Comparative study on DOTA-derivatized bombesin analog labeled with 90Y and 177Lu: in vitro and in vivo evaluation.

INTRODUCTION: The aim of the study was to compare in vitro and in vivo a novel DOTA-chelated bombesin (BN) analog of the amino acid sequence, QRLGNQWAVGHLM-CONH(2) (BN[2-14]NH(2)), labeled with (90)Y and (177)Lu, for its potential use in targeted radiotherapy of tumors expressing gastrin releasing peptide (GRP) receptors. The same amino acid sequence, but with different chelator, referred as BN1.1 (Gly-Gly-Cys-Aca-QRLGNQWAVGHLM-CONH(2)), has already been studied and reported; however, the DOTA-chelated one, suitable for labeling with M(+3) type radiometals, was not yet described. METHODS: The conditions for labeling of DOTA-BN[2-14]NH(2) with noncarrier added (90)Y and with (177)Lu [specific activity (SA), 15 Ci/mg Lu] were investigated and optimized to provide (90)Y-DOTA-BN[2-14]NH(2) and (177)Lu-DOTA-BN[2-14]NH(2) of high SA. The stability of the radiolabeled compounds in human serum was evaluated over a period of 24 h. The human prostate cancer cell line PC-3, known to express GRP receptors, was used for in vitro evaluation of radiolabeled peptide affinity to GRP receptors and for assessment of cytotoxicity of both nonlabeled and radiolabeled peptide. Biodistribution accompanied by receptor blocking was studied in normal Swiss mice. RESULTS: (90)Y-DOTA-BN[2-14]NH(2) and (177)Lu-DOTA-BN[2-14]NH(2) were obtained with radiochemical yield >98% and high SA (67.3 GBq (90)Y/mumol and 33.6 GBq (177)Lu/mumol, respectively). They were stable when incubated in human serum for up to 24 h. The binding affinities of DOTA-BN[2-14]NH(2) and both (nat)Y- and (nat)Lu-labeled analogs to GRP receptors were high (IC(50)=1.78, 1.99, and 1.34 nM, respectively), especially for the (nat)Lu-DOTA-BN[2-14]NH(2) complex. The cytotoxicity study of DOTA-BN[2-14]NH(2) to PC-3 cells revealed an IC(50)=6300 nM after 72 h of exposition, while the labeled derivatives showed no significant cytotoxic effect. The internalization rate to PC-3 cells was more rapid for (177)Lu-labeled peptide (84.87%) than for the (90)Y-labeled one (80.79%), while the efflux rate was slower for (177)Lu-DOTA-BN[2-14]NH(2) (46.8% vs. 61.74%). The biodistribution study of both derivatives in normal mice revealed a specific binding to GRP receptor-positive tissues, which could be blocked by coinjection of cold peptide. The effect of receptor blockage in vivo was also more pronounced for the (177)Lu-labeled peptide than that for the (90)Y-labeled (81% vs. 42%, respectively). CONCLUSIONS: Our studies demonstrated that DOTA-BN[2-14]NH(2) can be labeled with (90)Y (NCA) and (177)Lu (CA) with high radiochemical yields. The in vitro and in vivo comparison between (90)Y-DOTA-BN[2-14]NH(2) and (177)Lu-DOTA-BN[2-14]NH(2) indicated that the change of radiometal in the complex from Y to Lu influence the binding affinity to the GRP receptors with preference to the (177)Lu-labeled derivative.

Therapy of ovarian cancers with targeted cytotoxic analogs of bombesin, somatostatin, and luteinizing hormone-releasing hormone and their combinations.

The aim of this study was to investigate the effect of treatment of experimental ovarian cancers with targeted cytotoxic analogs as single compounds and in combination. Targeted cytotoxic analogs of bombesin (AN-215), somatostatin (AN-238), and luteinizing hormone-releasing hormone (AN-207) consisted of 2-pyrrolinodoxorubicin (AN-201) linked to the respective peptide carrier. AN-238 at 200 nmol/kg significantly inhibited growth of UCI-107, ES-2 and OV-1063 ovarian cancers. AN-215 alone at 200 nmol/kg and its combination with AN-238 at one-half of the dose were also able to inhibit the growth of UCI-107 tumors. A combination of AN-238 with AN-207at 50% of the dose strongly suppressed the proliferation of ES-2 and OV-1063 ovarian tumors. Cytotoxic radical AN-201 was toxic and had no significant effect on tumor growth. In contrast, the toxicity of the conjugated peptide analogs was low. Because ovarian cancers tend to acquire chemoresistance, we used real-time PCR to measure the mRNA expression of multidrug resistance protein 1, multidrug resistance-related protein 1, and breast cancer resistance protein after treatment. Low or no induction of multidrug resistance protein 1, multidrug resistance-related protein, and breast cancer resistance protein occurred after treatment with AN-238, AN-215, and the combination of AN-238 with AN-207 or AN-215. These results demonstrate that a therapy with cytotoxic analogs such as single agents and combinations is effective and nontoxic. Our work suggests that cytotoxic peptide analogs of luteinizing hormone-releasing hormone, somatostatin, and bombesin could be used for the therapy of ovarian cancers, considering the lack of induction of chemoresistance.

Targeting cytotoxic conjugates of somatostatin, luteinizing hormone-releasing hormone and bombesin to cancers expressing their receptors: a "smarter" chemotherapy.

Chemotherapy is one of the main modalities in the therapy of cancer. However, an improvement in the efficacy and a reduction in the toxicity of chemotherapeutic agents remains a great challenge to oncologists. A specific delivery of cytotoxic drugs to cancerous cells may help improving both aspects. Peptide hormones, for which receptors have been found in various human cancers, can serve as carriers for a local delivery of cytotoxic agents or radiopharmaceuticals to the tumors, as demonstrated by the successful clinical use of radiolabeled somatostatin analog Octreoscan for the detection and treatment of some somatostatin receptor-positive tumors. Thus, in recent years we developed a series of cytotoxic peptide hormone conjugates based on derivatives of hypothalamic hormones such as somatostatin and luteinizing hormone-releasing hormone (LHRH), and the brain-gut hormone bombesin. To create targeted conjugates with high cytotoxic activity, a derivative of doxorubicin (DOX), 2-pyrrolino-DOX (AN-201), which is 500-1, 000 times more active than its parent compound, was developed. This agent was coupled to somatostatin octapeptide RC-121 to form cytotoxic conjugate AN-238, and to [D-Lys6]LHRH carrier to produce analog AN-207. Cytotoxic bombesin hybrid AN-215 also contains AN-201. DOX was likewise linked to [D-Lys6]LHRH to form AN-152. A comprehensive testing of these cytotoxic conjugates in experimental models of various human and rodent cancers led to their selection as candidates for clinical trials.

RC-3095, a bombesin/gastrin-releasing peptide receptor antagonist, impairs aversive but not recognition memory in rats.

Bombesin and its mammalian equivalent, gastrin-releasing peptide (GRP), stimulate cell proliferation and are involved in the pathogenesis of several types of human cancer. Bombesin-like peptides also display neuroendocrine activities and regulate neural function. In the present study, we evaluated the effects of the bombesin/GRP receptor antagonist (D-Tpi(6), Leu(13) psi[CH(2)NH]-Leu(14)) bombesin-(6-14) (RC-3095), experimental antitumor drug, on memory in rats. Adult female Wistar rats were treated with an intraperitoneal injection of RC-3095 (0.2, 1.0 or 5.0 mg/kg) 30 min before training in either inhibitory avoidance or novel object recognition tasks. Retention test trials were carried out 1.5 (short-term memory) or 24 h (long-term memory) after training. RC-3095 at the doses of 0.2 or 1.0 mg/kg, but not at the dose of 5.0 mg/kg, impaired both short- and long-term inhibitory avoidance retention, but did not affect recognition memory. The memory-impairing effect of RC-3095 could not be attributed to alterations in sensorimotor functions. The results show that the antitumor drug/GRP antagonist RC-3095 impairs formation of aversive memory.

Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: implications for the design of preclinical studies.

Recently, we developed a series of cytotoxic peptide conjugates containing 14-O-glutaryl esters of doxorubicin (DOX) or 2-pyrrolino-DOX (AN-201). Serum carboxylesterase enzymes (CE) can partially hydrolyze these conjugates in the circulation, releasing the cytotoxic radical, before the targeting is complete. CE activity in serum of nude mice is about 10 times higher than in human serum. Thus, we found that the t(1/2) of AN-152, an analog of luteinizing hormone-releasing hormone (LH-RH) containing DOX, at 0.3 mg/ml is 19. 49 +/- 0.74 min in mouse serum and 126.06 +/- 3.03 min in human serum in vitro. The addition of a CE inhibitor, diisopropyl fluorophosphate (DFP), to mouse serum in vitro significantly (P < 0. 01) prolongs the t(1/2) of AN-152 to 69.63 +/- 4.44 min. When DFP is used in vivo, 400 nmol/kg cytotoxic somatostatin analog AN-238 containing AN-201 is well tolerated by mice, whereas all animals die after the same dose without DFP. In contrast, DFP has no effect on the tolerance of AN-201. A better tolerance to AN-238 after DFP treatment is due to the selective uptake of AN-238 by somatostatin receptor-positive tissues. Our results demonstrate that the suppression of the CE activity in nude mice greatly decreases the toxicity of cytotoxic hybrids containing 2-pyrrolino-DOX 14-O-hemiglutarate and brings this animal model closer to the conditions that exist in humans. The use of DFP together with these peptide conjugates in nude mice permits a better understanding of their mechanism of action and improves the clinical predictability of the oncological and toxicological results.

Suppression by apigenin of peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane in Wistar rats.

The effect of a naturally occurring flavonoid apigenin on the development of bombesin-enhanced peritoneal metastasis from intestinal adenocarcinomas induced by azoxymethane was investigated in male Wistar rats. From the start of the experiment, rats were given weekly s.c. injections of azoxymethane (7.4 mg/kg body weight) for 10 weeks and s.c. injection of bombesin (40 microg/kg body weight) every other day, and from week 16, s.c. injections of apigenin (0.75 or 1.5 mg/kg body weight) every other day until the end of the experiment in week 45. Bombesin significantly increased the incidence of intestinal tumors and cancer metastasis to the peritoneum in week 45. It also significantly increased the labeling index of intestinal cancers. Although administration of apigenin at either dose with bombesin had little or no effect on the enhancement of intestinal carcinogenesis by bombesin, the location, histologic type, depth of involvement, infiltrating growth patterns and labeling index, it was found to decrease significantly the incidence of cancer metastasis. Apigenin significantly decreased the incidence of lymphatic vessel invasion of adenocarcinomas, which was enhanced by bombesin. In vitro experiments revealed that apigenin inhibited bombesin-enhanced phosphorylation of mitogen-activated protein kinase (MAPK), but not matrix metalloprotease (MMP)-9 expression. Our findings indicate that apigenin inhibits cancer metastasis through inhibition of phosphorylation of MAPK.

Inhibitory effects of tetrandrine and hernandezine on Ca2+ mobilization in rat glioma C6 cells.

The effects of tetrandrine (TET), a Ca2+ antagonist of Chinese herbal origin, and hernandezine (HER), a structural analogue of TET, on Ca2+ mobilization were studied in rat glioma C6 cells. TET and HER alone did not affect the resting cytoplasmic Ca2+ concentration ([Ca2+]i). TET and HER inhibited the peak and sustained elevation of [Ca2+]i induced by bombesin and thapsigargin (TG), a microsomal Ca2+ ATPase inhibitor, in a dose-dependent manner. The doses of TET or HER needed to abolish the sustained and peak increase in [Ca2+]i induced by bombesin and TG were 30 microM and 300 microM, respectively. TET and HER did not increase inositol 1,4,5-trisphosphate (IP3) accumulation by themselves but inhibited IP3 accumulation elevated by bombesin. In permeabilized C6 cells, the addition of IP3 and TG released Ca2+ from intracellular stores. Pretreatment with TET or HER abolished Ca2+ release from intracellular stores induced by bombesin and TG. In the absence of extracellular Ca2+, the addition of 3 mM Ca2+ to extracellular medium slightly increased [Ca2+]i, which indicated Ca2+ entry due to leakage of Ca2+ at the plasma membrane but not Ca2+ influx through Ca2+ channels. TET and HER did not affect this leakage entry of Ca2+. The present results suggest that TET and HER inhibit Ca2+ release from intracellular stores as well as Ca2+ entry from extracellular medium evoked by bombesin and TG. In addition, TET and HER inhibit IP3 accumulation induced by bombesin in rat glioma C6 cells.

Triamterene suppresses bombesin-enhanced peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane.

The effects of combined administration of bombesin and the diuretic triamterene on the incidence of peritoneal metastasis of intestinal cancers induced by azoxymethane (AOM) and the labeling index of intestinal cancers were investigated in male inbred Wistar rats. From the start of the experiment, rats were given weekly s.c. injections of AOM (7.4 mg/kg body weight) for 10 weeks and s.c. injections of bombesin (40 micrograms/kg body weight) every other day, and from week 16, s.c. injections of triamterene (10 and 20 mg/kg body weight) every other day until the end of the experiment in week 45. Bombesin significantly increased the incidence of intestinal tumors and cancer metastasis to the peritoneum in week 45. It also significantly increased the labeling index of intestinal cancers. Although administration of both doses of triamterene with bombesin had little or no influence on the enhancement of intestinal carcinogenesis by bombesin, or the location, histologic type, depth of invasion, or labeling index of intestinal cancers, it significantly reduced the incidence of cancer metastasis. These findings indicate that triamterene suppresses cancer metastasis through a mechanism that does not affect the proliferation of intestinal cancers.

Suppression by amiloride of bombesin-enhanced peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane.

The effects of concomitant administration of bombesin and of the diuretic drug amiloride on the development of large and small intestinal tumors induced by azoxymethane (AOM), the incidence of their metastasis to the peritoneum and the labeling index of intestinal adenocarcinomas were investigated in inbred Wistar rats. From the start of the experiment, rats were given weekly s.c. injections of AOM for 10 weeks and s.c. injections of bombesin and/or a higher or lower dose of amiloride hydrochloride (amiloride) every other day until the end of the experiment in week 45. Administration of bombesin significantly increased the incidence of intestinal tumors and cancer metastasis to the peritoneum in week 45. It also significantly increased the labeling index of intestinal adenocarcinomas. Although administration of both doses of amiloride with bombesin had little or no influence on the enhancement of intestinal tumorigenesis by bombesin, the location, histological type, depth of involvement or labeling index of intestinal adenocarcinomas, a higher dose of amiloride significantly reduced the incidence of cancer metastasis to the peritoneum. Our findings indicate that amiloride possesses an anti-metastatic activity.

Bombesin enhances experimental carcinogenesis induced in rat colon by 1,2-dimethylhydrazine.

The effects of bombesin on the colonic mucosa and on the incidence, number, size and histology of colon cancers induced by 1,2-dimethylhydrazine (DMH) were studied in Fischer 344 rats. In experiment 1, rats were randomized into three groups to receive either saline or bombesin (10 or 30 micrograms/kg body wt) to determine the labeling index of normal colonic mucosa. In experiment 2, rats were given 20 weekly injections of DMH (20 micrograms/kg body wt) and received either saline or bombesin (10 or 30 micrograms/kg body wt) every other day for 24 weeks. Administration of bombesin significantly increased the labeling indices of colonic mucosa in a dose-dependent manner. Chronic administration of bombesin at both dosages with DMH caused significant increases in the incidence, number and depth of involvement of colon cancers; however, it did not affect the size and histological type of colon cancers. In addition, bombesin at the dose of 30 micrograms/kg significantly increased the labeling index of colon cancer. These results suggest that bombesin stimulates the cell proliferation of colonic mucosa and colon cancer and enhances colon carcinogenesis in rats.

Bombesin impairs spindle function in mitotic V79 Chinese hamster cells by a receptor-dependent mechanism.

Bombesin belongs to a family of peptides acting as local hormones with roles in growth regulation, neural function and secretion. Upon binding to its receptor bombesin primarily elicits an increase of inositolphosphates and diacylglycerol, events leading to increased [Ca2+]i and activation of protein kinase C. When asynchronously growing V79 Chinese hamster cells were treated with bombesin in the 10(-9)-10(-7) M concentration range their content of inositolphosphates increased and so did the frequency of mitotic cells with abnormal chromosomal arrangements (c-mitoses). Both effects were abolished by simultaneous addition of the synthetic peptide antagonist D-Arg1,D-Phe5,D-Trpu7,9-Leu11-substance P that binds to certain bombesin receptors. These results demonstrate that the V79 cells most probably have receptors for bombesin and that the weak but significant c-mitotic effect is mediated by such receptors.

Effects of cholecystokinin and bombesin on development of azaserine-induced pancreatic tumours in rats: modulation by the cholecystokinin receptor antagonist lorglumide.

Cholecystokinin and bombesin have been shown to promote pancreatic growth and development of azaserine-induced acidophilic atypical acinar cell nodules in rat pancreas after treatment for 16 weeks. Lorglumide, a specific cholecystokinin receptor antagonist, inhibited the stimulating effect of cholecystokinin, but not of bombesin. The present study was carried out to determine effects of cholecystokinin and bombesin, alone and in combination with lorglumide, on pancreatic growth and carcinogenesis after chronic treatment. The animals were killed 8 months after the start of treatment. Growth of the pancreas and the development of acidophilic atypical acinar cell nodules in exocrine pancreas was enhanced significantly by both cholecystokinin and bombesin, but the number of carcinomas was increased only by bombesin. Lorglumide inhibited the effects of cholecystokinin on both pancreatic growth and on the development of acidophilic nodules. The effects of bombesin on pancreatic growth and development of pancreatic lesions, except for adenomas, were not inhibited by lorglumide.

Enhancement by bombesin of colon carcinogenesis and metastasis induced by azoxymethane in Wistar rats.

The effects of bombesin on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), and on their metastases to the peritoneum and/or lymph nodes, were investigated in Wistar rats. Rats received weekly s.c. injections of AOM for 10 weeks, and s.c. injections of bombesin in depot form every other day until the end of the experiment in week 30. Administration of bombesin significantly increased the incidence of colon tumors in week 30. It had no influence on the histological features or depths of involvement of colon adenocarcinomas, but significantly increased the incidence of cancer metastasis to the peritoneum and/or lymph nodes. It also caused a significant increase in the labeling index of the colon epithelial cells. Our findings indicate that bombesin enhances the development and metastasis of colon tumors, and that this effect may be related to its effect in increasing proliferation of epithelial cells of the colon.

Diazepam reverses bombesin-induced gastric spasms and food intake reduction in the rat.

Bombesin (2-16 micrograms/kg, i.p.) produced abnormally large gastric contractions in intact rats consisting of increases in gastric pressure and motility. The effect was antagonized by diazepam (5 mg/kg, i.p.). The same dosage of diazepam abolished bombesin-induced food intake reduction. Diazepam by itself did not increase food intake above control levels. The time course of this behavioral antagonism was followed. There was no significant difference in the intake time course of diazepam-alone and diazepam-bombesin treatments, while there was a significant difference between the saline control and the bombesin-alone treatments. Intake of the saline control, the diazepam-alone and the diazepam-bombesin treatment terminated at the same level, while the bombesin-alone remained significantly different. Additionally, swift aversion to 8 micrograms/kg bombesin was obtained when flavored nutrient was substituted for flavored water, suggesting that the aversion developed as a consequence of interaction with the ingested diet. Abnormalities that result from intake reduction is due to the bombesin-produced intragastric abnormalities, and not by satiety.