SYNTHETIC PEPTIDES HAVING

PITUITARY GROWTH HORMONE RELEASING ACTIVITY

Background of the Invention

1. Field of the Invention

This invention relates to peptides which pos¬ sess pituitary growth hormone releasing activity and to combinations comprising at least one peptide and at least one growth promoting agent, which combination possesses synergistic pituitary growth hormone releasing activity.

2. Description of the Prior Art

Growth hormone, which is secreted from the pituitary, causes growth of all tissues of the body that are capable of growing. In addition, growth hormone is known to have the following basic effects on the meta¬ bolic process of the body:

1. Increased rate of protein synthesis in all cells of the body;

2. Decreased rate of carbohydrate utilization in cells of the body;

3. Increased mobilization of free fatty acids and use of fatty acids for energy.

A deficiency in growth hormone secretion can result in various medical disorders, such as some in¬ stances of dwarfis .

Various ways are known to release growth hor¬ mone. For example, chemicals such as arginine, L-3,4- dihydroxyphenylalanine (L-DOPA) , glucagon, vasopressin, and insulin induced hypoglycemia, as well as activities such as sleep and exercise, indirectly cause growth hor¬ mone to be released from the pituitary by acting in some fashion on the hypothalamus perhaps either to decrase

OM?l_

somatostatin secretion or to increase an unknown endog¬ enous growth hormone-releasing hormone or both.

Compounds which directly act on the pituitary to release growth hormone include prostaglandin E- _j and E2" theophylline, and cyclic nucleotides. However, these compounds neither specifically release growth hormone nor are they believed to act at the putative growth hormone- releasing hormone receptors in the peripheral membrane of the pituitary cell to initiate growth hormone release.

In addition, under special conditions certain chemically defined peptides, e.g., vasopressin, thyrotro- pin-releasing hormone (TRH) , luteinizing hormone-releas¬ ing hormone (LH-RH) , α-melanocyte-stimulating hormone (α- MSH) , glucagon, substance P, neurotensin; et-enkephalin, β-endorphin, chlorea-enterotoxin, and basic yelin pro¬ tein, act to release growth hormone from the pituitary. However, only TRH acts -directly on the pituitary to elicit this response. Furthermore, the above listed peptides release other pituitary hormones and under most experimental conditions do not release growth hormone. For example, TRH does not release growth hormone in nor¬ mal rats or in normal humans or from pituitaries of nor¬ mal rats or monkeys. In vitro, TRH releases growth hor¬ mone, prolactin, and thyroid stimulating hormone (TSH) in certain species, and, in vivo, TRH releases these hor- ^• mones from bovine pituitary.

Vasopressin's induced release of growth hormone is considered to be due to a non-specific response to stress caused by administration of high dosages of vaso¬ pressin.

Accordingly it would be highly desirable to have a compound or combination of compounds which

directly acts on the pituitary under normal experimental conditions to effect the release of growth hormone there¬ from. Such compound or combination of compounds would be useful in vitro, e.g., as unique. research tools for understanding how growth hormone secretin is regulated at the pituitary level and would also be useful in vivo, e.g., to treat symptoms related to growth hormone defi¬ ciencies, to increase the rate and extent of growth in commercial animals, to increase milk yield in commercial animals, and to reduce the number of mucosal erosions induced by hypoxemia.

Summary of the Invention In accordance with the present invention there is provided peptides which act directly on the pituitary under normal experimental conditions in vitro to release growth hormone therefrom. In addition, there is provided a combination of compounds which act directly on the pituitary under normal experimental conditions in vitro to synergistically release growth hormone therefrom.

These growth hormone releasing compounds and combinations can be utilized in vitro as unique research tools for understanding, inter alia, how growth hormone secretion is regulated at the pituitary level.

Also, the growth hormone releasing peptides of the instant invention can also be administered in vivo to increase growth hormone release.

More particularly, this invention encompasses novel peptides having the formulas I and II

1

^(x 2)a~ ^A 1-A2-A3-A4-A5-Y (I) ⁽ X3 ⁾ b

( ^χ 2 ' ) _a - ^A 1 - ^A 2- ^A 3- ^A 4- ^A 5- ^A 6-ϊ UD

( X3 ^r ) b

wherein X-j , χ ₂ , χ ₃ , and X* ' , 2*, and X3• are selected from a group consisting of N-terminal and ^' desamino alpha- carbon substitutions; a and b are 0 or 1 , provided that a and b are always 0 when A1 is a desamino residue; A ^* | ^an< 3 ^A 4 are selected from a group consisting of histidyl, arginyl, lysyl, α-naphthylalanyl, e-naphthylalanyl, iso- quinolyl, tyrosyl, tryptophyl, phenylalanyl, homologues and analogues thereof, and, with respect to A-j only, the desamino forms thereof; A ₂ and A5 are selected from a group consisting of D-histidyl, D-arginyl, D-lysyl, D-α- naphthylalanyl, D-β-naphthylalanyl, D-isoquinolyl, D- tyrosyl, D-tryptophyl, D-phenylalanyl, homologues and analogues thereof, and, with respect to A only, the descarboxy forms thereof; A3 is selected from a group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, seryl, threonyl, methionyl, aspartyl, glutamyl, asparaginyl,glutaminyl, histidyL^ D-alanyl, D-valyl, D- leucyl, D-isoleucyl, D-prolyl, D-seryl, D-threonyl, D- ethionyl, D-aspartyl, D-glutamyl, D-asparaginyl, D- glutaminyl, D-histidyl, and homologues and analogues thereof; Ag is selected from a group consisting of a ino acid redisues of the L- and D-configuration, homologues and analogues thereof, and the descarboxy forms thereof; and Y is selected from a group consisting of C-terminal and descarboxy alpha-carbon subtitutions; and the pharmaceutically acceptable salts thereof; provided that (a) when (1 } is 1 and b is 0 and X- _] and X2 are selected from the group consisting of tyrosyl, tryptophyl, and phenylalanyl; (3) A3 is selected from the group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, seryl, threonyl, methionyl, aspartyl, glutamyl,

asparaginyl, gluta inyl, and histidyl; and (4) Y is selected from the group consisting of - R- _] R2, -OR, and -CH ₂ OR, wherein R, R _j , and R2 are selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at least one of A ₂ and A5 is selected such that it is not from a group consisting of D-tyrosyl, D-tryptophyl, D- phenylalanyl, and, with respect to A ₅ . the descarboy forms thereof; (b) when (1) a is 1 and b is 0 and X1 ^{and x} 2 are selected from the group consisting of -H and -CH3; (2) A2 and A5 are selected from the group consisting of D-tyrosyl, D-tryptophyl, D-phenylalanyl, and, with respect to A5, the descarboxy forms thereof; (3) A3 is selected from the group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, seryl, threonyl, methionyl, aspartyl, glutamyl, asparaginyl, glutaminyl and histidyl; and (4) Y is selected from the group consisting of -NR-]R ₂ , -OR, and -CH20R- wherein R, W\ , and R ₂ are selected from a group consisting of hydrogen- and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at least one of A-| and A4 is selected such that it is not from a group-consisting of tyrosyl, tryptophyl, and phenylalanyl; (c) when (1) a is 1 and b is 0 and X-j ^« _an( ^* | χ ₂ - _are selected from the group consisting of -H, -CH3, and -CHOCH3; (2) A- and A4 are selected from the group consisting of tyrosyl, tryptophyl, and phenylalanyl; (3) A3 is selected from the group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, seryl, threonyl, methionyl, asparaginyl, and glutaminyl; (4) Ag i _s selected from the group consisting of asparaginyl, glutaminyl, glutamyl, arginyl, lysyl, seryl, threonyl, and the descarboxy forms thereof; and (5) Y is selected from the group consisting of -NR-|R2 - -OR, and -CH ₂ OR, wherein R, R-j , and R2 are selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at

least one of A ₂ and A5 is selected such that it is not from a group consisting of D-tyrosyl, D-tryptophyl, and D-phenylalanyl; and (d) when (1) a is 1 and b is 0 and

X-j ' and X ₂ ' are selected from the group consisting of -H, -CH3, and -CHOCH3; (2) A ₂ and A5 are selected from the group consisting of D-tyrosyl, D-tryptophyl, and D- phenylalanyl; (3) A3 is selected from the group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, seryl, threonyl, methionyl, asparaginyl, and glutaminyl;

(4 ⁾ g i _s selected from the group consisting of asparaginyl, glutaminyl, glutamyl, arginyl, lysyl, seryl, threonyl, and the descarboxy forms thereof; and (5) Y is selected from the group consisting of -NR1 2' -OR, and -CH ₂ OR, wherein R, R_ , and R ₂ are selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at least one of A- _] and A4 is selected such that it is not from a group consisting of tyrosyl, tryptophyl, and phenylalanyl.

In addition, this invention encompasses a com¬ bination of compounds comprising. (a) at least one peptide having the formula III

⁽ X2")a- l-A2- 3-A ₄ -A5-A6-Y (III) ⁽ X3" ⁾ b

wherein - " , χ ₂ ", and X3" are selected from a group con¬ sisting of N-terminal and desamino alpha—carbon substitu¬ tions; a and b are 0 or 1 , provided that a and b are always 0 when A-j is a desamino residue; AT and A4 are selected from a group consisting of histidyl, arginyl, lysyl, α-naphthylalanyl, β-naphthylalanyl, isoquinolyl, tryosyl, tryptophyl, phenylalanyl, homologues and ana¬ logues thereof, and, with respect to 1 only, the des-

-7- ammo forms thereof; A2 and A5 are selected from a group consisting of D-histidyl, D-arginyl, D-lysyl, D-α- naphthylalanyl, D-3-naphthylalanyl, D-isoquinolyl, D- tyrosyl, D-tryptophyl, D-phenylalanyl, homologues and analogues thereof, and, with respect to A5 only, the descarboxy forms thereof; A3 is selected from a group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, .prolyl, seryl, threonyl, methionyl, aspartyl, glutamyl, asparaginyl, glutaminyl, histidyl, D-alanyl, D-valyl, D- leucyl, D-isoleucyl, D-prolyl, D-seryl, D-threonyl, D- mthionyl, D-aspartyl, D-glutamyl, D-asparaginyl, D-gluta- inyl, D-histidyl, and homologues and analogues thereof; is selected from a group consisting of amino acid residues of the - and D-configuration, homologues and analogues thereof, and the descarboxy forms thereof; and Y is selected from a group consisting of C-terminal and descarboxy alpha-carbon substitutions; and the pharmaceutically acceptable salts thereof; and (b) at least one growth promoting agent.

Detailed Description of the Preferred Embodiments

The peptides of this invention have the amino acid residue sequence represented by formulas I-III, supra.

All amino acid residues identified herein are in the natural or L-configuration unless otherwise speci¬ fied

Abbreviations for amino acid residues are used in accordance with the following standard peptide nomen¬ clature:

Tyr L-tyrosyl He L-isoleucyl

D-Tyr D-tyrosyl D-Ile D-isoleucyl

Gly glycyl Leu L-leucyl

8- jfue L-phenylalanyl D-Leu D-leucyl

D-Phe D-phenylalanyl Thr L-threonyl

Met L-methionyl D-Thr D-threonyl

D-Met D-methionyl Val ^" L-valyl

Ala L-alanyl D-Val D-valyl

D-Ala D-alanyl Pro L-prolyl

Ser L-seryl D-Pro D-prolyl

D-Ser D-seryl Gin L-glutaminyl

Lys L-lysyl D-Gln D-glutaminyl

D-Lys D-lysyl Glu L-glutamyl

Asn L-asparaginyl D-Glu D-glutamyl

D-Asn D-asparaginyl Trp L-tryptophyl

His L-histidyl D-Trp D-tryptophyl L-Asp . L-aspartyl

D-His D-histidyl D-Asp D-aspartyl

Cys L-cysteinyl Arg L-arginyl

D-Cys D-cysteinyl D-Arg D-arginyl

Hypro L-hydroxypropyl

D-Hypro D-hydroxypropyl

Dopa L-3, -dihydroxy- phenylalanyl L-<Glu L-pyroglutamyl

D-Dopa D-3,4-dihydroxy- - phenylalanyl D-<Glu D-pyroglutamyl

Hylys L- .-hydroxylysyl Sar N-methylglycyl (sarcosyl)

D-Hylys D-δ-methylalanyl

Aib L-α-meth lalanyl α-Naphth L-α-naphth l-

(L-aminoisobutyryl) alan l

D-α-Naphth D-α-naphthyl- alanyl β-Naphth L-β-naphthyl- alan l

Iql L-isoquinolyl D-β-Naphth D-B-nap th l- D-Iql D-isoquinolyl alanyl

-9-

Virtually any suitable N-terminal and desamino alpha-carbon substitution can be used in the instant invention as represented by the .various structural formu¬ las set forth herein. Typical N-terminal and desamino alpha-carbon substitutions include, but are not limited to, those set forth in Table I.

TABLE I

N-Terminus and Desamino Alpha-Carbon Su bstitutions

N- -Terminus Substitutions fi i l . .1 . ^' J2.

^R 1 " R2- - 1 0

R1 - R2- R3- ¹ 1

0 11

R _{l r} Z- C- R2- . 0

RT HO- - 1 0

^R 2

R- -N- R ₃ - ^ - 0 z

IJ

R- -N - C- R ₃ - - - 1 0

Des amino Alpha-Carbon Substitutions

R- - 0 0

RZ- — 0 0

LEGEND: R, Ri , R2, and R3 are selected from a group consisting of hydrogen; straight and branched chain alkyl groups having from 1 to 6 carbon atoms; cyclo- alkyl groups having from 3 to 6 carbon atoms; benzyl; benzhydryl; trityl; aryl; alkoxybenzyl; al- koxybenzhydryl; alkoxytrityl; lower haloalkyl groups having from 1 to 6 carbon atoms; halobenzyl; halo- benzhydryl; halotrityl; haloaryl; and cyclohaloalkyl groups having from 3 to 6 carbon atoms. Preferably,

R, R-| , R ₂ , and R3 are selected from the group con¬ sisting of hydrogen and alkyl groups having from 1 to 6 carbon atoms. More preferably, R, R^j, R2- ^anc -- R3 are selected from the group consisting of hydro¬ gen and alkyl groups having 1 to 2 carbon atoms.

Z is selected from a group consisting of oxygen and sulfur. Z is preferably oxygen.

Virtually any suitable C-terminal and descar¬ boxy alpha-carbon substitution can be used in the instant invention as represented by the various structural formu¬ las set forth herein. Typical C-terminal and descarboxy alpha-carbon substitutions include, but are not limited to, those also set forth in Table II.

TABLE II

C-Terminus and Descarboxy Alpha-Carbon Substitutions C-Terminus Substitutions -OR

-N-R2 _- -R

^■ }•

-C-R ₂ R3

-

R1 R2° -N- N-C-R3

-1 1 -

Descarboxy Alpha-Carbon Substitutions -R

-C-OR2

R3 VRi

-C-VR ₂

R3

NH Ri

-C- N-R2

N-N

*

-C

\

N-N I

R

-C=N ⁺ -

I R1 2 O-R3

1. LEGEND: R, R-j _f R _{2 f an} d R3 are as defined in Table I, supra.

V is selected from a group consisting of oxy¬ gen, sulfur, and nitrogen. V is preferably oxygen.

The structure of amino acid residues employed in the peptides of this invention are set forth in Table III. Typical homologues and analogues of these amino acid residues which can also be employed in the peptides of this invention include, but are not limited to, those listed in Table III.

TABLE III L or D Amino Acid Residue

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES . ANALOGUES'

Ra Rb Re Rα Rb Re

Gly -H -H -H -H U Ui

=CH ₂ # U

Val -CH (CH ₃ ) ₂ -H ^• H ~CH(CH ₃ ) ₂ U u,

-C(CH ₃ ) ₃ u Ui

=C(CH ₃ ) ₂ - U

Leu -CH ₂ CH (CH ₃ ) ₂ -H -H -(CH ₂ ) _n+1 CH(CH ₃ ) ₂ U U,

-(CH ₂ ) _{n+ 1} C(CH ₃ ) ₃ U U,

=CHCH(CH ₃ ) ₂ u

=CH(CH2) _n CH(CH ₃ ) ₂ — u

TABLE III (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES & ANALOGUES'

CH, CH ₃ 1 1

He - ^■ CH-"-C ₂ H _{ - _j -H -H -"CH~-C HR u ^u ι

Nile —CH CH CH CH -H -H -(CH ₂ ) _n +|CH ₃ u ^υ ι =CH(CH ₂ ) _n CH ₃ u

Pro CH ₂ CH ₂ — CH ₂ CH ₂ (CH ₂ ) _n H

-H u -CH ₂ O

1 ²

CH _? CHOU- -CH?

1 1 ²

/ (pCil 1l ₂ . \) _n ,.., D u ~(CH ₂ )

1

Ser -CH ₂ OH -H -H -(CH ₂ ) _n+| OU l u ₂

Thr -CHOH-CH3 -H ~H -(CH > _n+ ,CHOU- -CH ₃ u ₂ ^υ '

TABLE (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES a ANALOGUES' R _α Rb R ₍ Rα ^• Rb Re

Cys -CH ₂ SH -H -H -(CH ₂ ) _n+ ,SU ^U l u ₂

-(CH ₂ ) _n + ιS0 ₃ H U ^U l

Met -CH ₂ CH ₂ SCH ₃ -H -H ^•* -(CH ₂ )|-n. | SCH3 U ^υ ι

-(CH ₂ )n+lS0 ₂ CH3 u U|

Asp -CH ₂ C0 ₂ H -H -H -(CH ₂ ) _n+ .C0 ₂ U U| u ₂

=CH(CH ₂ ) _n C0 ₂ U — U|

Glu ~(CH ₂ ) ₂ C0 ₂ H -H -H -(CH ₂ ) _n+ ,C0 ₂ U U| u ₂ Asn -CH ₂ CONH ₂ -H -H -(CH ₂ ) _n+1 CONR,R ₂ u U|

=CH(CH ₂ ) _n CONR _| R ₂ — u

TABLE HI (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES ANALOGUES ¹

Rα Rb Re Rα Rb Re

Lys -(CH ) ₄ NH ₂ -H -H ^• (CH ₂ ) _n+l N-U ₂ U U3 U|

TABLE III (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES a ANALOGUES'

R( Rb Re Rα Rb R<

TABLE - III (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES a ANALOGUES ¹

Rr Rb Re Rb Re

TABLE III (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES a ANALOGUES ¹

Rr Rb Rr R_ Rb e

TABLE III (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES a ANALOGUES ¹ Rα Rb e Rα b R<

cx-Nαphth

TABLE III (contd.)

SUBSTITUENTS OF

NAME NATURAL SUBSTITUENTS HOMOLOGUES a ANALOGUES ¹

j3-Nαphth

Sαr -H -H ^• CH- U U, -CH-

-20-

1. LEGEND: U, U- _| , U2, U3 and U4 are selected from a group consisting of hydrogen, alkyl groups having from 1-10 carbon atoms, and benzyl.

^B f ^1 , and B2 are selected from a group consisting of -N-D, 0, S.

^D - D-j , D2, and D3 are selected from a group consisting of hydrogen, methyl, ethyl, propyl, benzyl, formyl, and tosyl.

K1, K2, K3, K4, K5, K6, and K7 are N or -C-G, provided that adjacent positions are not both N. G is selected from a group consisting of hydrogen, halogen, -Oϋ, -0R _χ ,

^*SR x- -RX, -SO3RX,

-B(0H)2, -RNχSORγ, ~NRχRγ, -C≡N, -N(Rχ)C0Ry/ wherein

^R χ and Ry are selected from a group consisting of hydrogen and straight and branched alkyl groups containing 1-6 carbon atoms, and substituted straight and branched alkyl groups containing 1-6 carbon atoms, wherein the substituents .include, but are not limited to, one or more halo, hydroxy, amino, and mercapto groups. L is -N or -N+-D.

Rl and R2 are as defiϊed in Table I. n is an integer from 0 to 4.

^' -21 -

The term "pharmaceutically acceptable salts", as used herein, refers to the non-toxic alkali metal, alkaline earth metal and ammonium _^ salts commonly used in the pharmaceutical industry including, but not limited to, the sodium, potassium, lithium, calcium, magnesium, barium, ammonium and protamine salts which are prepared by methods well known in the art. The term also includes non-toxic acid addition salts which are generally pre¬ pared by reacting the compounds of this invention with a suitable organic or inorganic acid. Representative salt include, but are not limited to, the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, val- erate, oleate, laurate, borate, benzoate, lactate, phos¬ phate, tosylate, citrate, maleate,- fumarate, succinate, tartrate, napsylate, and the like.

Preferably, the peptides of formula I of this invention have the amino acid sequence wherein a is 0 or 1 , b is 0 and X-j and X2 are selected from a group con¬ sisting of -R, -OR, and RC(O)-, wherein R is selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms;

^A 1 and A4 are selected from the group consisting of his¬ tidyl, tryptophyl, phenylalanyl, tyrosyl, homologues and analogues thereof,' and, with respect to A- _j . the desamino forms thereof; A2 and A5 are selected from the group consisting of D-histidyl, D-tryptophyl, D-phenylalanyl, D-tyrosyl, homologues and analogue thereof, and, with respect to Ag . the descarboxy forms thereof; A3 is se¬ lected from the group consisting of glycyl, alanyl, seryl, asparaginyl, prolyl, D-alanyl, D-seryl, D- asparaginyl, D-prolyl, and homologues and analogues thereof; Y is selected from a group consisting of -CH2OH, -OR, and -NR-jR ₂ , wherein R, R-j , and R2 ^are selected from a group consisting of hydrogen and straight and branched

-22- chain alkyl groups containing 1-6 carbon atoms; and the pharmaceutically acceptable salts thereof.

Preferably, the peptides of formulas II and III of this invention have the amino acid sequence wherein a is 0 or 1, b is 0 and X _ ∑2* , XT', and X2" are selected from a group consisting of -R, -OR, and RC(O)-, wherein R is selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; A- and A4 are selected from the group con¬ sisting of histidyl, tryptophyl, phenylalanyl, tyrosyl, homologues and analogues thereof, and, with respect to

^A 1 , the desamino forms thereof; A2 and A5 are selected from the group consisting of D-histidyl, D-tryptophyl, D- phenylalanyl, D-tyrosyl, homologues and analogues there¬ of; _A 3 is selected from the group consisting of glycyl, alanyl, seryl, asparaginyl, prolyl, D-alanyl, D-seryl, D- asparaginyl, D-prolyl, and homologuess and analogues thereof; Ag is selected from the group consistng of ar- ginine, lysine, ornithine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, D-arginine, D- lysine, D-orthithine, D-histidine, D-aspartic acid, D- glutamic acid, D-asparagine, D-glutamine, D-arginine, homologues and analogues thereof, and the descarboxy forms thereof; Y is selected from a group consisting of

-CH2OH, -OR, and -NR1R2, wherein R, R-j , and R2 are se ^¬ lected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; and the pharmaceutically acceptable salts thereof.

More preferably, the peptides of (a) formula I and (b) formulas II and III of this invention have the amino acid sequence represented by formulas IV and V, respectively:

_________

-23 -

H I

( ^X 2 ) a-Al -D-Trp-A ₃ -A ₄ -A ₅ -Y ( IV)

H I ( X2 * ) - ι -D-Trp- A3-A4-A5-A6-Y (V)

wherein a is 0 or 1 ; X2 ^is selected from the group con¬ sisting of R- and RC(0)-; wherein R is selected from the group consisting of hydrogen and alkyl groups containing 1-2 carbon atoms; A-j i _Ξ selected from the group consist¬ ing of tyrosyl, 0-methyltyrosyl, histidyl, 3-N-methyl- histidyl, p-chlorophenylalanyl, and the desamino forms thereof; A3 is selected from the group consisting of alanyl, seryl, and D-alanyl; A ₄ ± _s selected from the group consisting of tryptophyl and tyrosyl; A5 s se¬ lected from the group consisting of D-phenylalanyl, D- histidyl, D-tyrosyl, and D-p-chlorophenylalanyl; Aζ s selected from the group consisting of arginine, homo- arginine, lysine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine, and D-lysine; and Y is se¬ lected from the group consisting of -OR and -NHR, wherein R is selected from the group consisting of hydrogen and alkyl groups containing 1-2 carbon atoms; and the pharma¬ ceutically acceptable salts thereof.

Peptides within the scope of the instant inven¬ tion include, but are not limited to, those set forth in Table IV and the desamino and/or descarboxy forms there¬ of, wherein the respective positions of (a) X- _j . χ , X3,

(b) T", X ₂ ', X3 ¹ , and (c) Xi", X ₂ ", X3" are set forth in formulas I-III, respectively.

TABLE IV (Xi-/ (X2-)a- ( 3-)b)-His-D-Trp-Ala-Trp-D-Phe-Y ^{x 1-/ (X2-)a/ (X3-)b)-His-D-5-Br-Trρ-Ala-Trp-D-Phe-Y ( l-, (X2-)a _# ( 3-)b ³ -His-D-Trp-Ala-5-Br-Trp-D-Phe-Y

-24-

( Xl - , ( 2- ) a _t ( 3- ) b ) - ¹ -N-Me-Hi s-D-Trp-Al a-Trp-D-Phe-Y

( ^x 1 - / ( 2~ ) a 1 ( ^x 3~ ) b) -3-N-Me-His-D-Trρ-Ala-Trp-D-Phe-Y

( Xl - , ( X ₂ - ) _a , ( X3- ) b - -Arg-D-Trp-Al a-Iql-D-Phe- Y

( Xl - , ( X ₂ - ) a, ( 3- ) b ) -Lys-D-Trp-Ala-Trp-D-Phe-Y

( X! - , ( X ₂ - ) _a , ( X3- ) b ) - ^Hi s-D-Trp-Ser-Trp-D-Phe-Y

( ^x 1 - , ( 2- ) a - ( ^x 3" ) b ) -Tyr-D-Trp-D-Ala-Trp-D-His-Y

( Xl - , ( 2- ) _{a r} ( X3- ) b - -Ty r-D-Trp-Al a-Trp-D- 1 -N-Me-His-Y

( ^χ 1 - 1 ( 2- ) a , ( 3- ) b ) -Tyr-D-Trp-Ala-Trp-D-3-N-Me-His-Y

( Xl - , ( X2- ) a _/ ( 3- ) b - "Ty r-D-Trp-Al a-Trp-D-Arg-Y

( ^x 1 - , ( X2- ) a 1 ( X3- ) b ) -Tyr-D-Trp-Ala-Trp-D-Lys-Y

( Xl - , ( X2- ) a ( 3- ) b • "Tyr-D-Trp-D-Ser-Trp-D-Lys-Y

^{( x} 1 - - ( 2- )ar( X3- ) b ) -His-D-Trp-Ala-Trp-D-His-Y

( Xi - , ( X ₂ - ) _a , ( X3- ) I.' -Arg-D-Phe-Val-Tyr-D-Lys-Y

^{( x} 1 - ( 2- ) a / ( ^x 3~ ) b ) -Tyr-D-Tyr-Met-Phe-D-Arg-Y

⁽ Xl~, ⁽ X2-) a- ⁽ X3- ⁾ b)~ ^]? - ^e ~ ^D ~- ^? - ^ιe ~ ^G - ^Ln ~ ^p - ^e ~ ^D ~ ^', ~ ^N ~ ^Me ~ ^H i ^s ~ ^γ

( ^x 1 - / ( X2- ) a r ( X3- ) b ) -His-D-Trp-Ile-Tyr-D-Trp-Y

( Xl - , ( X2" ) a _/ ( 3- ) b - - ^α - ^Na P ⁿ th-D-Trp-D-Ala-β -Naph th-D-Phe- Y

• ^x 1 - ( X2- ) a , ( X3- ) b ) - β-Naphth-D-Lys-D-His-Hj-S.-D-Arg-Y

( Xi ^• - , ( X2 ' - ) _a , ( X3 ' - ) b- -His-D-Trp-Ala-Trp-D-Phe-Ly s-Y

^{( x} 1 ' - , ( X2 ' - ) a - ( X3 ' - ) b ) -His-D-5-Br-Trp-Ala-Trp-D-Phe-Lys-Y

( i ' - , ( X2 ^« - ) _{a r} ( X3 ' -) b- -His-D-Trp-Ala-5-Br-Trp-D-Phe-Ly s-Y

(Xl*-,(X2 ^, -)a (X3 ^, -)b)-l -N-Me-His-D-Trp-Ala-Trρ-D-Phe- Asn-Y

( l *-, (X2'-)ar (X3'-)b)-3-N-Me-His-D-Trp-Ala-Trp-D-Phe- _ Lys-Y

( ^x 1 ' - ( X2 ' - ) a / ( X3 ' ~ ) b ) -Arg-D-Trp-Ala-Iql-D-Phe-Arg-Y ( x ^' ι ^• - , ( X2 ' - ) _{a t} ( 3 ' - ) b- ~ ^L y s-D-Trp-Ala-Trp-D-Phe-Glu-Y ( Xi * - , ( X2 ' - ) a t ( 3 ' - ) b) -His-D-Trp-Ser-Trp-D-Phe-Lys-Y ( Xi ^• - , ( X2 ' - ) a - ( X3 ' - ) b' -Tyr-D-Trp-D-Ala-Trp-D-His-Gln-Y

^{x 1 '-■ (X2 ^f -)a, (X3'-)b)-Tyr-D-Trp-Ala-Trρ-D-1-N-Me-His- Met-Y

( ^χ 1 '-/ (X2'-)a- (X3'-)b)-Tyr-D-Trp-Ala-Trp-D-3-N-Me-His- Pro-Y

( ^χ 1 '-/ (X2'-)a- (X3'-)b)-Tyr-D-Trp-Ala-Trp-D-Arg-His-Y ( Xi ^« - , ( X ₂ ' - ) _a r ( 3 ' ~ ) b- -Tyr-D-Trp-Ala-Trp-D-Lys-Ser-Y ^(x 1 --/ (X2'-)aF (X3'-)b)-Tyr-D-Trp-D-Ser-Trp-D-Lys-Phe-Y

-25-

(Xi '-,(X2'-) _{a r} ( 3'-)b)-His-D-Trp-Ala-Trp-D-His-Trp-Y ^(x 1 '-/(X2'-)a- ( ^x 3'~)b)-Arg-D-Phe-Val-Tyr-D-Lys-D-Lys-Y (Xl ^« -,(X2'-) _{a r} (X3'-)b--Tyr-D-Tyr-Met-Phe-D-Arg-D-Orn-Y

^(x 1 ^* - r(X2'-)a t( ^x 3'")b)-Phe-D-Phe-Gln-Phe-D-1-N-Me-His-D-

Asp-Y

(Xl ^, -,(X2 ^, -)a-(X3 ^, -)b)- ^H is-D-Trp-Ile-Tyr-D-Trp-D-Glu-Y

(Xi *-,(X2*-)a _/ (X3'-)b>-α-Naρhth-D-Trp-D-Ala-β-Naphth-D-

Phe-Val-Y

⁽ Xl '-,(X2'-)a ( 3'-)b)-S-Naphth-D-Lys-D-His-His-D-Arg- Thr-Y

The peptides of the instant invention can be prepared by solution methods known in the art or by using standard solid-phase techniques. The solid-phase syn¬ thesis, for example, can be commenced from the C-terminal end of the peptide using an α-amino protected amino acid. A suitable starting material can be prepared, for instance, by attaching the required α-amino acid to a chloromethyl resin, a hydroxymethyl resin, a. benzhydryl- amine (BHA) resin, or a p-methylbenzylhydrylamine (p-Me- BHA) resin. One such chloromethyl resin is sold under the tradename BIO-BEADS SX-1 by _Bio Rad Laboratories, Richmond, California. The preparation of the hydroxy¬ methyl resin is described by Bodansky et al., Chem. Ind. (London) 38, 1597 (1966). The BHA resin has been de¬ scribed by Pietta and Marshall, Chem. Commn. 650 (1970) and is commercially available from Beckman Instruments, Inc., Palo Alto, California in the hydrochloride form thereof (BHA-HC1).

In the solid-phase preparation of the compounds of this invention, a protected amino acid can be coupled to a resin with the aid of a coupling agent. After the initial coupling, the α-amino protecting group can be removed by a choice of reagents including trifluoroacetic acid (TFA) or hydrochloric acid (HC1) solutions in or-

- - ganic solvents at room temperature. After removal of the α-amino protecting group, the remaining protected amino acids can be coupled stepwise in the desired order. Each protected amino acid can be generally reacted in about a 3-fold excess using an appropriate carboxyl group activa¬ tor such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride(CH2CI2)-dimethylforma- ide (DMF) mixtures.

After the desired amino acid sequence has been completed, the desired peptide can be cleaved from the resin support by treatment with a reagent such as hydro¬ gen fluoride (HF) which not only cleaves the peptide from the resin, but also cleaves all remaining side-chain protecting groups. When a chloromethyl resin or hydroxy- methyl resin is used, HF treatment results in the forma¬ tion of the free peptide acids of formulas I-III (Y ^*** -C00H) . When the BHA or p-Me-BHA resin is used, HF treatment results directly in the free peptide amides of formulas I--III (Y = -CONH ₂ ). Alternatively, when the chloro ethylated or hydroxymethyl resin is employed, the side-chain protected peptide can be cleaved from the resin by treatment of the peptrde-resin with ammonia to give the desired side-chain protected amide or with an alkylamine to give a side-chain protected alkylamide or dialkylamide. Side-chain protection can then be removed in the usual fashion by treatment with HF to give the free peptide amides, alkylamides, or dialkylamides.

In preparing the esters of this invention, the resins used to prepare the acids of formulas I-III (Y = -C00H) can be employed and the side-chain protected pep¬ tide can be cleaved with a base and an appropriate alco¬ hol, i.e., methanol. Side-chain protecting groups can then be removed in the usual fashion by treatment with HF to obtain the desired ester.

-27-

The solid-phase procedure discussed above is well known in the art and has been essentially describe by Stewart and Young, Solid- Phase ^* -Peptide ^* -Synthesis, Freeman and Co., San Francisco (1969).

Some of the well known solution methods which can be employed to synthesize the peptides of the insta invention are set forth in Bodansky et al., Peptide Synthesis, 2nd Edition, John Wiley & Sons, New York, N. 1976).

Accordingly, also within the scope of the in¬ stant invention are intermediate compositions prepared during the synthesis of the novel peptides of formulas II. Intermediate compositions prepared via solid-phase techniques are the peptide-resin compounds of formulas VI-VII and intermediate compositions prepared via solu¬ tion techniques are the protected peptide-cqmpounds of formulas VIII-XIII:

^(] j ^r l-q

(?rι) _r -(X! ^1V

⁽ ? ι ) _r -(X ₂ ^V )

(Pr ₁ ) _r -(X ₂ '5-Y (VIII)

(Pr ₁ ) _r -(X2 ^V ) ₆ -Y (XI)

(p-πjg

( Pr ₁ ) _r _ ( X ₂ ^V ) ( XII )

Bι-B ₂ -B3-B4-B5-B' ₆ -Y (XIII)

wherein Pri s an α-amino protecting group; q, r, and s are each either 0 or 1; a and b are as defined above; m is either 0 or 1 ; Xι ^lv , X2 ^1V , 3* ^V r and Xι ^v , X ₂ ^V , and X ₃ ^V are selected from a group consisting of N-terminal and desamino alpha-carbon substitutions and radicals; Bi and ^B 4 are selected from a group consisting of histidyl, arginyl, lysyl, α-naphthylalanyl, 3-naphthylalanyl, iso- quinolyl, tyrosyl, tryptophyl, phenylalanyl, homologues

-29- and analogues thereof, the side-chain protected forms thereof, and, with respect to B-, , the desamino forms ^■ thereof; B2, B5, and B's are selected from a group con¬ sisting of D-histidyl, D-arginyl, D-lysyl, D-α-naphthyl- alanyl, D-β-naphthylalanyl, D-isoσuinolyl, D-tyrosyl, D- tryptophyl, D-phenylalanyl, homologues and analogues thereof, the side-chain protected forms thereof, and, with respect to B* ₅ the descarboxy forms thereof; B3 is selected from a group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, seryl, threonyl, methi¬ onyl, aspartyl, glutamyl, asparginyl, glutaminyl, histi¬ dyl, D-alanyl, D-valyl, D-leucyl, D-isoleucyl, D-prolyl, D-seryl, D-threonyl, D-methionyl, D-aspartyl, D-glutamyl, D-asparaginyl, D-glutaminyl, D-histidyl, homologues and analogues thereof, and the side-chain protected forms thereof; Bg and B'g are selected from a group consisting of amino acid residues of the L- and D- configuration, homologues and analogues thereof, the side-chain protection forms thereof; ® is a resin; Y is as defined above; and Pr2 is a carboxyl protecting group; provided that (a) when (1) a is 1 and b and m are 0 and 2 ^1V * ³ selected from the group consisting of -H and -CH3. (2) Bi and B4 are selected from the group consisting of tyrosyl, tryptophyl, phenylalanyl, and the side-chain protected forms thereof; (3) B3 s selected from the group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, seryl, threonyl, methionyl, aspartyl, glutamyl, asparaginyl, glutaminyl, histidyl and the side-chain protected forms thereof; and, with respect to formulas (VIII) and (X), (4) Y is selected from the group consisting of -NR1R2, -OR, and -CH2 ^0R * wherein each R, ^R l , and R2 is selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at least one of B ₂ , B5, and B*5 s selected such that it is not from a group consisting of D-tyrosyl, D-tryptophyl, D-phenylalanyl,

-30- and, with respect to B*5, the descarboxy forms thereof, and the side-chain protected forms thereof; (b) when (1) a is 1 and b and m are 0 and X2 ^{iv is} selected from the group consisting of -H and -CH _{3 }} (2) B2 and B5 or B's are selected from the group consisting of D-tyrosyl, D- tryptophyl, D-phenylalanyl, and, with respect to B'g, the descarboxy forms thereof, and the side-chain protected forms thereof; (3) B ₃ ± _s selected from the group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, seryl, threonyl, methionyl, aspartyl, glutamyl, asparaginyl, glutaminyl, histidyl, and the side-chain protected forms thereof; and, with respect to formulas (VIII) and (X), (4) Y is selected from the group consisting of -NR-,R ₂ -OR, and -CH2OR, wherein each R, l , and R2 i ^s selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at least one of Bi and B4 is selected such that it is not from a group consisting of tyrosyl, tryptophyl, phenylalanyl, and the side-chain protecte forms thereof; (c) ^' when (1) a is 1 and b and m are 0 and X2 ^{V is} selected from the group consisting of -H, -CH _{3/ a} nd -CHOCH3; (2) Bi and B4 are selected from the group consisting of tyrosyl, tryptophyl, phenylalanyl, and the side-chain protected forms thereof; (3) B3 is selected from the group consisting of glycyl, alanyl, valyl, leucyl, isoleucyl, seryl, threonyl, methionyl, asparaginyl, glutaminyl, and the side-chain protected forms thereof; (4) Bg is se¬ lected from the group consisting of asparaginyl, gluta¬ minyl, glutamyl, arginyl, lysyl, seryl, threonyl, and the side-chain protected forms thereof; and, with respect to formulas (XI) and (.XIII), (5) Y is selected from the group consisting of - R1R2, -OR, and -CH2 ^0R * wherein each ^R , ^R ι , and R2 is selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at least one of B ₂ and

-3 / --

B5 is selected such that it is not from a group consist¬ ing of D-tyrosyl, D-tryptophyl, D-phenylalanyl, and the side-chain protected forms thereof; and (d) when (1) a is 1 and - and m are 0 and -^ ⁷ is selected from the group consisting of -H, -CH3, and -CHOCH3; (2) B2 and B5 are selected from the group consisting of D-tyrosyl, D- tryptophyl, D-phenylalanyl, and the side-chain protected forms thereof; (3) B3 i _s selected from the group consist¬ ing of glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl, seryl, threonyl, methionyl, aspartyl, glutamyl, aspara¬ ginyl, glutaminyl, histidyl, and the side-chain protected forms thereof; (4) Bg i _s - selected from the group consist¬ ing of asparaginyl, glutaminyl, glutamyl, arginyl, lysyl, seryl, threonyl, and the side-chain protected forms thereof; and, with respect to formulas (XI) and (XIII), (5) Y is selected from the group consisting of -NR R2,

-OR, and -CH2OR, wherein each R, Ri , and R2 ^is selected from a group consisting of hydrogen and straight and branched chain alkyl groups containing 1-6 carbon atoms; then at least one of - and B4 is selected such that it is not from a group consisting of tyrosyl, tryptophyl, phenylalanyl, and the side-chain protected forms thereof.

Preferably, the peptide resins of formula VI, and the protected peptide-compounds of formulas VIII-X, have the amino acid sequence wherein Bi and B4 ^{are se} ~ lected from the group consisting of histidyl, tryptophyl, phenylalanyl, -tyrosyl, homologues and analogues thereof, the side-chain protected forms thereof, and, with respect to Bi , the desamino fornis thereof; B2, B5, and B*5 are selected from the group consisting of D-histidyl, D- tryptophyl, D-phenylalanyl, D-tyrosyl, homologues and analogues thereof, and, with respect to B-5, the descar¬ boxy forms thereof, and the side-chain protected forms thereof; and B3 i _s selected from the group consisting of glycyl, alanyl, seryl, asparaginyl, prolyl, D-alanyl, D-

O

-32- seryl, D-asparaginyl, D-prolyl, homologues and analogues thereof, and the side-chain protected forms thereof.

Preferably, the peptide resins of formula VII, and the protected peptide-compounds of formulas XI-XIII, have the amino acid sequence wherein Bi and B4 are se¬ lected from the group consisting of histidyl, tryptophyl, phenylalanyl, tyrosyl, homologues and analogues thereof, the side-chain protected forms thereof, and, with respect to Bi , the desamino forms thereof; B2 and B5 ^are selected from the group consisting of D-histidyl, D-tryptophyl, D- phenylalanyl, D-tyrosyl, homologues and analogues there¬ of, and the side-chain protected forms thereof; B3 i _Ξ selected from the group consisting of glycyl, alanyl, seryl, asparaginyl, prolyl, D-alanyl, D-seryl, D-aspara¬ ginyl, D-prolyl, homologues and analogues thereof, and the side-chain protected forms thereof, and Bg and B' _β are selected from the group consisting of arginine, ly- sine, ornithine, histidine, aspartic acid, gl-utamic acid, asparagine, glutamine, D-arginine, D-lysine, D-ornithine, D-histidine, D-aspartic acid, D-glutamic acid, D-aspara- gine, D-glutamine, D-arginine, homologues and analogues thereof, and the descarboxy forms thereof; and, with respect to B'6' ^fc - ^le descarboxy forms thereof, and the side-chain protection forms thereof.

More preferably, the peptide-resins of this invention are represented by formulas XIV and XV and the protected peptide-compounds are represented by formulas XVI-XXI:

< ^V l ⁾ m ( ^pr ₁ ) r- ( X ' ^V ₂ ) a Trp-B3-B4-B5-« ( χiV) b

-33-

( Pr i ) _r - ( χ ₂ (XV)

(Prι)q

( f ,»v-) m

^(Pr 1)r-(X2 ^lv -a-Bι-D-Trp-B3-B4-B ^, 5-Y (XVI) (X3 ^iv )b

( ^Pr 1 ) r- ( 2 ^iV (XVII) (έ ι) _s

^B l-D-Trp-B3-B4-B' 5-Y (XVIII)

( Pr ^■ ] ) _r _ ( χ ₂ ^v ) ^* g-Y ( XIX)

( ^p ι) _s

( ^Pr 1 ) r- ( (XX)

B ^* -D-Trp-B3-B4-B5-B6-0-Y (XXI)

^{"" "} -34- wherein B1 is selected from the group consisting of tyro¬ syl, O-methyltyrosyl, histidyl, 3-N-methylhistidyl, p- chlorophenylalanyl, the desamino forms thereof, and the side-chain protected forms thereof; B i _s selected from the group consisting of alanyl, seryl, D-alanyl and the side-chain protected forms thereof; B4 i _s selected from the group consisting of tryptophyl, tyrosyl and the side- chain protected forms thereof; B _{5 an<} ^* ι - ₃ -5 are selected from the group consisting of D-phenylalanyl, D-histidyl, D-tyrosyl, D-p-chlorophenylalanyl, and, with respect to B'π the descarboxy forms thereof, and the side-chain protected forms thereof and Bg _an a B'6 are selected from the group consisting of arginine, homoarginine, lysine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine, and D-lysine.

Suitable α-amino acid protecting groups Pr- _{j r} include, but are not limited to, tertiary-butyloxycar- bonyl (BOC) , isoamyloxycarbonyl (AOC) , o-nitrophenylsul- fenyl (NPS), fluoroenylmethyloxycarbonyl (FMOC), o-nitro- pyridinylsulfenyl (NPYS) , and biphenylproploxycarbonyl (BPOC).

Suitable carboxyl protecting groups, Pr in¬ clude, but are not limited to, salts (e.g., Li ⁺ , Na ⁺ CS ⁺ , etc.), methyl, ethyl, benzyl, benzhydryl, substituted benzyl, phthalimidomethyl, tertiary butyl, phenacyl, phenyl, 4-picolyl, 2-methylthioethyl, 2(p-toluenesul- fonyl)ethyl, ^" 2(p-nitrothiophenyl)ethyl, p-methylthio- phenyl, and hydrazides.

In addition to the resins, ®, noted above, other resins include, but are not limited to, phenyl- acetamidomethyl (PAM) , chloromethyl, and poly-N-acrylpyr- rolidine resins.

-35-

Virtually any suitable N-terminal and desamino alpha-carbon substitution and radical can be used in the instant invention. Typical N-terminal and desamino alpha-carbon substitutions and radicals include, but are not limited to, those set forth in Table V.

OT\ϊPI r

TABLE V N-Terminus and Desamino Substitutions and Radicals ¹

N-Terminus Substitutions and Radicals

R---(R,÷H) R ₂ -(R ₂ =fH) O O O O

R ₂ ^* 0 O 0 O

Rr -(R _j ^H) R ₂ -(R ₂ ≠H) R ₃ -(R ₃ -≠H) O O U) σ.

0 II

Rr -z- -c- R ₂ - 0 0 0 6

O

II

Rr -C- R ₂ - O O O O

TABLE V (Contd.)

^■ ?2

-N- — — o 0 0

-

R ₂ Z R _] -N-C-(R ₁ aR ₂ ≠H) R ₃ — (R ₃ -^H) — 0

U) cn

R--N-C— (R!aR ₂ ≠H) — — 0 0 0 I

Desαmino Substituents and Radicals

R- O O O O O

RZ-(R=^H) O O O O O

-37- The growth hormone releasing peptides of formu¬ las I and II and combinations thereof (including combinations of formula III) with at least one growth promoting agent are useful in ^* vi-tro as unique tools for understanding how growth hormone secretion is regulated at the pituitary level. This includes use in the evaluation of many factors thought or known to influence growth hormone secretin such as age, sex, nutritional factors, glucose, amino acids, fatty acids, as well as fasting and non-fasting states. In addition, the peptides of this invention can be used in the evaluation of how other hormones modify growth hormone releasing activity. For example, it has already been established that somatostatin inhibits growth hormone release. Other hormones that are important and in need of study as to their effect on growth hormone release include the gonadal hormones, e.g., testosterone, estradiol, and progesterone; the adrenal hormones, e.g., cortisol and other corticoids, epinephrine and .norepinephrine; the pancreatic and gastrointestinal hormones, e.g., insulin, glucagon, gastrin, secretin; the vasoactive intestinal peptides, e.g., bombesin; and the thyroid hormones, e.g., thyroxine and triiodothyronine. These peptides and combinations of this invention can also be employed to investigate the possible negative or positive feedback effects of some of the pituitary hormones, e.g., growth hormone and endorphin peptides, on the pituitary to modify growth hormone release. Of particular scientific importance is- the use of these peptides to elucidate the subcellular mechanisms mediating the release of growth hormone.

The peptides and combinations of this invention can also be administered to animals, including man, to release growth hormone in--vivo. For example, the peptides can be administered to commercially important

-38- animals such as swine, cattle, sheep and the like to accelerate and increase their rate and extent of growth, and to increase milk production in such animals. In addition, these peptides can be administered to humans i ^* π vivo as a diagnostic tool to directly determine whether the pituitary is capable of releasing growth hormone. For example, the peptides and combinations can be administered in* ^* vivo to children. Serum samples taken before and after such administration can be assayed for growth hormone. Comparison of the amounts of growth hormone in each of these samples would be a means for directly determining the ability of the patient's pituitary to release growth hormone.

Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the peptides or combinations of this invention in association with a pharmaceutical carrier or diluent. Optionally, the ac¬ tive ingredient of the pharmaceutical compostions. can comprise a growth promoting agent in addition to at least one of the peptides of formulas I or II or another compo¬ sition which exhibits a different activity, e.g., an tibiotic or other pharmaceutically active material.-

Growth promoting agents include, but are not limited to, TRH, diethylstilbesterol, theophylline, enke- phalins, E series prostaglandins, compounds disclosed in U.S. Patent 3,239,345, e.g., zeranol, and compounds dis¬ closed in U.S. Patent 4,036,979, e.g., sulbenox.

The peptides of this invention can be adminis¬ tered by oral, parenteral (e.g., intramuscular, intra- peritoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or topical

-39- routes of administration and can be formulated in dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is ad¬ mixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, addition¬ al substances other than inert diluents, e.g., lubricat¬ ing agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can addi¬ tionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert dilu¬ ents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adju¬ vants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

Preparations according to this- invention for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, or emulsions. Examples of. non-aqueous solvents or -vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for exam¬ ple, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the composi¬ tions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form

-40- of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

Compositions for rectal or vaginal administra¬ tion are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.

Compositions for nasal or sublingual adminis¬ tration are also prepared with standard excipients well known in the art.

The dosage of each active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable doage form is obtained. The se¬ lected dosage depends upon the desired therapeutic ef¬ fect, on the route of administration, and on ^" the duration of the treatment. Generally, dosage levels per active - ingredient of between 0.001 to 10 mg/kg. of body weight daily are administered to animals, e.g., mammals, to obtain effective release of growth hormone.

The following examples are provided for the purpose of further illustration only and are not intended to be limitations on the disclosed invention.

Example

Synthesis ^* p-f H2***His-D-Trp-Ala-Trp-D-Phe- ^* NH2

Pa a-methylbenzhydrylamine hydrochloride (p-Me- BHA.HCl) resin was placed in a reaction vessel. The following procedure, starting at step 6, was then em¬ ployed in conjunction with a Beckman brand Peptide Synthesizer Model No. 990 in preparing the peptide \\ - His-D-Trp-Ala-Trp-D-Phe-NH ₂ . _τhe synthesis was started

. - -4 1 - at step 6 because there was no amino acid present in the resin and one need only neutralize the resin which was initially in the HC1 form.

1. Wash with methylene chloride (CH2CI2) for 1.5 minutes, three times.

2. Treat with trifluoroacetic acid - methylene chloride (40% TFA/CH2CI2' ^{v v} ) containing 0.1% indole for 1.5 minutes.

3. Repeat Step 2 for 20 minutes.

4. Wash with chloroform (CHCI3) ^for 1 - ⁵ minutes, three times.

5. Wash with 30% ethanol-methylene chloride (30% EtOH/CH2Cl2' ^v / ^{v) for 1} - ⁵ minutes, two times.

6. Wash with CH ₂ ci ₂ for 1.5 minutes, three times.

7. Treat with 10% triethylamine in CH2CI2 C-0% TEA/CH ₂ Cl2, V/V) for 1.5 minutes.

8. Repeat Step 7 for 10 minutes.

9. .Wash with CH ₂ Cl2 for 1.5 minutes, three times.

10. Add to the washed resin 2.5 equivalents of the appropriate protected amino acid in dimethyl formamide- methylene chloride (D F-CH2CI2 ⁾ •

11. Add 0.5N dicyclohexylcarbodiimide in CH d

(DCC/CH2CI2) ^{* more} than 2.5 equivalents.

12. Rinse addition funnel with CH ₂ ( ^* ;ι _an< ^* | _a dd rinse to the reaction vessel.

13. Stir the reagents in Steps 10-12 for 2 hours or more.

14. Wash with CH ₂ ci ₂ for 1.5 minutes, three times.

15. Wash with D F for 1.5 minutes.

16. Wash with CH ₂ ci ₂ for 1.5 minutes, two times.

17. Test by ninhydrin reaction according to the procedure of Kaiser et al. , Anna -.- Biochem , 34;595 (1970).

18. If Step 17 shows complete reaction, repeat the above procedures starting from Step 1 employing the next

-42- protected amino acid. If Step 17 shows incomplete reac¬ tion, repeat Steps 7-17.

The above procedure was employed using the following sequence of amino acids:

Boc-D-Phe Boc-Trp Boc-Ala Boc-D-Trp Boc-His(Tos*) *Tos denotes p-toluenesulfonyl.

After completion of the synthesis of the de¬ sired peptide resin, the reaction vessel containing the peptide resin was then placed in a dessicator and dried overnight under a vacuum. The dried peptide resin was removed from the reaction vessel and placed in another vessel suitable for HF cleavage. This latter vessel also contained a magnetic stirring bar. A quantity of anisole sufficient to wet the peptide resin was added to this vessel. The vessel was next connected to an HF line and placed under a vacuum to remove any air therein. The vessel was then cooled to about -78°C. with a dry ice- acetone bath. Doubly distilled HF (about 10 ml/gm of peptide resin) was added to the vessel. The dry ice- acetone bath was then removed from the vessel and re¬ placed by an ice-water bath. The vessel's contents were vigorously stirred for about 45 minutes while the vessel remained immersed in the ice-water bath. Most of the HF in the vessel was then removed by water aspiration. After the majority of HF was removed by water aspiration, the remaining HF and anisole were removed via a vacuum pump.

-43-

The vessel's contents were washed with about 100 ml of ether to further remove any residual anisole.

The peptide was removed from the resin by ex¬ traction with aqueous acetic acid (aq»H0Ac). The aq-HOAc was lyophilized off to yield a fluffy peptide powder.

The peptide was then purified by partition chromatography or counter current distribution (CCD) employing a butanol:HOAc:water (4:1:5) system. When further purification was necessary, a Pharmacia LH-20 brand chromatography column was also employed.

E- ample--2 Synthesis-:o-f-Η2'-Tyr"D--*Trp--Ala-Tτp-- ⁱ D--HiS-NH2 The procedure set forth in Example 1 can be employed to synthesize the peptide H ₂ -Tyr-D-Trp-Ala-Trp- D-His-NH2 employing the following sequence of amino acids:

Boc-D-His(Tos) Boc-Trp Boc-Ala Boc-D-Trp Boc-Tyr(BrZ*) *BrZ denotes o-bromobenzyloxycarbonyl

Example 3

Sy ^j thesi^ ^.. of. ^, H ₂ -Hi-s-D-Trp-Ala-Trp-D-Tyr-NH2 The procedure set forth in Example 1 can be employed to synthesize the peptide H2- ^His - ^D - ^Tr P- ^Ala ~ ^Tr P" D-Tyr- ^N H ₂ employing the following sequence of amino acids:

Boc-D-Tyr (BrZ) Boc-Trp

-44-

Boc-Ala Boc-D-Trp Boc-His (Tos)

E-χamp ^* le ^** 4 Synthesl ^* s--o-f ^* Η -^H-is--D"Trp-Ala-Trp-D-Hi s mi2 The procedure set forth in Example 1 can be employed to synthesize the peptide H -His-D-Trp-Ala-Trp- D-His-NH2 employing the following sequence of amino acids:

Boc-D-His (Tos) Boc-Trp Boc-Ala Boc-D-Trp Boc-His (Tos)

Example--5

Synthesis--o ^* f- ^* H^"Tyr^D-Trp--A-la--T ρ-"D^ --Cl--Ph-e---- H . The procedure set forth in Example 1 can be employed to ^' synthesize the peptide H ₂ -T -D-Trp-Ala-Trp- D-p-Cl-Phe-NH2 employing the following sequence of amino acids:

Boc-D-p-Cl-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-Tyr(BrZ)

Example- 6 Synthesis of H2-Tyr-D-Trp--D-A a-Trp-D-Phe-NH2 The procedure set forth in Example 1 can be employed to synthesize the peptide H ₂ -Tyr-D-Trp-D-Ala- Trp-D-Phe-NH2 employing the following sequence of amino acids :

-45-

Boc-D-Phe Boc-Trp Boc-D-Ala Boc-D-Trp Boc-Tyr ( BrZ )

Example- 7

Synthesis- of ^• H2-p- ¹ Cl-Phe-D-Trp-Ala-Trp-D"-Phe^-NH-2 • The procedure set forth in Example 1 can be employed to synthesize the peptide H ₂ _p_ci-phe-D-Trp-Ala- Trp-D-Phe-NH2 employing the following sequence of amino acids:

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-p-Cl-Phe

Example--8

Synthesis- ^* of ^* H-de-samiπoTyT-D-Trp^-Al-a-Trp^-D^-Ph-e-NH^ The procedure set forth in Example 1 can be employed to synthesize the peptide H-desaminoTyr-D-Trp-

Ala-Trp-D-Phe-NH2 employing the following sequence of amino acids:

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

3(p-OH-phenyl)propanoic acid

-46- Ex ample-- 9

H S. th sis. of..CK ₃ co--Ty --D--Trp--Al-a--Trp-D _' -Phe-4NH-2 The procedure set forth in Example 1 can be employed with several modifications to synthesize the peptide H

^CH 3CO-Tyr-D-Trp-Ala-Trp-D-Phe-NH2 employing the following sequence of amino acids:

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-Tyr(BrZ)

The modifications consisted of the following additional steps after the last protected amino acid, Boc-Tyr(BrZ) was added to the peptide resin: ^' -

19. The Boc group was removed from the peptide resin by TFA.

20. The resulting peptide ^" resin was washed with

CH2CI2 **- ^{or '} '•--' minutes, two times.

21. Acetic anhydride (2.5 molar excess) and 2.5 molar excess of pyridine were added and stirred for about 10 minutes.

22. Repeat Step 20.

The same drying and purification steps as used in Example 1 were then employed to obtain the desired peptide.

Example ^* 10 S-yn ^. th ^. es ^. is..of H ₂ -Q-Me-Tyr-D-Trp-Ala-Trp-D-Phe-NH2 The procedure set forth in Example 1 can be

-47- employed to synthesize the peptide H2-0-Me-Tyr-D-Trp-Ala- Trp-D-Phe-NH ₂ employing the following sequence of amino acids:

Boc-D-Phe Boc-Trp Boc-Ala Boc-D-Trp Boc-O-Me-Tyr(BrZ)

Example- ^* 1 ^* 1 Synthesis*-of ^* H2"Tyr--D-Trp-- ⁱ A-la-Trρ-D-'Phe---Met- H2 BHA*HCl resin was placed in a reaction ves¬ sel. The following procedure was then employed in con¬ junction with a Beckman brand Peptide Synthesizer Model No. 990 in preparing the hexapeptide H ₂ -τyr-D-Trp-Ala- Trp-D-Phe-Met-NH2 ^:

1. Methylene chloride (CH ₂ ci2; about. ^' IO ml/gm of BHA*HCl resin) was added to the reaction vessel. The BHA* HCl resin was washed with vigorous stirring for about 1.5 minutes. The CH2CI2 solution wa^ then drained from the reaction vessel. This washing -step was repeated once.

2. A triethyla ine solution ( (Et3 )/CH2Cl2 (10:90); about 10 ml/gm BHA*HCl resin) was added to the washed BHA-HCl resin in the reaction vessel. The result¬ ing mixture was vigorously stirred for about 1.5 min¬ utes. The solution was then drained from th reaction vessel.

3. Another Et ₃ /CH2Cl ₂ (10:90) solution (about 10 ml/gm BHA*HCl) was added to the reaction vessel. The BHA*" HCl resin was neutralized by vigorous stirring for about 20 minutes. The solution was then drained from the reaction vessel.

4. CH ₂ cl ₂ (about 10 ml/gm of BHA-HCl resin) was added to the reaction vessel. The resulting mixture was

422D-154

-48- vigorously stirred for about 1.5 minutes. The solution was then drained from the reaction vessel. This pro¬ cedure was repeated an additional two times.

5. Tertiarybutyloxycarbonyl-methionine (Boc-Met; about 2.5 times the theoretical amount of the total bind¬ ing capacity of the BHA-HCl resin originally placed in the reaction vessel) in about 50 ml of dimethylformamide- methylene chloride solution (DMF-CH ₂ ci2 (1:9)) was added to the reaction vessel. The resulting mixture was vigor¬ ously stirred for about 1.5 minutes.

6. A 0.5 molar (M) dicyclohexylcarbodiimide (DCC) in CH ₂ ci2 solution (about 2.5 times the theoretical amount of total binding capacity of the BHA*HCl resin originally placed in the reaction vessel) was added to the reaction vessel. The resulting mixture was vigorous¬ ly stirred until a negative ninhydrin test was obtained (about 12.0 minutes). The solution was. then drained from the reaction vessel.

7. CH ₂ Cl2 (about 10 ml/gm of BHA-HCl resin) was added to the reaction vessel. The resulting solution was vigorously stirred for about 1.5 minutes. The solution was then drained from the reaction vessel. This washing procedure was repeated once.

8. DMF (about 10 ml/gm of BHA-HCl resin) was added to the reaction vessel. The resulting mixture was stirred for about 1.5 minutes. The solution was then drained from the reaction vessel.

9. CH ₂ ci2 (about 10 ml/gm of BHA-HCl resin) was added to the reaction vessel. The resulting mixture was vigorously stirred for about 1.5 minutes. The solution was then drained from the reaction vessel. This washing procedure was repeated an additional two times.

10. A trifluoroacetic acid/methylene chloride solu¬ tion ⁽ TFA/CH ₂ cl ₂ (40:60); about 10 ml/gm of BHA-HC1 resin) was added to the reaction vessel. The resulting mixture was vigorously stirred for about _. 1.5 minutes.

422D-154

-49-

The solution was then drained from the reaction vessel. ^*

11. Another TFA/CH ₂ ci ₂ (40:60) solution (about 10 ml/gm of BHA.HCl resin) was added to the reaction ves¬ sel. The resulting mixture was vigorously stirred for about 20 minutes. The solution was then drained from the reaction vessel.

12. CH ₂ ci ₂ (about 10 ml/gm of BHA-HCl resin) was added to the reaction vessel. The resulting solution was vigorously stirred for about 1.5 minutes. The solution was then drained from the reaction vessel. This washing procedure was repeated once.

13. A triethylamine solution ( (Et3 )/CH2Cl2 (10:90); about 10 ml/gm BHA HCl resin) was added to the washed BHA*HC1 resin in the reaction vessel. The result¬ ing mixture was vigorously stirred for about 1.5 min¬ utes. The solution was then drained from the reaction vessel.

14. Another Et ₃ /CH2Cl2 (10:90) solution (about 10 ml/gm BHA-HCl) was added to the reaction vessel. The BHA-

HCl resin was neutralized by vigorous stirring for about 20 minutes. The solution was then drained from the reaction vessel.

15. Chloroform (CHC1 _3; about 10 ml/gm of BHA-HCl resin) was added to the reaction vessel. The resulting mixture was vigorously stirred for about 1.5 minutes. The solution was then drained from the reaction vessel.

16. An ethanol/methylene chloride solution (EtOH/CH2Cl2 (30:70); about 10 ml/gm of BHA-HC1 resin) was added to the reaction vessel. The resulting mixture was vigorously stirred for about 1.5 minutes. The solu¬ tion was then drained from the reaction vessel. This washing step was repeated once.

Steps 4 through 16 were then repeated employing the following sequence of amino acids:

-50- Boc-Trp Boc-Ala Boc-D-Tr . Boc-Tyr (BrZ*) *BrZ denotes o-bromobenzyloxycarbonyl

After completion of the synthesis of the de¬ sired peptide resin, the reaction vessel containing the peptide resin was then placed in a dessicator and dried overnight under a vacuum. The dried peptide resin was removed from the reaction vessel and placed in another vessel suitable for HF cleavage. This latter vessel also contained a magnetic stirring bar. A quantity of anisole sufficient to wet the peptide resin was added to this vessel. The vessel was next connected to an HF and placed under a vacuum to remove any air therein. The vessel was then cooled to about -78 ^* C. with a dry ice- acetone bath. Doubly distilled HF (about 10 -.ml/gm of peptide resin) was added to the vessel. The dry ice- acetone bath was then removed from the vessel and re¬ placed by an ice-water bath. The vessel's contents were vigorously stirred for about 45 inutes while the vessel remained immersed in the ice-water bath. Most of the HF in the vessel was then removed by water aspiration, the remaining HF and anisole were removed via a vacuum pump.

The vessel's contents were washed with about 100 ml of ether to further remove any residual anisole.

The peptide was removed from the resin by ex¬ traction with 30% aqueous acetic acid (aq.HOAc) . The aq.HOAc was lyophilized off to yield a fluffy peptide powder.

-51-

The peptide was then purified by partition chromatography or counter current distribution (CCD) employing a butanol: HOAc: water (4:1:5) system. When further purification was necessary, a Pharmacia LH-20 brand chromatography column was also employed.

Example--12 Synthesis .of..H. ₂ _τ _y . _r -D-Tr-p-Ala-Trp-D-Phe-Thr-NH-2

The procedure set forth in Example 11 was em¬ ployed to synthesize the hexapeptide H2~ ^τ y ^r ~ ^D ~ ^Tr P'" ^Ala ~ Trp-D-Phe-Thr-NH ₂ employing the following sequence of amino acids:

Boc-Thr(Bzl*) Boc-D-Phe Boc-Trp Boc-Ala Boc-D-Trp Boc-Tyr(BrZ) *Bzl denotes benzyl.

Example- 13

Synthesis- of -H ₂ ^ _Tyr ._ ^, _D -_> _Tr p-Ri _a - _Tr p-- _D -phe-GIn--OH

The procedure set forth in Example 11 was em¬ ployed to synthesize the hexapeptide H2~ ^τ y ^r ~ ^D ~ ^Tr P~ ^Ala ~ Trp-D-Phe-Gln-OH employing the following sequence of amino acids:

Boc-Glu-α-benzyl ester

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-Tyr(BrZ)

-52-

Exam le--V4

Syn h ^* esis--o ^* f-Η2^^ ^{r E} ^'^P" ^A ^ ^g ' ^* '^^F ^{^E} ^^ ^e*,* ' ⁶ ^ ^nw N- ^H

The procedure set forth in Example 11 was em¬ ployed with several modifications .to synthesize the hexa¬ peptide H2~Tyr-D-Trp-Ala-Trp-D-Phe-Gln-NH2- ^τhe modifi ^¬ cations consisted of:

(1) Omitting steps 2-4 of Example 11 and replacing them with a single step which entailed dissolving Boc-Gln (about 5 times the theoretical amount of the total bind¬ ing of the BHA-HCl resin originally placed in the reac¬ tion vessel) in 10 ml of CH ₃ ci ₂ -DMF solution (3:2) pres¬ ent in a round bottom flask. The flask and its contents was cooled in ice and then DCC (about 1.25 times the theoretical amount of the total binding capacity of the BHA-HC1 resin originally placed in the reaction vessel) was added to the flask. The resulting mixture was stirred in an ice bath for about 25-30 minutes. The DCC urea precipitate formed by the reaction between Boc-Gln and DCC was separated from the supernatant via filtra¬ tion. The supernature comprising the CH2Cl2~ ^DMF solution having a symmetrical anhydride (also formed by the reac¬ tion between Boc-Gln and DCC) dissolved therein was added to the reaction vessel. The resulting mixture was stirred until a negative ninhydrin test was obtained. The solution was then drained from the reaction vessel.

(2) After completing steps 7 through 16 of Example - 11, steps 4 through 16 of Example 11 were then repeated, without modification, employing the following sequence of amino acids:

Boc-D-Phe Boc-Trp Boc-Ala Boc-D-Trp Boc-Tyr(BrZ)

OMPI

-53-

Examp e ^** 1 ^* 5 Synthesis -o-f--H ^* -* ^* ry-r*-*D-Tτρ--'A-la ^* --Tτp^D--Phe-Α n-NΗ7

The procedure set forth in Example 11 was em¬ ployed with several modifications ^" to synthesize the hexa peptide H2-Tyr-D-Trp-Ala-Trp-D-Phe-Asn-NH2- ^τhe modifi ^¬ cations consisted of:

(1) Omitting steps 2-4 of Example 11 and replacing them with a simple step whic entailed dissolving Boc-Asn (about 5 times the theoretical amount of the total bind¬ ing capacity of the BHA-HC1 resin originally placed in the reaction vessel) in about 50 ml of DMF in a suitable flask. The resulting solution was added to the reaction vessel to form a mixture. The mixture was vigorously stirred until a negative ninhydrin test was obtained. The solution was then drained from the reaction vessel.

(2) After completing steps 7 through 16 of Eample 11, steps 4 through 16 of Example 11 were then repeated, without modification, employing the following- sequence of amino acids:

Boc-D-Phe Boc-Trp~ Boc-Ala Boc-Tyr(BrZ)

Example--16 Synthesis--of ^• H2'-His-D--Trp-D-Phe-Lys-NH7 Para-methylbenzhydrylamine hydrochloride (p-Me-

BHA.HC1) resin was placed in a reaction vessel. The following procedure, starting at step 6, was then em¬ ployed in conjunction with a Beckman brand Peptide

Synthesizer Model 990 in preparing the peptide H2 ^~His_D~ Trρ-Ala-Trp-D-Phe-Lys-NH ₂ . _T he synthesis was started at step 6 because there was no amino acid present in the

-54- resin and one need only neutralize the resin which was initially in the HCl form.

1. Wash with methylene chloride (CH2CI2) for 1.5 minutes, three times.

2. Treat with trifluoroacetic acid - methylene chloride (40% TFA/CH2CI2- ^v / ^v - containing 0.1% indole for 1.5 minutes.

3. Repeat Step 2 for 20 minutes.

4. Wash with chloroform (CHCI3) ^for • - ⁵ minutes, three times.

5. Wash with 30% ethanol-methylene chloride (30% EtOH/CH2Cl2' ^{v v} ) f° ^r 1-5 minutes, two times.

6. Wash with CH ₂ ci ₂ for 1.5 minutes, three times.

7. Treat with 10% triethylamine in CH2CI2 ^(10% - TEA/CH ₂ ci ₂ , v/V) for 1.5 minutes.

8. Repeat Step 7 for 10 minutes.

9. Wash with CH ₂ ci2 for 1.5 minutes, three times.

10. Add to the washed resin 2.5 equivalents of the appropriate protected amino acid in dimethyl formamide- methylene chloride (DMF-CH2CI2 ⁾ •

11. Add 0.5N dicyclohexylcarbodiimide in CH ₂ ci2 (DCC/CH2C12-- ' more than 2.5 equivalents.

12. Rinse addition funnel with CH ₂ ci2 and add rinse to. the reaction vessel.

13. Stir the reagents in Steps 10-12 for 2 hours or more.

14. Wash with CH ₂ ci ₂ for 1.5 minutes, three times.

15. Wash with DMF for 1.5 minutes.

16. Wash with CH ₂ ci ₂ for 1.5 minutes, two times.

17. Test by ninhydrin reaction according to the procedure of Kaiser et al., Annal.--Biocheπr. , 34:595 (1970) .

18. If Step 17 shows complete reaction, repeat the above procedures starting from Step 1 employing the next

-55- protected amino acid. If Step 17 shows incomplete reac¬ tion, repeat Steps 7-17.

The above procedure was- employed using the following sequence of amino acids:

Boc-Lys(ClZ ⁺ )

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-His(Tos*) +C1Z denotes o-chloro-benzyloxycarbonyl. *Tos denotes p-toluenesulfonyl.

After completion of the synthesis of the de¬ sired peptide resin, the reaction vessel containing the peptide ^' resin was then placed in a dessicator and dried overnight under a vacuum. The ^' dried peptide. resin was removed from the reaction vessel and placed in another vessel suitable for HF cleavage. This latter vessel also contained a magnetic. stirring bar. A quantity of anisole sufficient to wet the peptide resin was added to this vessel. The vessel was next connected to an HF line and placed under a vacuum to remove any air therein. The vessel was then cooled to about -78°C. with a dry ice- acetone bath. Doubly distilled HF (about 10 ml/gm of peptide resin) was added to the vessel. The dry ice- acetone bath was then removed from the vessel and re¬ placed by an ice-water bath. The vessel's contents were vigorously stirred for about 45 minutes while the vessel remained immersed in the ice-water bath. Most of the HF in the vessel was then removed by water aspiration. After the majority of HF was removed by water aspiration, the remaining HF and anisole were removed via a vacuum pump.

Oϊv-

-56-

The vessel's contents were washed with about 100 ml of ether to further remove any residual anisole.

The peptide was removed from the resin by ex¬ traction with aqueous acetic acid (aq.HOAc). The aq.HOAc was lyophilized off to yield a fluffy peptide powder.

The peptide was then purified by partition chromatography or counter current distribution (CCD) employing a butanol:HOAc: ater (4:1:5) system. When further purification was necessary, a Pharmacia LH-20 brand chromatography column was also employed.

Example--17

Synthesis ^*• o-f ^* -H2 Tyr-D--Trp-A-la-^Trp-^D^Phg-Lys--NH-?

The procedure set forth in Example 16 can be employed to synthesize the peptide H2-Tyr-D-Trp-Ala-Trp- D-Phe-Lys—NH2 employing the following sequence of amino acids:

Boc-Lys(ClZ) Boc-D- _~ Phe Boc-Trp Boc-Ala Boc-D-Trp Boc-Tyr(BrZ*) *BrZ denotes o-bromobenzyloxycarbonyl

Example ^* -18

Synthesis ^, of..H ₂ _.Tyr-D~Trp-Ala~Trp-D-Phe-Glu~NH-2

The procedure set forth in Example 16 can be employed to synthesize the peptide H2- ^τ Y ^r - ^D - ^r P ^~Ala ~ ^Tr " D-Phe-Glu-NH ₂ employing the following sequence of amino acids

£ --_

-57-

Boc-Glu-γ-Bzl* Boc-D-Phe Boc-Trp Boc-Ala . Boc-D-Trp Boc-Tyr(BrZ) *γ-Bzl denotes γ-benzyl ester

Example"19

Synthe is..of. ^. H ₂ --p _yr - _D - _Tr .p-Al-a-Trp-D--Phe-Phe-NH-2

The procedure set forth in Example 16 can be eraployed to synthesize the peptide H2~ ^τ y ^r-D ~ ^Tr ~ ^A -*- ^a ~'--' ^{r -} D-Phe-Phe-NH ₂ employing the following sequence of amino acids:

Boc-Phe

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-Tyr(BrZ)

Example ^* 20 Synthesls-'Q-f-H2 ^* -Tyr---D--^Trp^Gly-- ^r I_ ^* rp^D--Phe--^G-ln--NH2

The procedure set forth in Example 16. can be employed with one modification to synthesize the peptide

H2-Tyr-D-Trp-Gly-Trp-D-Phe-Gln-NH2 employing the follow ^¬ ing sequence, of amino acids:

Boc-Gln-ONP* Boc-D-Phe Boc-Trp Boc-Gly Boc-D-Trp Boc-Tyr(BrZ) *ONP denote p-nitrophenyl ester.

θ:v.?l

-58-

The sole modification was made just in the procedure for coupling Boc-Gln-ONP to the resin. This modification consisted of the omission of step 11 of Example 16 in this one coupling procedure.

Example 21 Synthesis of H ₂ -His-D-Trp-Ala-Trp-D-Phe-Lys-OH

The procedure set forth in Example 16 can be employed with several modifications to synthesize the peptide H ₂ _His-D-Trp-Ala-Trp-D-Phe-Lys-OH employing the following sequence of amino acids:

Boc- ys(ClZ)

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-His(Tos) -. ^' -- ^''

The modifications were as follows:

1. A hydroxymethyl resin-was employed instead of the p-ME-BHA-HCl resin.-

2. Steps 6-18 were modified as follows just in the procedure for.coupling Boc-Lys(C1Z) to the hydroxymethyl resin: a. Step 6 was the same; b. Steps 7 through 9 were omitted; c. Steps 10 and 11 were the same; d. Step 12 was omitted and the following procedure was substituted therefor:

Add 2.5 equivalents of N,N-dimethylaminopyridine (DMAP). e. Steps 13 through 16 were the same;

-59- f. Steps 17 and 18 were omitted and the fol¬ lowing procedure was substituted therefor:

Dry the amino acid-resin under vacuum until a con¬ stant weight is obtained. If the ^" final constant weight is equal to the sum of the weights of the resin and Boc- amino acid added to the reaction vessel, then add 10 equivalents of benzoyl chloride and 10 equivalents of pyridine to deactivate any unused hydroxymethyl resin. Wash as set forth in steps 15 and 16. Then employ steps 1-18 of Example 16 for remaining protected amino acids.

If the final constant weight is not equal to the sum of the weights of the resin and Boc-amino acid added to the reaction vessel, then repeat steps 10 to end as modified above in this example.

Example 22

Synthesis of H ₂ -His-D-T ^' rp-Ala-Trp-D-Phe-Arg-NH2

The procedure set forth in Example- 16 can be employed to synthesize the peptide H2~ ^H i ^s "" ^D "~ ^Tr P ^-A --- ^a "~ ^Tr P" D-Phe-Arg-NH ₂ employing the following sequence of amino acids:

Aoc*-Arg(Tos) Boc-D-Phe Boc-Trp Boc-Ala Boc-D-Trp Boc-His(Tos) *Aoc denotes isoamyloxycarbonyl

Example 23 Synthesis of. H ₂ -His-D-Trp-Ala-Trp-D-Phe-Gln-NH ₂

The procedure set forth in Example 16 can -be employed with one modifcation to synthesize the peptide

- .

H2-His-D-Trp-Ala-Trp-D-Phe-Gln-NH2 employing the follow¬ ing sequence of amino acids:

Boc-Gln-ONP

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-His(Tos)

The sole modification was made first in the procedure for coupling Boc-Gln-ONP to the resin. This modification consisted of the omission of step 11 of Example 16 in this one coupling procedure.

Example 24

Synthesis of H ₂ -His-D-Trρ-Ala-Trp-D-Phe-Glu-NH2

The procedure set forth in Example--16 can be employed to synthesize the peptide H2"" ^H is-D-Trp-Ala-Trp- D-Phe-Glu-NH ₂ employing the following sequence of amino acids:

Boc-Glu-γ-Bzl

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-His(Tos)

Example 25

^S ynthesis of H ₂ _His-D-Trp-Ala-Trp-D-Phe-HomoArg-NH2 The procedure set forth in Example 16 can be employed to synthesize the peptide H -His-D-Trp-Ala-Trρ- D-Phe-HomoArg-NH ₂ employing the following sequence of amino acids:

422D-154

-61-

Boc-HomoArg(Tos)

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-His(Tos)

Example 26

^■ Synthesis of H -3-N-Me-His-D-Trp-Ala-Trp-b-Phe-Lys-NH2

The procedure set forth in Example 16 can be employed to synthesize the peptide H2~ ³ ~ ^N ~ ^Me "" ^His ~ ^D ~ ^Tr P~ Ala-Trρ-D-Phe-Lys-NH ₂ employing the following sequence of amino acids ^" :

Boc-Lys(ClZ)

Boc-D-Phe

Boc-Trp

Boc-Ala

Boc-D-Trp

Boc-3-N-Me-His

Example 27

Synthesis of H ₂ -His-D-Trp-Ala-Trp-D-Phe- ys-NHCH2CH3

The procedure set forth in Example 16 can be employed with several modifications to synthesize the peptide H ₂ _His-D-Trp-D-Ala-Trp-D-Phe-Lys-NHCH2CH3 employ¬ ing the following sequence of amino acids:

Boc-Lys(ClZ)

Boc-D-Phe

Boc-Trp

Boc-D-Ala

Boc-D-Trp

Boc-His(Tos)

_OMPI_ WHO

-62-

The following ethylamine (CH ₃ CH2NH2) cleavage procedure was employed in place of the HF cleavage pro¬ cedure of Example 16:

After completion of the synthesis of the de¬ sired peptide resin, the reaction vessel containing the peptide resin was then placed in a dessicator and dried overnight under a vacuum. The dried peptide resin was removed from the reaction vessel and placed in another vessel suitable for ethylamine cleavage. This latter vessel also contained a magnetic stirring bar. The ves¬ sel was placed in an ice bath and gaseous ethylamine was condensed into the vessel. The contents of the vessel was stirred overnight.

This cleavage procedure was then followed by the extraction and purifications procedures as set forth in Example 16.

Example 28

Synthesis of H ₂ -His-D-Trp-Ala-Trp-D-Phe-Orn-NH2

The procedure set forth in Example 16 can be employed to synthesize the peptide H2- ^His - ^D_Tr P- ^Ala - ^Tr ~ D-Phe-Orn-NH ₂ employing the following sequence of amino acids:

Boc-Orn(Z*) Boc-D-Phe Boc-Trp Boc-Ala Boc-D-Trp Boc-His(Tos) *Z denotes benzyloxycarbonyl

-63- Exa ple 29

Synthesis. of -H ₂ -His-D-Trp-Val-Trp-D-Phe- ys-NH2

The procedure set forth .in Example 16 can be employed to synthesize the peptide H2~ ^H i ^s ~ ^D ~ ^Tr P~ ^Va l~ ^Tr P" D-Phe-Lys-NH employing the following sequence of amino acids:

Boc-Lys(ClZ)

Boc-D-Phe

Boc-Trp

Boc-Val

Boc-D-Trp

Boc-His(Tos)

Example 30 Synthesis, of. H ₂ -His-D-.Trp-Ser-Trp-D-Phe-. ys-NH2

The procedure set forth in Example 16 can be employed to synthesize the peptide D-Phe-NH ₂ employing the following sequence of amino acids:

Boc- ys-(ClZ)

Boc-D-Phe

Boc-Trp

Boc-Ser(Bzl)

Boc-D-Trp

Boc-His(Tos)

Example 31

In Vitro Growth Hormone Release Study Female rats of the CD-1 strain were housed in a constant temperature room at 24°C. with 14 hours light and 10 hours darkness. The rats were fed Purina brand rat chow ab libitum. All studies were started between 0800 and 1000 hours.

-6 *-

Pituitaries were removed from 20 day old female rats. In each polytetrafluoroethylene beaker (10 ml) was incubated two pituitaries at 36 ^β C. in 1 ml of lactated Ringer's solution in a Dubnoff Shaker (90 cycles/min.) . Three beakers were emplyed for each dosage shown in Table VI. All medium in each beaker was removed each hour (e.g., P-] _f _>2 , I ₃ , .I4, I5) and then fresh medium was added back to each beaker. Each medium removed was as¬ sayed for GH, in duplicate, by a standard radioimmuno- assay (RIA) .

The growth hormone agonist of Example 1 was not added to the incubation mediums employed during the first hour of the incubation period (P- _j ) or to the incubation mediums employed during the second hour of the incubation period (P ). The growth hormone agonist of Example 1 was dissolved in dimethylsulfoxide (DMSO; 10:1, agonist:DMSO) , added to each incubation medium employed during the third hour of the incubation period (13)? to each medium employed during the third hour of the incuba¬ tion period (I3)/ to each medium employed during the fourth hour of the incubation period (I4) and, when per¬ formed, to each medium employed _^ -during the fifth hour of the incubation period (I5). The release of growth hor¬ mone was recorded as ΔGH values obtained from the three beakers per doage level measured at I _r ^ _r and, when performed, I5 are set forth in Table VI.

Example 32

In Vitro Growth Hormone Releae Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example 2 and the results therefrom are set forth in Table VII.

-65-

Exa ple 33 In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the • peptide of Example 3 and the results therefrom are set forth in Table VIII.

Example 34

In Vitro Growth Release Study The procedure set forth in Example 31 was em¬ ployed in an in- vitro growth hormone release study of the peptide of Example 4 and the results therefrom are set forth in Table IX.

Example 35 In Vitro Growth Hormone Release Study The ^' procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example 5 and the results therefrom are set forth in Table X.

Example 36

In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in- itro growth hormone release study of the peptide of Example 6 and the results therefrom are set forth in Table XI.

Example 37 In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example 7 and the results therefrom are set forth in Table XII.

OVSΪ

- -

Example- 38 In Vitro Growth Hormone Release Study The procedure set forth ?n Example 31 was em¬ ployed in an in ^* vitro growth hormone release study of the peptide of Example 8 and the results therefrom are set forth in Table XIII.

Example- 39

In Vitro Growth Hormone- Release Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth release study of the peptide of Example 9 and the results therefrom are set forth in Table XIV.

Example 40

In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example 10 and the results therefrom are set forth in Table XV.

Example- 41 In Vitro Growt Hormo-ήe Release Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of thepeptide of Example 11 and the results therefrom are set forth in Table XVI.

Examle 42

In Vitro- Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example 12 and the results therefrom are set forth in Table XVII.

OMFI

-67-

Examole 43 In- Vitro Growth- Hormone -Release Study The procedure set forth in Example 31 ws era- ployed in an in-vitro growth hormone release study of the peptide of Exmaple 13 and the results therefrom are set forth in Table XVIII.

Example 44

In -Vitro Growth Release- Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hor-none release study of the peptide of Example 14 and the -results therefrom are set forth in Table XIX.^

Examole 45

In Vitro Growth Hormone- Release Study

The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example, 15 and the results therefrom are set forth in Table XX.

Example 46

In- Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example 16 and the results therefrom are set forth in Table XXI.

Example 47

In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the peptide of Example 17 and the results therefrom are set forth in Table XXII.

-68-

Example 48 In Vitro Growth- Hormone- elease Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the peptide of Exmaple 18 and the results therefrom are set forth in Table XXIII.

Example 49 In- Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the peptide of Example 19 and the results therefrom are set forth in Table XXIV.

Example- 50 In- Vitro- Growth- Hormone- Release Study The procedure set forth in Example 31 was em¬ ployed in an in itro growth hormone release study of the peptide of Example 20 and the results therefrom are set forth in Table XXV.

Example 51

In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of th peptide of Example 21 and the results therefrom are set forth in Table XXVI.

Example 52

In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of th peptide of Exmaple 22 and the results therefrom are set forth in Table XXVII.

- -

Exa ple--53 In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in- itro growth hormone release study of the peptide of Exmaple 23 and the results therefrom are set forth in Table XXVIII.

Example 54 In Vitro- Growth Hormone Release Studv

The procedure set forth in Example 31 was em¬ ployed in an in- itro growth hormone release study of the peptide of Example 24 and the results therefrom are set forth in Table XXIX.

Example ^-* 55 In--Vitro ^* Growth- Hormone Release Study The procedure set.forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the peptide of Example 25 and the results therefrom are set forth in Table XXX;

Example 56 In Vitro Growth Hormone Release Studv

The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the peptide of Example 26 and the results therefrom are set forth in Table XXXI.

Example ^* 57 In--Vitro- Growth Hormone Release- Studv

The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the peptide of Example 27 and the results therefrom are set forth in Table XXXII.

OMPI -

- -

Examcrle - 58

In ^* Vitro- Growth ^* -Hormone- Re e-ase St-udy The procedure set forth in Example 31 was em¬ ployed in an m-vi ro growth hormone rlease study of thepeptide of Example 28 and the results therefrom are set forth in Table XXXIII.

Example 59

In Vitro Growth Hormone Release Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the peptide of Example 29 and the results therefrom are set forth in Table XXXIV.

Example- 60

In--Vitro Growth--Hormone- Release Study The procedure set forth in Example 31 was em¬ ployed in an in- itro growth hormone release study of the peptide of Example 30 and the results therefrom are set forth in Table XXXV.

TABLE VI

IN VIT O GEOWffl HORMONE RELEASE

Dosage' ¹ ΔGH ¹ p Value

H9-His-D-Trp-Ala-Trp-D-Phe-NH _?

-167 +_ 114 ——_.

1 206 +_ 168 NS ³

3 288 _+ 79 <0.01

10 2,005 _+ 203 <0.001

30 3,046 + 93 <0.001

1. The mean of 9 assays gi'ven in terms of ng/ml incubation medium + _^ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE VII

IN VITRO GROWTH HORMONE RELEASE

Dosage^ ΔGH' p Value

H-)-Tyr-I T^Ala-Trp-l -His-NIl-)

^■ 167 +_ 114

1 ^• 179 +_ 323 NS ³

3 •192 +_ 120 ' NS

10 802 _+ 302 NS ro

30 611 + 103 <0.001

1. ϊhe mean of 9 assays giyen in terms of ng/ml incubation medium jf standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE VTII

IN VITRO GROWTH HORMONE RELEASE

Dosage 2. ΔGH ¹ p Value^

H -His-_ Trp-Ala-Trp-D-Tyr-NH^

-236 +_ 125 —-—

1 -126 _+ 253 NS ³

10 -99 +_ 230 ι NS

100 -238 _+ 133 NS

1,000 2,598 + 284 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE IX

IN VITRO GROWTH HORMONE RELEASE

Dosage' ^* ΔGH 1 p Value ⁴

H _? -His-D-Trp-Ala-Trp-D-His-NH _?

-236 125 _-.—

1 -166 _+ 277 NS ³

10 -369 ^ 152 . NS

100 43 +_ 185 NS

1,000 1,501 + 222 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE X

IN VITRO GROWTH HORMONE RELEASE

Dosage'- ΔGH 1 p Value

H -Tyr-C^Trp-Ala-Trp-D-p-Cl-Ph&-NIl _?

28 +_ 54 __.„.

10 -87 _+ 81 NS ³

30 124 +_ 123 ι NS

300 103 _+ 77 NS u ^*

3,000 531 +_ 42 <0.001

30,000 489 + 138 <0.01

1. The mean of 9 assays given in terms of ng/ml incubation medium + standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XI

IN VITRO GROWTH HORMONE ^• RELEASE

Dosage ^* ' • ΔGH ¹ p Value

H ₉ -Tyr-I Trp-p--Ala-Trp-'D--Phe--NH-)

-257 +. 97

300 648 _+ 210 <0.02

* ^' 3,000 435 +_ 143 <0.02

30,000 ^{^} 1,136 + 190 <0.001

1. The mean of 6 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml,incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XII

IN VITRO GROWTH HORMONE RELEASE

Dosage ¹ - ^• ΔGH p Value ⁴

H ₉ -p-Cl-Phe-I Trp-Ala-Trp-D-Phe-NH _?

-218 +_ 161

30 -260 _+ 27 '1 NS ³

300 367 _+ 159 • <0.05

10,000 1 ,326 + 143 <0.001

1. The mean of 6 assays given in terms of ng/ml incubation medium _+ standard error of the mean (SEM) ι

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XIII

IN VITRO GROWTH HORMONE RELEASE

Dosage ^* ' ΔGH ¹ p Value ³

H7-desaminoTyr-I Trp-Ala-Trp-D-Phe- l--i

28 +_ 54 _

30 231 +_ 65 «-0.02

300 432 +_ 10^ <0.01

3,000 700 +_ 201 <0.01

-j

00

20,000 861 + 13 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium _+_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XIV

IN VITRO GROWTH HORMONE RELEASE

ΔGH ¹ p Value ⁴

— -175 +_ 58

10 • -297 +_ 79 NS ³

30 -118 +_ 97 NS

300 5 _+ 29 0.02 C

3,000 1 ,594 +_ 385 <0.001

30,000 1,607 +_ 250 <0.001

• i

1. The mean of 9 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

422D-154

TABLE XV

IN VITRO-GROWTH HORMONE RELEASE

Dosage 2 , ΔGH p Value

H -Q-Me-Tyr-D-Trp-Ala-Trρ-D-Phe-NH-->

-9 _+ 66

300 177 +_ 166 NS ³

3,000 1,138 _+ 266 . <0.001

30,000 892 + 388 <0.05

1. The mean of 9 assays given in terms of ng/ml incubation medium _+_ standard error of the mean (SEM)

2. Given in terms of ng/ml. incubation medium 3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XVT

IN VITRO GROWTH HORMONE RELEASE

Dosage'- ΔGH ¹ p Value ⁴

_H -Tyr-p-;Trp-Ala-Trp-_ Phe--Met-NH^

-148 -h 137

30 • 456 _+ 151 NS ³

300 1,260 245 <0.01

1,000 1,832 + 441 <0.01 00 H

1. The mean of 6 assays given in terms of ng/ml incubation medium + standard error of the πean (SEM)

I

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XVII

IN VITRO GROWTH HORMONE ^• RELEASE

Dosage ΔGH ¹ p; Value ⁴

Ho-Tyr-D-Trp-Ala-Trp-D-Phe-Thr-NHό

-40 £ 35 —.—

10 94 _+ 69 NS ³

100 650 _+ 320 NS

1 ,000 2,167^591 <0.01

10,000 2,957 + 834 <0.01

1 . The π-ean of 6 assays given in terms of ng ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium 3. NS denotes not significant 4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XVIII

IN VITRO GROWTH HORMONE RELEASE

Dosage* ΔGH ¹ p Value-

H ₉ -Tyr-D-Trp-Ala-Trp-D-Phe-Gln-OH

.'

-40 _+ 35

100 ^' 753 _+ 559 ^' NS ³

1,000 2,759 _+ 515 <0.001

10,000 3,994 _+ 1,214 <0.01

1. The mean of 6 assays given in terms of ng/ml incubation medium £ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium 3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XIX

IN VITRO GROWTH HORMONE RELEASE

^{1 • • •} Dosage* ΔGH ¹ p Value ⁴

H^-Tyr-D-Trp-Ala-Trp-D-Phe-Gln-NH-)

-40 _+ 35

10 240 £ 96 NS ³

100 2,000 +_ 523 <0.01

1 ,000 4,235 jh 785 <0.001

10,000 4,141 + 576 <0.001

1. The mean of 6 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XX

IN VITRO ^• GROWTH ^• HORMONE RELEASE

•0

. ... - - ^■ Dosage ΔGH ¹ p Value-*

H^-Tyr-D-Trp-Ala-Trp-D-Phe-Asn- -NH ^r j -89 _+ 51

30 244.+_ 58 <0.01

100 528 _+ 184 <0.01

1,000 1,670 + 126 <0.001

1. The mean of 6 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels m medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXI

IN VITRO GROWTH HORMONE RELEASE

Dosage ^{2 ■} ΔGH ¹ p Value ³

H -His-D-Trp-Ala-Trp-D-Phe-Lys-NIl^

—167 £ 114 -_-_-._-.

1 955 +_ 272 <0.01 3 1 ,603 £ 305 <0.001

10 2,244 _+ 173 <0.001 30 2,198 + 358 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium £ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXII

IN VITRO GROWTH HORMONE ^• ELEASE

Dosage* . ΔGH 1 p Value ^*

H- _? -Tyr-E)-Trp-Ala^Trp^-D-Phe-Lys-'NH9

-29 _+ 59 _

30 1,346 £ 328 <0.001 300 2,160 _+ 166 <0.001 3,000 1,630 _+ 135 <0.001 00 30,000 1,318 + 247 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium £ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXIII

IN-VITRO GROWTH HORMONE'RELEASE

Dosage ² ΔGH ¹ p Value"

H^-Tyr-D-Trp-Ala-Trp-D-Phe-Glu-NH-)

-29 £ 59

3,000 1 ,404 179 <0.001 0 0

30,000 1 ,909 + 270 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium + standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

Q

^" Η > *T.

TABLE XXIV

IN VITRO GROWTH HORMONE RELEASE

Dosage* ΔGH ¹ p Value ⁴

H9-τyr-D-Trp7^a-Trp-D-Phe-Phe-NH-)

29 £ 164

30 10 _+ 57 NS ³

300 330 +_ 43 NS

3,000 710 _+ 67 •j-0.01

30,000 1,484 + 135 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium _+ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium ^' without the agonist

TABLE XXV

I VITRO ^• GROWTH HORMONE-RELEASE

Dosage* ΔGH- p-Value ⁴

H^-Tyr-D-Trp-Ala-^Trp^D-Phe-'Gln^NIl^

-89 £ 50

30 103 +_ 248 NS ³ '

100 167 £ 65 <0.02

1,000 1,092 + 42 <0.001

1. The mean of 6 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXVT

IN- VITRO-GROWTH"HORMONE RELEASE

Dosage* ΔGH ¹ p Value"

Hή-His-D-Trp-Ala-Trp-D-Phe-Lys-OH

-146 +_ 67 , .

1 467 178 <0.01

10 330 _+ 167 <0.02

C 100 2,441 + 353 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium + standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. - Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXVTI

I VITRO GROWTH HORMONE RELEASE Dosage* ΔGH ¹ p- Value ³

H ₉ -His-->-TrF^Ala-TγprD-Phe-Arg-NH2

-88 +_ 56 _---_-.

1 805 +_ 152 <o ^« ooi

3 997 £ 249 <0.001

10 2,064 + 346 <0.001

CD to

1. The mean of 9 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XVTII

IN- VITRO GROWTH HORMONE RELEASE

Dosage ^* - ΔGH ¹ p Value

H?-His-D-Trp^Ala-"Trr^I>-Phe-Gln-NH^

-288 +_ 182 | .. || .. ,

3 140 _+ 168 NS

10 275 + 66 ^0.02

CD

1. The mean of 6 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXIX

I VITR -GROWTH HORMONE RELEASE

^• Dosage 2. ΔGH ¹ p Value ⁴

H ₉ -His-D-T_η -Ala-Trp-D--Phe--Glu-NIl-->

-251 £ 105 ., || ,

1 -78 ± 72 NS ³

3 -294 £ 75 NS 10 -243 _ 71 NS. 30 117 + 41 <0.01

1. The mean of 9 assays given in terms of ng/ml incubation medium £ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXX

IN VITRO GROWTH HORMONE RELEASE

Dosage ^* - ΔGH p Value

H ^His-D-T-^Ala-T^D-Phe-Homo rg^NH-)

-553 +_ 105

10 1,096 + 341 <0.001

CD

1. The mean of 6 assays given in terms of ng/ml incubation medium £ standard cπ error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXXI

IN VITRO ^• GROWTH ^• HORMONE-RELEASE

Dosage* ΔGH 1 p-Value ^•

H-3-N--Me-D-Trp-Ala-Trρ-D-Phe-Lys^NIl>

— 435 +_ 176

1 165 +_ 136 NS ³

10 1,857 -r 254 <0.01

1. Tlie mean of 9 assays given in terms of ng/ml incubation medium_+ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation nredium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist •

TABLE XXXII

IN VITRO GROWTH HORMONE RELEASE

^• - Dosage* ΔGH p-Value ⁴

H ₉ -His-D-Trp--Ala-Trp-D-Phe-Lys-NHCH ₉ CHq 435 +_ 176

1 726 _+ 115 NS-

10 1 ,688 + 300 <0.01

C

1. The mean of 9 assays given in terms of ng/ml incubation medium £ standard - erro of the mean (SEM)

2. Given in terms of ng/ml incubation medium l

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXXIII

IN VITRO GROWTH HORMONE RELEASE

Dosage ² ΔGH ¹ p Value ⁴

H ₉ ^His-D-Trp-Ala-T^_ Phe^Orn-NH^

-553 +_ 105 «-,_-__.

1 -295 +_ 93 NS ³ 10 356 + 92 <0.001

CD 00

1. The mean of 6 assays given in terms of ng/ml incubation medium f standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE XXXIV

IN VITRO ^• GROWTH ^* HORMONE-RELEASE

Dosage* ΔGH ¹ p-Value ³

H ₉ --His^l Trp-Val^Tι_p^EHPhe"Lys"NH^

-553 +_ 105

30 -118 + 65 <0.01

1. The mean of 6 assays given in terms of ng/ml incubation medium _ standard error of the mean (SEM) ^{" "} co

CD

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in iredium containing growth hormone agonist analog to the GH levels in medium without the agonist

TABLE-XXXV

IN'VITRO GROWTH HORMONE"RELEASE

^• Dosage ^* ' ΔGH' p- Value ^■'

H ₉ -His-D-Trp-Ser-Trp-D-Phe-Lys--NH ₉ -

•125 +_ 132 ___

10 161 +_ 157 NS ³ 30 705 + 149 <0.01

1. The mean of 6 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

_ \ 0 \ .

The results set forth in Tables VI-XXXV demonstrate that peptides within the scope of the instant invention can induce a significant in-vitro release of growth hormone from the pituitary.

By introducing various other hormones, e.g., so atostatin, testosterone, cortisol, insulin, etc., into the incubation medium of Examples 11-30, one can study what effect these latter hormones have on the regulation of growth hormone secretion.

Example 61

In-Vivo Growth Hormone Release Study Female rats of the CD-1 strain were housed in a constant temperature room at 24°C. with 14 hours light and 10 hours darkness. The rats were fed Purina brand rat chow ab libitum. All studies were started between 0800 and 1000 ^' hours.

Each female rat (21 days old; eight rats per dosage level shown in Table XVI) was intraperitoneally injected with a desired dosage of the peptide of Example 1. Approximately 15 minutes• af-ter injection, the rat was guillotined. A blood sample was collected from the guil¬ lotined rat. The blood sample was centrifuged and a serum sample was collected therefrom. Each serum sample was assayed for GH, in duplicate, by a standard radio- immunoassay (RIA) . The mean of the GH values obtained per dosage level are set forth in Table XXXVI.

Example 62 In Vivo Growth Hormone Release Studv

The procedure set forth in Example 61 was em¬ ployed in an in- ivo growth hormone release study of the peptide of Example 21 and the results therefrom are set forth in Table XXXVII.

OM

422D-154

-102-

Example--63

In*Vivo--Growth ^* Hormone Release ^* Study The procedure set forth ^* in Example 61 was em¬ ployed in an in- vivo growth hormone release study of the peptide of Example 22 and the results therefrom are set forth in Table XXXVIII.

Example 64

In Vivo Growth Hormone Release Study The procedure set forth in Example 61 was em¬ ployed in an in vivo growth hormone release study of the peptide of Example 23 and the results therefrom are set forth in Table XXXIX.

Example 65 In-Vivo Growth-Hormone Release Studv

The procedure set forth in Example 61 was em¬ ployed in an in vivo growth hormone release study of the peptide of Example 24 and the results therefrom are set forth in Table XXXX.

Example .66 In Vivo Growth Hormone Release Studv

The procedure set forth in Example 61 was em¬ ployed in an in- ivo growth hormone release study of the peptide of Example 25 and the results therefrom are st forth in Table XXXXI.

Example 67

In Vivo Growth Hormone Release Study The procedure set forth in Example 61 was em¬ ployed in an in vivo growth hormone release study of the peptide of Example 26 and the results therefrom are set forth in Table XXXXII.

__ .

Example -68 In- Vivo -Growth Hormone Release Study The procedure set forth in Example 61 was em¬ ployed in an iπ-vivo growth hormone release study of the peptide of Example 27 and the results therefrom are set forth in Table XXXXIII.

Example 69

In Vivo- Growth Hormone Release Study The procedure set forth in Example 61 was em¬ ployed in an in vivo growth hormone release study of the peptide of Example 16 and the results therefrom are set forth in Table XXXXIV.

422D- 1 54

TABLE -XXXVT

IN VIVO -GROWTH HORMONE RELEASE Dosage ² ΔGH ¹ p Value ⁴

H ₉ -His-D^Trp-Ala-Trp-D-Phe--Nfl -

Control ₄ 1

1 .0 2 +_ 2 NS ³

10.0 ± 1 NS

100.0 82 + 18 <0.001

1 . The mean of 8 assays -given in terms of ng/ml intraperitoneal f standard error of the mean (SEM)

2. Given in terms of μg/ml

3. NS denotes not significant

4. GH levels in serum of rats intraperitonally injected with peptide compared to the Ql levels in serum of control rats

TABLE XXXVTI

IN- VIVO GROWTH HORMONE RELEASE - Dosage ² :- -- ΔGH ^■ p Value ³

H -His-D-Trp-Ala-Trp-D-Phe-Lys-OH

Control 1.7 +_ 1

3.0 76 _+ 24 <0.01

10.0 119 £ 25 <0.001 t-*

30.0 226 + 43 <0.001 o cπ

1. The mean of 8 assays given in terms ^* of ng/ml subcutaneous medium +_ standard error of the. mean (SEM)

I

2. Given in terms of yg/ml serum

3. GH levels in serum of rats subcutaneously injected with peptide compared to the GH levels in serum of control rats

TABLE XXXVIII

IN VIVO GROWIH HORMONE' RELEASE

Dosage - ΔGH p Value

H ₉ -His-D^Trp^Ala^Trp^-D-Phe-Arg'-NH

Control 5.7 2.2

1.0 9.2^ 3.4 NS

3.0 32.2 +_ 5.5 <0.001

10.0 87.5 ^ 31 0.02

30.0 31.1 + 3.4 <0.001

1. The mean of 8 assays given in terms of ng/ml subcutaneous _+_ standard error of the mean (SEM)

2. Given in terms of μg/ml serum

3. NS denotes not significant

4. GH levels in serum of rats subcutaneously injected with peptide compared to the GH levels in serum of control rats

TABLE XXXIX

IN VIVO GROWTH HORMONE -RELEASE

^• Dosage ^* ^ ΔGH 1 p Value ⁴

H ₉ -His-p-Trp-Ma-Trp-D-Phe-Gln-NH ₉ ;

Control 4.9 _+ 0.9

1.0 1.4 +_ 0.9 NS ^J

3.0 3.8 + _^ 2.1 NS

10.0 32.1 + 11.4 <0.05

1. The mean of 9 assays given in terms of ng/ml subcutaneous +_ standard error of the mean (SEM)

I I

2. Given in terms of μg/ml serum

3. NS denotes not significant

4. GH levels in serum of rats subcutaneously injected with peptide compared to the GH levels in serum of control rats

J ⁴ .BLE XXXX

- IN- VIVO GROWTH HORMONE RELEASE

1

ΔGH ¹ p Value ⁴

H^His-- Trp-Ala*Trp--D--Phe--Glu-'NH?

Control 0.34 +. 0, 19

1 .0 0.10 _+ 0 NS ³

1. The assays given in terms of ng/ml subcutaneous +_ standard error of the mean (SEM) '

2. Given in terms of μg/ml serum

3. NS denotes ^' not significant

4. GH levels in serum of rats subcutaneously injected with peptide compared to the GH levels in serum of control rats

TABLE XXXXI

IN- VIVO -GROWTH HORMONE RELEASE

Dosage* ΔGH p-Value ⁴

H ₉ -His-p-Trp-A-la-^Tι_p^- Phe--H

Control 0 + 0

0.3 ³ ± ² NS 3

10.0 62 + 8 <0.001 r-* rzs

1. The mean of 6 assays given in terms of ng/ml subcutaneous + standard error of the mean (SEM)

2. Given in terms of μg/ml serum 3. NS denotes not significant

4. GH levels in serum of rats subcutaneously injected with peptide compared to the GH levels in serum of control rats

TABLE XXXXII

IN VIVO GROWTH HORMONE- RELEASE

Dosage-' ΔGH ^• p- Value ³

H^-3^N-Me-His-p-Trp-Ala-Trp-p^Phe-Lys^NH-) Control 1.1 +_ 0.6 0.3 45.0^ 10 ^0.001 10.0 128 + 16 <0.001

I

1. The mean of 6 assays given in terms of ng/ml subcutaneous j+ standard o- ¹ error of the mean (SEM)

2. Given in terms of μg/ml serum ι

3. GH levels in serum of rats subcutaneously injected with peptide compared to the GH levels in serum of control rats

TABLE XXXXIII

IN- VIVO -GROWTH HORMONE RELEASE

Dosage* ΔGH 1 p Value-

H -His-D-Trp-Ala-Trρ-D-Phe-Lys-NHCH _? CH^

Control ¹ *- ± °- ⁶ —

0.3 • 13.0 +_ 5 <0.05

10.0 119 + 12 <0.001

H

1. The mean of 6 assays given in terms of ng/ml subcutaneous _+_ standard H t- ^o error of the mean (SEM)

2. Given in terms of μg/ml serum

I

3. GH levels in serum of rats subcutaneously injected with peptide compared to the GH levels in serum of control rats

TABLE XXXXIV

IN VIVO 'GROWTH' HORMONE -RELEASE

ΔGH 1 p- Value ⁴

H -His-D-Trp-Ala-Trp-D-Phe-Lys-NH ₉

Control 0

0.3 ^• 6 _ 4 NS ³

1.0 36 +_ 15 <0.04

10.0 100 +_ 19 <0.001

30.0 122 + 4 <0.001

1. The mean of 6 assays given in terms of ng/ml subcutaneous +_ standard error of the mean (SEM)

2. Given in terms of μg/ml serum

Tne results set forth in Tables XXVI-XXXXIV demonstrate that some peptides within the scope of this invention can induce a significant in- vivo release of growth hormone.

Example- 70 In Vitro-Growth 'Hormone Release- Studv

The procedure set forth in Example 31 was em¬ ployed in an in vitro growth hormone release study of the growth promoting agent zeranol, and the results therefrom are set forth in Table XXXXV.

Example 71

In Vitro Growth Hormone Releas-e Study The procedure set forth in Example 31 was em¬ ployed in an in-vitro growth hormone release study of the peptide of Example 21 in combination with the growth promoting agent zeranol, C-gH26θ5, and the results there¬ from are set forth in Table XXXXVI. The results s ^" et forth in Table XXXXVI, when compared to the results set forth in Tables XXVI and XXXXV, demonstrate that a combi¬ nation within the scope of the instant invention can induce a synergistic in vitro release of growth hormone from the pituitary.

c.:.?

TABLE XXXXV

^• I VITRO GROWTH HORMONE ^• RELEASE DOS ige ² ΔGH ¹ p- Value ³ Zeranol,- C _1R H ^l ?60r.,

— -115 +_ 49

300 83 _+ 63 0.02

3,000 87 +_ 59 <0.02

30,000 683 +.187 <0.001

100,000 -133 + 98 NS ³ H

1. The mean of 9 assays given in terms of ng/ml incubation medium + standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. NS denotes not significant

4. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium without the agonist

-S

t\Q "

TABLE XXXXVI

IN VITRO GROWTH HORMONE RELEASE

ΔGH ¹ p Value'

H ₂ -His-D-Trp ^rr Ala-Trp-D-Phe-Lys-NH2

——- -309 _+ 39

3,000 A + 1B 2,183 +.399 <0.001

3,000 A + 3B 2,114 +.336 <0.001 t-* t-*

30,000 A + IB 2,266 +_ 327 <0.001

30,000 A + 3B 2,761 + 356 <0.001

1. The mean of 9 assays given in terms of ng/ml incubation medium +_ standard error of the mean (SEM)

2. Given in terms of ng/ml incubation medium

3. Comparison of the GH levels in medium containing growth hormone agonist analog to the GH levels in medium- ithout the agonist

_ 116 -

Based on this disclosure, many other modifica¬ tions and ramifications will naturally suggest themselves to those skilled in the art. These are intended to be comprehended as within the scope of this invention.

CI.