Peptides,Vol. 13, pp. 401--407, 1992

0196-9781/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.

Printed in the USA.

Metabolic Stability and Tumor Inhibition of Bombesin/GRP Receptor Antagonists T. P. D A V I S , * S. C R O W E L L , * J. T A Y L O R , I " D. L. C L A R K , * D. COY,:~ J. S T A L E Y § A N D T. W. M O O D Y §

* Department of Pharmacology and the Arizona Cancer Center, The University of Arizona, College of Medicine, Tucson, A Z 85 724 -?Biomeasure Inc., Department of Neurosciences, Hopkinton, MA O1748 ~. Tulane University School of Medicine, New Orleans, LA 70112 § Department of Biochemistry and Molecular Biology, George Washington University Medical School, Washington, DC 2003 7 R e c e i v e d 12 A u g u s t 1991 DAVIS, T. P., S. CROWELL, J. TAYLOR, D. L. CLARK, D. COY, J. STALEY AND T. W. MOODY. Metabolicstabilityand tumor inhibition of bombesin/GRP receptor antagonists. PEPTIDES 13(2) 401-407, 1992.--SmaU cell lung cancers (SCLC) synthesize and secrete bombesin/gastrin releasing peptide (BN/GRP). The autocrine growth cycle of BN/GRP in SCLC can be disrupted by BN/GRP receptor antagonists such as [PsP3'~4]BN. Here several BN analogues were solid-phase synthesized and incubated with intact SCLC cells at 37°C in RPMI medium in a time-course fashion (0-1080 minutes) to determine enzymatic stability. The proteolytic stability of the compounds was determined by subsequent H PLC analysis. The metabolic half-life ranged from 154 minutes to 1388 minutes for the six analogues studied. [Psi~3.~4]BNwas found to be very stable to metabolic enzymes (T½ = 646 mm) and also inhibited SCLC xenograft formation in vivo in a dose-dependent manner. When [Psi~3,~4]BNwas incubated with NCI-H345 cells, it inhibited 'zSI-GRP binding with an IC5o value of 30 nM. These data suggest that BN/GRP receptor antagonists such as [Psit3'~4]BNmay be useful for the treatment of SCLC. Bombesin

Small cell lung cancer

Gastrin releasing peptide

MOST small cell lung cancer (SCLC) cell lines contain the autocrine peptide bombesin/gastrin releasing peptide (BN/GRP) (4,14,19,25). BN/GRP is synthesized in and secreted from SCLC cell lines (14). High affinity binding sites for BN or GRP are present in SCLC cell line NCI-H345 (13,17). Also, BN causes phosphatidylinositol turnover and elevates cytosolic Ca 2+ in NCIH345 (11,18). It has also been shown that BN stimulates the clonal growth of SCLC cell line NCI-H345 or NCI-N592 in vitro (6,9) and xenograft formation in nude mice in vivo (1). Recently, high affinity antagonists for BN/GRP have been identified (5,7,8,16,29,30). These include reduced peptide bond analogues such as [Psi13"14]BN and [des-Metl4]BN analogues (5,7,16). These BN antagonists inhibit binding to BN/GRP receptors with high affinity, inhibit the increase in cytosolic Ca + caused by BN, and also inhibit the clonal growth of SCLC cells in culture (7,8,16,29). Little is known about the metabolism of these BN analogues. Previously we identified degradative enzymes associated with SCLC cells ( 1 l), and in the present study the stability of these BN analogues was determined in vitro. Also, the biodistribution and growth inhibitory effect of [Psil3.t4]BN in nude mice bearing SCLC xenografts were investigated.

Xenografts

Half-life

function as SCLC BN receptor antagonists using SCLC cell lines NCI-H345 and -N592. NCI-H345 was cultured in SIT medium (RPMI-1640 containing 3 × 10-8 M NaSeO3, 5/~g/ml insulin, and 10 t~g/ml transferrin) (Sigma Chemical Co., St. Louis, MO) supplemented with 2.5% fetal bovine serum (GIBCO). NCI-N592 was cultured in RPMI-1640 containing 10% fetal bovine serum. The cells were cultured at 37°C in 95% air/5% COz and were used when they were in exponential growth phase. The ability of the peptides to function as SCLC BN receptor antagonists was determined in binding assays, second messenger assays, and growth assays in vitro.

Binding Studies NCI-H345, 1500 BN receptors/cell, was harvested 1 day after a medium change. The cells (2 X 106) were incubated with 0.25 nM 'ESI-GRP (2200 Ci/mmol) (Amersham Corp., Arlington Heights, IL) at 25 °C for 30 minutes in receptor binding medium [SIT containing 0.25% bovine serum albumin (Gibco) and 100 ~tg/ml bacitracin (Sigma)] in the presence or absence of unlabeled peptide. Bound ~zSI-GRP was separated from free using the centrifugation techniques described previously (17).

Second Messenger Studies

METHOD

NCI-H345 was harvested and the cells (2.5 X 106/ml) loaded with 5 uM Fura-2AM (Calbiochem, La Jolla, CA) at 37°C for 30 minutes as previously described (18). The cells which contained loaded Fura-2 were centrifuged at 150 X g for 10 minutes

Cell Culture Conditions BN analogues were synthesized using the methods described previously (7,8). The peptides were tested for their ability to 401

402 and resuspended at the same concentration in new SIT medium. The fluorescence intensity was continuously monitored using a spectrofluorometer equipped with a magnetic stirring mechanism and temperature-regulated (37°C) cuvette holder prior to and after the addition of peptide as described previously (18).

In Vitro Growth Studies Cell lines NCI-H345 and -N592 were harvested and tested in the agarose cloning system described previously (9,11). The base layer consisted of 3 ml of 0.5% agarose (Gibco) in SIT medium containing 5% fetal bovine serum in 6-well plates (Falcon, Oxnard, CA). The top layer consisted of 3 ml of HITES medium (0.3% agarose), the peptide(s) and/or protease inhibitor thiorphan (100 uM) doubly concentrated, and 6 × l 0 4 single viable cells. After 2 weeks, I ml of 0.1% p-iodonitrotetrazolium violet was added and after 16 hours at 37°C, the plates were screened for colony formation (colonies _>42 um in diameter were counted) using an Omnicon image analysis system (Bausch and Lomb, Rochester, NY) (9,11).

Proteolytic Enzyme Assays Assays were performed to quantitate the activities of neutral endopeptidase 24.11 (E.C. 3.4.24.11), metalloendopeptidase 24.15 (E.C. 3.4.24.15), carboxypeptidase H (E.C. 3.4.17.10), trypsin-like activity, and aminopeptidase (E.C. 3.4.11.1) activity in SCLC cell lines NCI-H345, NCI-N417, and NCI-N592. For all assays, cell pellets were resuspended in 50 mM Tris buffer (pH 7.4), sonified, and then centrifuged. Both supernatant and membrane pellet were analyzed for protein content. Neutral endopeptidase 24.11 was measured using a modified version of the method of Bateman and Hersh (2). Incubation samples were prepared in triplicate containing 10 ug protein per tube, 0.1 mM substrate (glutaryl-Ala-Ala-Phe-4-methoxy-2naphthylamide), and 0.05 M MES buffer (2-[N-morpholino]ethanesulfonic acid, pH 6.4) in a total volume of 0.25 ml per sample. In control samples (2 additional tubes), 6 #M phosphoramidon was used to block neutral endopeptidase 24.11 activity. The phosphoramidon reagent also contained aminopeptidase, so these tubes received 2.5 ug aminopeptidase M. Samples were incubated at 37°C for 1 hour, or longer in the case of samples with low activity. Neutral endopeptidase 24.11 activity was halted at the end of the incubation period in the samples which had not received phosphoramidon by addition of 10 #1 o f a phosphoramidon/aminopeptidase mixture, resulting in a 6 #M phosphoramidon concentration and 2.5 #g aminopeptidase per tube. All samples were then incubated at 37°C for 20 minutes for conversion of Phe-4-methyoxy-2-naphthylamide to 4-methoxy-2-naphthylamine (MNA). Samples were diluted with 1.0 ml H20, and MNA was quantified in 4 ml cuvettes with an Aminco-Bowman spectrophotofluorometer set at 340 nm excitation and 425 nm emission. MNA standards were prepared and activity of 3.4.24.11 was calculated in nanomoles product/ mg protein/hour from standard curve fluorescence response after subtracting phosphoramidon controls. The metalloendopeptidase 24.15 assay employed the substrate benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoicacid using a modification of the method of Orlowski, Michaud and Chu (22). Incubations were performed at 37°C for 1 hour with 25 to 50 #g cytosolic or washed membrane protein per tube. For each 24.15 activity measurement, five sample tubes were prepared. Two of the tubes contained the metalloendopeptidase 24.15 inhibitor N-[1-(RS)]carboxy-3-phenylpropyl]-Ala-Ala-Phe-pABat a 100 #M concentration, while the remaining three tubes were without 24.15 inhibitor. Sample tubes also contained 200 uM phosphoramidon, 250 #M dithiothreitol, and 1 mM substrate in 0.16

DAVIS ET AL. M Tris buffer (pH 7.0). Incubations were performed in a total volume of 0.2 ml per tube. Endopeptidase 24.15 activity was stopped by boiling for 2 minutes. After cooling, 10 ug aminopeptidase M was added per tube, and samples were incubated at 37°C for 2 hours to liberate p-aminobenzoic acid from the cleaved substrate. Free p-aminobenzoic acid in the samples and in prepared standards was diazotized and conjugated to N-(1naphthyl)-ethylenediamine and absorbance measured at 555 nm according to the method of Orlowski et al. (22). The difference in p-aminobenzoic acid content in sample tubes with and without 24.15 inhibtor was calculated from the standard curve, and expressed as picomoles product/mg protein/minute. Carboxypeptidase H (E.C. 3.4.17.10) was measured in SCLC cell lines using a modification of the method of Stack, Fricker and Snyder (28), utilizing [benzoyl-2,5-3H(N)]-L-Phe-L-Ala-LArg-OH as the substrate and guanidinoethane mercaptosuccinic acid (GEMSA) as the inhibitor. Following protein determination, membrane and supernatant aliquots were diluted with ice-cold sterile 80 mM sodium acetate, pH 5.6. Soluble (supernatant) fractions were assayed using 2.5-5 ug protein/tube and membrane fractions used 10 #g protein/tube. Assay tubes (1.5 ml microcentrifuge tubes) were prepared for samples and blanks. Each sample required one set of triplicate tubes without GEMSA and one set of duplicate blanks containing GEMSA. Each tube received sample protein, cobalt chloride, and sodium acetate buffer. Inhibited samples also received GEMSA. Samples were refrigerated for 30 minutes to allow GEMSA equilibration with the enzyme. Upon addition of radiolabeled substrate, reagent concentrations were 1 mM cobalt chloride, 80 mM sodium acetate, 0 or 10 #M GEMSA, and 60,000 cpm substrate in a total volume of 100 ul per tube. Samples were incubated for 10 minutes at 37°C. Enzymatic activity was terminated by placing samples on ice and rapidly adding 50 #1 of 1 N HCI with mixing. Chloroform (1.0 ml) was added to each tube which was then vortexed for 30 seconds. Aliquots (0.5 ml) of the cholorform layer from each sample were transferred to scintillation vials and evaporated to complete dryness in an N-Evap (Organomation Associates, South Berlin, MA). Samples were dissolved in 10 ml scintillation cocktail and counted along with triplicate samples of the 3H substrate (20 #1 in 10 ml of scintillation cocktail). Trypsin-like activity was analyzed using Boc-Glu-Lys-Lys-4methyl-coumaryl-7-amide as substrate, 7-amino-4-methyl coumatin as reference product, and p-chloromercuriphenyl sulfonic acid to inhibit activity of lysosomal (thiol) cathepsin proteases as described by Lindberg, Yang and Costa (15). Results were calculated and expressed as picomoles of amino methyl coumatin produced/mg protein/minute for both the soluble and membrane-bound trypsin-like activity. Aminopeptidase (E.C. 3.4.11.1) activity was measured using L-leucine-p-nitroanilide as the substrate and quantitating the formation ofp-nitroaniline (nmol/ml) using a spectrophotometer set at 405 nm as previously described (11).

In Vivo D&tribution and Growth Studies Female athymic Balb/c nude mice (4-5 weeks old) were housed in a pathogen-free temperature-controlled isolation room and were exposed to a light regimen of 0700 to 1900. The diet consisted of autoclaved rodent chow and water given ad lib. NCI-N592 cells (1 X 107) were injected subcutaneously into the right flank of each mouse. Palpable tumors were observed in approximately 80% of mice after 7 days. Phosphate-buffered saline (PBS) or PBS containing BN analogue was injected daily into the subcutaneous tissue adjacent to the tumor nodule. The tumor volume (height X weight X depth) was determined weekly

BN/GRP RECEPTOR ANTAGONISTS AND SCLC by calipers. After 4 weeks, the injections were discontinued. At week 5 t25I-[Tyr4-Psi13"4Leut4]BN (1 X 106 cpm) was injected IV. At the indicated times (i.e., 1 hour and 2 hours), the mice were sacrificed and the distribution of radiolabel determined in various organs as determined. Time-Course Metabolism

403 TABLE 1 STRUCTURE OF BN-LIKE PEPTIDES AND BN ANTAGONISTS

Ala-Pro-Val-Ser-Val-Gly-Gly-Thr-Val-LeuAla-Lys-Met-Tyr-Pro-Arg-Gly-Asn-HisYrp-Ala-Val-Gly-His-Leu-Met-NH2 Gly-Asn-Leu-Trp-Ala-Val-Gly-His-LeuMet-NH2 Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-AlaVal-Gly-His-Leu-Met-NH2 Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-AlaVal-Gly-His-Leu-Leu-NH2 D-Nal-Gln-Trp-Ala-Val-Gly-HisLeu= Phe-NH2 D-Phe-Gln-Trp-Ala-Val-Gly-HisLeu=propyl-NH2 D-Phe-Gln-Trp-Ala-Val-Gly-HisLeu=Cpa-NH2 DFPhe-Gln-Trp-Ala-Val-D-Ala-His-LeuoME

GRP NMB

NCI-H345 cells were maintained in HITES medium containing RPMI-1640 with L-glutamine (2 mM) supplemented with penicillin (100 units/ml), streptomycin (100 #g/ml), insulin (5 jag/ml), human transferrin (10 #g/ml), hydrocortisone (10 nM), /3-estradiol (10 nM), sodium selenite (30 nM), and 2% fetal bovine serum as described previously (11). NCI-H345 cells were harvested by centrifugation at 1250 X g for 10 minutes, then resuspended in sterile RPMI-1640 (37 °C) to a cell concentration of 2.0 × 106 viable cells/ml. Intact cells (0.4 × 106/tube) were incubated at 37°C with 50 uM bombesin, bombesin antagonist (Biomeasure, Inc., Hopkinton, MA), and/or thiorphan (100 uM) in a time-course fashion (0-1080 minutes) as described previously (10,11). Enzymatic activity was stopped by the addition of 17.4 M glacial acetic acid (25 #1) and boiling for 5 minutes. Samples were centrifuged at 3000 × g for 10 min and the supernatant was analyzed by reversed-phase HPLC (10). RP-HPLC Analysis was accomplished using a Vydac (25 cm × 4 ram) C18 column (5 u particle) (Separations Group, Hesparia, CA) at 40°C, a LC65T detector/oven (Perkin Elmer, Norwalk, CT) and two Waters pumps (Waters Associates, Milford, MA) (models 6000A and 510), a Waters Associates solvent programmer, and a Hewlett Packard (Berkeley, CA) 3390 A integrator. The proteolytic stability of the compounds was determined using a curvilinear elution gradient of 10-35% acetonitrile (Burdick and Jackson, Chicago, IL) over 45 minutes versus 100 mM NaHzP04 (Sigma Chemical Co., St. Louis, MO) (pH 2.4). UV absorbance was detected at 210 nm and the flow rate was maintained at 2 ml/min as described previously (10). RESULTS

Binding Studies Table 1 shows the amino acid sequence ofGRP, NMB, BN, and five BN analogues studied. Figure 1 shows that BN, NMB, and [FA]BN(6-13)Me inhibited specific ~2SI-GRP binding to SCLC cell line NCI-H345 in a dose-dependent manner. Little specific ~25I-GRP binding was inhibited by 0.1 nM [FA]BN(613)Me, whereas almost all specific binding was inhibited by 1000 nM [FA]BN(6-13)Me. The IC5o value for [FA]BN(6-13)Me was 9 nM. BN was slightly more potent than was [FA]BN(6-13)Me, with an IC5o value of I nM, whereas NMB was less potent, with an IC5o value of 200 nM. The IC5o value for GRP, [Psi'3a4]BN, [N Psi P]BN(6-14), [P]BN(6-13)PA, [P Psi C]BN(6-14), and BN(1-12) was 5, 30, 5, 3, and 10 nM, respectively. These data indicate that the five BN analogues studied bound to the the SCLC BN/GRP receptor with high affinity.

BN [Psit3,14]BN [N Psi P]BN(6-14) [P]BN(6-13)PA [P Psi C]BN(6-14) [FA]BN(6-13)Me

Normal peptide bond (C=O-NH), -; reduced peptide bond (CH2NH); =. Abbreviations are: =, reduced peptide bond; Nal, napthylalanine; PA, propyl amide; Cpa, chlorophenyl alanine; Psi is denoted by =; oME, methy ester; FA, fluoryl amide.

[FA]BN(6-13)Me is a BN/GRP receptor antagonist. Similar data was obtained for [Psil3a4]BN, IN Psi P]BN(6-14), [P]BN(613)PA, and [P Psi C]BN(6-14) (Table 2). In contrast, GRP and NMB strongly and weakly elevated (Ca2+)i, respectively, whereas BN( 1-12) was inactive. In Vitro Growth Studies BN (10 nM) significantly increased the SCLC colony number in NCI-H345 cells by 41% (Table 3). At a dose of 1000 nM, the BN/GRP receptor antagonists alone had no effect on colony formation, but did inhibit the number of colonies formed in the presence of 10 nM BN (Table 3). When [Psi'3'14]BN was incubated at 2 doses (1 #M and 10 ~M) in the presence of the neutral endopeptidase 3.4.24.11 inhibitor thiorphan (50 t~M), there was a significant (p < 0.01)

~a n.-" ~ lOO

_.1

50

._o u

Second Messenger Studies The ability of the BN analogues to alter cytosolic calcium [(Ca2+)i] was investigated using SCLC cell line NCI-H345. Figure 2A shows that 10 nM BN elevated the (Ca2+)i from 150 to 190 nM. Figure 2B shows that 10 nM BN [FA]BN(6-13)Me halfmaximally inhibited the ability of BN to elevate (Ca/+)i. In contrast, 1000 nM [FA]BN(6-13)Me blocked the ability of 10 nM BN to elevate (Ca2+)i (Table 2). These data indicate that

/J

-10

i

-9

~8

-7

-6

-5

(Peptide), Log M

FIG. 1. Inhibition of binding. The % specific t25I-GRPbinding to NCIH345 cells is indicated as a function of unlabeled [FA]BN(6-13)Me (©), BN (e), GRP (B), and neummedin B (A). The mean value _+SE of 3 determinations each repeated in duplicate is indicated.

404

D A V I S ET AL.

10 nM 10 nM (FA)BN6-13ME BN

1000 nM 10 nM (FA)BN6-13ME BN

2 0 0 I i NnM

f'"

15(

~

.I 4 min --~

4 min-~

FIG. 2. Alteration of cytosolic calcium (Ca2+)i. Fura-2-1oaded NCI-H345 cells were treated with (A) 10 nM BN, (B) 10 nM [FA]BN(6-13)Me followed by 10 nM BN, and (C) 1000 nM [FA]BN(6-13)Me followed by 10 nM BN. This experiment is representative of two others.

e n h a n c e m e n t in the inhibition o f SCLC colonies formed, as s h o w n in Table 3. This effect was also seen at b o t h doses o f [Psi13,14]BN studied.

In Vivo Growth Studies The ability o f the B N / G R P receptor antagonists to slow the growth o f SCLC xenografts was investigated a n d the biodistrib u t i o n o f radiolabeled [Psil3'14]BN was also determined. Figure 3 shows t h a t after 1 week a palpable t u m o r formed using cell line NCI-N592. The t u m o r grew exponentially a n d after 4 weeks the t u m o r v o l u m e was approximately 2000 m m 3. In some mice, [Psil3,14]BN was injected daily subcutaneously a r o u n d the t u m o r site. T h e t u m o r v o l u m e in mice receiving 0.1 ttg [PsiJ3'14]BN was not significantly different from the control. In mice receiving 10 #g o f [Psi ~3'I4]BN, however, the t u m o r growth was decreased by approximately 50%. These data indicate that [PsiZ3'~4]BN inhibits xenograft f o r m a t i o n in a dose-dependent m a n n e r . T h e t r e a t m e n t o f the n u d e mice was discontinued at week 4. At week 5, '25I-[Tyr4-Psi13'14]BN was injected by tail vein into mice with large t u m o r s (approximately 2000 m m ) . After 1 a n d 2 hours, various organs were harvested a n d counted.

TABLE 2 POTENCY OF BN ANALOGUES IN NCI-H345 CELLS Peptide

IC5o* (nM)

Ca2~ Responset

Growth Response

GRP NMB BN BN(I-12) [Psi13,14]BN [N Psi P]BN(6-14) [P]BN(6-13)PA [P Psi C]BN(6-14) [FA]BN(6-13)Me

5 200 1 >10,000 30 5 3 10 9

++ + ++ ------

++ ND ++

* The mean value of 3 ICso determination is indicated, each repeated in duplicate. t Peptides were added at a 1 #M dose and the ability to inhibit Ca 2+ and growth stimulated by 10 nM BN was determined. ++, stimulation of response; - - , inhibition of response; -, inactive; ND, not determined.

Proteolytic Enzymes in NCI-H345 Cells In a n a t t e m p t to d e t e r m i n e if SCLC cancer cells contain m e m b r a n e - a s s o c i a t e d proteases capable of degrading peptide receptor antagonists, we analyzed duplicate populations o f SCLC cell line N C I - H 3 4 5 for several proteolytic enzymes. Table 4 shows the results for several proteases shown previously to degrade peptides such as BN or G R P .

Time-Course Stability To d e t e r m i n e the stability of BN a n d the B N / G R P receptor antagonists, we time-course i n c u b a t e d the peptides with NCITABLE 3 EFFECT OF BOMBESIN (BN), BOMBESIN + [Psi~3a4]BNAND BOMBESIN + [PsiI3,I4BN + THIORPHAN ON THE IN VITRO CLONAL GROWTH OF HUMAN SCLC CELL LINE NCI-H345

Agent(s) Added Media control BN 10 nM control Thiorphan 0.01, 10, 100 uM BN 10 nM + thiorphan 50 uM BN 10 nM + [Psi~3'I4]BN 1 ttM BN 10 nM + [PsiJ3'Ia]BN 10 uM BN 10 n M + [Psi'3'J4]BN 10 ~M + thiorphan 50 ttM BN 10 nM + [Psi13'14]BN 1 t t M + thiorphan 50~tM

NCI-H345 Colony Count*

% Control

616 + 45 868 _+ 54 :!: 890 _+ 60 600 + 20 657 -+ 52

100 141t 100t 144t 69§ 76¶

121 _+ 10

14#

356 _+ 9

41#

* Mean _+ SEM for _>42/,m in diameter colonies, n = 5-6 plates per treatment. Each plate received 20,000 NCI-H345 cells. t§¶# There are two controls used for these data. tData is calculated against media control. §¶#Data is calculated against BN 10 nM control. tSignificantly different from media control, p < 0.01; §significantly different from BN l0 nM control alone, p < 0.01; ¶significantly different from BN l0 nM control alone, p < 0.01; #significantly different from BN l0 nM control and from same dose of [PsiI3,'4]BN without thiorphan, p < 0.01. :~ Thiorphan was assayed alone at 3 concentrations (0.01, 10, 100 taM) in three separate experiments and resulted in no significant effect on NCI-H345 growth.

BN/GRP RECEPTOR ANTAGONISTS AND SCLC

? O x

2 E E E

o E

1

1

2

3

4

Time, weeks

FIG. 3. Inhibition of xenograft formation. NCI-N592 cells (10 7) were injected over the left flank of nude mice and the mean tumor volume _+SE of 3 mice is indicated. After week 1 the mice were injected (100 ~tl PBS, SC) with ((3) no peptide, (O) 0.1 ~g of [Psil3'14]BNand (&) 10 #g of [Psi~3:4]BNdaily. This experiment is representative of 1 other.

H345 cells. After incubation, each sample was analyzed using reversed-phase HPLC as shown in Fig. 4. The metabolic halflife for BN and the five BN/GRP receptor antagonists is shown in Table 5. The half-life of BN, [Psil3.t4]BN, IN Psi P]BN(614), [P]BN(6-13)PA, and [P Psi C]BN(6-14) ranged from 154646 minutes. Surprisingly, [FA]BN(6-13)Me, which has a DAla at the 11 position of BN instead of Gly, had a significantly longer half-life of 1388 minutes. Because [FA]BN(6-13)Me may be resistant to degradation by neutral endopeptidase (E.C. 3.4.24.11 ), we tested if a 3.4.24.11 inhibitor ( 100 ~M thiorphan) altered the half-life of BN and [Psit3,t4]BN. When added to the incubation solution, thiorphan slightly increased the half-life of BN (Table 5). However, when thiorphan was incubated with the BN/GRP receptor antagonist [Psi~3.14]BN, a significant increase in the metabolic half-life was observed from 646 minutes to 907 minutes (Table 5). DISCUSSION

Previously, BN/GRP receptor antagonists were identified which bound with high affinity to SCLC cells, altered BN-induced second messenger production, and inhibited SCLC growth in vitro (7,16,17,29,30). The potency of peptides such as [Psi~3,14]BN in vivo, however, is also dependent on their biodistribution and metabolism. Here the potency of [Psi13:4]BN was investigated using nude mice containing SCLC tumors. In vitro binding studies indicated that BN and GRP bound with high affinity to NCI-H345 (IC50 values of 1 and 5 nM). Similarly, the five BN/GRP receptor antagonists inhibited tzsIGRP binding with high affnity (IC50 values of 3-30 nM). In particular, [FA]BN(6-13)Me had an IC50 value of 9 nM even though it lacked the N-terminal heptapeptide of BN and the Cterminal methionine. Previously, we found that [P]BN(6-13)Me, which has a D-phenylalanine at the 6 position instead of Dfluorophenylalanine and glycine at the 11 position instead of Dalanine, had an IC50 value of 5 nM (29). Therefore, substitution

405 of the electron withdrawing fluorine on the D-phenylalanine ring or substitution of D-alanine for glycine does not dramatically alter the ability of [des-Met14]BN analogues to bind to the BN/ GRP receptor. Therefore, the BN/GRP receptor antagonists bind with similar affnity as does BN to SCLC cells (3,25). Second messenger studies indicate that BN increased the (Ca2+)i on NCI-H345. [FA]BN(6-13)Me (1000 nM) blocked the increase in (Ca2+)i caused by 10 nM BN. Similar data were obtained for [Psil3:a]BN, [N Psi P]BN(6-14), [P]BN(6-13)PA, and [P Psi C]BN(6-14). BN stimulates phosphatidylinositol turnover in SCLC cell line NCI-H345, and the PIP2 released may subsequently be metabolized by phospholipase C to yield inositol1,4,5-trisphosphate, which causes release of calcium from intracellular pools (18,19). Therefore, each of the five BN analogues tested functioned as SCLC antagonists in vitro and inhibited the ability of BN to elevate (Ca2+)i. Previous studies indicate that BN stimulated c-fos and c-myc protooncogene production in Swiss 3T3 cells and 3H-thymidine uptake (23,24,26). Here the ability of BN/GRP receptor antagonists to inhibit the clonal growth of SCLC cells was investigated. BN (10 nM) significantly stimulated the number of NCI-H345 colonies by 41% from 636 to 868. [Psi~3'14]BN (1 and 10 #M) inhibited the total number of colonies formed in the presence of 10 nM BN by 31 and 24%, respectively. Similarly, [FA]BN(613)Me and [P]BN(6-13)PA (1 ~aM) significantly inhibited the number of colonies formed in the presence of 10 nM BN. In the presence of 50 ~tM thiorphan, however, 10 ~tM [Psil3.t4]BN decreased the number of colonies formed by 86% to 121 colonies. These data suggest that in the absence ofthiorphan [Psit3:4]BN may be degraded by endogenous membrane-associated enzymes on the SCLC cells, resulting in limited growth inhibition. Ten #M [Psi~3.~4]BN in the presence of thiorphan inhibited the increase in colony formation caused by 10 nM BN plus much of the basal colony formation as well. These data suggest that BN/ GRP is an important autocrine growth factor in SCLC. The metabolism of the BN/GRP receptor antagonists by the SCLC cells was investigated. Previously, BN was demonstrated to be metabolized by a phosphoramidon-sensitive enzyme (i.e., neutral endopeptidase 3.4.24.11) in dog plasma (30). After incubation with NCI-H345 cells, the half-life of [Psi~3:4]BN, [N Psi P]BN(6-14), [P]BN(6-13)PA, [P Psi C]BN(6-14), and [FA]BN(6-13)Me was 646, 197, 559, 154, and 1388 minutes, respectively. Neutral endopeptidase E.C.3.4.24.11, which is thiorphan sensitive, was detected in NCI-H345 membranes as well as metalloendopeptidase E.C. 3.4.14.15, trypsin-like enzymes, and aminopeptidase. Because the BN analogues have blocked amino terminals and lack basic amino acid residues, they cannot readily be degraded by aminopeptidase and trypsin-like enzymes, respectively. The histidine bond at position 12 of BN, however, may be a site for metabolism by E.C. 3.4.24.11. Because this enzyme is inhibited

TABLE 4 P R O T E O L Y T I C E N Z Y M E A C T I V I T Y IN N C I - H 3 4 5

Proteases

Soluble

MembraneBound

Neutral endopeptidase (E.C. 3.4.24.11) Metalloendopeptidase (E.C. 3.4.24.15) Trypsin-like activity Aminopeptidase (E.C. 3.4.11.1)

ND 12.16" 4.56 NT

36.90 1.66* 779.50 5.67*

* Values × 103; ND = not detectable; NT = not tested. Triplicate measurements (picomoles/mg protein/minute).

406

DAVIS ET AL. HPLC

of B N a n d

Psi - 1 3 , 1 4

~ o

.008,

60 rain Incubation with Bombeeln

60 minute Incubation with (Pill 't" Leu")BN

Z

S 360 mln Incubation with Bomballn

~

360 minute Incubetion with (Pill '~" L e u " ) B N

FIG. 4. Reverse-phase HPLC of bombesin or [Psi]3']4]BNtime-course incubated with intact NCI-H345 cellsfor 60 minutes and 360 minutes. Peaks were identified by comigration with authentic peptide standards.

by thiorphan, we investigated ifthiorphan increased the stability of BN and [Psi~3'14]BN. Thiorphan slightly increased the halflife of BN but significantly increased the half-life of [Psi ]3,]4]BN. Replacement of the glycine at position 1 1 by a D-alanine, however, greatly increased the half-life of [FA]BN(6-13)Me exposed to SCLC cells. Previously, it was found that BN stimulated SCLC xenograft formation in nude mice whereas inactive compounds such as BN( 1-12) had no effect (1,12). [Psi TM]4]BNinhibited NCI-N592 xenograft formation when 10 ug per day was in-

TABLE 5 HALF-LIFE VALUESFOR BOMBESINAND BOMBESINANALOGUES WHEN INCUBATEDWITH INTACTSCLC CELL LINE NCI-H345 Compound

Ti/2

Bombesin? Bombesin + thiorphan (100 ~M)~ [PsW,14]BNt [Psi~3:4]BN + thiorphan (100 #M)? [N Psi P]BN(6-14)* [P]BN(6-13)PA* [P Psi C]BN(6-14)t [FA]BN(6-13)Met

417 minutes 489 minutes 646 minutes 907 minutes 197 minutes 559 minutes 154 minutes 1388 minutes

* Incubated 0-360 minutes. t Incubated 0-1080 minutes.

jected subcutaneously adjacent to the tumor. The effects ot [Psi]3:4]BN were dose dependent, in that 0. I #g/day had no effect. The plasma half-life of the BN/GRP receptor antagonists remains unknown. Approximately 3% and 2% of the ]25I-[Tyr4,Psi~3:4]BN injected intravenously is associated with the stomach and intestine after 1 and 2 hours, respectively. Using in vitro autoradiographic techniques, ]25I-[Tyr4]BN binding sites were localized to the circular muscle layer of the gastric fundus and antrum (20) and the submucosal layer. In the colon, grains were present in the longitudinal and circular muscle and the submucosa in the rat small intestine (21). Approximately 1% of the ]251-[Tyr4]BN injected intravenously localized to the tumor, whereas the heart, kidney, liver, lung, and spleen had < 1%. These data suggest that some of the BN/ GRP receptor antagonists may bind to BN/GRP receptors on the tumor. In summary, reduced peptide bond analogues of BN, such as [Psi]3,14]BN, and [des-Met~4]BN analogues, such as [FA]BN(613)Me, function as SCLC BN/GRP receptor antagonists in that they bind with high affinity, inhibit the BN-induced elevation of cytosolic Ca 2+, and inhibit SCLC clonal growth in vitro. Also, [Psi]3']4]BN inhibits tumor growth in vivo using nude mice. ACKNOWLEDGEMENTS This research was supported in part by grants CA-53477 and CA44869 from the National Cancer Institute and grant DK36289 and MH42600 from the National Institutes of Health of the U.S.P.H.S.

B N / G R P R E C E P T O R A N T A G O N I S T S A N D SCLC

407

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