Bradykinin analogues, their synthesis and their use in therapy

Abstract: Bradykinin analogues containing a non-peptide bond are described; their formula: some of the analogs exhibit antagonist activity rendering them useful therapeutically, e.g., for the relief of vasodilation-­ induced swelling and/or itching; others exhibit agonist activity and are useful e.g. for the treatment of hypertension.

Description: This invention relates to bradykinin analogues. Martinez et al. observed in studies on the mode of action of gastrin that changes in the peptide backbone (replacement of a carbonyl group with a methylene group) could provide analogues to gastrin with significantly altered biological activity, as agonists or antagonists (Martinez et al., J. Med. Chem., 28 : 1874-1879 (1985)). Yet peptide bond replacement does not systematically provide for antagonist activity as Sasaki et al. found in a study of somatostatin analogues (Sasaki et al., J. Med. Chem., 30 :1162-1166 (1987)), and Martinez et al. discovered in a study on the effects of changes to peptide bond of the C-terminal heptapeptide of cholecystokinin, a peptide which has much the same biological activity and the same final four amino acids as gastrin (Rodriquez et al., J. Med. Chem., 30 :1366-1373 (1987)). Abbreviations (uncommon): pGlu = Nle = Pal = 3-pyridyl-alanine Nal = naphthylalanine HYP = hydroxyproline In general, the invention features a bradykinin analog of the formula (1): Q⁰-A¹-A²-A³-Gly-A⁵-A⁶- A⁷-A⁸-A⁹-Z¹⁰ wherein Q⁰ = L-Arg, D-Arg, L-homo-Arg, H, D-homo-Arg, L-Lys, D-Lys, lower (1-5 carbon atoms) Σ-N-alkyl-L-Lys, lower Σ-N-alkyl-D-Lys, lower Σ-N-alkyl-L-His, lower Σ-N-alkyl-D-His, lower Σ-N-alkyl-D-His, lower Σ-N-alkyl-L-Pal, lower Σ-N-alkyl-D-Pal, lower acyl, or lower α-N-alkyl; A¹ and A⁹ (independently) = Arg, homo-Arg, Lys, lower Σ-N-alkyl-Lys, His, or Pal; A² = Pro, hydroxy-Pro, or N-Me-Ala; A³ = Pro, hydroxy-Pro, or N-Me-Ala; A⁵ and A⁸ (independently) = Phe, thienylalanine, His, Trp, Nal, Pal, or P-X-Phe (X = F, Cl, Br, OH, or CH₃); A⁶ = Ser, Thr, Ala, Leu, Ile, Val, or Tyr; A⁷ = Pro, hydroxy-Pro, N-Me-Ala, D-Phe, or D-thienylalanine; Z¹⁰ = OH, COOH, NH₂, or lower alkylamide; provided that, for each of the residues A⁵, A⁶, A⁷, and A⁸, independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon (i.e., at least one of the subject peptide CONH bonds must be replaced by a non-peptide CH₂NH bond); or a pharmaceutically acceptable salt thereof. Preferred bradykinin analogs of the invention have non-peptide bonds joining residue A⁸ to A⁹, or A⁵ to A⁶. (In the examples given below, non-peptide bonds are symbolized by "ψ[CH₂NH]".) Preferred analogs are: Arg-Pro-Pro-Gly-Phe-ψ[CH₂NH]-Ser-Pro-Phe-Arg; and Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-ψ[CH₂NH]-Arg-OH. The present bradykinin analogues, which act as bradykinin antagonists are useful for preventing increased vascular permeability that occurs during non-infectious inflammation, as analgesics, for preventing edema due to pulmonary brain trauma, in preventing shock due to hemorrhage, or for any condition characterized by vasodilation-induced swelling and/or itching. The invention also provides a method for synthesizing a bradykinin peptide analogue of formula (1), said method comprising providing an amino terminal amino acid attached to a resin support and coupling successive amino acids to said amino terminal amino acid to produce said peptide analog, removing said peptide analog from said resin, and purifying said peptide analog. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. We now describe the structure, synthesis, and use of the preferred embodiments of the invention. The bradykinin analogs of the invention all have a non-peptide bond in at least one of the indicated positions. By non-peptide bond is meant that the carbon atom participating in the bond between two residues is reduced from a carbonyl carbon to a methylene carbon. The peptide bond reduction method which yields this non-peptide bond is described in Coy et al., U.S. Patent No. 4,803,261, the disclosure of which is hereby incorporated by reference. These bradykinin analogues can be provided in the form of pharmaceutically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids, e.g., acetic lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluenesulfonic, or pamoic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as the hydrohalic acids, e.g., hydrochloric acid, sulfuric acid, or phosphoric acid. The synthesis of Boc-Arg(tosyl)Pro-Pro-Gly-Phe-ψ[CH₂NH]-Ser(benzyl)-Pro-Phe-Arg(nitro)-O-resin is carried out as follows. Boc-Arg(nitro)-polystyrene resin (Vega Biochemicals) (0.86 gm, 0.5 mmole) is placed in the reaction vessel of an Advanced ChemTech ACT 200 peptide synthesizer programmed to perform the following reaction cycle; (a) methylene chloride wash; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each); (c) methylene chloride wash; (d) 10% triethylamine in dimethyformamide; (e) methylene chloride wash. The neutralized resin is stirred with Boc-Phe and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 h and the resulting amino acid resin is then cycled through steps (a) to (e) in the above wash program. The Boc group is then removed by TFA treatment and the following amino acid derivatives (1.5 mmole) are then coupled successively by the same procedure: Boc-Phe, Boc-Pro, Boc-Ser(benzyl). Boc-Phe aldehyde (1.5 mmoles), prepared by the method of Fehrentz and Castro, Synthesis, p. 676 (1983) is dissolved in 5 ml of dry DMF and added to the resin TFA salt suspension followed by the addition of 400 mg (8 mmoles) of sodium cyanoborohydride (Coy et al., U.S. Patent No. 4,803,261). After stirring for 1 h, the resin mixture is found to be negative to ninhydrin reaction (1 min) indicating complete derivitization of the free amino group. After removal of the Boc group, Boc-Gly-p-nitrophenyl ester (3.0 mmoles) is then coupled in dimethylformamide. The following amino acids (1.5 mmole) are then coupled successively by the carbodiimide procedure: Boc-Pro, Boc-Pro, Boc-Arg(nitro). After drying, the peptide resin weighs 1.23 g. The synthesis of H-Arg-Pro-Pro-Gly-Phe-ψ [CH₂NH]-Ser-Pro-Phe-Arg-OH follows. The resin, as described above, (1.23 g, 0.5 mmole) is mixed with anisole (5 ml) and anhydrous hydrogenfluoride (35 ml) at 0°C and stirred for 45 min. Excess fluoride is evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide is dissolved in a minimum volume of 2M acetic acid and eluted on a column (2.5 x 95 cm) of Sephadex G-25. Fractions containing a major component by uv absorption (254 nm) and thin layer chromatography are then pooled, evaporated to a small volume and applied to a column (1.5 x 50 cm) of Vydac octadecylsilane (10-15µM). The peptide is eluted with a linear gradient of 10-35% acentonitrile in 0.1% trifluoroacetic acid in water. Fractions are examined by thin layer chromatography and analytical high performance liquid chromatography and pooled to give maximum purity. Repeated lyophilization of the solution from water gives 104 mg of the product as a white,fluffy powder. The product is found to be homogeneous by hplc and tlc. Amino acid analysis of an acid hydrolysate confirms the composition of the octapeptide. The presence of the Pheψ[CH₂NH] peptide bond is also demonstrated by fast atom bombardment mass spectrometry. Other peptides can be prepared in similar yields in an analogous fashion by appropriately modifying the above procedure. A linear bradykinin analog can be tested for effectiveness as either an agonist or antagonist of bradykinin using the following method. Bradykinin-Stimulated Cyclic GMP Formation Described below is a method that employs cultured neuroblastoma cells, which have high-affinity receptor sites for bradykinin and respond to bradykinin with an increase in intracellular levels of cyclic GMP. Murine neuroblastoma cells (clone NlE-115) were cultured in Dulbecco's modified Eagle's medium without antibiotics and supplemented with 10% (vol/vol) fetal calf serum. Cells were grown in wells of a 24 multiwell plastic tray in an atmosphere of 10% CO₂-90% air at 37°C. Cell Culture Described below is an assay method that employs cultured neuroblastoma cells, which have high-affinity receptor sites for bradykinin and respond to bradykinin with an increase in intracellular levels of cyclic GMP. Mouse neuroblastoma cells (Clone NlE-115) were obtained from Dr. Elliott Richelson (Mayo Clinic and Foundation, Rochester, MN, Snider and Richelson, 1984, J. Neurochem. 43 :1749-1754) and cultured in Dulbecco's modified Eagle's medium (Sigma Chem., St. Louis, MO) without antibiotics and supplemented with 10% fetal calf serum (Sigma Chem.). The stock cultures were grown in an atmosphere of 10% CO₂/90% humidified air. Bradykinin-Stimulated cyclic GMP Formation The assay of cyclic GMP formation was modified from the procedure described by Snider and Richelson (1984). The cells were initially grown for five days in 24-well culture plates. On the day of assay the culture medium was then removed, and the cells were washed twice with a phosphate-buffered saline solution (PBS) containing 110 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 2.0 mM Na₂HPO⁴, 25 mM glucose, and 70 mM sucrose (pH 7.4, 335-340 mmol). The cells were labeled with [³H]guanine (4 µCi/ml, 0.6 µM) in PBS for 45 min at 37°C. After the [³H]guanine incubation the PBS/[³H]guanine solution was removed and each well was washed an additional time with PBS. PBS (240 µl) and antagonists (30 µl) were added, and the cells were pre-incubated for 10 min (37°C). To assay for agonist activity, either Bradykinin or the test compound (30 µl) (Bachem Inc., Torrence, CA) was added, and the incubation was continued for an addtional 30 sec. (37 C). To test for antagonist activity, labeled cells were preincubated for 5 min. with the test antagonist before the addition of Bradykinin (indicated as "+" in results of antagonist assays, below). The Bradykinin or test compound stimulation was terminated by the addition of 30 µl of 50% (w/v) trichloroacetic acid, and the contents of each well were transferred to (0.8 x 8.0 cm) AG50 W-X2 ion exchange column which had been equilibrated with 0.1 N HCl. The columns were then sequentially washed with 4.4 ml of 0.1 N HCl (eluate discarded), 1.0 ml H₂O (eluate discarded), and 1.5 ml H₂O which was collected into 12 x 75 mm plastic culture tubes. To this last eluate, equal volumes (30 µl) of 2.67 M ZnSO₄ and 2.67 M Na₂CO₃ were added to further precipate any residual [³H]GDP or [³H]GTP. After the precipitate had been removed by centrifugation, the supernatant was transferred to 7 ml of Scint A (Packard) and the radioactivity determined by liquid scintillation spectrometry. [³H] Bradykinin Binding Experiments Membranes from the cells were prepared by homogenizing the NlE-115 cells in ice-cold buffer with a Brinkman Polytron (seting 6, 15 sec) and centrifuging twice (39,000 g, 10 min.) with an intermediate resuspension in fresh buffer. Aliquots of the membrane preparation were incubated with 1.0 nM (competition experiments) or 0.3 - 5 nM (saturation experiments) [³H]bradykinin (88.7 Ci/mmol, New England Nuclear) for 90 min (25°C) in a final volume of 1.0 ml. The binding assay was terminated by rapid filtration through Whatman GF/B filters that had previously soaked in 0.1% aqueous polyethyleneimine. Each assay tube and filter were immediately washed three times with 5 ml aliquots of ice-cold buffer, and the bound radioactivity trapped on the filters were counted by liquid scintillation spectrometry. As shown in Table I, incubation of N l E-115 cells with bradykinin produced a dose-dependent stimulation of cyclic GMP formation. Maximum stimulation of cyclic GMP formation (to ∼ 6 pmoles) occurred at approximately 100 nM of bradykinin. Results of Assays of Test Peptides A true antagonist molecule binds to a specific receptor (i.e. the Bradykinin receptor) with the same affinity as an agonist but lacks biological activity. An example of a true antagonist is BIM-31006, as shown below. (Where bradykinin analog numbers are also referred to in Table I, amino acid formulas are also given in the table.) Assay for antagonist activity of BIM-31006 Condition cGMP response (dpm) basal 327 bradykinin (3 nM) 2459 +BIM-31006 (1 nM) 2004 +BIM-31006 (10 nM) 1201 +BIM-31006 (100 nM) 418 Assay for agonist activity of BIM-31006 Condition cGMP response (dpm) basal 437 BIM-31006 (0.3 nM) 424 BIM-31006 (1.0 nM) 335 BIM-31006 (3.0 nM) 448 BIM-31006 (10 nM) 266 BIM-31006 (30 nM) 333 BIM-31006 (100 nM) 427 bradykinin (3 nM) 3019 The results show that BIM-31006 does have antagonist activity in the concentration range of 1 - 100 nM, and does not have agonist activity in the same concentration range. A molecule may appear to be an antagonist, but not be a true antagonist, if it acts as a superagonist by causing over-stimulation of its target receptor and thus desensitizes the receptor to stimulation. This is a mechanism by which a molecule that is a superagonist may appear to be an antagonist, An example of this type of pseudo-antagonist is D-Arg⁰,HYP³ψ[CH₂NH]Phe⁸BK, or BIM-31012. BIM-31012 is an example of an antagonist which acts by desensitization of the receptor. The antagonist and agonist activities was shown in the following experiment: Assay for antagonist activity of BIM-31012 Condition cGMP response (dpm) basal 279 bradykinin (3 nM) 1929 +BIM-31012 (0.1 nM) 1818 +BIM-31012 (1 nM) 516 +BIM-31012 (10 nM) 401 +BIM-31012 (100 nM) 296 Therefore, in concentrations between 1 - 100 nM, BIM-31012 is an antagonist. Assay for agonist activity of BIM-31012 Condition cGMP response (dpm) basal 279 BIM-31012 (1 nM) 1379 BIM-31012 (10 nM) 4608 BIM-31012 (100 nM) 5938 These results show that, in the same concentration range, BIM-31012 is also an agonist. To determine if the apparent antagonistic activity of BIM-31012, shown above, was a result of agonist-induced receptor desensitization, the cells were preincubated with bradykinin for 5 min. at 37°C before assaying for Bradykinin-stimulated cGMP formation. Condition cGMP response (dpm) basal 385 bradykinin (3 nM) 2085 +bradykinin (1 nM) 1655 +bradykinin (10 nM) 1591 +bradykinin (100 nM) 496 +bradykinin (1000 nM) 348 The results demonstrate that pre-incubation of cells with bradykinin results in apparent antagonistic activity for bradykinin itself, which is normally an agonist. This suggests that the initial exposure to bradykinin over-stimulated the receptor and thus desensitized it, which also explains the apparent antagonism of BIM-31012. Other bradykinin analogs may be mixed antagonist/agonists; i.e., may be true antagonists over a given concentration range, but agonists at a different concentration range. BIM-31005, Phe⁸ψ[CH₂NH]BK may also be a mixed antagonist/agonist, as shown by the following experiments. Assay for agonist activity of BIM-31005 Condition cyclic GMP response (dpm) basal 265 BIM-31005 (0.1 nM) 246 BIM-31005 (0.3 nM) 260 BIM-31005 (1.0 nM) 306 BIM-31005 (3.0 nM) 227 BIM-31005 (10 nM) 1481 BIM-31005 (30 nM) 3705 Bradykinin (3.0 nM) 3821 The results show tht BIM-31005 is less potent than Bradykinin as an agonist. BIM-31005, however, binds with higher affinity to the receptor than Bradykinin. The Ki for BIM-31005 is 0.38 nM, and for bradykinin is 1.0 nM. Table I and the assay below show antagonist activity of BIM-31005. Condition cyclic GMP response (dpm) basal 327 bradykinin (3.0 nm) 2594 +BIM-31005 (1.0 nM) 2459 +BIM-31005 (10 nM) 1390 +BIM-31005 (100 nM) 538 These results suggest that the antagonistic activity of BIM-31005 begins to occur at 1.0 nM; at this concentration, no agonist activity is observed. This suggests a mxture of agonist/antagonist activities. BIM-31005 may also act antagonistically by desensitizing the receptor at those concentrations at which it appears to act as an antagonist. Another example of a mixed antagonist/agonist is D-Arg⁰,HYP³ψ[CH₂NH]Phe⁵BK, or BIM-31011. The results for the BIM-31011 analog are shown by the following experiment: Assay for antagonist activity of BIM-31011 Condition cGMP response (dpm) %inhibition basal 308 -- Bradykinin 6968 -- +BIM-31011(1 nm) 4757 33 (10 nm) 4619 35 (100 nm) 2886 61 (1000 nm) 320 99 Assay for agonist activity of BIM-31011 Condition cGMP response (dpm) basal 308 BIM-31011(0.1 nm) 238 (1.0 nm) 325 (10 nm) 314 This compound shows antagonist activity in the range of 1 - 1000 nM and no agonist activity up to 10 nM. The sharp drop in antagonist activity seen above between 100 nM and 1000 nM was investigated further. Assay for antagonist activity of BIM-31011 Assay for agonist activity of BIM-31011 Condition cGMP response (dpm) basal 179 BIM-31011 (100 nM) 146 BIM-31011 (300 nM) 281 BIM-31011 (1000 nM) 1987 bradykinin (3 nM) 1336 The results show that antagonist activity of BIM-31011 starts to decrease at a concentration of 300 nM and completely disappears at 1000 nM, whereas agonist activity is markedly stimulated at 1000 nM. BIM-31011 is therefore a mixed agonist/antagonist. Inhibition of bradykinin-stimulated cyclic GMP formation (to ∼ 2.4 pmoles) by the bradykinin analogues, Phe⁵ψ[CH₂NH]-bradykinin (BIM-31002) and Phe⁸ψ(CH₂NH)-bradykinin (BIM-31005) occurred at approximately 3 - 10 nM, as shown in Table I. The Phe⁵- and Phe⁸-ψ[CH₂NH]-bradykinin antagonists are as potent as other analogues, such as those developed by Schachter et al., 1987, Br. J. Pharmac. 92 :851-855; Rifo et al., 1987, Eur. J. Pharmac. 142 :305-312; Vavrek and Stewart, 1985, Peptides 6 :161-164; and Steranka et al., 1987, Eur. J. Pharmac, 136 :261-262). In addition, Drapeau et al., 1988, Eur. J. Pharm. 155:193, reported that [Phe⁸ψ[CH₂NH]Arg⁹]BK is a very potent agonist. Bradykinin Antagonists - Receptor Binding and Biological Activity Compound Structure Receptor Binding Ki (nM) cyclic GMP Antagonism Ki (nM) BIM-31002 Phe⁵ψ[CH₂NH]BK 1.0 ± 0.36 8.4 ± 3.8 BIM-31003 Gly⁴ψ[CH₂NH]BK 2215 ± 298 >10000 BIM-31004 Pro⁷ψ[CH₂NH]BK 11 ± 1.3 97 ± 56 BIM-31005 Phe⁸ψ[CH₂NH]BK 0.38 ±0.07 9.1 ± 2.1 BIM-31006 [D-Arg⁰,Hyp³, D-Phe⁷]BK 0.24 ±0.05 14 ± 1.7 BIM-31007 Pro³ψ[C₂NH]BK 48 ± 8.6 >10000 BIM-31008 Lys 1,9 ,Phe⁸ ψ[CH₂NH]BK 2294 >10000 BIM-31009 Phe 5,8 ψ[CH₂NH]BK >10000 >10000 The described bradykinin analogues may be administered to a mammal, particularly a human, in one of the traditional modes (e.g., orally, parenterally, transdermally, or transmucosally), in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels and liposomes. The bradykinin analogues which act as antagonists are suitable for the treatment of medical disorders characterized by unwanted vascular dilation or permeability, e.g., common cold symptoms, vascular permeability-induced pain, edema caused by brain trauma, hemorrhage-induced shock, poison ivy, or other non-infectious swelling or itching, and are also suitable for the treatment of pain and arthritis. Bradykinin agonists can be used to treat hypertension, or to increase blood flow e.g., caused by poor circulation in the extremities. The bradykinin analogues can be administered (most preferably, topically) to a human patient in a dosage of 0.5µg/kg/ day to 5 mg/kg/day, preferably 10-1000 µg/kg/day.

Claims: A bradykinin analog of the formula: Q⁰-A¹-A²-A³-Gly-A⁵-A⁶- A⁷-A⁸-A⁹-Z¹⁰ wherein Q⁰ = L-Arg, D-Arg, L-homo-Arg, H, D-homo-Arg, L-Lys, D-Lys, lower (1-5 carbon atoms) Σ-N-alkyl-L-Lys, lower Σ-N-alkyl-D-Lys, lower Σ-N-alkyl-L-His, lower Σ-N-alkyl-D-His, lower Σ-N-alkyl-L-Pal, lower Σ-N-alkyl-D-Pal, lower acyl, or lower α-N-alkyl; A¹ and A⁹ (independently) = Arg, homo-Arg, Lys, lower Σ-N-alkyl-Lys, His, or Pal; A² = Pro, hydroxy-Pro, or N-Me-Ala; A³ = Pro, hydroxy-Pro, or N-Me-Ala; A⁵ and A⁸ (independently) = Phe, thienylalanine, His Trp, Nal, Pal, or P-X-Phe, (X = F, Cl, Br, OH, or CH₃); A⁶ = Ser, Thr, Ala, Leu, Ile, Val, or Tyr; A⁷ = Pro, hydroxy-Pro, N-Me-Ala, D-Phe, or D-thienylalanine; Z¹⁰ = OH, COOH, NH₂, or lower alkylamide; provided that, for each of the residues A⁵, A⁶, A⁷, and A⁸, independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon; or a pharmaceutically acceptable salt thereof. The bradykinin analogue or its salt of Claim 1, wherein, for each of the residues A⁵ or A⁸, independently, the carbon atom participating in the amide bond between that residue and the nitrogen atom of the alpha amino group of the adjacent amino acid residue may be a carbonyl carbon or may be reduced to a methylene carbon, provided that at least one such carbon atom must be reduced to a methylene carbon. The bradykinin analog of Claim 1, having the amino acid formula: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-ψ[CH₂NH]-Arg-OH, or a pharmaceutically acceptable salt thereof. The bradykinin analog of Claim 1, having the formula: Arg-Pro-Pro-Gly-Phe-ψ[CH₂NH]-Ser-Pro-Phe-Arg-OH, or a pharmaceutically acceptable salt thereof. A method for synthesizing a bradykinin peptide analog as defined in any of Claims 1 to 4, said method comprising providing an amino terminal amino acid attached to a resin support and coupling successive amino acids to said amino terminal amino acid to produce said peptide analog, removing said peptide analog from said resin, and purifying said peptide analog. A bradykinin analogue or its salt according to any of Claims 1 to 4 for use in therapy. A bradykinin analogue or its salt according to any of Claims 1 to 4 for use in the relief of conditions characterised by unwanted vascular dilation or permeability, or for the treatment of pain or arthritis. A bradykinin analogue or its salt according to any of Claims 1 to 4 for use in the treatment of hypertension, poor circulation, or in any condition where increased blood flow is required. Use of a bradykinin analogue or its salt according to any of Claims 1 to 4 for the manufacture of a medicament for use in the relief of conditions characterised by unwanted vascular dilation or permeability, or for the treatment of pain or arthritis. Use of a bradykinin analogue or its salt according to any of Claims 1 to 4 for the manufacture of a medicament for use in the treatment of hypertension, poor circulation, or in any condition where increased blood flow is required.

Inventors: COY DAVID H, MOREAU JACQUES-PIERRE, TAYLOR JOHN E, COY, DAVID H., MOREAU, JACQUES-PIERRE, TAYLOR, JOHN E.
Applicants: UNIV TULANE, THE ADMINISTRATORS OF THE TULANE UNIVERSITY EDUCATIONAL FUND
Application year: 1989
Main classification: C07K7
IPC codes: C07K14, C07K7, A61K38