J. Endocrinol. Invest. 16: 793-798,1993

New Gaba-containing analogues of human Growth Hormone­Releasing Hormone (1-30)-amide: I. Synthesis and in vitro biological activity I. Mez6*, M. Kovacs**, B. Sz6ke*, E.Z. Szab6*, J. Horvath**, G.B. Makara***, Gy. Rappay***, J. Tamas****, and I. Teplan* *Semmelweis University Medical School, Budapest, **University Medical School, Pecs, ***Institute of Experimental Medicine, Budapest, ****Hungarian Academy of Sciences, Central Institute for Chemistry, Budapest, Hungary

ABSTRACT. Analogues of human growth hormone­releasing hormone (1-30)-amide have been devel­oped. All analogues have been modified in position 27 with Nle and with Gaba in position 30. Additional D-amino-acids have been inserted in the GHRH(1-30)-NH2 sequence: A-1741: Nle27, Gaba30-GH-RH (1-30)-NH2 A-495: D-Ala2, Nle27, Gaba30-GH-RH (1-30 )-NH2 A-515: D Ala2, Leu15, Nle27, Gaba30-GH-RH (1-30)-NH2 A-527: D-Ala2, D-Arg11, Leu15, Nle27 , Gaba30-GH­RH (1-30)-NH2' Our analogues were synthesized by solid phase peptide synthesis and were tested is two different in vitro systems and in rat pituitary cell cultures. A-495 and A-1741 were found to be the most active in releasing GH, however they showed different activ­ities in the two different test systems. A-495 exhib­ited higher potency in the superfusion system (1.63 fold potency of the GHRH (1-29)-amide), while A-

INTRODUCTION

Controlling GH-secretion is of great importance both in human and veterinary medicine, because GH can influence not only cellular growth, but the metabolism of the whole organism. Since the natu­rally occurring hGHRH has an important role in con­trolling GH-secretion, intensive research was initi­ated after the isolation of hGHRH (1, 2) to synthe­size superactive and long-acting analogues. This requirement is crucial in the medical application of GHRH, because its biological half-life does not ex­ceed a few min (3, 4). However numerous data

Key-words: GHRH analogues superfusion, cell culture, Gaba, GH-re­lease.

Correspondence: Imre Mezb, 1 st. Institute of Biochemistry, Semmelweis University Medical School, 1444 Budapest 8. PO Box 260, Hungary.

Received February 22, 1993; accepted July 27, 1993.

793

1741 evoked higher GH release from cultured pitu­itary cells (1.5-2.5 times higher than the GH-RH(1-44)-amide). The other analogues (A-515 and A-527) were found to be equipotent to the standard molecule. We can conclude that Nle27 and Gaba30 substitutions appeared to be a good modification in in vitro test systems, and Gaba30 substitution served as a good spacer during the synthesis, since it made the coupling of the C-terminal amino acids easier and produced quantitative coupling. In addi­tion to the advantageous properties in the synthesis these modifications with or without D-Ala at the N­terminus increased the in vitro biological activity to 1.5-2.5 fold of the GHRH molecule. The additional substitution of Gly15 with Leu and Arg11 with D-Arg did not improve the in vitro GH-releasing activity of our analogues. A detailed in vivo investigation, which is essential for the future clinical use, has been performed and written in Part" of this paper.

show that GHRH may play an important role in cor­recting certain clinical disorders (5, 6) and in in­creasing milk and meat (7) production in veterinary applications. A major step in structure-biological activity studies of GHRH was, when it was proved that hGHRH (1-29)-amide exhibited full biological activity (2) and it was almost as active as the na­tive hGHRH (1-44)-amide. Substitutions of L amino acids with their D-isomers - in the N-terminal region of the molecule - resulted inthe first generation of superactive analogues (8). The efforts to synthesize more potent and long act­ing analogues included certain substitutions of the natural sequence with the appropriate D-isomers (9-11), or with other amino acids (12). Synthetic derivatives of amino acids especially in the C-, and N-terminus (13, 14), and special moieties in the side chains of the amino acids (15) were used to develop GHRH analogues having the mentioned

I. Mezo, M Kovacs, B. Szoke, et al.

advantageous effects. On the basis of these in­vestigations GHRH (1-29)-amide and its analogues are widely used in human (16, 17) and veterinary experimental medicine (18,19). We reported ear­lier that the elongation of GHRH(1-29)-amide ana­logues with Gaba30-amide resulted in potent GHRH analogues, and Gaba served as a very good spacer on the resin in solid phase peptide synthesis (SPPS) (20, 21) as well. In this paper we publish our experiments on the synthesis, charac­terization and in vitro biological assays of our new­ly developed Gaba30-analogues of GHRH (1-30)­amide.

MATERIALS AND METHODS Peptide synthesis

Analogues were synthesized by solid phase methodology on a Beckman 990 B automatic syn­thesizer. Peptides were prepared on benzhydry­lamine resin (BHA-resin; 1 % cross linkage) (Bachem, Feinchemikalien AG, Bubendorf, Switzerland). Boc-protected amino acids were pur­chased from Reanal (Factory for Fine Chemicals, Budapest, Hungary), with three exceptions: Boc­Lys (2-CI-Z)-OH (was purchased from Bachem) while N-Boc-Aspartic acid B-cyclohexyl ester [Boc­Asp (OChx)-OH] and Boc-gamma-amino-butyric acid (Boc-Gaba-OH) were synthesized in our lab­oratory. Solvents were purchased from Reanal. N,N-dimethylformamide (DMF) and dichlorome­thane (DCM) were redistilled prior to use. Triethylamine (TEA), anisole, trifluoracetic acid (TFA), diisopropyl-carbodiimide (DIC) and 1-hy­droxy-benzotriazol (HOBT) were purchased from Fluka Chemie A.G. (Buchs, Switzerland). Side-

Table 1 - MPLC purification of analogues.

Procedure Conditions

Purification Column Synchroprep RP-P 30~m resin (C-18), 30x1 cm

Eluent A: 0.25 N Teap (Triethylammonium phosphate), pH 3.0 solution

B: 60% CH3CN (acetonitrile) and 40% A solution

Gradient from 40% to 65% B, 400-400 ml, flow rate 2-2.5 ml/min.

Desalting Column: Synchroprep RP-P 30 ~m resin (C-18), 30x1 cm

Eluent A: 400 ml isopropanol- 10% AcOH (1 :3) B 400 ml isopropanol - 10% AcOH (12)

Gradient from 0% B to 100% B.

794

chain protection was benzyl for Glu, Tyr, Ser, Thr; tosyl for Arg and 2-CI-Z for Lys. Boc strategy was used for SPPS as it was described before (22). Cleavage of Boc-group was carried out by 33% TFA containing 5% anisole as scavenger. 3 molar excess of the amino acid derivatives were coupled in the presence of DIC and 1-hydroxy-benzotria­zole. Couplings were monitored by Kaiser test (23) and repeated when necessary. BOC-asparagine and Boc-glutamine were coupled as their p-nitro­phenyl esters. Cleavage of peptides from resin was performed by anhydrous hydrogen fluoride con­taining anisole and dithiothreitol. Crude GHRH products were purified first on Sephadex G-50 col­umn, eluted with 15% acetic acid, then by medium pressure liquid chromatography (MPLC) on re­versed phase column with gradient elution. Desalting was the final purification step which was carried out on the same reversed phase column (Table 1). Purity of fractions was estimated by thin layer chromatography (TLC) and analytical high performance liquid chromatography (HPLC). Fractions containing pure GHRH analogues,(purity >92%) were pooled and liophylized.

Characterization of the analogues

Final purity of the analogues was checked by HPLC, TLC (Table 2). Amino acid analysis of the analogues correlated well with the theoretical val­ues. Fast Atom Bombardment-Mass Spectrometry mea­surement of the analogues were carried out on a VG ZAB-2SEQ type hybrid tandem mass spec­trometer, equipped with a Cs ion gun (30 keV). Peptides were dissolved in dimethyl sulfoxide and mixed with a FAB matrix solvent (glycerol+ TFA). The obtained mass spectral data of the molecular peaks are given in Table 3. CD studies will be discussed in Part II. of this paper.

Table 2 - Codes and Analytical Oata of NIe27, Gaba30-hGRF (1-30)-NH2 (A-17 41) analogues.

Substitutions Code Rf1 Rf2 K' tR min

A-1741 040 044 4.37 11.9

D-Ala2, A-495 0.39 045 4.55 12.3

D-Ala2, Leu 15, A-495 041 046 4.68 12.6

D-Ala2, D-Arg11, Leu15, A-527 0.38 045 3.52 10.0

k': capacity factor, tR: retention time TLC solvent system: nBuOH: AcOH: H20=4: 1: 2 Rf( Merck, 5553, DC-Alufolien Kieselgel 60 Rfi Merck, 5724, DC-Fertigplattern Kieselgel 60 Column: Vydac 218TP54, 5 flm, 250 x 4.6 mm Eluent: 30% CH3CN/70% 0.25 N Teap, pH: 2.25 Flow rate: 1.3 ml/min Detection: 215 nm, 0.1 AUFS

Table 3 - Mass spectral data for compounds.

Monoisotopic mass of M+H Average mass of M+H Compound measured calculated measured calculated

A-1741 3424.3 3423.9 3426.2 3426.0

A-495 3424.2 3423.9 3426.1 3426.0

A-527 3480.0 3479.9 3481.7 3482.1

A-515 3480.3 3479.9 3481.9 3482.1

Biological assays

For biological assays hGHRH(1-29)-amide and hGHRH(1-44)-amide were purchased from Peninsula Laboratories, Inc. (Belmont, CA, USA).

Superfused pituitary cell system

A dynamic in vitro assay, the superfused rat anteri­or pituitary cell system (24) was used to test the GH­releasing effect of the peptides. Anterior pituitaries of 6 male animals were removed, cut into small pieces, incubated with collagenase (Type I, 0.5% Worthington, USA) for 50 min in a metabolic shaker, dispersed, gently mixed with 1 ml of swollen Sephadex G-10 which had been equilibrated with oxygenated tissue culture medium (Medium 199, Sigma, Germany), and transferred into the superfu­sion chamber. To assure stable baseline values the cells (approx. 5x106 in each chambers) were per­fused with the enzyme-free Medium 199 overnight before collecting fractions. The standard hGHRH(1-29)-amide (Peninsula Laboratories, Inc., Belmont, Ca, U.S.A.) and the tested materials were adminis­tered alternately into the system every 30 min. One ml of the appropriate concentrations of the peptides was pumped onto the cells for 3 min, and 1 ml frac­tions were collected every 3 min. The effect of Gaba alone on the GH release was also tested in con­centrations of 1, 10, 100, and 1000 nM. GH-releas­ing activities of the analogues were calculated by integrating the areas under the superfusion curves and were based on 5 nM and 10 nM concentrations.

Cultured anterior pituitary cells

A static in vitro assay, the cultured rat anterior pi­tuitary cell system was also used (25) to test the GH releasing activity of our analogues. In this assay system hGHRH( 1-44 )-amide (Peninsula Laborato­ries, Inc., Belmont CA, U.S.A.) was used as stan­dard. The anterior pituitary cells (50,000 cells/well) were cultured in 96-well culture plate for 4 days. On the day of testing the cells were preincubated in serum-free medium for 1 h, then were incubated for 2 h with the appropriate concentrations of the ana­logues or the standard molecule.

795

Synthesis of GHRH analogues

GH concentration of the fractions from the super­fusion system and from the tissue cultures was de­termined by RIA using materials supplied by the National Hormone and Pituitary Program. Interassay variation was less than 15% and intraassay varia­tion was 10% or less.

Statistical analysis

Statistical analysis of data was performed by Duncan's multiple range test.

RESULTS Peptide synthesis

Earlier we found that the coupling of the bulky Boc­Arg(Tos)OH to BHA-resin was very difficult in our hand; two or more recouplings were necessary for negative Kaiser test. Using BocGaba-OH as C-ter­minal amino acid, quantitative coupling was ob­served, therefore Gaba proved to be a good spac­er in the synthesis, too. Similarly to others (26), in the course of the syntheses we learned that recouplings and/or cappings were necessary for complete cou­plings or to prevent the formation of defective se­quences, respectively, in the region from Val 13 to Leu17. On the basis of these results we found that it was easy to synthesize pure Nle27 , Gaba30-hGH­RH(1-30)-amide analogues by SPPS methodology.

Superfused pituitary cell system

The GH-releasing activities of our analogues in the superfusion system are seen in Table 4. Our ana­logues showed equal, or slightly higher potency in releasing GH than the parent molecule. A-495 was found to be the most active, showing 1.63 fold the potency of the hGHRH(1-29)-amide (p<0.001). The GH-releasing potency of A-515 and A-1741 was

Table 4 - In vitro GH-releasing Effects of GHRH Analogues Relative to GHRH(1-29)-amide in the Superfused Rat Pituitary Cell System.

Peptides GH-releasing activity% Potency 5nM 10 nM

hGH-RH(1-29) amide 100±9.2 100±8.9 1.00

A-495 142± 11 *** 183±31 *** 1.63

A-515 90±4.2 159± 13*** 1.25

A-527 82±7.0 95±6.0 0.89

A-1741 115±8.0* 112± 10 1.14

GH-releasing activities were calculated from the results of four experi­ments in the superfused pituitary cell system and are reported as the mean±SE relative to GHRH (1-29)-amide. GH-releasing potencies were based on 5 nM and 10 nM doses and were calculated by the factorial analYSis of Bliss and Marks with 95% confidence limits (28). (*p<O.05 *** p<O.001).

I. Mezo, M. Kovacs, B. Szoke, et a/.

calculated to be slightly higher than the standard hormone (1.25 and 1.14 fold, respectively), and sig­nificant difference was found only at 5 nM concen­trations of A-1741 (p<0.05) and at 10 nM concen­trations of A-515 (p<0.001). No significant differ­ence could be detected between the activity of A-527 and the hGHRH standard (Table 4) . GH re­sponses induced by A-495 in one of these experi­ments are shown in Figure 1. Gaba alone in con­centrations from 1 nM to 1).lM did not have any ef­fect on the GH secretion in vitro.

Cultured pituitary cell system

All analogues, except A-17 41, evoked GH release equal to that released by the standard hGHRH (1-44 )-amide at 1 and 10 pM concentrations. At these concentrations A-1741 was 1.5-2.5 times more ac­tive in releasing GH than the hGHRH (1-44)-amide (p<0.01) . At 100 pM and 10 nM concentrations all analogues were found to be equipotent to the stan­dard. However, at 1 nM concentration A-495 showed slightly lower activity than the standard hor­mone (0.8 fold, p<0.05). No difference was found

GH ng/ml A 1500

900

300

B

900

300

I I I I I I I I I II 2 3 4 5 6 7 8 9 10 11 12

I I I I I I I I I II 2 3 4 5 6 7 8 9 10 1112

Fig. 1 - Representative experiment on the GH-releasing effect of A-495 in the superfused rat pituitary cell system. Bars ~) in­dicate 3 min pulses of GHRH(1-29)-amide (1,3, 5, 7,9, 11) and A-495 (2, 4, 6, 8, 10) in 5 nM (A) and 10 nM (B) concentrations. Bar 12 indicates a 3 min pulse of 50 mM KCI at the end of the experiment to control the amount of relasable GH in the pitu­itarycells.

796

GH fE'lease lng/weil l

600

500

300

200

100

Control

Fig. 2 - GH releasing effect of GHRH(1-30)-amide analogues in the cultured anterior pituitary cell system. Cells were incu­bated for 2 h with Medium 199 (control) or with increasing con­centrations of A-495 (6.--6.), A-515 ( • .. .• ), A-527 (A-. ­.. A), A-1741 (0 -.-.-0 ) and the standard GHRH(1-44)-amide (open bars). Plotted values are means ± SEM of six different ex­periments . •• p<0.01, • p<0.05.

between the activity of the other analogues and the standard GHRH (1-44)-amide.

DISCUSSION

In solid phase peptide synthesis spacers are wide­ly used, but these spacers are removed from the peptide molecule during the cleavage of peptides from the resins. In our case the Gaba spacer is an integral part of the analogues improving the effica­cy of the peptide synthesis. Our newly developed Gaba30 analogues of hGH­RH( 1-30)-amide were found to have 1-2 times high­er GH-releasing activity than the hGHRH molecule, testing them in the in vitro superfused pituitary cell system and in pituitary cell cultures. Results from the two tests were essentially similar in the case of A-515, A-527, and A-1741. At 10nM concentrations these analogues showed potency equal to the stan­dard molecule or slightly higher (A-515) than the hGHRH(1-29)-amide (superfusion system) or the hGHRH( 1-44 )-amide (cell culture system). On the other hand some differences were found between the GH-releasing activities of A-495 in the two dif­ferent systems. This analogue evoked GH release 1.8 times higher than the hGHRH(1-29)-amide molecule in the superfusion system, but slightly low­er (0.8 times) than the hGHRH(1-44)-amide in tissue cultures. This difference may be explained by the different incubation periods of the two methods. In the superfusion system we detect the effect of the test-materials after 3-min pulses, while in tissue cul­tures the incubation lasts for 2 h. In the pituitary cell

culture system A-495 might have caused higher GH release in the first minutes of the incubation than later, and this way the average GH concen­tration after the 2h incubation could be lower than the GH released in the first 3 min. The decrease in the releasing effect could be due to enzymatic degradation, decomposition or changes in the con­figuration of the peptide. These changes are un­likely to occur in the superfusion system, even dur­ing a long exposure to the peptides, because fresh, enzyme-free medium is perfused onto the cells continuously. Based on our result we can conclude that Gaba30

substitution served as a good spacer during the synthesis, and together with other modifications of the hGHRH(1-30)-amide molecule, resulted in ana­logues with in vitro GH-releasing activity 1-2 times higher than the parent molecule. The in vitro meth­ods are useful to screen the analogues for select­ing the ineffective peptides and to determine their pure biological activities, independently from all those factors which would influence the final effects of the molecule in vivo. Since some peptides, es­pecially the GHRH hormones are susceptible to de­composition and loose their activities due to ab­sorption to subcutaneous tissues (4, 14,27), none of the in vitro tests alone is sufficient to determine the real biological activities of the GHRH ana­logues. To evaluate the authentic GH-releasing po­tency, which is essential for the future clinical use, we performed a detailed in vivo investigation of our analogues. Results from these in vivo experiments including intravenous, intramuscular and subcuta­neous administration will be described in Part II of our paper.

ACKNOWLEDGMENTS This work was supported by the grants of the Hungarian Academy of Sciences (OTKA 1-600-2-86-1-492.) and the Ministry of Health (OTKA-SZEM 676/88). We thank Ms. V. Elias (Univ. Med. School, Pecs) for skilful experimental assistance and the National Hormone and Pituitary Program (NHPP) for the gifts of materials used in radioimmunoassays. We are thankful to Ms. V. Garamvolgyi for valuable help and the Tissue Culture and Hormone Laboratory of the Institute of Experimental Medicine of the Hungarian Academy of Sciences for in vitro bioassays. We thank Dr. H. Medzihradszky-Schweiger (Department of Organic Chemistry of the Eotvos L6rand University, Budapest) for amino acid analyses. We thank Dr. Marianna Mak (Hung. Acad. Sci. Central Institute for Chemistry) for the FAB-MS spec­tra.

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