Combinations Having Synergistic Growth Hormone Releasing Activity and Methods for Use Thereof

Immature female Sprague-Dawley rats were obtained from the Charles River Laboratories (Wilmington, Mass.). After arrival they were housed at 25.degree. C. with a 14:10 hr light:dark cycle. Water and Purina rat chow were available ad libitum. Pups were kept with their mothers until 21 days of age.

Normal saline with 0.1% gelatin was the vehicle for subcutaneous (s.c.) and intravenous (i.v.) injections of the peptides. In some experiments in which the peptides were very insoluble, DMSO was used to dissolve the compounds, with dilutions then being made to the specified concentration with normal saline with 0.1% gelatin (compounds for which DMSO was needed to effect solution are so noted in the Tables). The unanesthetized rats, weighing 55-65 grams, were injected i.v. with the quantity of growth hormone releasing compounds indicated in Tables 1-4. Injection was made as a 0.2 ml solution via the tail vein. All animals were sacrificed by guillotine 10 min after the final test injection unless specified otherwise. Trunk blood for the determination of blood GH levels was collected following decapitation. The blood was allowed to clot, centrifuged and the serum separated from the clot. Serum was kept frozen until the day of sampling for radioimmunoassay (RIA) determination of growth hormone levels according to the following procedure, as developed by the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases (NIADDK).

Reagents are generally added to the RIA analysis tubes at a single sitting, at refrigerator temperature (about 4.degree. C.) in the following sequence:

(a) buffer,

(b) "cold" (i.e., non-radioactive) standard or unknown serum sample to be analyzed,

(c) radio-iodinated growth hormone antigen, and

(d) growth hormone antiserum.

Reagent addition is generally carried out so that there is achieved a final RIA tube dilution of about 1:30,000 (antiserum to total liquid volume: vol:vol).

The mixed reagents are then typically incubated at room temperature (about 25.degree. C.) for about 24 hours prior to addition of a second antibody (e.g., goat or rabbit anti-monkey gamma globulin serum) which binds to and causes precipitation of the complexed growth hormone antiserum. Precipitated contents of the RIA tubes are then analyzed for the number of counts in a specified period of time in a gamma scintillation counter. A standard curve is prepared by plotting number of radioactive counts v. growth hormone (GH) level. GH levels of unknowns are then determined by reference to the standard curve.

Serum GH was measured by RIA with reagents provided by the National Hormone and Pituitary Program.

Serum levels in Tables 1-4 are recorded in ng/ml in terms of the rat GH standard of 0.61 International Units/mg (IU/mg). Data is recorded as the mean .+-./- standard error of the mean (SEM). Statistical analysis was performed with Student's t-test.

The results with combinations of polypeptide compounds from Groups 2 and 3 are shown in Tables 1-3. The results with combinations of polypeptide compounds from Groups 1 and 3 are shown in Table 4. The results with combinations of polypeptide compounds from Groups 1 and 2 are shown in Table 4. In each of the four Tables, the results shown are the average of studies with six rats. In Vivo GH Release (ng/ml) of Group 2 and Structurally Similar Compounds Plus Group 3 Compounds in Rats Column A GH Released GH Released Compound in GH Released Compound in Group 2 and by Compound by Column A + by Column A + Structurally Dose Control in Column A #8801* #8801* #9218* #9218* Number Similar Compounds (.mu.g) GH ng/ml ng/ml (Group 3) (Groups 2 & 3) (Group 3) (Groups 2 & 3) 8310 DAla--His--DTrp--Ala--Trp-- 100 3 .+-. 0.5 16 .+-. 2 313 .+-. 100(b) DPhe--Lys--NH2** 8937 (beta)Ala--His--DTrp--Ala-- 10 10 .+-. 1.4 32 .+-. 4 49 .+-. 7(a) 87 .+-. 35(a) Trp--DPhe--Lys--NH2** 8588 Ala--His--DPal--Ala--Trp-- 100 8 .+-. 8 19 .+-. 5 109 .+-. 36(a) 115 .+-. 27(b) DPhe--Lys--NH2** 8323 Ala--His--DTrp--Ala--Pal-- 100 3 .+-. 0.5 18 .+-. 1 421 .+-. 101(b) DPhe--Lys--NH2** 9308 Ala--Cys--DTrp--Ala--Trp-- 10 14 .+-. 2.0 10 .+-. 1 178 .+-. 78 98 .+-. 11 DPhe--Lys--Cys--NH2** 9887 Ala--His--DTrp--Ala--Trp-- 10 13 .+-. 1.0 11 .+-. 1 161 .+-. 63 27 .+-. 4 DPgl--Lys--NH2** 9090 Ala--His--DTrp--DLys--Trp-- 10 7 .+-. 2.0 14 .+-. 3 68 .+-. 4(a) 78 .+-. 13(a) DPhe--Lys--NH2** 10265 Ala--His--DArg--Ala--Trp-- 10 13 .+-. 2(b) 14 .+-. 2 229 .+-. 26 364 .+-. 165 DPhe--Lys--NH2** 10351 DDopa--Ala--His--DTrp--Ala-- 10 12 .+-. 2 13 .+-. 2 135 .+-. 27 288 .+-. 62 Trp--DPhe--Lys--NH2*** 8758 Cys--Ala--His--DTrp--Ala-- 10 8.0 .+-. 2.0 37 .+-. 8 398 .+-. 196(a) 1070 .+-. 211(a) Trp--DPhe--Lys--Cys--NH2*** 8758 Cys-- Ala--His--DTrp--Ala-- 10 14.0 .+-. 2.0 26 .+-. 9 178 .+-. 78(a) 588 .+-. 152(a) Trp--DPhe--Lys--Cys--NH2** 8114 Ala--His--DTrp--Ala--Trp-- 10 3.0 .+-. 0.5 93 .+-. 30 648 .+-. 176(b) 1143 .+-. 270(b) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 17.0 .+-. 3.0 129 .+-. 46 DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 10.0 .+-. 1.4 52 .+-. 8 49 .+-. 7(a) 791 .+-. 50(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 7.0 .+-. 2.0 54 .+-. 7 68 .+-. 4(a) 746 .+-. 43(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 12.0 .+-. 1.0 72 .+-. 9 37 .+-. 12(a) 172 .+-. 6(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 14.0 .+-. 2.0 119 .+-. 28 178 .+-. 78(a) 2697 .+-. 427(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 7.3 .+-. 2.0 55 .+-. 13 43 .+-. 8(a) 1155 .+-. 288(a) 32 .+-. 10(a) 1633 .+-. 679(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 2.2 .+-. 0.6 152 .+-. 40 92 .+-. 31(b) 1490 .+-. 245(b) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 18.0 .+-. 11.0 149 .+-. 62(a) 2035 .+-. 472(a) 131 .+-. 67(a) 1223 .+-. 384(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 14.0 .+-. 1.0 132 .+-. 36 54 .+-. 16(a) 1265 .+-. 284(a) 38 .+-. 9(a) 566 .+-. 25(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 9.0 .+-. 4.0 105 .+-. 37 205 .+-. 29(a) 1472 .+-. 307(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 7.0 .+-. 0.6 117 .+-. 36 309 .+-. 88(b) 1386 .+-. 224(b) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 2.0 .+-. 0.2 115 .+-. 50 448 .+-. 175(b) 1176 .+-. 347(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 6.0 .+-. 0.7 169 .+-. 60 60 .+-. 16(a) 1390 .+-. 419(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 9.0 .+-. 1.0 210 .+-. 42 49 .+-. 9(a) 1589 .+-. 313(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 18.0 .+-. 3.0 255 .+-. 73 367 .+-. 79(a) 1921 .+-. 284(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 10.0 .+-. 1.0 315 .+-. 65 177 .+-. 53(a) 1169 .+-. 269(a) DPhe--Lys--NH2 8114 Ala--His--DTrp--Ala--Trp-- 10 30.0 .+-. 1.0 114 .+-. 20 226 .+-. 70(a) 2006 .+-. 333(a) DPhe--Lys--NH2 8866 Tyr--Ala--Gly--Thr--Ala--His-- 10 8.0 .+-. 2.0 177 .+-. 38 398 .+-. 196(a) 1698 .+-. 281(a) DTrp--Ala--Trp--DPhe--Lys--NH2 8866 Tyr--Ala--Gly--Thr--Ala--His -- 30 8.0 .+-. 2.0 144 .+-. 38 398 .+-. 196(a) 1130 .+-. 172(a) DTrp--Ala--Trp--DPhe--Lys--NH2 8866 Tyr--Ala--Gly--Thr--Ala--His-- 10 14.0 .+-. 1.0 292 .+-. 42 38 .+-. 9(a) 1567 .+-. 350(a) DTrp--Ala--Trp--DPhe--Lys--NH2 9106 Tyr--DAla--Phe--Gly--Ala--Hi s-- 100 14.0 .+-. 1.0 116 .+-. 42 38 .+-. 9(a) 1227 .+-. 348(a) DTrp--Ala--Trp--DPhe--Lys--NH2 9216 Tyr--DAla--Gly--Phe--Ala--His-- 30 12.0 .+-. 1.0 46 .+-. 12 37 .+-. 12(a) 506 .+-. 174(a) DTrp--Ala--Trp--DPhe--Lys--NH2 9216 Tyr--DAla--Gly--Phe--Ala--Hi s-- 100 14.0 .+-. 1.0 196 .+-. 43 38 .+-. 9(a) 1365 .+-. 276(a) DTrp--Ala--Trp--DPhe--Lys--NH2 8938 His--DTrp--Ala--Trp-- 10 3.0 .+-. 0.5 70 .+-. 29 648 .+-. 176(b) 2375 .+-. 312(b) DPhe--Lys--NH2 9036 His--DTrp--Ala--Trp--DPhe--Lys-- 10 10.0 .+-. 1.0 52 .+-. 7 177 .+-. 53(a) 873 .+-. 201(a) Ala--Tyr--NH2 9020 Abu--His--DTrp--Ala--Trp--DPhe-- 10 18.0 .+-. 11.0 85 .+-. 24 131 .+-. 67(a) 1770 .+-. 444(a) Lys--NH.sub.2 9868 DOPA--Ala--His--DTrp--Ala--Trp-- 10 13.0 .+-. 1.0 123 .+-. 30 161 .+-. 63(a) 1791 .+-. 284(a) DPhe--Lys--NH.sub.2 10276 Leu--His--DTrp--Ala--Trp--DPhe-- 10 13.0 .+-. 2(b) 190 .+-. 58 229 .+-. 26 1748 .+-. 233 Lys--NH.sub.2 10276 Leu--His--DTrp--Ala--Trp--DPhe-- 10 13.0 .+-. 1.0 74 .+-. 25 166 .+-. 44 1602 .+-. 403 Lys--NH.sub.2 10321 Val--His--DTrp--Ala--Trp--DPhe-- 10 13.0 .+-. 2(b) 137 .+-. 28 229 .+-. 26 3093 .+-. 382 Lys--NH.sub.2 10337 Phe--Ala--His--DTrp--Ala--Trp-- 10 12.0 .+-. 2.0 68 .+-. 11 135 .+-. 27 1049 .+-. 203 DPhe-- Lys--NH.sub.2 10337 Phe--Ala--His--DTrp--Ala--Trp-- 10 14.0 .+-. 2.0 132 .+-. 45 2855 .+-. 642 DPhe--Lys--NH.sub.2 10391 Ala--His--DTrp--Ala--Trp--DPhe-- 10 12.0 .+-. 2.0 36 .+-. 6 135 .+-. 27 1830 .+-. 583 Lys--Gly--Tyr--NH.sub.2 (c) 10391 Ala--His--DTrp--Ala--Trp--DPhe-- 10 14.0 .+-. 2.0 155 .+-. 59 135 .+-. 27 1544 .+-. 554 Lys--Gly--Tyr--NH.sub.2 (c) 10321 Val--His--DTrp--Ala--Trp--DPhe-- 1 5.0 .+-. 0.4 11 .+-. 2 223 .+-. 95 464 .+-. 76 Lys--NH.sub.2 10321 Val--His--DTrp--Ala--Trp--DPhe-- 3 5.0 .+-. 0.4 129 .+-. 13 223 .+-. 95 849 .+-. 134 Lys--NH.sub.2 10321 Val--His--DTrp--Ala--Trp--DPhe-- 10 5.0 .+-. 0.4 68 .+-. 29 223 .+-. 95 1259 .+-. 286 Lys--NH.sub.2 10855 Ala--His--(formyl)DTrp--Ala-- 10 6.0 .+-. 1.0 19 .+-. 6 144 .+-. 46 558 .+-. 154 Trp--DPhe--Lys--NH.sub.2 10814 Dopa--His--DTrp--Ala--Trp--DPhe-- 3 6.0 .+-. 1.0 67 .+-. 17 144 .+-. 46 1427 .+-. 418 Lys--NH.sub.2 10814 Dopa--His--DTrp--Ala--Trp--DPhe-- 10 6.0 .+-. 1.0 76 .+-. 20 144 .+-. 46 3631 .+-. 459 Lys--NH.sub.2 10957 Trp--His--DTrp--Ala--Trp--DPhe-- 10 7.0 .+-. 1.0 127 .+-. 23 107 .+-. 21 1819 .+-. 243 Lys--NH.sub.2 (d) 10957 Trp--His--DTrp--Ala--Trp--DPhe-- 30 7.0 .+-. 1.0 268 .+-. 58 107 .+-. 21 2293 .+-. 319 Lys--NH.sub.2 (d) 10873 Met--His--DTrp--Ala--Trp--DPhe-- 10 7.0 .+-. 1.0 65 .+-. 18 107 .+-. 21 2005 .+-. 428 Lys--NH.sub.2 (d) 10873 Met--His--DTrp--Ala--Trp--DPhe-- 30 7.0 .+-. 1.0 161 .+-. 47 107 .+-. 21 2052 .+-. 577 Lys--NH.sub.2 10973 Ala--His--DTrp--Ser--Trp--DPhe-- 10 7.0 .+-. 0.5 30 .+-. 7 156 .+-. 39 558 .+-. 131 Lys--NH.sub.2 (d) 11009 Lys--His--DTrp--Ala--Trp--DPhe-- 3 5.0 .+-. 0.4 57 .+-. 9 115 .+-. 57 1063 .+-. 231 Lys--NH.sub.2 (d) 11009 Lys--His--DTrp--Ala--Trp--DPhe-- 10 5.0 .+-. 0.4 217 .+-. 64 115 .+-. 57 1514 .+-. 322 Lys--NH.sub.2 (d) 11012 Asp--His--DTrp--Ala--Trp--DPhe-- 10 7.0 .+-. 0.5 128 .+-. 48 156 .+-. 39 1267 .+-. 306 Lys--NH.sub.2 11012 Asp--His--DTrp--Ala--Trp--DPhe-- 10 12.0 .+-. 0.5 107 .+-. 26 110 .+-. 55 1052 .+-. 174 Lys--NH.sub.2 18988 Met(O)--His--DTrp--Ala--Trp-- 10 14.0 .+-. 0.5 65 .+-. 17 173 .+-. 52 2126 .+-. 420 DPhe--Lys--NH.sub.2 18988 Met(O)--His--DTrp--Ala--Trp-- 10 5.0 .+-. 0.4 90 .+-. 25 115 .+-. 57 1448 .+-. 183 DPhe--Lys--NH.sub.2 11553 Tyr--Ala--His--DTrp--Ala--Trp-- 10 6.5 .+-. 0.5 170 .+-. 38 252 .+-. 46 2354 .+-. 469 DPhe--Lys--NH.sub.2 11561 Lys--His--DTrp--Ala--Trp--DPhe-- 3 8.0 .+-. 1.0 9 .+-. 1 84 .+-. 37 444 .+-. 167 Asp--NH.sub.2 11561 Lys--His--DTrp--Ala--Trp--DPhe-- 10 8.0 .+-. 1.0 11 .+-. 1 84 .+-. 37 530 .+-. 237 Asp--NH.sub.2 11562 Arg--His--DTrp--Ala--Trp--DPhe-- 3 8.0 .+-. 1.0 50 .+-. 17 84 .+-. 37 473 .+-. 53 Lys--NH.sub.2 11562 Arg--His--DTrp--Ala--Trp--DPhe-- 10 8.0 .+-. 1.0 87 .+-. 27 84 .+-. 37 1448 .+-. 319 Lys--NH.sub.2 11603 Lys--Ala-- His--DTrp--Ala--Trp-- 3 16.0 .+-. 3.0 41 .+-. 5 170 .+-. 42 1008 .+-. 272 DPhe--Lys--NH.sub.2 11603 Lys--Ala--His--DTrp--Ala--Trp-- 10 16.0 .+-. 3.0 82 .+-. 19 170 .+-. 42 1437 .+-. 226 DPhe--Lys--NH.sub.2 11603 Lys--Ala--His--DTrp--Ala--Trp-- 30 16.0 .+-. 3.0 135 .+-. 38 170 .+-. 42 1859 .+-. *#8801 is Tyr--DAla--Phe--Gly--Tyr--Pro--Ser--NH.sub.2 and #9218 is Tyr--DArg--Phe--Gly--NH.sub.2 **compounds so designated are structurally similar to the Group 2 compounds defined in the text, but do not display the synergistic respons promoted by compounds within Group 2. ***contains cyclic disulfide bridge between Cys groups (a) 30 .mu.g dose (b) 100 .mu.g dose (c) Group 2 compound initially dissolved in 10 mM acetic acid (d) Group 2 compound initally dissolved in 10 DMSO

In Table 1, Group 2 compounds are shown to act synergistically with two different Group 3 compounds. Several different assays of the compound, #8114, with compounds #8801 and/or #9218 are shown, in which synergism is evident. Many additional combinations of Group 2 compounds were also assayed when administered in combination with compounds #8801 and/or #9218. In every case for Group 2 compounds, when the amount of GH released by the individual compound is added to the amount of GH released by the second compound when given alone, the result is far less than the amount of GH released when the two compounds have been administered to the rat either simultaneously, or at different locations on the rat. Thus, synergism is seen for all such combinations. It is also clear that the synergism observed is not a function of premixing the compounds, but is a result of their combined action on the GH releasing mechanism, requiring both compounds to be present to exert the remarkable synergistic effect.

Several examples are also presented in Table 1 of compounds which are structurally similar to, but outside the definition of Group 2 compounds set forth in the text. The first nine entries in Table 1, denoted by **, which are all structurally similar to the Group 2 compounds, display no synergistic response when administered in combination with either compound #8801 or #9218. It is thus seen that the surprising synergistic response observed in the practice of the present invention is not a general phenomenon, but is instead obtained only with specific compounds as set forth in the text under the definition of Group 1, Group 2 and Group 3 polypeptides.

In Tables 2 and 3, we show the enhanced release of GH into the blood caused by a number of different tetra-, penta-, and heptapeptides of Group 3 in combination with #8114 (Group 2). Even low dosages are shown to achieve synergistic responses. Also shown in Table 3 are a number of compounds (those denoted by *) which are structurally similar to the Group 3 compounds, but which do not exhibit a synergistic response in combination with Group 1 and/or Group 2 compounds. These results demonstrate that the surprising synergistic response observed in the practice of the present invention is obtained only with specific compounds as set forth in the text.

Table 4 shows the results of combining members of Groups 1 and 2, Groups 1 and 3, and all three groups of compounds together. Since the response to the three-component combination is well above the sum of the three individual responses, low doses effect significant GH responses. In fact, at equal doses of 3 .mu.g (3+3+3) as shown in Table 4, one would expect a release of only several hundred ng/ml of GH released if the result were only additive. However, the three-component mixture actually caused release of approximately 2000 ng/ml of GH at these low dosages.

In Table 5 is shown a study in which the rats were treated for ten days using a combination of His-DTrp-Ala-Trp-DPhe-Lys-NH.sub.2 (compound #8938, Group 2) +hGHRH (Human Growth Hormone Releasing Hormone; compound #144-NH.sub.2, see Group 1). Saline or 10 .mu.g each of #8938+#144-NH.sub.2 was administered once daily via the tail vein to conscious female rats. Injections were begun when the rats were 21 days of age.

Table 5. Effects of Once Daily i.v. Treatment with #8938+#144-NH.sub.2 on Body Weight in Rats.

The body weight change of 11.7% increase over control is conclusive evidence for the effect of the combination therapy.

Female lambs (17-22 kg) were housed in individual cages in a constant temperature room at 24.degree. C. with 12h-12h light-dark cycle. The lambs were fed a maintenance diet containing chick cracked corn (43.3%), soybean meal (7.1%), cottonseed hulls (38.1%), molasses (8.6%) and premix consisting of vitamins and minerals (2.9%).

Various doses of the dermorphin (compound #8801) in combination with various doses of compound #8114 were mixed and dissolved in 200 .mu.l of 10mM acetic acid and brought to 5 ml with phosphate buffered saline (PBS). Lambs were catheterized via the jugular vein. Intravenous infusions were performed by using a multichannel infusion pump (Model 600-900, Harvard Apparatus Co., Inc., Dover, Mass.) preset at a flow rate of 1.36 ml/min. Sampling of blood was performed every 20 minutes starting 1 hour prior to treatment and continuing until 3-4 hours after treatment. Additional samples were taken at -10 min., +5 min. and +10 min. Blood samples were drawn and deposited into EDTA-treated tubes for plasma preparation. EDTA treated plasma was analyzed for GH using a standard double antibody RIA, according to the following procedure:

Iodination grade ovine GH was obtained from the National Pituitary Center, and was used for iodination (Chloramine T method) and standard. The anti-ovine GH serum was also obtained from the National Pituitary Center. The results are presented in Tables 6 and 7.

In Tables 6 and 7 a Group 2 compound (#8114) is shown to act synergistically with a Group 3 compound (#8801) in the sheep. Each assay shown is an average result of studies on two sheep. In each case, when an effective dose of the combination is administered to the animal together, the resulting amount of growth hormone released is far greater than the amount of growth hormone released by #8114 and #8801 when given individually.

Lactating female goats (46-60 kg) were housed in individual cages in a constant temperature room at 24.degree. C. with 12h-12h light-dark cycle. The goats were fed a commercial goat chow containing 16% crude protein, 3.25% crude fat and 8% crude fiber and alfalfa hay.

A mixture of 5.87 .mu.g/kg body weight of compound #8801 and identical quantity of compound #8114 was dissolved in 10mM acetic acid and brought to 5 ml with phosphate buffered saline. Goats were catheterized via the jugular vein. Intravenous infusions of the entire 5 ml of phosphate buffered saline solution were performed by using a multichannel infusion pump (Model 600-900, Harvard Apparatus Co., Inc., Dover, Mass.) preset at a flow rate of 1.36 ml/min. Blood GH levels were determined as described in the previous example. The results are presented in Table 8.

The enhanced levels of serum growth hormone levels at times of +5 up to +40 minutes compared to serum growth hormone levels at times of -10 and 0 (control value) provides strong evidence for the beneficial effect of administration of the combination of growth hormone releasing compounds (#8114 and #8801) to lactating ruminant mammals.

The synergistic release of growth hormone caused by Group 1, Group 2 and Group 3 compounds was examined in male and female subhuman primates. The weight range for the female rhesus monkeys was 5.5.+-.0.3 kg and 7.8.+-.0.5 kg for the males. The ages of the females were 8.0.+-.1.2 yrs and the males were 8.7.+-.0.9 yrs. The monkeys were maintained on a diet of Purina Hi Pro Monkey Chow.

After an overnight fast the animals were anesthetized between 9:00 and 11:30 a.m. with ketamine hydrochloride at a dosage of 10 mg/kg body weight. In equal dosages of 15, 40, 120 or 360 .mu.g each, compound #144-NH.sub.2 (Group 1), compound #8938 (Group 2) and compound #8801 (Group 3) were administered to groups ranging from 6-18 adult male and 6-21 female rhesus monkeys.

Blood was drawn at 0 time and the test peptides injected i.v. via a sterile butterfly inserted into a saphenous vein. The test peptides were dissolved in 0.1% sterile gelatin saline and injected in 0.5 ml volume. Blood was then collected at +10 and +20 minutes after the injection via the same vein. All animals were observed for one hour after injection.

The serum was allowed to clot and frozen until analyzed for GH. The samples were assayed for GH with an ICN (Immuno Nuclear) human GH (hGH) kit obtained from Immuno Nuclear Corporation, Stillwater, Minn. The results were read against the hGH kit standard (1.0-30 ng/ml) and appropriate dilutions were made with assay buffer when necessary.

Results are presented in Tables 9 and 10.

In Tables 9 and 10 various binary and ternary combinations of a compound from Group 1 (#144-NH.sub.2), Group 2 (#8938) and Group 3 (#8801) are shown to act synergistically to release growth hormone in either the male or female monkey. The total number of animals in each test group is indicated in the individual table.

The synergistic effect of the same combination of Group 1, Group 2 and Group 3 compounds as was tested in Example 5 for causing the release growth hormone in rhesus monkeys was again tested, this time in male and female rats. The experimental protocol set forth in Example 1 was employed for these tests. Results are presented in Tables 11 and 12.

In Tables 11 and 12 various binary and ternary combinations of a compound from Group 1 (#144-NH.sub.2), Group 2 (#8938) and Group 3 (#8801) are shown to act synergistically to release growth hormone in either the male or female rat. Each assay shown is the average result of studies on six rats.

The data set forth in Tables 9-12 clearly demonstrate that the synergistic effect of the combination of the Group 1 compound (#144-NH.sub.2), the Group 2 compound (#8938) and the Group 3 compound (#8801) is not dependent upon the individual species being examined, nor upon the sex of the animal being examined. Instead, the synergistic response to combinations in accordance with the present invention is seen to be broadly applicable with respect to species and gender.

Various dosage combinations of any two or all three of the above peptides (i.e., #144-NH.sub.2, #8938 and #8801) showed a dramatic synergistic response in both the subhuman primate (both male and female) and the rat (both male and female).

Four non-lactating Holstein cows (mean body weight 543 kg) were housed outside in pasture and brought into the large animal laboratory for experimental studies in conventional stanchions. Cow diet was hay, grass and 1x/day 5 lbs. of Omolene (wheat, oats, corn, soybean, molasses with vitamins and trace minerals)--Purina. Cows were in the large animal laboratory and off grass for 1-1.5 hours before initiation of experiments. Catheters were inserted into the jugular vein for withdrawal of blood samples and IV injections of peptides. Fifteen to twenty ml of saline was flushed through the catheter after each blood drawing and a slow IV [i.v. ?] drip of saline was used to keep the blood from clotting in the catheter. Ten ml blood samples were collected between 9 AM and 2 PM at -40, -30, -10, 0, +5, +10, +15, +20, +25, +30, +40, +60, +90, +120, +150, +180. Normal saline with 0.1% gelatin or peptides dissolved in 0.1% gelatin saline was injected IV through the catheter at 0 time to the unanesthetized cows. The saline/peptide was infused over a 3 minute period in a 5.0 volume. The blood was allowed to clot, centrifuged and the serum separated from the clot. Serum was kept frozen until the day of sampling for radioimmunoassay (RIA) of growth hormone. Serum GH was measured by RIA with reagents provided by the NIADDK. The GH levels are reported in terms of ng/ml of a bovine GH reference preparation, NIH-GH-B18, which is equivalent to 3.2 IU/mg. Data is recorded as the mean.+-. the standard error of the mean (SEM). Statistical analysis was performed with the Student's t-test.

The data presented in Table 13 demonstrates the synergistic response in a non-lactating dairy cow for combinations of Group 1 plus Group 2 compounds; Group 2 plus Group 3 compounds; and Group 1 plus Group 2 plus Group 3 compounds.

The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.