Regulatory Peptides, 41 (1992) 185-193 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
185
REGPEP 01227
High potency of a new bombesin antagonist (RC-3095) in inhibiting serum gastrin levels; comparison of different routes of administration J a c e k Pinski, T e t s u Y a n o , Z o l t a n Rekasi, R e n - Z h i Cai, Sinisa Radulovic and A n d r e w V. Schally Endocrine, Polypeptide and Cancer Institute, VA Medical Center and Department of Medicine, Section of Experimental Medicine, Tulane University School of Medicine, New Orleans, LA (USA) (Received 20 March 1992; revised version received 2 July 1992; accepted 7 July 1992)
Key words." Bombesin antagonist; Pulmonary inhalation; Gastrin
Summary This study was performed to evaluate the efficacy and duration of action of a new bombesin antagonist D-Tpi6,Leu 13 ~ (CH2NH)Leu14-bombesin (6-14) (RC-3095), given by different routes of administration, in suppressing gastrin releasing-peptide (GRP(14-27))-stimulated gastrin release in rats. First, we showed that GRP(14-27) itself was highly active when administered by different routes. GRP(14-27), given to rats at a dose of 25 gg/100 g b.w. significantly increased serum gastrin levels 3 and 6 min after intravenous and for more than 30 min after subcutaneous administration or pulmonary inhalation. RC-3095 was then injected subcutaneously, intravenously and also delivered by pulmonary inhalation at a dose of 10 gg/100 g b.w. in each case to seven male rats 2, 30, 60 or 120 min prior to i.v. administration of 5 lag GRP(14-27). RC-3095 administered 2 min prior to GRP(14-27) decreased the gastrin response to GRP(14-27), measured as area under the curve, by 81 ~o in the intravenously injected group and 64~o in the pulmonary inhalation group in the first 6 min. When G R P ( 1 4 27), was given 30 min after administration of RC-3095, the gastrin response was decreased by 52~o in the subcutaneous group, 49~o in the pulmonary inhalation group and 11~o in the intravenous group during the first 6 min. RC-3095 delivered subcutaneously or by pulmonary inhalation 1 h before GRP(14-27) was also able to significantly inhibit gastrin release. Analysis of the data revealed that the bioavailability of RC-3095 given by the pulmonary inhalation route was about 69~o of the s.c. route. Our Correspondence to." A.V. Schally (151), VA Medical Center, 1601 Perdido Street, New Orleans, LA 70146, USA.
186
results indicate high activity of bombesin antagonist RC-3095, administered by different in vivo routes, in suppressing serum gastrin in rats. The administration by pulmonary inhalation evaluated here in rats, may prove to be practical and useful in the clinical setting.
Introduction
Bombesin (BN) is a tetradecapeptide originally isolated from the skin of the frog Bombina bombina by Espamer and colleagues in 1970 [ 1]. The mammalian counter-
part of bombesin, gastrin-releasing peptide (GRP), has the same carboxyl-terminal sequence as bombesin, and displays biological effects similar to bombesin. Bombesin/ GRP and related peptides were found to stimulate the secretion of gastroenteropancreatic hormones [2,3], pancreatic juice [4], and the contraction of various smooth muscles [5]. Several studies demonstrated biological effects of bombesin-like peptides not only in the periphery, but also on the central nervous system [6-10]. CNS effects of bombesin/GRP may be involved in thermoregulation, control of gastric acid secretion and satiety [6-10]. Interest in the development of potent, competitive bombesin/GRP receptor antagonists has been stimulated by the discovery that bombesin acts as an autocrine growth factor in human small cell lung carcinoma (SCLC) in vitro and in vivo [11 ]. Various antagonists of bombesin/GRP were reported previously [ 12-18 ]. Recently, more than 40 pseudo [ 13-14] bombesin (6-14) antagonists with different modifications at position 6,7 and 14, including D-Tpi6,Leu 13 ~0(CH2NH)Leu14-bombesin (6-14) (RC-3095), have been synthesized in our laboratory [ 19]. These antagonists inhibited the binding of iodinated Tyr4 bombesin to Swiss 3T3 and SCLC H-345 cells and suppressed the response to GRP(14-27), as based on incorporation of [3H]thymidine into DNA of these cells [19]. Nanomolar concentrations of RC-3095 also reduced GRP(14-27)stimulated amylase release from rat pancreatic acini in a superfusion assay [ 19]. Recently, we have shown that this antagonist inhibited the growth of nitrosamine-induced pancreatic cancer in hamsters [20], MXT estrogen-independent mammary cancer in mice [21] and HT-29 human colon cancer and PC-82 human prostate cancer in nude mice [22,23]. The aim of this study was to examine of effect of intravenous, subcutaneous and intrabronchial administration of gastrin-releasing peptide (GRP(14-27)) and its antagonist RC-3095 on gastrin release in rats.
Materials and Methods Animals
Male Sprague-Dawley rats weighing 230-250 g were used in all the experiments. The animals were allowed standard rat diet and tap water ad libitum, and were maintained
187
under controlled conditions: 12 h light, 12 h dark schedule at 24 + 2 o C. Groups of 7-8 rats were used in each experiment.
Peptides The bombesin antagonist D-Tpi6,Leu 13 tp (CH2NH)Leu14-BN (6-14) (RC-3095), was synthesized by solid phase methods on benzhydrylamine resin [ 19]. Introduction of the reduced peptide bond was carried out by a method described by Sasaki and Coy [24]. Purification of the crude material by reverse-phase H P L C on a Vydac C 18 support yielded a product whose purity was over 95 ~o by analytical HPLC. Amino acid analyses were consistent with expected values. RC-3095 used in this study was provided by Asta-Pharma Frankfurt/M, Germany. GRP(14-27) was synthesized by solid phase methods in our laboratory.
RIA All blood samples were centrifuged and kept at - 7 0 ° C until assayed. Serum gastrin levels were measured by double antibody RIA with a kit provided by Becton Dickenson and Company (Orangeburg, New York 10962-1294). The interassay variation was less than 7.3 ~o, and the intraassay variation was less than 4.0~o. Statistical significance was assessed by Duncan's new multiple range test [25]. The areas under the gastrin response curves were calculated using a computer program developed in our laboratory.
Experimental procedure Experiment 1 This experiment was performed to evaluate the effect of GRP(14-27) itself, given by different routes of administration, in stimulating gastrin release in rats. Rats were deprived of food, but not water, for 18 h prior to the experiment. The rats were anesthetized with sodium pentobarbital (30 mg/kg b.w.) and half of the initial pentobarbital dosage was given at 60 min intervals to maintain anesthesia. G R P ( 1 4 27) was administered intravenously, subcutaneously and by pulmonary inhalation in each case to groups of 7 rats at a dose of 25 ~tg/100 g b.w.. For pulmonary inhalation, a modified 26 gauge hypodermic needle was inserted into the upper part of the trachea. The peptide was delivered as a fine spray, after a brief compression of the chest. Blood samples were taken from the jugular vein before and 3, 6, 30, 60, 120 and 180 min after GRP(14-27) stimulation for measurements of serum gastrin levels. Experiment 2 This experiment was performed to evaluate the efficacy and duration of effect of bombesin antagonist RC-3095, given by different routes of administration, in suppressing GRP(14-27)-stimulated serum gastrin release in rats. Sodium pentobarbital was given at 60 min intervals to maintain anesthesia. RC-3095 was administered intravenously, subcutaneously and by pulmonary inhalation at a dose of 10 ~tg/100 g b.w. to 7 male rats 120, 60, 30 or 2 rain prior to GRP(14-27) (5 tag/100 g b.w.). Blood samples were taken from the jugular vein before, 3 and 6 min after GRP(14-27) stimulation. The procedure for pulmonary inhalation was the same as for experiment 1.
188 450-
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:
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Control GRP 25 fig i.v. GRP 25 /~g s.c. GRP 25 g,g i.h.
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Fig. 1. Effect of GRP(14-27), given by different routes of administration at a dose of 25 lag/100 g b.w. (i.v., intravenous; s.c., subcutaneous; i.h., inhalation), on serum gastrin release in rats. * P < 0.05 and **P
185
REGPEP 01227
High potency of a new bombesin antagonist (RC-3095) in inhibiting serum gastrin levels; comparison of different routes of administration J a c e k Pinski, T e t s u Y a n o , Z o l t a n Rekasi, R e n - Z h i Cai, Sinisa Radulovic and A n d r e w V. Schally Endocrine, Polypeptide and Cancer Institute, VA Medical Center and Department of Medicine, Section of Experimental Medicine, Tulane University School of Medicine, New Orleans, LA (USA) (Received 20 March 1992; revised version received 2 July 1992; accepted 7 July 1992)
Key words." Bombesin antagonist; Pulmonary inhalation; Gastrin
Summary This study was performed to evaluate the efficacy and duration of action of a new bombesin antagonist D-Tpi6,Leu 13 ~ (CH2NH)Leu14-bombesin (6-14) (RC-3095), given by different routes of administration, in suppressing gastrin releasing-peptide (GRP(14-27))-stimulated gastrin release in rats. First, we showed that GRP(14-27) itself was highly active when administered by different routes. GRP(14-27), given to rats at a dose of 25 gg/100 g b.w. significantly increased serum gastrin levels 3 and 6 min after intravenous and for more than 30 min after subcutaneous administration or pulmonary inhalation. RC-3095 was then injected subcutaneously, intravenously and also delivered by pulmonary inhalation at a dose of 10 gg/100 g b.w. in each case to seven male rats 2, 30, 60 or 120 min prior to i.v. administration of 5 lag GRP(14-27). RC-3095 administered 2 min prior to GRP(14-27) decreased the gastrin response to GRP(14-27), measured as area under the curve, by 81 ~o in the intravenously injected group and 64~o in the pulmonary inhalation group in the first 6 min. When G R P ( 1 4 27), was given 30 min after administration of RC-3095, the gastrin response was decreased by 52~o in the subcutaneous group, 49~o in the pulmonary inhalation group and 11~o in the intravenous group during the first 6 min. RC-3095 delivered subcutaneously or by pulmonary inhalation 1 h before GRP(14-27) was also able to significantly inhibit gastrin release. Analysis of the data revealed that the bioavailability of RC-3095 given by the pulmonary inhalation route was about 69~o of the s.c. route. Our Correspondence to." A.V. Schally (151), VA Medical Center, 1601 Perdido Street, New Orleans, LA 70146, USA.
186
results indicate high activity of bombesin antagonist RC-3095, administered by different in vivo routes, in suppressing serum gastrin in rats. The administration by pulmonary inhalation evaluated here in rats, may prove to be practical and useful in the clinical setting.
Introduction
Bombesin (BN) is a tetradecapeptide originally isolated from the skin of the frog Bombina bombina by Espamer and colleagues in 1970 [ 1]. The mammalian counter-
part of bombesin, gastrin-releasing peptide (GRP), has the same carboxyl-terminal sequence as bombesin, and displays biological effects similar to bombesin. Bombesin/ GRP and related peptides were found to stimulate the secretion of gastroenteropancreatic hormones [2,3], pancreatic juice [4], and the contraction of various smooth muscles [5]. Several studies demonstrated biological effects of bombesin-like peptides not only in the periphery, but also on the central nervous system [6-10]. CNS effects of bombesin/GRP may be involved in thermoregulation, control of gastric acid secretion and satiety [6-10]. Interest in the development of potent, competitive bombesin/GRP receptor antagonists has been stimulated by the discovery that bombesin acts as an autocrine growth factor in human small cell lung carcinoma (SCLC) in vitro and in vivo [11 ]. Various antagonists of bombesin/GRP were reported previously [ 12-18 ]. Recently, more than 40 pseudo [ 13-14] bombesin (6-14) antagonists with different modifications at position 6,7 and 14, including D-Tpi6,Leu 13 ~0(CH2NH)Leu14-bombesin (6-14) (RC-3095), have been synthesized in our laboratory [ 19]. These antagonists inhibited the binding of iodinated Tyr4 bombesin to Swiss 3T3 and SCLC H-345 cells and suppressed the response to GRP(14-27), as based on incorporation of [3H]thymidine into DNA of these cells [19]. Nanomolar concentrations of RC-3095 also reduced GRP(14-27)stimulated amylase release from rat pancreatic acini in a superfusion assay [ 19]. Recently, we have shown that this antagonist inhibited the growth of nitrosamine-induced pancreatic cancer in hamsters [20], MXT estrogen-independent mammary cancer in mice [21] and HT-29 human colon cancer and PC-82 human prostate cancer in nude mice [22,23]. The aim of this study was to examine of effect of intravenous, subcutaneous and intrabronchial administration of gastrin-releasing peptide (GRP(14-27)) and its antagonist RC-3095 on gastrin release in rats.
Materials and Methods Animals
Male Sprague-Dawley rats weighing 230-250 g were used in all the experiments. The animals were allowed standard rat diet and tap water ad libitum, and were maintained
187
under controlled conditions: 12 h light, 12 h dark schedule at 24 + 2 o C. Groups of 7-8 rats were used in each experiment.
Peptides The bombesin antagonist D-Tpi6,Leu 13 tp (CH2NH)Leu14-BN (6-14) (RC-3095), was synthesized by solid phase methods on benzhydrylamine resin [ 19]. Introduction of the reduced peptide bond was carried out by a method described by Sasaki and Coy [24]. Purification of the crude material by reverse-phase H P L C on a Vydac C 18 support yielded a product whose purity was over 95 ~o by analytical HPLC. Amino acid analyses were consistent with expected values. RC-3095 used in this study was provided by Asta-Pharma Frankfurt/M, Germany. GRP(14-27) was synthesized by solid phase methods in our laboratory.
RIA All blood samples were centrifuged and kept at - 7 0 ° C until assayed. Serum gastrin levels were measured by double antibody RIA with a kit provided by Becton Dickenson and Company (Orangeburg, New York 10962-1294). The interassay variation was less than 7.3 ~o, and the intraassay variation was less than 4.0~o. Statistical significance was assessed by Duncan's new multiple range test [25]. The areas under the gastrin response curves were calculated using a computer program developed in our laboratory.
Experimental procedure Experiment 1 This experiment was performed to evaluate the effect of GRP(14-27) itself, given by different routes of administration, in stimulating gastrin release in rats. Rats were deprived of food, but not water, for 18 h prior to the experiment. The rats were anesthetized with sodium pentobarbital (30 mg/kg b.w.) and half of the initial pentobarbital dosage was given at 60 min intervals to maintain anesthesia. G R P ( 1 4 27) was administered intravenously, subcutaneously and by pulmonary inhalation in each case to groups of 7 rats at a dose of 25 ~tg/100 g b.w.. For pulmonary inhalation, a modified 26 gauge hypodermic needle was inserted into the upper part of the trachea. The peptide was delivered as a fine spray, after a brief compression of the chest. Blood samples were taken from the jugular vein before and 3, 6, 30, 60, 120 and 180 min after GRP(14-27) stimulation for measurements of serum gastrin levels. Experiment 2 This experiment was performed to evaluate the efficacy and duration of effect of bombesin antagonist RC-3095, given by different routes of administration, in suppressing GRP(14-27)-stimulated serum gastrin release in rats. Sodium pentobarbital was given at 60 min intervals to maintain anesthesia. RC-3095 was administered intravenously, subcutaneously and by pulmonary inhalation at a dose of 10 ~tg/100 g b.w. to 7 male rats 120, 60, 30 or 2 rain prior to GRP(14-27) (5 tag/100 g b.w.). Blood samples were taken from the jugular vein before, 3 and 6 min after GRP(14-27) stimulation. The procedure for pulmonary inhalation was the same as for experiment 1.
188 450-
4-00-
"~-
350-
T** 'IF / 'l\ \
500-
/ T ,~,
200-
/•\
0--0: 0---0: A---A: • -- •
:
/ _
/ / z ~ / / \ A.
~-_
/**
o:."~ ~ i ,
S ~so-// / 100-///
Control GRP 25 fig i.v. GRP 25 /~g s.c. GRP 25 g,g i.h.
•
-¢*.
-- z~IZ~'-'-~ai.~a
5oi~0--0
~
0 3
6
.3'0 Time (Minutes)
6'0
1'20
180
Fig. 1. Effect of GRP(14-27), given by different routes of administration at a dose of 25 lag/100 g b.w. (i.v., intravenous; s.c., subcutaneous; i.h., inhalation), on serum gastrin release in rats. * P < 0.05 and **P
