Proc. Natl. Acad. Sci. USA Vol. 77, No. 1, pp. 577-579, January 1980 Medical Sciences

[Phe4]Somatostatin: A potent, selective inhibitor of growth hormone release (peptides/pituitary/bioassay/receptors/somatotropin) CHESTER A. MEYERS, DAVID H. Coy, WILLIAM A. MURPHY, TOMMIE W. REDDING, AKIRA ARIMURA, AND ANDREW V. SCHALLY Department of Medicine, Tulane University School of Medicine, and Veterans Administration Medical Center, New Orleans, Louisiana 70112 Contributed by Andrew V. Schally, October 4, 1979

ABSTRACT [Phe4jSomatostatin was twice as active as so- MATERIALS AND METHODS matostatin (SS) in suppressing rat growth hormone release in vitro but had only weak activity toward inhibition of insulin Amino acids are of the L configuration unless otherwise noted. and glucagon release in vivo. The ability of this analogue to Amino acid derivatives were purchased from Bachem Fine inhibit growth hormone release more actively than SS was Chemicals (Torrance, CA). confirmed in vivo by two separately designed bioassays. Further Solid-Phase Synthesis. The peptides were synthesized structure/activity studies of position 4 were carried out with stepwise as described for SS analogues (3) with the modifications [Glu4]SS, [Thr4JSS, and des-Lys4-SS, all of which had negligible inhibiting activity in the pituitary and pancreas. In this context, described by Meyers et al. (4). The standard polystyrene-1% the strikingly selective activity of [Phe4]SS suggests a funda- divinylbenzene resin was used. Amino acids were coupled as mental difference in the SS receptors of pituitary and pancreas, their Na-tert-butyloxycarbonyl (Boc) derivatives, except that and the normal side-chain basicity of position 4 appears to be alanine and glycine were coupled in the form of the carbo- more important for action in pancreas than in pituitary. [Phe4JSS benzoxy-protected dipeptide (we have consistently isolated a has properties that may be useful in the development of agents major contaminant from the completed peptides with low in- for the treatment of acromegaly or other disorders associated corporation of alanine as judged by amino acid analysis when with increased growth hormone levels. glycine and alanine were added individually as their t-Boc derivatives). Reactive amino acid side-chains were protected Systematic changes in the structure of the somatostatin (SS) as follows: cysteine, 4-methylbenzyl (3); threonine, serine, and tetradecapeptide (Fig. 1) have resulted in the identification of glutamate, benzyl; lysine, 2-chlorocarbobenzoxy. After at- certain amino acid residues that are particularly important to tachment of the first protected amino acid to the resin (5), each the expression of biological activity in several tissues. Some succeeding amino acid was coupled in a Beckman model 990 analogues of SS with substitutions in position 4 have already peptide synthesizer in the presence of diisopropylcarbodiimide been tested for inhibition of insulin, glucagon, and growth and, in the case of Boc-Asn, 1 molar equivalent of 1-hydroxy- hormone (GH), but a systematic investigation into the effects benzotriazole. Removal of Boc protection at each step was ac- of decreasing the basicity or lipophilicity of the side-chain in complished by treatment (30 min) with 33% (vol/vol) trifluo- this position has not been undertaken. roacetic acid in CH2Cl2 containing 0.2% 1,2-ethanedithiol. The Although the position 4 analogues so far reported are gen- completed peptides were cleaved from the resin and depro- erally much less active than SS in the pituitary and pancreas tected with 10% anisole in HF (1 hr, 0°C). The free disulfhydryl (with the exception of the basic [Arg4]SS analogue), [Ala4]SS peptides were cyclized in dilute aqueous solution with potas- retained significant activity (20%) in these tissues, and [D- sium ferricyanide as described (6), with the exception that the Lys4]SS showed a tendency toward selective action on the pi- insoluble [Phe4]SS was cyclized in 50% dimethylformamide and tuitary (1). To investigate further the importance of position lyophilized after removal of the dimethylformamide in vacuo 4 in maintaining the biological activity of SS peptides and to at 35°C. exploit the possibility of developing selective analogues, we Purification and Homogeneity. The crude cyclic peptides systematically replaced the Lys4 residue by aromatic (Phe), were purified by gel filtration on columns (2.5 X 95 cm) of alcoholic (Thr), and acidic (Glu) amino acid residues, and we Sephadex G-15 in 50% AcOH and Sephadex G-25 (fine) in 0.2 also prepared the des-Lys4-SS peptide. Preliminary results of M AcOH, followed by partition chromatography on a column this work have appeared (2). (1.5 X 140 cm) of Sephadex G-25 (fine) in the biphasic solvent system 2 M AcOH/1-BuOH, 1:1 (vol/vol). [Phe4]SS'required The publication costs of this article were defrayed in part by page additional purification by partition chromatography in 0.5 M charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: SS, somatostatin; GH, growth hormone (somato- this fact. tropin). I H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 FIG. 1. Structure of SS. 577 Downloaded by guest on September 25, 2021 578 Medical Sciences: Meyers et al. Proc. Natl. Acad. Sci. USA 77 (1980)

Table 1. Physicochemical properties of SS analogues Thin-layer chromatography, Substituent in RF in various systems [a]25 in [a]23 in Yield,* position 4 A B C D 0.1 M AcOH 50% AcOH % Phe 0.34 0.67 0.82 0.78 - t (c 0.38) 8 Thr 0.18 0.48 0.48 0.64 -30 (c 0.66) -18 (c 0.66) 11 des-Lys 0.23 0.52 0.57 0.67 -26 (c 0.38) -24 (c 0.51) 8 Glu 0.27 0.54 0.60 0.76 -28 (c 0.50) -21 (c 0.48) 6 * Yields based on final products compared with total starting BOC-amino acid esterified to the resin. t Not detectable at this concentration.

AcOH/1-BuOH, 1:1 (vol/vol). [Glu4]SS required further pu- (2.5 mg/100 g) into the jugular vein to increase the GH levels. rification on a column (1.5 X 145 cm) of silica gel G in solvent Blood samples were collected by decapitation 15 min after in- system C. jection of test substance, and serum GH was determined by the The homogeneity of the final peptides was demonstrated by double-antibody radioimmunoassay described above. thin-layer chromatography on silica gel plates (Brinkman SIL Experiment B. Male Charles River CD rats (300-350 g) with G-25) in the following solvent systems: A, upper phase of 1- free access to food and water were anesthetized with Nembutal BuOH/AcOH/H20, 4:1:5 (vol/vol); B, 2-PrOH/1 M AcOH, (5 mg/100 g intraperitoneally). After 30 min, saline or test 2:1; C, 1-BuOH/AcOH/H20/EtOAc, 1:1:1:1; D, EtOAc/ peptide was injected subcutaneously. Blood samples (1 ml) were Pyr/AcOH/H20, 5:5:1:3. Solvent fronts were allowed to travel drawn from the jugular vein 15 min after the injection of the 12-14 cm and spots (loads, 20-40 jig) were visualized with test substance, and the plasma was separated and assayed for ninhydrin and Ehrlich's reagents. RF values are given in Table GH as described above. 1 along with optical rotations and yields. Amino acid analyses RESULTS AND DISCUSSION were performed on a Beckman 119 amino acid analyzer on samples that were hydrolyzed (1100C, 18 hr) in 4 M meth- The biological activities of the previously reported position 4 anesulfonic acid containing 0.2% 3-(2-aminoethyl)indole in analogues (1) were markedly decreased except for [Arg4]SS, in sealed, evacuated tubes. Results appear in Table 2. which the normally basic character of the side chain was pre- Insulin and Glucagon Bioassays. Each analogue was tested served (Table 3). However, the decreased but significant in- for its ability to inhibit the release of insulin and glucagon in hibiting activity in the pituitary and pancreas (20%) of [Ala4]SS, vivo in male rats (CD strain, Charles River) weighing 300-350 which has a neutral, hydrophobic side chain, indicated the lack g. The rats were kept in controlled temperature (24°C) and light of an absolute requirement for a basic or polar side chain in (0500-1900 hr) conditions for 1 week before the assay. They position 4. This prompted our present investigation with pre- were anesthetized with urethane (150 mg/100 g intraperito- viously untested position 4 SS analogues featuring an aromatic neally) and then arginine was infused into the jugular vein alone (Phe), acidic (Glu), or alcoholic (Thr) moiety in the side chains or with SS or analogue as described (3). Blood samples were of these residues. collected from the jugular vein or by decapitation, and the Table 3 shows a clearly decreasing order of in vitro GH- plasma was separated and stored at -20°C until assayed for inhibiting activity as the L amino acid residue in position 4 of insulin and glucagon (7). Plasma insulin was determined by SS changes from basic (Lys and Arg, 100%) to neutral (Ala, 20%) double-antibody radioimmunoassay using porcine insulin (Eli to acidic (Glu, 5%). Although this suggests a role for side-chain Lilly) for labeling and a guinea pig antiserum generated against basicity in this position for expression of biological activity in porcine insulin (8). Plasma glucagon was determined by the the pituitary, the effect probably does not involve a direct in- method of Faloona and Unger (9) using crystalline glucagon teraction of this residue with the pituitary receptors because (Eli Lilly) and rabbit antiserum 30 K against glucagon (Unger [Phe4]SS was more potent than SS in this assay. pool 2, lot 34). Duncan's new multiple range test was used to [Phe4JSS was twice as active as SS on the inhibition of in vitro compare the values obtained in each four-point assay (10). GH release but exhibited substantially less activity than SS In Vitro GH Bioassay. The analogues were compared with (similar to all of the other nonbasic L amino acid position 4 SS SS for their ability to inhibit the release of radioimmunoas- analogues) toward inhibition of insulin and glucagon release sayable GH in vitro from enzymatically dispersed rat anterior in vivo (Table 3). To determine whether [Phe4]SS was truly a pituitary cells as described (3, 7, 11). GH was determined by potent, selective inhibitor of GH release or merely rendered double-antibody radioimmunoassay for rat GH by using the ineffective in vivo, we examined its potency to inhibit GH re- NIAMDD Rat GH kit (12). lease in vivo (Table 4). [Phe4]SS appeared to be even more ac- In Vivo GH Bioassay. Experiment A. Male Charles River tive in inhibiting GH release in vivo than in vitro. Thus, a basic CD rats (150-200 g) were fasted overnight and then anesthe- side chain in position 4 appears to be considerably more im- tized with urethane (150 mg/100 g intraperitoneally). After portant for biological activity in the pancreas than in the pi- 45 min, saline or test peptide was injected subcutaneously, tuitary, which may reflect a fundamental difference in their followed immediately by intravenous injection of Nembutal respective receptors. Differences in the receptor types are

Table 2. Amino acid analyses of SS analogues Substituent in position 4 Ala Gly 1/2-Cys Glu Lys Asp Phe Trp Thr Ser NH:, 1Phe 1.05 1.00 1.73 - 0.93 1.08 3.73 0.73 1.92 0.93 1.37 Thr 1.06 1.00 1.90 - 0.96 1.01 2.99 1.00 2.79 0.92 1.28 des-Lys 0.99 1.00 1.81 - 1.06 1.02 2.98 0.92 1.91 0.89 1.58 Glu 1.00 0.98 1.87 1.08 1.03 1.08 2.98 0.86 1.87 0.85 1.20 Downloaded by guest on September 25, 2021 Medical Sciences: Meyers et al. Proc. Natl. Acad. Sci. USA 77 (1980) 579 Table 3. GH, insulin, and glucagon release-inhibiting activities of 1) may reflect this conformational shift. The possible contri- position 4 SS analogues bution of a steric effect by the position 4 phenyl ring to the % inhibiting activity* observed biological actions of this analogue cannot be ruled Substituent in In vivo In vitro out. position 4 Insulin Glucagon GH The activity observed for [Phe4]SS would not ordinarily be explained by resistance to enzymatic degradation because the Lys(SS) 100 100 100 was Phe 2.8 (0.14-55) 15.5 (3.6-66) 203 (92-449) trypsin-sensitive lysyl residue replaced by the chymo- Thr 1.6 (0.12-20) <5 <1 trypsin-sensitive phenylalanyl residue. Nevertheless, prelimi- des-Lyst 1.5 (0.20-11) 2.7 (1.1-6.8) nary results (unpublished observations) indicate that [Phe4]SS Glu 1.1 (0.05-23) 4.9 (2.9-8.4) has long-acting properties, and its ability to inhibit GH release Ala§ 20 20 20 with apparently greater activity in vivo than in vitro is con- D-Lys§ 1 1 22 sistent with this observation. Replacement of Lys4 by hydro- Arg§ 60 100 100 phobic, endopeptidase-resistant residues could, however, still be important to the development of potent * 95% confidence limits are shown in parentheses. analogues with t This peptide has been reported (1), and our observed activities agree prolonged activity. well with the literature values. In the determination of GH release-inhibiting activity in vivo, No glucagon release-inhibiting activity was observed at doses of 10 no significant differences in the final results were found be- and 100 mg/kg. tween the assay using fasted rats under urethane anesthesia § From Rivier et al. (1). (Exp. A) or fed rats under Nembutal anesthesia (Exp. B) (Table 4). However, Nembutal-stimulated GH levels in the control further suggested by the retention of moderate Gli-inhibiting animals of Exp. B were higher than in those of Exp. A. activity by [D-Lys4]SS which was almost devoid of activity in the pancreas (Table 3). This research was supported by National Institutes of Health Grants The highly selective inhibition of GH by [Phe4JSS could make AM 18370 (D.H.C.) and AM 22156 (C.A.M.) and by the Veterans this or similar analogues useful in treating patients who have Administration. GH-secreting tumors (e.g., acromegalics) or other disorders associated with increased plasma GH levels (e.g., diabetes 1. Rivier, J., Brown, M., Rivier, C., Ling, N. & Vale, W. (1977) in mellitus). Other SS analogues with selective actions on the pi- Peptides 1976, ed. Loffet, A. (Editions de l'Universite de Brux- tuitary have been reported (13-15), although none was more elles, Brussels, Belgium), pp. 427-451. active than SS. 2. Meyers, C. A. & Coy, D. H. (1978) Endocrinology 102, 239A. It is remarkable that such a profound replacement as phe- 3. Meyers, C. A., Arimura, A., Gordin, A., Fernandez-Durango, R., nylalanine for lysine in position 4 produced a highly active Coy, D. H., Schally, A. V., Drouin, J., Ferland, L., Beaulieu, M. & Labrie, F. (1977) Biochem. Biophys. Res. Commun. 74, GH-inhibitory analogue when so many other modifications in 630-636. this position resulted in peptides with less activity than SS. 4. Meyers, C. A., Coy, D. H., Huang, W. Y., Schally, A. V. & Whether this high activity can be attributed more to lipophil- Redding, T. W. (1978) Biochemistry 17,2326-2331. icity or to electronic effects (7r electron interactions) imparted 5. Gisin, B. F. (1973) Helv. Chim. Acta. 56, 1476-1482. by the aromatic phenylalanine side-chain is uncertain. The 6. Coy, D. H., Coy, E. J., Arimura, A. & Schally, A. V. (1973) Bio- observed biological effects, however, are almost certainly the chem. Biophys. Res. Commun. 54, 1267-1273. result of a pronounced change in the overall conformation of 7. Gordin, A., Meyers, C. A., Arimura, A., Coy, D. H. & Schally, A. the SS peptide, which ip the case of [Phe4]SS appears to be fa- V. (1977) Acta Endocrinol. 86,833-841. vorable to activity in the pituitary and deleterious in the pan- 8. Starr, J. I. & Rubenstein, A. H. (1974) in Methods of Hormone creas. The dramatic loss of optical activity in [Phe4]SS (Table Radiolmmunoassay, eds. Jaffe, B. & Behrman, H. R. (Academic, New York), pp. 289-315. 9. Faloona, G. R. & Unger, R. H. (1974) in Methods of Hormone Table 4. Inhibition of GH release in vivo by SS and [Phe4]SS Radloimmunoassay, eds. Jaffe, B. & Behrman, H. R. (Academic, New York), pp. 317-330. Dose, Plasma GH, ng/ml* 10. Steel, R. G. & Torrie, J. H. (1960) Principles and Procedures of Peptide ,ug/100 g Exp. A Exp. B Statistics (McGraw-Hill, New York), pp. 107-109. None 67 16 344 21 11. Vale, W., Grant, G., Amoss, M., Blackwell, R. & Guillemin, R. SS 0.5 48± 9 87 26 (1972) Endocrinology 91, 562-572. 12. Birge, C. A., Peake, G. T., Mariz, I. K. & Daughaday, W. H. 5.0 8± 4 54 13 (1967) Endocrinology 81, 195-204. [Phe4]SSt 0.1 45 ± 17 123 ± 24 13. Grant, N., Clark, D., Garsky, V., Jaunakais, I., McGregor, W. & 1.0 19± 4 38± 6 Sarantakis, D. (1976) Life Sci. 19, 629-632. * Shown as mean ± SEM. Exp. A, n = 5 rats per group; Exp. B, n = 14. Garsky, V. M., Clark, D. E. & Grant, N. H. (1976) Biochem. 6. Biophys. Res. Commun. 73,911-916. t Percent inhibiting activity with 95% confidence limits: Exp. A, 374 15. Sarantakis, D., Teichman, J., Clark, D. E. & Lien, E. L. (1977) (96-1465); Exp. B, 342 (78-1503). Biochem. Biophys. Res. Commun. 75,143-148. Downloaded by guest on September 25, 2021