Inhibitory effect of novel somatostatin peptide analogues on human cancer cell growth based on the selective inhibition of DNA polymerase ß.

The present study was designed to investigate the anticancer activity of novel nine small peptides (compounds 1-9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, compounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1-naphthylamide), 5 (Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1-naphthylamide) containing tyrosine (Tyr) but no carboxyl groups, selectively inhibited the activity of rat pol ß, which is a DNA repair-related pol. Compounds 3-6 strongly inhibited the growth of human colon carcinoma HCT116 p53(+/+) cells. The influence of compounds 1-9 on HCT116 p53(-/-) cell growth was similar to that observed for HCT116 p53(+/+) cells. These results suggest that the cancer cell growth suppression induced by these compounds might be related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol ß and cancer cell growth among the nine compounds tested. This compound specifically inhibited rat pol ß activity, but had no effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sulfonate (MMS) treatment synergistically suppressed HCT116 p53(-/-) cell growth compared with MMS alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results, the influence of compound 4, a specific pol ß inhibitor, on the relationship between DNA repair and cancer cell growth is discussed.

Preparation, characterization and related in vivo release, safety and toxicity studies of long acting lanreotide microspheres.

The goal of this project was to prepare long-acting lanreotide acetate poly(lactic-co-glycolic acid) (PLGA) microspheres and to analyze the in vivo and in vitro release, safety and toxicology of these preparations. Long-acting lanreotide acetate PLGA microspheres that exhibited a 5-week slow-release period were prepared by a multiple-emulsion solvent evaporation method. Physical characterization, as well as the analysis of the in vivo and in vitro release, safety, acute toxicity and chronic toxicity of the lanreotide microspheres, were conducted in animal models in rats, guinea pigs, rabbits and beagle dogs. The lanreotide acetate PLGA microspheres prepared by multiple-emulsion solvent evaporation had smooth surfaces, uniform particle size and stable lanreotide loading. In vivo and in vitro experiments showed that the lanreotide acetate PLGA microspheres could continuously release lanreotide for 5 weeks. The safety of these long acting lanreotide microspheres was good in the following animal models: active systemic anaphylaxis test in guinea pigs, passive cutaneous anaphylaxis test in rats, hemolytic test in rabbits, local skin irritation test after subcutaneous administration in rabbits and muscle stimulation test in rabbits. Furthermore, no significant acute toxicity or chronic toxicity was observed after administration of lanreotide acetate PLGA microspheres in beagle dogs at dosages up to 22 mg/kg. The lanreotide acetate PLGA microspheres that were prepared in this study exhibited beneficial characteristics in apparent property and structural stability, as well as in release trends in vivo and in vitro.

Somatostatin analog lanreotide in the treatment of castration-resistant prostate cancer (CRPC).

Prostate cancer is a common disease affecting males. Despite initial sensitivity to hormone treatment, prostate cancer eventually progresses to a castration-resistant stage (CRPC), which carries an ominous prognosis. Lanreotide is a long-acting somatostatin analog with the same properties with the native peptide. It has been shown to be highly efficacious in treating various hypersecretoty disorders and tumors. Lanreotide has been administered to patients with CRPC within a novel treatment concept, with the aim of targeting not only cancer cells but also various factors secreted in the tumor cell milieu that confer protection from apoptosis. Within this concept, lanreotide has been administered as part of the "antisurvival factor therapy" in combination with dexamethasone and a gonadotropin releasing hormone (GnRH) analog. It has also been given combined with oestrogens in patients with CRPC. The so far published series have documented a clinical response in many patients treated along with significant improvement in parameters related to quality of life. In view of these promising results, large-scale, randomized, controlled trials are warranted to clearly define the exact role of lanreotide and other somatostatin analogs in the treatment of patients with CRPC.

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.

Study of inhibitory effects of an antiangiogenic somatostatin-camptothecin conjugate on laser-induced choroidal neovascularization in rats.

PURPOSE: To evaluate the ocular toxicity and efficacy of intravitreal camptothecin-somatostatin conjugate (JF-10-81), a somatostatin type 2 receptor-directed, antiangiogenic compound. METHODS: Part 1: New Zealand albino rabbits (except for controls) were injected intravitreally with conjugate at various concentrations. Preoperative and postoperative ocular examinations and electroretinography were performed until animals were killed for histology. Part 2: Long-Evans pigmented rats had choroidal neovascularization (CNV) induced by argon laser. One eye per animal was injected with concentration 10M (safe dose), whereas the other eyes were controls and received 30 microL of sterile water at different time intervals after laser application. Fluorescein angiography was performed at various time points to evaluate the lesions and confirm presence of CNV. Animals were euthanized. The eyes were immediately enucleated and prepared for histologic examination. RESULTS: Part 1: No clinical changes were seen in groups receiving 10(-8)M, 10(-7)M, 10(-6)M, and 10(-5) M of conjugate. Electroretinography showed decreasing b-wave amplitude in groups receiving 10(-4) M and 10(-3) M; cataracts also developed in these eyes. Part 2: Fluorescein angiography revealed that intravitreal injection of somatostatin conjugate JF-10-81 favorably affected the development of CNV when the treatment was performed at least 1 week after the laser application. These results were statistically significant. Histologic analysis results of eyes treated 2 weeks after laser application also showed significant benefit. CONCLUSIONS: Part 1: Camptothecin-somatostatin conjugate injected intravitreally appeared safe at concentrations of 10(-5)M or less. Part 2: Conjugate JF-10-81 at a concentration of 10(-5)M administered intravitreally 1 to 3 weeks after laser demonstrated statistically significant efficacy in the treatment of choroidal neovascularization.

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.

Induction of experimental allergic encephalomyelitis in a low-susceptible Albino Oxford rat strain by somatostatin analogue SMS 201-995.

OBJECTIVE: The effect of the somatostatin analogue SMS 201-995 (octreotide; OCT) on the course of experimental allergic encephalomyelitis (EAE) in the relatively resistant Albino Oxford (AO) strain of rats was studied. METHODS: Animals were actively immunized with bovine brain homogenate in complete Freund's adjuvant. OCT was given subcutaneously in the hind legs on days 7, 8 and 9 after immunization, at a dose of 3 x 5 microg/kg/day. Rats in control groups were treated with saline or were left untreated. EAE was scored clinically and immunophenotypically, estimating by flow cytometry the changes in the popliteal lymph nodes (PLN) and spleen and monitoring immunohistologically the brain sections of rats recovered from disease. RESULTS: In control AO rats, EAE was induced in only 2 of 22 rats (9%). In OCT-treated rats, however, EAE developed in 11 of 20 rats (55%), in comparison with 3 of 17 saline-treated animals (17%) (p <0.05). In PLN of OCT-treated rats during the clinical course of EAE, a decreased proportion of OX8+ cells was seen, followed by increases in OX39+ and W3/25+ cells on days 17 and 26. In spleen, OCT decreased the proportion of OX1+, OX39+ and OX8+ cells (on days 12 and/or 17), and increased the proportion of OX39+ cells on days 26 and 31. In the brain sections of saline-treated rats recovered from EAE, numerous Mac-1+, Mac-3+ and OX8+ cells were found. These cells were, however, absent in OCT-treated rats; instead, several W3/25+ cells were noticed. CONCLUSIONS: These data imply that OCT increases the susceptibility of AO rats to EAE, interfering with specific and/or nonspecific defense mechanisms operating in both the initial and recovery phase of EAE.

Effects of mexiletine on algogenic mediator-induced nociceptive responses in mice.

To clarify the possible mechanism of the antinociceptive effect of mexiletine, the effects of the agent on formalin- and algogenic mediator-induced nociceptive responses were examined as compared to lidocaine. Subcutaneous (s.c.) injection of 0.5% formalin into the hindpaw caused an acute nociceptive response that lasted about 5 min (first phase). This response then disappeared completely for about 5 min and then recurred lasting about 20 min (second phase). Intraperitoneal (i.p.) administration of mexiletine (10 and 30 mg/kg) significantly and dose-dependently reduced the durations of the first and second phases of formalin-induced nociceptive response. On the other hand, although i.p. administration of lidocaine (10 and 30 mg/kg) had no significant effect on the first phase of formalin-induced nociceptive response, the duration of the second phase response was significantly and dose-dependently reduced. Pretreatment with mexiletine resulted in a significant and dose-dependent inhibition of the nociceptive response produced by intrathecal (i.t.) injection of substance P (0.1 nM), somatostatin (1.0 nM), bradykinin (1 microgram/mouse) and prostaglandin (PG) F2 alpha (1 microgram/mouse). Although lidocaine had no significant effect on the substance P- or somatostatin-induced nociceptive response, bradykinin- and PGF2 alpha-induced nociceptive responses were inhibited. These results suggest that the antinociceptive effect of mexiletine involves the inhibition of substance P-, somatostatin-, bradykinin- and PGF2 alpha-mediated nociceptive transmission in the spinal cord. Furthermore, it is possible that the weaker antinociceptive effect of lidocaine as compared with that of mexiletine may be due to the lack of its inhibitory effect on substance P- and somatostatin-mediated nociceptive transmission in the spinal cord.

Increased density of somatostatin binding sites in respiratory nuclei of the brainstem in sudden infant death syndrome.

Sudden infant death syndrome is the primary cause of mortality in children aged one to six months in industrialized countries. Although the etiology of this syndrome is still unknown, subtle abnormalities in the neuronal circuitry involved in the control of respiratory activity are suspected. Since stereotaxic administration of somatostatin in the brainstem of rat and cat produces fatal apnea, we have compared the densities of somatostatin binding sites in the respiratory centers of 11 cases of sudden infant death syndrome and six control infants without neuronal disease. The density of binding sites was measured in 17 structures of the pons and medulla oblongata by means of quantitative in vitro autoradiography using iodinated [Tyr0,D-Trp8]somatostatin-14 as a radioligand. The density of somatostatin binding sites was significantly higher in the medial and lateral parabrachial nuclei in the sudden infant death syndrome group than in the control group. In six other nuclei, the median of the receptor density was higher in the sudden infant death syndrome group than the maximum values measured in the control group. The presence of high concentrations of somatostatin binding sites in several respiratory nuclei of the brainstem in approximately half of the sudden infant death syndrome victims suggests that the decrease in receptor density that normally occurs during ontogeny was delayed in these infants. In particular, the high level of somatostatin binding sites in the medial and lateral parabrachial nuclei of sudden infant death syndrome suggests that the delayed maturation of these receptors may be associated with a deficit of the hyperventilatory response to hypoxia.

In vitro antineoplastic activity of a novel lanthionine-containing peptide.

It has been a long-term goal to discover peptides that can kill tumor cells while sparing normal tissues. Lan-7 is a novel, chemically stable peptide structurally related to somatostatin that contains a lanthionine bridge between the two cysteines in the peptide; TT-232 is a less stable analogue containing a disulfide bridge. The antitumor activity of Lan-7 was examined, relative to that of TT-232 and the clinically used analogue octreotide, against a panel of malignant human tumor cell lines and normal human hematopoietic precursors. Lan-7 was cytotoxic to all four tumor cell lines, with IC50 values ranging over a 2-fold range from 16 to 36 microM. The potency of Lan-7 was comparable to that of TT-232, and both of these agents were two to three times more potent than octreotide. At concentrations that were highly cytotoxic to tumor cells, Lan-7 produced no significant toxicity to normal human hematopoietic precursors. Lan-7 induced apoptosis in human ovarian carcinoma 2008 cells over the same concentration range at which it produced cytotoxicity, but it did so without activating G1, S, or G2 checkpoints, given that it produced no perturbation of cell cycle phase distribution. Cells engineered to overexpress P-glycoprotein were not more resistant to Lan-7 than isogeneic cells not expressing the mdr1 gene. These results make Lan-7 of interest as a potential cancer chemotherapeutic agent.

Phase I-II study of the somatostatin analogue lanreotide in hormone-refractory prostate cancer.

Lanreotide (BIM 32014), a somatulin analogue, was found to be as effective as castration in a rat prostate tumor model. Therapeutic benefit was also demonstrated in the hormone-resistant phase of this tumor model. The activity of lanreotide may be due to a reduction in the levels of growth factors such as insulin growth factor 1 (IGF1). A total of 30 patients with hormone-refractory prostate cancer were treated with a slow-release formulation of lanreotide. The mean age was 71 years. Patients were treated with one intramuscular injection of 30 mg BIM 23014 once a week and were followed for prostate-specific antigen (PSA) level evolution until disease progression or WHO grade 3 or 4 toxicity and for survival. The patients were treated for a mean duration of 12 weeks (range, 2-60 weeks). The performance status and bone pain were improved in 40% and 35% of patients respectively. In all, 20% of the patients had a decrease of > or = 50% in PSA levels and 16% showed a stabilization. The biological response was correlated with clinical improvement. The 1-year global survival rate was 72%, with the rate being 89% in the group of patients who were responders on PSA plasma level and 64% in patients with progressive disease. The response duration ranged from 16 to 60 weeks. Toxicity was minor, with transient grade I digestive side effects being noted in a few patients. Lanreotide given at 30 mg once a week to patients with metastatic hormone-refractory prostate cancer was well tolerated. The response rate was higher than that reported in recent published series. Higher doses of lanreotide should be evaluated.

[Effect of intracerebral administration of somatostatin and neurotensin on posture and movement disorders]. / Pozo-rukhovi porushennia pry vnytrishnómozkovykh vvedenniakh somatostatyny ta neirotenzynu.

Experiments were performed on male Wistar rats to study posture-movement disorders after intracerebroventricular, intraamygdalar, intrahippocampal and intranigral (pars reticulata) injections of somatostatin and neurotensin. Development of movement disturbances characterized by changes in the posture-movement syndrome was observed after somatostatin and neurotensin administration. Manifestation of movement disorders, their duration and the posture-movement syndrome structure depended on the structure to which neuropeptides were injected as well as on the neuropeptides themselves. Prolonged changes characterized by appearance of the so called "Sphinx" posture in animals developed after somatostatin intranigral administration. The obtained data permit concluding that somatostatin and neurotensin may have pathogenic significance in cataleptic syndrome development.

Toxic effects of somatostatin in the cerebellum and vestibular nuclei: multiple sites of action.

This study demonstrates that somatostatin (SRIF), an endogenous peptide in vestibular nuclei and cerebellum, can produce both a dose-dependent death of Purkinje cells in distinct sagittal regions of cerebellar cortex and vascular infarcts centered selectively in the inferior vestibular nucleus. Alert, adult male rats were given a 5 microliters intracerebroventricular (i.c.v.) bolus of either SRIF alone (20 or 40 micrograms) or a combined dose of SRIF plus either arginine-vasopressin (AVP, 1 micrograms) or an AVP V1 antagonist, (1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(O-methyl)-tyrosine)-arginine 8-vasopressin (mcAVP, 1 micrograms), through an implanted cannula. After a 4-5 day survival, the brains were stained with the cupric-silver selective degeneration method. Two types of dose-dependent lesions were observed in the cerebellar and vestibular nuclei of these animals: degeneration of Purkinje cell responses in the cerebellar cortex and vascular infarcts in vestibular nuclei. These toxic responses were unaffected by application of AVP or mcAVP; hence, they can be attributed to actions of SRIF. The distribution of Purkinje cell degeneration varied with the SRIF dose in different cerebellar regions. Purkinje cell responses in lobules I-III were equivalent at both SRIF doses, and degeneration in the copula pyramis, paraflocculus and paramedian lobule emerged at the higher SRIF dose. Purkinje cells in the medial aspect of lobules IX-X had an intermediate sensitivity to SRIF intoxication. Degenerating Purkinje cells tended to be arranged in parasagittal bands in each region, suggesting parasagittal zonal variations in susceptibility to SRIF intoxication. By contrast, infarctions in the vestibular nuclei only appeared at the higher SRIF dose. These infarcts could be unilateral or bilateral and always involved the inferior vestibular nucleus at the level of the caudal margin of the acoustic tubercle; they often extended into the medial and lateral vestibular nuclei. The infarcts had a necrotic core that was infiltrated by non-neuronal elements. Thus, they appear to reflect a direct or neurally-mediated vascular response to the peptide.

The effect of somatostatin 201-995 on the early course of porcine pancreaticoduodenal allotransplantation.

This study was undertaken to determine the effects of somatostatin 201-995 (SMS) on the maintenance dose of intravenous cyclosporine and on graft blood flow, exocrine secretion, and rejection after porcine pancreaticoduodenal allotransplantation (PDA). For seven days, 12 pigs (6 control, 6 SMS-treated) were studied to determine the effects of SMS on serum CsA concentrations. Twenty-six pigs (14 control, 12 SMS) with streptozocin-induced diabetes underwent PDA. Blood flow was measured through graft celiac and superior mesenteric arteries 15 and 60 min after reperfusion. SMS (75 micrograms s.c.) was given after the 15-min blood-flow measurement in the SMS group. Sixteen pigs (8 control, 8 SMS) were followed postoperatively with daily measurements of serum glucose and amylase concentrations, and urine amylase and trypsin activities. All pigs were immunosuppressed with azathioprine, prednisone, and i.v. CsA. SMS pigs also received SMS (75 micrograms s.c.) every 8 hr. SMS had no effect on maintenance dose of CsA or on serum amylase, urine amylase, or urine trypsin activities. Mean days to rejection were also not affected. Intraoperative graft blood flow was significantly decreased by SMS, but incidence of graft thrombosis was unchanged. These results suggest that in the porcine PDA model, SMS does not appear to inhibit exocrine secretion and potentially may adversely affect the early course of PDA by decreasing graft blood flow.

Intrathecal somatostatin in cat and mouse studies on pain, motor behavior, and histopathology.

Effects of intrathecal (i.t.) somatostatin (SST) on nociception, motor function, and spinal cord pathology were evaluated in cats and mice. Cats chronically implanted with i.t. lumbar catheters received either a single injection of 2 mg SST i.t. (group I, N = 4), four repetitive injections on consecutive days of 2 mg SST i.t. (group II, N = 4) or saline i.t. (group III, N = 2). No analgesic effects were observed following single or repeated SST injections as evaluated by the skin twitch response. However, significant impairment of hind leg motor function ranging from unbalanced gait to paralysis was observed following the first SST i.t. injection. Histological examination of spinal cords six days after the first SST injection in group II showed multiple pyknotic neurons in all cats. Some cats showed focal demyelination in the posterior column of the spinal cord white matter. Mice received a single percutaneous injection of 50 micrograms SST i.t. (group I, N = 7), 5 micrograms SST i.t. (group II, N = 3), or saline i.t., (group III, N = 5). No analgesic effects were observed in groups II and III as evaluated by the hot plate (HP) and tail flick (TF) tests. Injection of 50 micrograms SST i.t. (group I) caused reversible flaccid hind leg paralysis in all mice and concomitant increases in HP and TF latencies. Histologic examination revealed focal demyelination in the spinal cord in three out of seven mice in this group. Present data substantiate neurotoxic effects of i.t. SST and lack of behaviorally defined antinociception at innocuous dosages.