USE OF CERTAIN COMPOUNDS IN LIVESTOCK FOOD AS GROWTH PROMOTANTS

Abstract: A method is described in which the feed utilization by livestock is improved by the incorporation in the feed or drinking water of the said livestock of certain compounds, with resultant increase in body weight, decrease in body fat and increase in body protein for the same food intake.

Description: TITLE OF THE INVENTION THE USE OF CERTAIN COMPOUNDS IN LIVESTOCK FOOD AS GROWTH PROMOTANTS FOR BETTER FEED UTILIZATION AND IMPROVED CARCASS COMPOSITION STATEMENT OF THE INVENTION This invention relates to a method of improving feed utilization and carcass composition by livestock which comprises the incorporation in the food or drinking water furnished to said livestock of from 0.01 to 100 parts per million of at least one compound selected from the group described in Table A. More specifically, it comprises a method of improving feed utilizatior and carcass composition in which the compounds of structures I, IV and IX are used. More specifically also, it relates to animal feed compositions for administration to livestock which comprises from 0.01 to 100 parts per million of a compound listed in Table A especially compounds I, IV and I. e invention also relates to ruminants RS slow release rumen boluses releasing doses of from 0.1 to 100 mg per head per day of at least one compound selected from the group listed in Table A especially compounds I, IV and IX. The invention also relates to all livestock as systemic implants that provide doses of 0.001 to 10 mg per head per day. TABLE A I. Zinterol EMI2.1 II. Z 1170 EMI2.2 III. QH25B EMI2.3 IV. L-644,969 EMI2.4 TABLE A CONT'D V. Bitolterol EMI3.1 VI. Reproterol EMI3.2 VII. Pirbuterol EMI3.3 VIII. AA497 EMI3.4 IX. Formoterol EMI3.5 X. Colterol EMI3.6 Compound X, Colterol, while similar to the other compounds in its growth promoting activity, differs in that very little activity is found when administered orally. It is however substantially active when administered parenterally. It will be appreciated by those skilled in the art that the above compounds have one or more asymmetric centers and may exist as separable optical isomers or as equal mixtures of optical isomers which would be optically inactive racemic mixtures. The foregoing structures are presented in planar form, however, all possible optical isomers contained therein are intended to be included within the ambit of this invention. One skilled in the art will also realize that separated optical isomers may often have different activities, one from the other, and such differences are also to be considered within the instant invention. As an example of this, it is noted that compound IV has two centers of asymmetry, the first (referred to as "a") and third (referred to as "1") carbon atoms to the right of the pyridine ring carrying a hydroxy and a methyl substituent respectively. This will give rise to four possible optical isomers when the hydroxy and methyl are individually either above (ss) or below (a) the plane of the molecule. In particular the isomer wherein both the hydroxy and methyl are ss (which is more exactly referred to as the a-R,lR compound) has been found to be significantly more active than the most active of the other isomers. BACKGROUND OF THE INVENTION There is a need in animal husbandry for methods of improving the utilization of food. It is important not only that the growth of the animals be promoted but that the utilization of the food be directed in a different way, namely, to produce less fat and more protein. Some compounds have been described which enhance the utilization of food and change the carcass composition in some species of food livestock. Baker et al. have described (Fed. Proc. 42 No. 4 p. 816, No. 3 p. 668) that a compound called clenbuterol functions in this way. However, while this may be the best such agent published in the scientific literature, clenbuterol is not as active as other compounds in poultry, sheep or swine, especially compared to the compounds used in this invention. DESCRIPTION OF THE INVENTION We have found that the compounds listed in Table A are highly active in promoting the utilization of food by livestock intended for human consumption. They provide more growth and better utilization of the feed producing more protein and less fat. As can be seen in Table A the compounds involved differ widely in chemical structure. They also differ drastically from clenbuterol. While some of the active compounds listed in Table A are reported to have B-adrenergic activity, many compounds reported to be ss-adrenergic agonists have very little or no growth promoting activity. Thus, accurate prediction of growth promoting activity on the basis of chemical structure or ss-adrenergic activity is not possible. Of a special use in this invention are the three compounds listed in Table A as I, IV, V and IX. All of these share high activity in all species under consideration. However, all of these compounds of Table A enhance the food utilization in some species by promoting growth and providing more protein and less fat for the same food intake. In particular, the high level of activity of Compound V, bitolterol, combined with its very low level of toxicity, makes it a highly preferred growth promotant agent. EXAMPLE 1 IN VITRO TESTING Adipose tissue collection and preparation. Fat was collected from freshly killed animals as follows: epididymal and perirenal fat from 150 to 200 g male rats; perirenal fat from 20 to 25 kg wether lambs, and perirenal and omental fat from 35 to 50 kg gilts. Immediately after collection, fat was placed in a siliconized beaker (all glassware was siliconized) containing Krebs-Ringer bicarbonate solution (KRB; pH 7.4) at 370C. Fat was transferred to a clean beaker and finely chopped with scissors. Chopped fat was then used as such (whole adipose preparation) or treated with collagenase (isolated adipocytes). Isolation of adipocytes. Chopped fat was placed in a shaker containing 70 ml cell dispersion medium (CDM; 5 mM glucose, .1% collagenase, 4% bovine serum albumin (BSA), in KRB) and incubated at 370C for 1 hour with gentle shaking. Fat was then filtered to remove clumps and washed 4 times with washing medium (WM; 2.5 mM glucose, 1% BSA, in KRB) and 1 time with final suspension medium (FSM; 2.5 mM glucose, 2% essentially fatty acid free BSA, in KRB). Isolated adipocytes were then resuspended in the required amount of FSM (1 ml/flask, 3 flasks/treatment). Determination of lipolytic activity in adipose tissue or adipocytes. Whole adipose tissue from the rat (.3 ml) or isolated adipocytes from sheep or swine (1 ml) were placed in plastic scintillation vials containing FSM (1.7 or 1 ml, respectively) and test compounds, and incubated at 370C for 2 hours. Compounds were tested at concentrations of .01, .05, .25 and 1.25 pM. Isoproterenol, at the same concentrations, was used as a positive control. After incubation, media was removed and assayed for glycerol by a fluorometric modification of the method of Wieland (1974. Glycerol Method. pp. 1404. In: Bergmeyer (Ed.) Methods of Enzymatic Analysis. Verlag Chemie Weinham, Academic Press, New York). Determination of anti-lipogenic activity in hepatocytes. Hepatocytes were isolated from chicken liver using the procedure for adipocytes and incubated for 2 hours at 370C in the presence of 14C acetate and test compounds. Compounds were tested at concentrations of .01, .05, .25 and 1.25 WM. Isoproterenol, at the same concentrations, was used as a positive control. After incubation, 8 ml Doles reagent (iponropanol:hexane:HSO4r 40:10:1) was added and incubation vials were shaken at room temperature for 30 minutes. Vial contents were poured into 50 ml screw-cap tubes. Vials were then rinsed with 6 ml hexane which was poured into the appropriate 50 ml tube. Tubes were shaken by hand, 10 ml H20 was added, and tubes were shaken again. After phase separation, 2 ml of the upper (hexane) phase was removed to scintillation vials. Hexane was evaporated by a gentle air flow and the remaining lipids were resuspended in 10 ml Scinti Verse II. 14C was counted in a Packard liquid scintillation 14 counter. Data are reported as CPM of C acetate incorporated into total lipids. Results of in vitro testing. All nine compounds listed in Table A had significant antilipogenic activity. The three compounds identified as I, IV and IX appear to have five times the antilipogenic activity of isoproterenol and greater than 125 times the antilipogenic activity of clenbuterol in chicken hepatocytes. The nine compounds were tested for lipolytic activity in isolated swine (Table C) and ovine (Table D) adipocytes. All nine compounds significantly stimulated lipolysis in isolated swine or ovine adipocytes. Eight of the nine compounds were significantly more active than clenbuterol in stimulating lipolysis in isolated adipocytes. The lack of activity of clenbuterol in swine adipocytes should especially be noted. The compounds were also tested (Table E) for lipolytic activity using chopped adipose tissue from rats and all nine compounds were found to be active. While rats are not a target species for the product, they are a good model for testing in vivo. EXAMPLE 2 IN VIVO TESTING Eighty intact, male Charles River CD rats, 75-90 g, were housed and maintained on rat chow and water ad libitum. After four days acclimation ten rats per group were randomized by weight to treatment and pen. Compounds to be tested were premixed and added to ground rat chow using doses equivalent to 20 ppm of clenbuterol on a molecular weight basis. It had previously been determined 10 ppm of clenbuterol was the minimum dose needed to give maximum improvements in gain and feed conversion. Therefore, the results of any compound tested should cause responses similar to clenbuterol if the compound has half or more the activity of clenbuterol. The results are summarized in Table F and demonstrate that all nine compounds listed in Table A are capable of improving rate of gain and feed conversion and shifting carcass composition of growing animals from fat toward protein. TABLE B Comparative Activity of Inhibitors of Lipogenesis by Isolated Chicken Hepatocytes (Measured by 14C acetate incorporation in fatty acids and expressed as percent change compared to control) COMPOUND LEVEL 0.01 0.05 0.25 1.25 Test 1 Isoproterenol -29.1 -48.6 -70.3 -74.4 Clenbuterol -4.2 -16.4 -24.6 -24.7 1 -45.2 -61.3 -66.0 -63.3 IV -33.7 -54.3 -63.2 -65.1 IX -50.5 -67.2 -72.3 -72.6 III -9.5 -35.5 -54.4 -62.4 V 0 -34.0 -64.6 -68.8 VI 0 0 -8.6 -31.2 II 0 0 -7.9 -18.4 Test 2 Isoproterenol -21 -15 -49 -61 Clenbuterol -8 -18 -16 1 IX -30 -56 -63 -64 VIII 26 -9 -34 -63 VI 3 -12 -25 -28 TABLE C Comparative Activity of Stimulators of Lipolysis by Isolated Swine Adipocytes (Measured by glycerol production and expressed as percent increase compared to control) COMPOUND LEVEL WM 0.01 0.05 0.25 1.25 Test 1 Isoproterenol 40 140 660 333 Clenbuterol 27 -20 -27 -27 I 87 100 53 60 IV 260 333 80 53 IX 207 267 187 180 VIII 13 147 180 60 V 7 47 147 80 III 20 33 53 40 VI 20 -7 0 140 II 27 -7 -40 -27 Test 2 Isoproterenol 175 196 820 800 Clenbuterol 0 -20 4 17 IX 340 600 844 896 VIII 61 59 460 580 VI 8 21 3 160 TABLE D Comparative Activity of Stimulators of Lipolysis by Isolated Ovine Adipocytes (Measured by glycerol production and expressed as percent increase compared to control) COMPOUND LEVEL gM 0.01 0.05 0.25 1.25 Test 1 Isoproterenol 670 1510 1820 2080 Clenbuterol inactive 240 325 305 I 730 960 1775 2400 IV 820 1160 1540 1645 IX 675 695 1525 2400 III 465 845 1775 2255 VIII 135 445 565 720 V inactive 435 1170 2140 VI " 130 465 2010 II <RTI ID=12.2> " inactive 325 180 Test 2 Isoproterenol 600 1614 1800 2157 Clenbuterol 186 857 1171 1914 IX 2014 2100 2043 2271 VIII 71 3143 1800 2143 VI -14 129 700 1800 TABLE E Comparative Activity of Stimulators of Lipolysis by Rat Adipose Tissue (Measured by glycerol production and expressed'as percent increase compared to control) COMPOUND LEVEL pM 0.01 0.05 0.25 1.25 Test 1 Isoproterenol 45 173 290 272 Clenbuterol 12 26 21 15 I 90 129 281 334 IV 114 219 334 318 IX 42 91 258 322 V 17 33 111 288 III 194 203 334 288 VI 63 86 86 361 II 51 15 35 91 VIII 19 26 21 15 Test 2 Isoproterenol 49 154 216 237 Clenbuterol 10 10 16 43 IX 16 70 145 222 VI 13 16 3 46 VIII -7 4 7 40 TABLE F In vivo Testing in Rats Feed feed Carcass Composition CcToound Dose Weight Gain Intake Conversion % of Change over Control Test 1 n g/day g/day g/dawy Fat Protein Control 0 191.8 7.3 18.6 2.55 - - Clenbuterol 10 198.6 7.8 19.2 2.48 -11.3 11.2 VII 15.3 (a) 200.9 8.0 19.8 2.48 -8.7 3.6 V 38.5 (a) 192.2 7.3 18.7 2.58 -4.3 3.1 III 24.3 (a) 201.7 8.0 19.4 2.41 -7.0 6.1 I 26.4 (a) 199.1 7.8 19.2 2.46 -10.4 7.3 IX 21.8 (a) 199.4 7.9 19.1 2.44 -10.4 10.6 VIII 15.7 (a) 202.6 8.1 19.5 2.41 -6.1 6.4 Test 2 Control 0 202.3 7.5 19.3 2.63 - Clenbuterol 10 216.9 8.2 19.9 2.46 -11.9 4.3 II 19.0 (a) 215.9 8.4 21.1 2.51 -9.3 3.8 VI 27.0 (a) 213.4 8.3 21.2 2.57 2.5 0.5 IV 23.0 (a) 214.7 8.4 20.7 2.48 -16.1 5.0 (a) Dose based on molecular weight equivalent of 20 ppm clenbuterol. EXAMPLE 3 Compound V (bitolterol) was compared to clenbuterol for effects on growth, feed conversion and carcass composition of broilers raised in floor pens for 7 weeks. Bitolterol at 0.5, 1.0 and 2.0 ppm in the diet improved rate of gain, feed conversion and carcass protein in a dose-related manner. Bitolterol at the highest'dose improved rate of gain 2.6% (P .01), feed conversion 3.8% (P .05) and carcass protein 4.9% (P .10). The latter responses were better than those observed with clenbuterol at 0.5 ppm. Bitolterol at these doses did not reduce carcass fat. Clenbuterol at 0.5 ppm in the diet was administered for 7 weeks to one group and only during the last 4 weeks to another group. Administration of clenbuterol for 7 weeks was slightly more effective for improving feed conversion and carcass protein than clenbuterol for 4 weeks. Clenbuterol given for 7 weeks improved feed conversion 3.5% (P .05) and appeared to improve (not significant) rate of gain 0.4%, carcass weight 0.7% and carcass protein 4.0% and to decrease (not significant) carcass fat 7.1%. DOSAGE UTILIZATION The method of this invention is intended to increase the rate of body weight gain and/or feed efficiency and to decrease the carcass fat and increase the carcass protein of livestock such as cattle, sheep, poultry, pigs, etc. The compounds described herein in Table A can be administered in doses of from 0.01 to 100 ppm in feed or drinking water. In addition, they may be administered to ruminants as slow release rumen boluses at doses of 0.1 to 100 mg per head per day or to all livestock as parenteral formulations such as systemic implants providing doses of 0.001 to 10 mg per head per day. The boluses and subcutaneous implants typically are designed to provide for continuous administration of the active growth promoting compound for about 100 days.

Claims: WHAT IS CLAIMED IS 1. Application of a compound selected from compounds I through X inclusive as set forth in the above specification, as livestock growth promotants for better feed utilization and improved carcass composition. 2. A method of improving the growth of livestock which comprises incorporating into the feed or drinking water furnished to said livestock from 0.01 to 100 parts per million of the active compound ; into a ruminal bolus which provides from 0.1 to 100 mg per head per day of the active compound ; wherein the active compound consists of at least one compound selected from Compounds I through IX inclusive as set forth in the above specification ; or into a parenteral formulation which provides from 0.001 to 10 mg per head per day of the active compound wherein the active compound consists of at least one compound selected from Compounds I through X inclusive as set forth in the above specification. 3. The method of claim 2 in which the compounds are selected from the group consisting of compounds I, IV, V and IX. 4. The method of claim 3 in which the compound is Compound I. 5. The method of claim 3 in which the compound is Compound IV. 6. The method of Claim 2 in which the compound is Compound V. 7. The method of Claim 3 in which the compound is Compound IX. 8. The method of Claim 2 in which the compound is Compound II. 9. The method of Claim 2 in which the compound is Compound III. 10. The method of Claim 2 in which the compound is Compound VI. 11. The method of Claim 2 in which the compound is Compound VII. 12. The method of Claim 2 in which the compound is Compound VIII. 13. The method of Claim 2 in which the compound is Compound X. 14. Animal feed compositions for administration to livestock which comprises 0.01 to 100 parts per million of at least one compound selected from the group of compounds described in the above specification as Compounds I through IX inclusive. 15. Animal feed compositions of Claim 14 which comprises at least one compound selected from the group consisting of Compounds I, IV, V and IX. 16. A slow release ruminal bolus capable of continuously administering from 0.1 to 100 mg per head per day of at least one compound selected from the group comprising those described in the above specification as Compounds I through IX inclusive. 17. The bolus of Claim 16 containing a compound selected from the group consisting of Compounds I, IV, V and IX. 18. A parenteral formulation capable of continuously administering from 0.001 to 10 mg per head per day of at least one compound selected from the group comprising those described in the above specification as Compounds I through X inclusive. 19. The parenteral formulation of Claim 18 which is a subcutaneous implant. 20. The subcutaneous implant of Claim 19 containing a compound selected from the group consisting of Compounds I, IV, V and IX.

Inventors: MUIE, LARRY A.
Applicants: MERCK & CO. INC.
Application year: 1985
Main classification: A23K1
IPC codes: C07C219, A61K31, C07C311, A61P3, C07C213, A23K1, A61D7, C07D473, C07C215, C07C231, C07D213, C07C233, C07C301, C07C67, C07C217, C07D261