597B2/en not_active Ceased
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