Proc. Nati. Acad. Sci. USAVol. 91, pp. 12298-12302, December 1994Medical Sciences

Synthesis and biological activities of highly potent antagonists ofgrowth hormone-releasing hormone

(inhibitory growth hormone-releasing hormone analogs/structure-activity relationships)

M. ZARANDI*t, J. E. HORVATH**, G. HALMOS*, J. PINSKI*, A. NAGY*, K. GROOT§, Z. REKASI*,AND A. V. SCHALLY*§¶§Veterans Affairs Medical Center, Endocrine, Polypeptide and Cancer Institute, and *Department of Medicine, Tulane University School of Medicine, NewOrleans, LA 70146

Contributed by A. V. Schally, August 30, 1994

ABSTRACT In the search for antagonists ofhuman growthhormone-releasing hormone (hGERH) with high activity, 22analogs were synthesized by solid-phase methods, purified, andtested biologically. Within the N-terminal sequence of 28 or 29amino acids of hGHRH, all the analogs contained D-Arg2,Phe(4-Cl)6 (par-chlorophenylalanine), Abu's (a-aminobutyricacid), and Nle" and most of them had Agm9 (agmatine)substituents. All the peptides, except one, were acylated at theN terminus with different hydrophobic acids-e.g., isobutyricacid (Ibu) or 1-naphthylacetic acid (Nac) in order to study theeffect ofN-terminal acylation on the antagonistic activity. In thesuperfused rat pituitary cell system, all the analogs inhibitedmore powerfully the GHRH-induced growth hormone (GH)release than the standard GHRH antagonist [Ac-Tyr',D-Arg2]hGHRH-(1-29)NH2. Antagonists [Ibu0,D-Arg2,Phe(4-Cl)6, Abuls,NMe27JhGHRH-(1-28)Agm (MZ-4-71), [Nac°,D-Arg2,Phe(4-CI)6,Abuls,Nle2]hGHRH-(1-28)Agm (MZ-4-243),[Nac0,D-Arg2,Phe(4-Cl)6,Abuls,Nle27]hGHRH-(1-29)NH2(MZ-4-169), [Nac0-His',D-Arg2,Phe(4-Cl)6,Abul',Nle271-hGHRH-(1-29)NH2 (MZ-4-181), and [NacO,D-Arg2,Phe(4-Cl)6,Abuls,Nle27,Aspe]hGHRH-(1-28)Agm (MZ-4-209) inhib-ited GH release at 3 x 10-9 M. Among these peptides, MZ-4-243, MZ-4-169, and MZ-4-181 were also long acting in vitro.Antagonist MZ-4-243 inhibited GH release 100 times morepowerfully than the standard antagonist and was the most potentin vitro among GHRH antagonists synthesized. Analogs withhigh inhibitory effects in vitro were also found to have highainities to rat pituitary GEIRH receptors. In experiments invivo, antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abul5,Nle271-hGHRH-(1-28)Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abuls,Nle27]hGHRH-(1-29)NH2 (MZ-4-169), and [Nace-H lSD-Arg2,Phe(4-CI)6,Abu1s,NIe27]hGHRH-(1-29)NH2 (MZ-4-181) induced a significantly greater inhibition of GH releasethan the standard antagonist. In view of their high antagonisticactivity and prolonged duration of action, some of these antag-onists ofGHRH may find clinical applications, including treat-ment of certain endocrine disorders and insulin-like growthfactor I-dependent tumors.

Since the isolation and structural elucidation of growthhormone-releasing hormone (GHRH) (1, 2), numerous ana-logs of GHRH have been synthesized. Most of them weredesigned as agonists with very high activity intended forpotential clinical and veterinary applications.

Specific antagonists ofhuman (h)GHRH are necessary forthe study of the mechanism of action ofGHRH and may alsobe useful clinically for the treatment of disorders caused byexcessive secretion of growth hormone-e.g., acromegalyand diabetic retinopathy. More importantly, GHRH antago-

nists could find applications in cancer therapy. Theoretically,by suppressing GH secretion, antagonists of GHRH shoulddecrease the synthesis of insulin-like growth factor I (IGF-I)by the liver and other tissues. GHRH antagonists could alsolower the autocrine or paracrine production of IGF-I byvarious tumors and lead to inhibition of cancer proliferation(3). GHRH antagonists might also inhibit the production ofIGF-II (4). There is a clinical need for antagonists of GHRHsince somatostatin analogs alone do not adequately suppressGH and IGF-I levels (5). The advantage of GHRH antago-nists over somatostatin analogs would also be based on thefact that GHRH antagonists could be used for suppression ofgrowth oftumors that do not have somatostatin receptors likeosteosarcomas (5). GHRH antagonists could be given aloneor together with somatostatin analogs. The use of a combi-nation of both analogs could achieve more complete sup-pression of IGF-I levels (4).

In the course of synthesis of various analogs of hGHRH-(1-29)NH2, it was found that the replacement of Ala2 byD-Arg2 led to antagonists (6). [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2 was able to inhibit the GHRH-stimulated adenylatecyclase activity in rat pituitary cells (6, 7). This antagonistwas also reported to block endogenous and GHRH-stimulated GH secretion after injection to rats (8, 9) and toinhibit GH release in cell cultures (10). Since D-Arg atposition 2 appears to be essential for generating GHRHantagonistic activity, all GHRH antagonists contain D-Arg atposition 2 (6-15). Some modifications in hGHRH-(1-29)NH2had been shown to increase the affinity of the molecule to theGHRH receptor (11) and the use of these substitutions in[D-Arg2]hGHRH-(1-29)NH2 led to more potent antagonists(12).

This paper reports the synthesis and biological evaluationof a series of GHRH antagonists containing different acylgroups at the N terminus and D-Arg at position 2 combinedwith other substitutions. The contribution of N-terminalacylation to biological activity was also studied.

MATERIALS AND METHODSSynthesis. GHRH antagonists were prepared by solid-

phase methodology (16) using manual solid-phase peptide

Abbreviations: Abu, a-aminobutyric acid; Agm, agmatine; Aqc,anthraquinone-2-carbonyl; Boc, tert-butoxycarbonyl; BrProp, bro-mopropionyl; IAc, iodoacetyl; Ibu, isobutyryl; Nac, 1-naphthyl-acetyl; 2-Nac, 2-naphthylacetyl; Npt, naphthoyl; Phe(4-Cl), para-chlorophenylalanine; GH, growth hormone; GHRH, GH-releasinghormone; h, human; IGF-I, insulin-like growth factor I.tOn leave from: Department of Medical Chemistry, Albert Szent-Gyorgyi Medical University, H-6720 Szeged, Dom ter 8, Hungary.tOn leave from: Department of Anatomy, University MedicalSchool, Pecs, H-7643 Hungary.$To whom reprint requests should be addressed at: Veterans AffairsMedical Center, 1601 Perdido Street, New Orleans, LA 70146.

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Proc. Natl. Acad. Sci. USA 91 (1994) 12299

synthesis equipment. For peptides with an amidated C ter-minus, para-methylbenzhydrylamine resin (0.57 mmol/g;Bachem) was used. For antagonists with C-terminal agmatine(1-amino-4-guanidinobutane) (Agm), Boc-Agm-SPA-para-methylbenzhydrylamine resin (Boc, tert-butoxycarbonyl;SPA, sulfophenoxy) (0.30 mmol/g; California Peptide Re-search, Napa, CA) was used as the starting material.

Protected amino acids used in the synthesis were of theL-configuration unless stated otherwise. The a-amino functionwas protected with the Boc group, and the reactive side chainfunctional groups were protected as follows: para-toluenesulfonyl for Arg, cyclohexyl for Asp and Glu, benzyloxyme-thyl for His, 2-chlorobenzyloxycarbonyl for Lys, benzyl forSer and Thr, and 2,6-dichlorobenzyloxycarbonyl for Tyr. Theside chains of Asn and Gln were unprotected. The couplingreaction was achieved with a 3-fold excess ofBoc amino acid,using N,N'-diisopropylcarbodiimide or benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) as activating agent in dichloromethane, dime-thylformamide, or mixtures thereof. Boc-Asn and Boc-Glnwere coupled with preformed 1-hydroxybenzotriazole ester.After a coupling time of 1 hr, the completeness of acylationwas monitored at each stage by the standard ninhydrin test(17). In cases where incomplete coupling was found, thecoupling procedure was repeated or the acetylation was car-ried out before removal ofthe Na-amino protecting group priorto the coupling of the next amino acid. Acetylation wasperformed with 30%6 (vol/vol) acetic anhydride in dichlo-romethane for 10 and 20 min. Intermediate deblocking wasachieved with 50o (vol/vol) trifluoroacetic acid in dichlo-romethane followed by neutralization with 5% (vol/vol) di-isopropylethylamine in dichloromethane.

After completion of the synthesis and removal of theNa-Boc protecting group from Tyr', His', or Glu', mostpeptides were acylated with acetic acid (Ac), anthraquinone-2-carboxylic acid (Aqc), bromopropionic acid (BrProp), io-doacetic acid (IAc), isobutyric acid (Ibu), 1-naphthylaceticacid (Nac), or naphthoic acid (Npt) using a symmetricalanhydride method. Final deprotection as well as cleavage ofthe peptide from the resin was performed with anhydroushydrogen fluoride in the presence of 10% m-cresol at 0°C for60 min. After removal of the hydrogen fluoride under astream of nitrogen and in vacuo, the free peptides wereprecipitated with diethyl ether, filtered, washed with diethylether and ethyl acetate, extracted with 50%o (vol/vol) aqueousacetic acid, diluted with water, and lyophilized.

Purffication. Crude peptides were purified by using aMacRabbit HPLC system (Rainin, Woburn, MA) equippedwith a Vydac (Hesperia, CA) model 228TP column (10 x 250mm) packed with C8 silica gel (pore size, 300 A; particle size,10 ,m) (Rainin). The column was eluted with a solventsystem consisting of 0.1% aqueous trifluoroacetic acid (A)and 0.1% trifluoroacetic acid in 70% aqueous acetonitrile (B)in a linear gradient mode (e.g., 30-65% B in 140 min). Theeluent was monitored at 220 nm. The fractions were checkedby analytical HPLC, and those with purity higher than 95%were pooled and lyophilized.

Analytical HPLC. The HPLC analyses of crude and puri-fied peptides were carried out with a Hewlett-Packard liquidchromatograph (model 1090) using a W-Porex C18 reversed-phase column (4.6 x 250 mm; pore size, 300 A; particle size,5 ,um) (Phenomenex, Belmont, CA) and isocratic and/orgradient elution with the two solvent systems describedabove at a flow rate of 1.2 ml/min. The peaks were monitoredat 220 and 280 nm.Amino Acid Analysis. Amino acid analyses of the purified

peptides were carried out on a Beckman 6300 amino acidanalyzer after hydrolysis of the samples in 6 M hydrochloricacid at 110HC for 20 hr in tubes sealed under vacuum.

Determination of GHRH Antagonistic Activity in Vitro.Antagonistic activities of the peptides on GH release weredetermined by using a superfused rat pituitary system (18,19). First, the antagonistic peptides were perfused throughthe cells for 9 min (3 ml) at various concentrations (10-7-10-9M). After this 9-min preincubation, the cells were immedi-ately exposed to a mixture ofthe GHRH antagonists and 10-9M hGHRH-(1-29)NH2 for an additional 3 min (1 ml) (0-minresponse). To check the duration of the antagonistic effect ofthe analog, 10-9M hGHRH-(1-29)NH2 was applied 30 and 60min later for 3 min (30- and 60-min responses). GH contentof the 1-ml fractions collected was determined by RIA. Netintegral values of the GH responses were evaluated with aspecially designed computer program (20). GH responseswere compared to and expressed as percentage ofthe originalGH response induced by 10-9 M hGHRH-(1-29)NH2. Thepotencies of the modified antagonists were compared to thatof [Ac-Tyr',D-Arg2]hGHRH-(1-29)NH2 (standard antago-nist).

Evaluation of Receptor Binding in Vitro. The binding ofGHRH antagonists to membrane receptors of rat anteriorpituitary cells was determined by using 1251-labeled [His',-Nle27]hGHRH-(1-32)NH2 as radioactive ligand (21). In com-petitive binding analysis 125I-labeled [His',Nle27]hGHRH-(l-32)NH2 (0.2 nM) was displaced by GHRH antagonists at10-610-12 M. The final binding affinities were estimated byKi (dissociation constant of the inhibitor-receptor complex)(22) and were calculated by using the ligand PC computerizedcurve-fitting program ofMunson and Rodbard as modified byMcPherson (23). Relative affinities compared to [Ac-Tyr',D-Arg2]hGHRH-(1-29)NH2 (standard antagonist) were calcu-lated as the ratio of Ki of the tested GHRH antagonists to theKi of the standard antagonist.

Inhibition of GH Release in Vivo. Adult male Sprague-Dawley rats were anesthetized with pentobarbital (6 mg per100 g of body weight, i.p.). Blood samples were taken fromthe jugular vein 30 min after the injection of pentobarbital(0-min blood sample). One group of seven animals receivedhGHRH-(1-29)NH2 as control (3 gg per kg of body weight).Other groups of rats were injected with [Ac-Tyr',D-Arg2]hGHRH-(1-29)NH2 as standard antagonist (100 and 400ug per kg of body weight) or with modified antagonists (20and 80 ,Ig per kg of body weight) 30 sec prior to administra-tion of hGHRH-(1-29)NH2. Blood samples were taken fromthejugular vein 5 and 15 min after injection of the antagonistsfor measurement of GH levels. Potencies of the antagonistswere calculated by the factorial analysis of Bliss and Marksas outlined by Pugsley (24) with 95% confidence limits.Statistical significance was assessed by Duncan's new mul-tiple range test.RIA for GH. Rat GH levels in aliquots of superfusion

samples and in serum were measured by double-antibodyRIA using materials supplied by the National Hormone andPituitary Program, Baltimore. Interassay variation was<15% and intraassay variation was <10%.

RESULTSIn a search for potent GHRH antagonists, a series ofhGHRH-(1-29)NH2 analogs was synthesized, purified, andtested both in vitro and in vivo. The structure of the newpeptides is shown in general formula A.

A = [X-R',R2,Phe(4-Cl)6,Abul5,Nle27,R28,R29]-GHRH(1-29) [Phe(4-Cl), para-chloro-phenylalanine; Abu, a-aminobutyric acid]

WhereX = Ibu, BrProp, lAc, 1- or 2-Nac, 1- or 2-

Npt, Aqc, Ac, or H

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R1 = Tyr, His, Glu, or X-R1 = Glt (glutaryl)

R2 = D-Arg or Ala

R28 = Ser or Asp

R29 = Agm or Arg-NH2

The 22 GHRH antagonists were prepared by solid-phasepeptide synthesis (16). After purification by preparativereversed-phase HPLC, the purity of peptides was examinedby analytical HPLC and found to be >95%. Amino acidanalyses of the acid hydrolysates of the pure productsshowed the expected amino acid compositions.The peptides were tested for their ability to inhibit GH

release from dispersed rat pituitary cells induced by hGHRH-(1-29)NH2 in vitro. Table 1 shows the structures of22 GHRHantagonists and their inhibitory effects, at different doses, onGH release induced by hGHRH-(1-29)NH2 in vitro. All theanalogs contained D-Arg2 except for peptide 22, in which Ala2

substitution was used. All the analogs also contained Phe(4-Cl)6, Abu15, and Nle27 and most of them had Agm at the Cterminus. Peptides 1-12, 13-17, and 19-21 had Tyr', His', orGlul, respectively, acylated with different monocarboxylicacids with the exception of analog 6, which has a free aminogroup at the N terminus. The series of GHRH antagonistscontaining different acyl groups at the N terminus wassynthesized in order to evaluate the contribution of N-ter-minal acylation to the biological activity. As can be seen fromthese data, acylation with Nac or Ibu of the analogs thatcontain D-Arg2 substitution combined with Phe(4-Cl)6,Abu15, Nle27, and Agm29 causes a great increase in inhibitoryactivity on GH release in vitro (Table 1) as well as in bindingaffinity to the receptors (Table 2). Among peptides acylatedwith Nac or Ibu those containing Tyr' showed greater an-tagonistic potencies than the corresponding analogs with His'or Glul.

Antagonists 1, 4-6, 8, 16, and 17 caused a significantdecrease in GH release even at 3 x 10-9 M (Table 1). The

Table 1. Structures of antagonists [X-R',R2,Phe(4-Cl)6,Abul5,Nle27,R28,R29]hGHRH-(1-29) and their inhibitory effects on GH release insuperfused rat pituitary system

Peptide % inhibition of GH release

No. Code X R1 R2 R28 R29 Dose, M 0 min 30 min 60 min

Standard antagonist*1 MZ-4-71

2 MZ-4-75

3 MZ-4-794 MZ-4-243

5 MZ-5-42

6 MZ-5-14

7 MZ-4-169

8 MZ-4-209

9 MZ-4-239

10 MZ-4-23511 MZ-5-12

12 MZ-4-237

13 MZ-4-18314 MZ-4-17715 MZ4-12516 MZ-4-181

17 MZ-4-231

18 MZ-4-18519 MZ-4-129

20 MZ4.17521 MA-4-17322 MZ-5-02t

*[Ac-Tyrl,D-Arg2]hGHRH-(1-29)N]tPeptide contains Phe6; prolonged r

10-7Ibu Tyr D-Arg Ser Agm 10-7

3 x 10-810-8

3 x 10-9BrProp Tyr D-Arg Ser Agm 10-7

3 x 10-810-8

IAc Tyr D-Arg Ser Agm 3 x 10-7Nac Tyr D-Arg Ser Agm 10-8

3 x 10-910-9

3 x 10-1sAc Tyr D-Arg Ser Agm 3 x 10-8

3 x 10-9H Tyr D-Arg Ser Agm 3 x 10-8

3 x 10-9Nac Tyr D-Arg Ser Arg-NH2 3 x 10-8

10-83 x 10-9

Nac Tyr D-Arg Asp Agm 3 x 10-83 x 10-9

2-Nac Tyr D-Arg Asp Agm 3 x 10-83 x 10-9

1-Npt Tyr D-Arg Asp Agm 3 x 10-82-Npt Tyr D-Arg Ser Agm 3 x 10-8

3 x 10-9Aqc Tyr D-Arg Asp Agm 3 x 10-8

3 x 10-9Ac His D-Arg Ser Arg-NH2 3 x 10-8Ibu His D-Arg Ser Arg-NH2 3 x 10-8IAc His D-Arg Ser Arg-NH2 3 x 10-8Nac His D-Arg Ser Arg-NH2 3 x 10-8

3 x 10-9Nac His D-Arg Asp Agm 3 x 10-8

3 x 10-9Glt D-Arg Ser Arg-NH2 3 x 10-8

Ibu Glu D-Arg Ser Arg-NH2 3 x 10-710-8

IAc Glu D-Arg Ser Arg-NH2 3 x 10-8Nac Glu D-Arg Ser Arg-NH2 3 x 10-8Nac Tyr Ala Ser Agm 3 x 10-8

H2. Glt, glutaryl.release of GH.

51.99582.7706286.45856.289.396.677.656.311.097.345.696.272.496.190.318.189.351.583.60

84.590.923.764.92423.180.71792.689.398.945.317.973.914.259.188.4

074.740.718.216.47519.335.232.997.283.456.745.053.219.171.112.2959031.585.156.664.433.33250.114.648.931.26

16.40

86.481.28.2

12.40

2520.80

46.5

36.79.613.9

62.835.351.8

97.175.341.315.638.824.963.516.092.187.117.171.632.56.70

42.745.722.642.321.6

00

81.466.953.225.1

4551.67.3

43.9

Proc. Natl. Acad. Sci. USA 91 (1994)

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Proc. Natl. Acad. Sci. USA 91 (1994) 12301

Table 2. Ki values and relative affinities of hGHRH antagoniststo membrane receptors on rat anterior pituitary cells

Peptide Relative

No. Code Ki,* nM affinitytStandard antagonist 3.22 ± 0.12 11 MZ4.71 0.12 ± 0.04 26.182 MZ-475 0.99 ± 0.12 3.274 MZ-4243 0.04 ± 0.01 85.127 MZ-4-169 0.04 ± 0.01 82.5616 MZ-4-181 0.05 ± 0.01 67.0819 MZ-4-129 1.35 ± 0.02 2.3920 MZ4175 0.91 ± 0.01 3.5421 MZ4173 0.87 ± 0.01 3.72

*Dissociation constant of the inhibitor-receptor complex.tExpressed relative to [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2 (stan-dard antagonist) = 1.0.

duration of the inhibitory effect of the antagonists after threeconsecutive exposures to GHRH at 30-min intervals wasvariable. The standard antagonist at 10-7M inhibited GHRH-induced GH release by 51.9%o; however, it had no effect 30min later. Antagonistic activities of some analogs (e.g.,peptides 1-3, 5, 6, 10-13, 17, and 19-21) decreased rapidly.However, other analogs such as peptide 4 [Nac0,D-Arg2,Phe(4-Cl)6,Abul5,Nle27]GHRH-(1-28)Agm (MZ-4-243),7 [Nac0,D-Arg2,Phe(4-C1)6,Abul5,Nle27]GHRH-(1-29)NH2(MZ-4-169), and 16 [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abul5,-Nle27]GHRH-(1-29)NH2 (MZ-4-181) were extremely longacting in vitro. The inhibition of 10-9 M GHRH-induced GHrelease was =90% of the control value even 4.5 hr afterincubation with these antagonists at 3 x 10-8 M doses (to bereported elsewhere). The inhibition ofGH release by peptide4 (MZ-4-243) was found to be 56.3%, 56.7%, and 41.3% after0, 30, and 60 min, respectively, at 10-9 M. This inhibitoryeffect was 100 times greater at 0 min than that of [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2 (standard antagonist). AntagonistMZ-4-243 at 10-8M completely blocked the GHRH responseand was found to be the most potent in vitro among theGHRH antagonists synthesized. The inhibitory effects ofantagonists 1, 5-8, and 16 at 3 x 10-9 M were found to besimilar to those of the standard antagonist at 10-7M at 0 min.The binding affinities of analogs 1, 4, 7, and 16 to mem-

brane receptors on rat anterior pituitary cells were 26.18,85.12, 82.56, and 67.08 times greater than that ofthe standardGHRH antagonist, respectively (Table 2). Antagonists 1, 4,7, and 16 showed Ki values in the range 0.04-0.12 nM as

compared to the standard antagonist, which had a Ki of 3.2nM.To assess the inhibitory activity in vivo and its duration of

action for peptides containing Ibu° or Nac° combined withArg-NH2 or Agm at the C terminus and other substitutions inthe molecule, antagonists 1, 7, and 16 were injected i.v. torats at doses of 20 and 80 ,g per kg of body weight prior tostimulation with GHRH and blood samples were drawn atvarious time intervals. Table 3 shows serumGH levels in ratsresulting from pretreatment with these GHRH antagonists, ascompared to that produced by the standard antagonist. Theinhibiting potencies of these peptides as compared to thestandard antagonist are also listed. Peptides 1, 7, and 16induced a significantly greater and longer-lasting inhibition ofthe GH response to hGHRH-(1-29)NH2 than the standardantagonist. In these in vivo tests, antagonist 1 (MZ471)inhibited GH release 18.9 times more powerfully than thestandard antagonist. This compound showed the highestinhibitory potency among peptides tested in vivo. The in vivoevaluation of other analogs remains to be done.

DISCUSSION

Structure-activity studies on GHRH analogs indicate thatD-Arg at position 2 is essential for antagonistic activity. In theseries of analogs of GHRH with single D-amino acid substi-tution at position 2, only [D-Arg2]hGHRH-(1-29)NH2 showssignificant antagonistic activity. [L-Arg2]hGHRH-(1-29)NH2does not show such activity, indicating the importance of theD-configuration for the Arg residue (13). The design andsynthesis of our GHRH antagonists were based on findingsthat the incorporation of a hydrophobic residue into analogsof peptide hormones often yields a very active agonist orantagonist. Our peptides were also designed to increase theaffinities of the analogs to the receptor, improve metabolicstability, and maximize the amphiphilic secondary structureof the molecules. Acylation at the N terminus ofthe peptidescan enhance the receptor binding affinities as well as thestability against aminopeptidases. The metabolic stability ofthe antagonists was also improved by replacement of Arg29with Agm and by acylation of the N terminus. The hydro-phobic character of the modified GHRH analogs was en-hanced by acylation of the N terminus with hydrophobicacids and substitution of Phe(4-Cl)6 for Phe6. Replacement ofGly"5 with Abu"5 increases the regional amphiphilicity andalso stabilizes the central a-helical structure of the GHRHanalogs. In addition, in order to avoid oxidative inactivationof the analogs, Met27 was replaced by Nle27 in every analog.

Table 3. Serum GH levels and relative inhibiting potencies of different antagonistsDose, Serum GH level,* Inhibiting

Exp. Substance pg/kg ng/mi potencyt1 Saline 89.0 ± 17.7

hGHRH-(1-29)NH2 3.0 956.7 ± 113.6Standard antagonistt 100.0 738.3 + 34.7 1.0

400.0 439.7 ± 47.3§Antagonist 1 (MZ-4-71)t 20.0 451.8 ± 42.2§ 18.90

80.0 155.0 ± 38.2§ (11.0-32.47)1Antagonist 7 (MZ-4-169)t 20.0 641.2 ± 81.4§ 6.09

80.0 470.0 46.1§ (3.11-11.96)2 Saline 41.8 8.2

GHRH-(1-29)NH2 3.0 884.3 ± 77.2Antagonist 16 (MZ4.181)t 80.0 359.5 ± 65.1

*Mean ± SEM (seven rats per group) 5 min after injection of GHRH.tExpressed relative to [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2 (standard antagonist) = 1.0.tRats were pretreated i.v. with the GHRH antagonists 30 sec prior to stimulation with hGHRH-(1-29)NH2 (3 Ag/kg).§P < 0.01 vs. GHRH-(1-29)NH2.¶95% limits.

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Our previous results with GHRH agonists (25) showed thatreplacement of Ser28 with Asp28 increased the GH-releasingactivity both in vitro and in vivo. After i.v. injection, anAsp28-containing analog showed the highest GH-releasingpotency in rats (25). To determine whether substitution ofAsp28 can lead to antagonists ofGHRH with high potencies,some peptides contained this modification. Among peptidescontaining Asp28, antagonists 8-10 and 17 caused 80-90%inhibition of GH release in vitro at 3 x 10-8 M at 0 min.Antagonists 8 and 17 at 3 x 10-9M also decreased GH releaseby 50o at 0 min.

Studies on the effect of N-terminal acetylation of GHRHanalogs on the inhibition of GH secretion in vitro and onbinding affinity to rat anterior pituitary cells have yieldedconflicting results (10, 11, 15). For instance, one studyshowed that N-terminal acetylation resulted in decreasedinhibitory potency of [D-Arg2]hGHRH-(1-29)NH2, but thesame derivatization increased the antagonistic activity ofanalogs of rat [D-Arg2]GHRH-(1-29)NH2 (15). In contrast, inanother study, it was found that Na-acetyl derivatives ofbothhGHRH-(1-29)NH2 and rat GHRH had lower affinity toGHRH binding sites than their parent peptides with a freeamino group at the N terminus (11). It was concluded thatmodifications to increase the hydrophobic character of the Nterminus of hGHRH-(1-29)NH2 do not constitute a suitableapproach to increase receptor affinity (11). Nevertheless, ourwork shows that acylation ofGHRH antagonists with Nac orIbu not only increases the receptor binding affinity but alsoaugments biological effects ofthe analogs in vitro and in somecases also in vivo.

Affinity constants for binding of some antagonists to mem-brane receptors in the pituitary of rats are given in Table 2.The high affinities of analogs 1, 4, 7, and 16 for pituitaryreceptors coincide with their powerful inhibitory effects inthe superfusion assay. These properties can be explained bythe presence of highly hydrophobic acyl groups at the Nterminus. While antagonist 1 showed an affinity to thepituitary receptors 26 times higher than the standard antag-onist and, in accordance with this, exerted a strong inhibitoryeffect both in vitro and in vivo, the closely related analog 2had an affinity 8 times lower, although it was only 30%o lessactive in vitro than peptide 1. Higher affinities were found forantagonists 4 and 16, which were more active in vitro thancompound 1 but less potent in vivo (Table 3).The high and long-lasting antagonistic activity of our

analogs is believed to be due to the combination of asubstitution of D-Arg at position 2 with a hydrophobic acylgroup at the N terminus and other substitutions in themolecule. Acylation of the N terminus of the GHRH antag-onists with hydrophobic acyl groups resulted in analogs withhigh binding affinities to the pituitary GHRH receptorsthrough hydrophobic interactions. Binding data of the ana-logs suggest that the GHRH receptor protein contains anonpolar or aromatic group at the region that binds the Nterminus of the hormone. In conclusion, our structure-activity studies on GHRH antagonists show that substitu-tions in GHRH-(1-29)NH2 at positions 1, 2, 6, 15, 27, 28, and29 paired with a hydrophobic, acylated N terminus producehigh antagonistic potency.

Antagonists ofGHRH are needed clinically since somato-statin analogs do not adequately suppress GH and IGF-Ilevels. Both types of analogs may have to be used together inorder to obtain maximum suppression ofGH release. GHRHantagonists may be useful clinically for the treatment of

disorders caused by excessive secretion of GH-e.g., ac-romegaly and diabetic retinopathy. More importantly,GHRH antagonists could find applications clinically in can-cer therapy of IGF-I-dependent tumors.

We are grateful to Dr. A. Beck for Mass Spectra and to ProfessorH. Binz and Dr. C. Andreoni (Pierre Fabre Medicaments, Center ofImmunology and Biotechnology Pierre Fabre, St. Julien En Ge-nevois, France) for useful exchange of information. The gifts ofmaterials used in RIA from the National Hormone and PituitaryProgram of the National Institute of Diabetes and Digestive andKidney Diseases are greatly appreciated. We thank Ms. Katalin C.Halmos and Ms. Lisa Karczewski for their excellent technicalassistance. The work described in this paper was supported by agrant from Pierre Fabre Medicaments to Tulane University School ofMedicine and by the Medical Research Service of the VeteransAffairs Department (to A.V.S.).

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