Abstract:
Satiety inducing composition comprises Neuropeptide Y analogues which are effective for inducing satiety and for treating diseases and conditions associated with a lacking of, or having lowered, satiety.

Description:
PRIOR PATENT APPLICATIONS 
     This is a divisional application of U.S. patent application Ser. No. 08/422,839, filed Apr. 17, 1995, by the same inventors, and now U.S. Pat. No. 6,075,009. 
    
    
     This invention was made with Government support under Grant No. RO1 CA47217-06 from the National Cancer Institute. The Government may have certain rights in this invention. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention concerns novel Neuropeptide Y analogs, pharmaceutical formulations containing the same and methods of lowering blood pressure and inducing satiety in a subject employing the same. 
     2. Description of the Background 
     Neuropeptide Y is a 36 amino acid member of the pancreated polypeptide family. It is highly concentrated in both the central and peripheral mammalian nervous system. It apparently serves important functions in the control of blood pressure, satiety, and other responses. The amino acid sequence of Neuropeptide Y is known. See, e.g., K. Tatemoto,  Proc. Natl. Acad. Sci. USA  79, 5485-5489 (1982). 
     Neuropeptide Y analogs are disclosed in U.S. Pat. No. 5,026,685 to Boublik et al. The compounds are indicated to be active in lowering the blood pressure of mammalian subjects. The compounds are indicated to be 18-20 amino acids in length, and include amino acid residues 19 to 36 of human NPY (see, e.g., Column 2, lines 25-35 therein). Additional Neuropeptide Y analogs are disclosed in U.S. Pat. No. 5,328,899 to Boublik et al. 
     A recent study proposed two modified fragments of NPY, called PYX 1  and PYX 2 , to be specific NPY receptor antagonists. Both compounds are analogs of the 27-36 residue C-terminal fragment. Both compounds have a D-amino acid substitution at Thr 32 . [3-(2,6-dichlorobenzyl)] is substituted at Tyr 27  on PYX 1 , and is substituted at both Tyr 27  and Tyr 36  in PYX 2 . See K. Tatemoto,  Ann. NY Acad. Sci.  611, 1-6 (1990); K. Tatemoto et al.,  Proc. Natl. Acad. Sci. USA  89, 1174-1178 (1992). 
     SUMMARY OF THE INVENTION 
     The present invention is based on the a new group of Neuropeptide Y analogs. Active Neuropeptide Y (NPY) analogs. The compounds preferably include amino acids 28-35 of human NPY, and include a D-Thr amino acid substitution at the Thr 32  position. 
     The present invention relates to a method of lowering blood pressure in a subject in need of such treatment by administering to the subject the active compounds given above in an amount effective to lower blood pressure. 
     The present invention relates to a method of inducing satiety in a subject in need of such treatment by administering to the subject the active compounds given above in an amount effective to induce satiety. 
     The present invention relates to a pharmaceutical formulation comprising the active compounds given above in combination with a pharmaceutically acceptable carrier. The active compound maybe included in the formulation in a pharmaceutically effective amount i.e., an amount effective to lower blood pressure; an amount effective to induce satiety. 
     The invention also applies the active compounds given above to the preparation of a medicament for lowering blood pressure in a mammalian subject, or for inducing satiety in a mammalian subject. 
     The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth hereinbelow. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. Where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented, unless otherwise indicated. 
     The methods of the present invention are concerned primarily with the treatment of human subjects, but may also be employed for the treatment of other mammalian subjects, such as dogs, cats and cows, for veterinary purposes. Subjects may be those subjects in need of such treatment for any reason for which lowering of blood pressure would be of therapeutic benefit, including but not limited to those subjects afflicted with hypertension or high blood pressure. 
     The active compounds of the present invention are, in general, NPY analogs that are NPY fragments. The compounds preferably include amino acids 28-35 of NPY, and include a D-Thr amino acid substitution at the Thr 32  position. The compounds are preferably at least 8, 9 or 10 amino acids in length, and are preferably not more than 15, 16, 17 or 18 amino acids in-length. 
     Specific examples of active compounds of the present invention are as follows: 
      I: D-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2   (SEQ ID NO:1); 
     
       
         I Ac : Ac-D-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2   (SEQ ID NO:2); 
       
     
     
       
         IIB: D-Asp-Pro-Lys-Ser-Pro-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2   (SEQ ID NO:3); 
       
     
     
       
         IIB Ac : Ac-D-Asp-Pro-Lys-Ser-Pro-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2   (SEQ ID NO:4); 
       
     
     
       
         III: D-Phe(NO 2 )-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(NO 2 )-NH 2   (SEQ ID NO:5); 
       
     
     
       
         III Ac : Ac-D-Phe(NO 2 )-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(NO 2 )-NH 2   (SEQ ID NO:6); 
       
     
     
       
         IV: D-Phe(pF)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pF)-NH 2   (SEQ ID NO:7); 
       
     
     
       
         IV Ac : Ac-D-Phe(pF)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pF)-NH 2   (SEQ ID NO:8); 
       
     
     
       
         V: D-Phe(pCl)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pCl)-NH 2   (SEQ ID NO:9); 
       
     
     
       
         V Ac : Ac-D-Phe(pCl)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pCl)-NH 2   (SEQ ID NO:10); 
       
     
     
       
         VI: D-Phg-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phg-NH 2   (SEQ ID NO:11); 
       
     
     and 
     
       
         VI Ac : D-Phg-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phg-NH 2   (SEQ ID NO:12). 
       
     
     The terms used herein have their standard meanings. The term “Ac” means acetyl; the term “Phe(NO 2 )” refers to phenylalanine substituted by —NO 2  on the phenylalanine ring, preferably at the para position, the term “Phe(pCl)” refers to phenylalanine substituted by —Cl on the phenylalanine ring at the para position, and the term “Phe(pF)” refers to phenylalanine substituted by —F on the phenylalanine ring at the para position. 
     The compounds of the invention may be prepared in accordance with known techniques, such as solid phase-chemistry. See, e.g., U.S. Pat. No. 4,415,558 to Vale et al. 
     The active compounds disclosed herein may be prepared in the form of their pharmaceutically acceptable salts. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b) salts formed from elemental anions such as chlorine, bromine, and iodine, and (c) salts derived from bases, such as ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium, and salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine. 
     Pharmaceutical compositions for use in the present method of lowering blood pressure include those suitable for inhalation, oral, rectal, topical, (including buccal, sublingual, dermal and intraocular) parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular) and transdermal administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art. The formulations may conveniently be presented in unit dosage form and may be prepared.by any of the methods well known in the art. 
     The dose of active compound administered will vary according to the route of administration, the manner of formulation, the condition of the subject, and the dose at which adverse pharmacological effects occur. One skilled in the art will take such factors into account when determining dosage. In general, in one preferred embodiment, the dosage will be from 400 or 500 up to about 1000, 2000, or 4000 nM/kg subject body weight. 
     In the manufacture of a medicament according to the invention (a “formulation”), active agents or the physiologically acceptable salts thereof (the “active compound”) are typically admixed with, among other things, an acceptable carrier. The carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.5% to 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention (e.g., the formulation may contain one or more additional anti-tubercular agents as noted above), which formulations may be prepared by any of the well known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory therapeutic ingredients. 
     Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder. Formulations for oral administration may optionally include enteric coatings known in the art to prevent degradation of the formulation in the stomach and provide release of the drug in the small intestine. 
     Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia. 
     Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The formulations may be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. 
     Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture. 
     Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. 
     Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g.,  Pharmaceutical Research  3, 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. 
    
    
     The following examples are provided to more fully illustrate the present invention and should not be construed as restrictive thereof. In the following examples, temperatures are given in degrees centigrade unless otherwise indicated. 
     EXAMPLES 
     Example 1 
     Synthesis of NPY27-36 (D-Tyr 27,36 , D-Thr 32 ) 
     NPY27-36 (D-Tyr 27,36 , D-Thr 32 ) was synthesized using Fmoc-BOP chemistry in accordance with known techniques. The automated BIOSEARCH model 9600 peptide synthesizer was used to produce the peptide. The amino acid derivatives were Arg (Mtr), Ile, Leu, Thr (tBu), tYR (tBu), Asn (Tmob) and Gln (Tmob). Tyr 27 , Thr 32  and Tyr 36  were D isomers. To remove the side chain protecting group and separate the pepetide from the resin (PAL resin), TFA/thioanisol/ethanedithiol/anisol was used in molar excess (10 ml/g). After deprotection of the final product, the peptide was purified over a 2″×25 cm VYDAC 15-20 micron C 18  column using a Waters model 600-E HPLC with 0.1% TFA and 60% acetonitrile in 0.1% TFA applying a linear gradient over 45 minutes. The primary peak detected by UV absorbance at 215 nm was collected and repeatedly lyophilized. The purity of the end product, having the sequence D-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2  (SEQ ID NO:1) (M.W. 1336.57), was ≧95%. 
     Example 2 
     Synthesis of Additional NPY Analogs 
     The following compounds were synthesized in essentially the same manner as given in Example 1 above to yield the indicated compounds: 
     
       
         I Ac : Ac-D-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2   (SEQ ID NO:2) (M.W. 1378.62); 
       
     
     
       
         IIB: D-Asp-Pro-Lys-Ser-Pro-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2   (SEQ ID NO:3) (M.W. 1864.14); 
       
     
     
       
         IIB Ac : Ac-D-Asp-Pro-Lys-Ser-Pro-Tyr-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Tyr-NH 2   (SEQ ID NO:4) (M.W. 1906.19); 
       
     
     
       
         III: D-Phe(NO 2 )-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(NO 2 )-NH 2   (SEQ ID NO:5) (M.W. 1396.58); 
       
     
     
       
         III Ac : Ac-D-Phe(NO 2 )-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(NO 2 )-NH 2   (SEQ ID NO:6(M.W. 1438.63); 
       
     
     
       
         IV: D-Phe(pF)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pF)-NH 2   (SEQ ID NO:7) (M.W. 1342.57); 
       
     
      IV Ac : Ac-D-Phe(pF)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pF)-NH 2   (SEQ ID NO:8) (M.W. 1342.57); 
     
       
         V: D-Phe(pCl)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pCl)-NH 2   (SEQ ID NO:9) (M.W. 1359.02); 
       
     
     
       
         V Ac : Ac-D-Phe(pCl)-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phe(pCl)-NH 2   (SEQ ID NO:10) (M.W. 1401.07); 
       
     
     
       
         VI: D-Phg-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phg-NH 2   (SEQ ID NO:11) (M.W. 1306.54); 
       
     
     and 
     
       
         VI Ac : D-Phg-Ile-Asn-Leu-Ile-D-Thr-Arg-Gln-Arg-D-Phg-NH 2   (SEQ ID NO:12) (M.W. 1348.59). 
       
     
     Example 3 
     In Vivo Activity of NPY27-36 (D-Tyr 27,36 , D-Thr 32 ) 
     Male Sprague-Dawley rats obtained from Charles River Laboratories, Wilmington, Mass., and weighing 276-300 g, were group housed at 22° C. and kept on 12 hour light/dark cycle. Food and water were provided ad lib. 
     Animals were anesthetized with sodium pentobarbital (65 mg/kg) and under sterile conditions a one-inch incision was made in the fold of the left leg to expose the femoral blood vessels. Two lengths of PE-50 tubing filled with 10% heparin were inserted into the left femoral artery and left femoral vein for measurement of blood pressure and for drug delivery, respectively. The tubings were tunneled under the skin and exposed through the mid-scapular region. Animals were allowed at least 24 hours recovery prior to experiments. 
     A force transducer (Spectramed Model P23XL, Grass Instruments, Quincy, Mass., USA), and tachograph (Model 7P44D, Grass Instruments) was used for recording of systolic and diastolic pressures and heart rate from the femoral artery catheter. Patency of catheters was verified by flushing with either saline or 10% heparin. 
     A dose of either 30, 100, 500, 800 or 1000 nM/kg (nanomoles per liter per kilogram) of NPY27-36 (D-Tyr 27,36 , D-Thr 32 ) (prepared as described in Example 1 above), or saline was delivered to the animal via the catheter in the left femoral vein. Changes in systolic and diastolic pressures and heart rater were recorded from the catheter in the left femoral artery over a 10 minute time interval. 
     NPY27-36 (D-Tyr 27,36 , D-Thr 32 ) produced a dose-dependent decrease in mean arterial pressure and an increase in heart rate when given intraveneously to the normotensive Sprague-Dawsley rats. Doses of 30 and 100 nM/kg failed to produce changes that were significantly different from saline treatment. When given in doses of 500, 800, and 1000 nM/Kg, however, significantly different decreases in mean arterial pressure were seen ad is as compared to saline-treated controls. Further, doses of 500 and 1000 nM/kg produced significantly different increases in heart rate as compared to saline controls. 
     The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.