CBL0137 increases the targeting efficacy of Rovalpituzumab tesirine against tumour-initiating cells in small cell lung cancer.

Small cell lung cancer (SCLC) is characterised by high relapse rates. Tumour-initiating cells (TICs) are responsible for drug resistance and recurrence of cancer. Rovalpituzumab tesirine (Rova-T), a potent humanised antibody-drug conjugate, selectively targets delta-like protein 3, which is highly expressed in SCLC TICs. The experimental drug CBL0137 (CBL) inhibits the histone chaperone FACT (facilitates chromatin transcription), which is required for the expression of transcription factors that are essential for TIC maintenance. Rova-T and CBL each target SCLC TICs as single agents. However, acquired or intrinsic resistance to single agents is a major problem in cancer. Therefore, we investigated the potential effect of combining Rova-T and CBL in SCLC to eradicate TICs more effectively. Our preclinical studies report a novel and highly translatable therapeutic strategy of dual targeting TICs using Rova-T in combination with CBL to potentially increase survival of SCLC patients.

Gastric Corpus Mucosal Hyperplasia and Neuroendocrine Cell Hyperplasia, but not Spasmolytic Polypeptide-Expressing Metaplasia, Is Prevented by a Gastrin Receptor Antagonist in H+/K+ATPase Beta Subunit Knockout Mice.

Proton pump inhibitor use is associated with an increased risk of gastric cancer, which may be mediated by hypergastrinemia. Spasmolytic polypeptide-expression metaplasia (SPEM) has been proposed as a precursor of gastric cancer. We have examined the effects of the gastrin receptor antagonist netazepide (NTZ) or vehicle on the gastric corpus mucosa of H+/K+ATPase beta subunit knockout (KO) and wild-type (WT) mice. The gastric corpus was evaluated by histopathology, immunohistochemistry (IHC), in situ hybridization (ISH) and whole-genome gene expression analysis, focusing on markers of SPEM and neuroendocrine (NE) cells. KO mice had pronounced hypertrophy, intra- and submucosal cysts and extensive expression of SPEM and NE cell markers in the gastric corpus, but not in the antrum. Numerous SPEM-related genes were upregulated in KO mice compared to WT mice. NTZ reduced hypertrophia, cysts, inflammation and NE hyperplasia. However, NTZ neither affected expression of SPEM markers nor of SPEM-related genes. In conclusion, NTZ prevented mucosal hypertrophy, cyst formation and NE cell hyperplasia but did not affect SPEM. The presence of SPEM seems unrelated to the changes caused by hypergastrinemia in this animal model.

A translocator protein 18 kDa ligand, Ro5-4864, inhibits ATP-induced NLRP3 inflammasome activation.

Ro5-4864 and PK11195, prototypical synthetic ligands of translocator protein 18 kDa (TSPO), have shown anti-inflammatory effects in several models of inflammatory diseases; however, their biochemical mechanisms remain poorly understood. Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation as a part of the innate immune system, has been implicated in a variety of inflammatory diseases. Here, we demonstrate for the first time that TSPO ligands, especially Ro5-4864, potently suppressed ATP-induced NLRP3 inflammasome activation in THP-1 and BMDM cells. Detailed action mechanism was further investigated in THP-1 cells. Ro5-4864 efficiently attenuated NLRP3 translocation to mitochondria, inflammasome assembly/oligomerization, activation of caspase-1, and subsequent secretion of the mature forms of interleukin-1ß and -18. Ro5-4864 also reduced the production of mitochondrial superoxide and preserved the mitochondrial membrane potential in ATP-treated cells, suggesting that Ro5-4864 may act on mitochondria or more upstream targets in NLRP3 inflammasome signaling. We also observed the distinct effects of the TSPO ligands between THP-1 monocytes and macrophages, which suggested different NLRP3 inflammasome signaling depending on cell type. Collectively, our novel findings demonstrate that Ro5-4864 effectively inhibited ATP-induced NLRP3 inflammasome activation through the prevention of mitochondrial perturbation. Our results indicate Ro5-4864 as a promising candidate for the treatment of NLRP3 inflammasome-related diseases.

Topotecan synergizes with CHEK1 (CHK1) inhibitor to induce apoptosis in ovarian cancer cells.

BACKGROUND: Topotecan (TPT) is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of TPT is myelosuppression. This led us to seek a combination treatment to augment TPT anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1 (CHK1), a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers. METHODS: We evaluated the cellular effects of TPT in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of TPT in the absence and presence of CHEK1 inhibitor, PF477736. Synergism between TPT and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2AX. RESULTS: Non-HGS ovarian cancer cells were generally more sensitive to TPT treatment compared to HGS ovarian cancer cells. When combined with CHEK1 inhibitor, TPT potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis. CONCLUSIONS: Our findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to TPT. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining TPT with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit.

Effect of netazepide, a gastrin/CCK2 receptor antagonist, on gastric acid secretion and rabeprazole-induced hypergastrinaemia in healthy subjects.

AIMS: To compare gastric acid suppression by netazepide, a gastrin/CCK2 receptor antagonist, with that by a proton pump inhibitor (PPI), and to determine if netazepide can prevent the trophic effects of PPI-induced hypergastrinaemia. METHODS: Thirty healthy subjects completed a double-blind, randomized, parallel group trial of oral netazepide and rabeprazole, alone and combined, once daily for 6 weeks. Primary end points were: basal and pentagastrin-stimulated gastric acid and 24 h circulating gastrin and chromogranin A (CgA) at baseline, start and end of treatment, gastric biopsies at baseline and end of treatment and basal and pentagastrin-stimulated gastric acid and dyspepsia questionnaire after treatment withdrawal. RESULTS: All treatments similarly inhibited pentagastrin-stimulated gastric acid secretion. All treatments increased serum gastrin, but the combination and rabeprazole did so more than netazepide alone. The combination also reduced basal acid secretion. Rabeprazole increased plasma CgA, whereas netazepide and the combination reduced it. None of the biopsies showed enterochromaffin-like (ECL) cell hyperplasia. Withdrawal of treatments led neither to rebound hyperacidity nor dyspepsia. CONCLUSIONS: Netazepide suppressed pentagastrin-stimulated gastric acid secretion as effectively as did rabeprazole. The reduction in basal acid secretion and greater increase in serum gastrin by the combination is consistent with more effective acid suppression. Despite our failure to show rabeprazole-induced ECL cell hyperplasia and rebound hyperacidity, the increase in plasma CgA after rabeprazole is consistent with a trophic effect on ECL cells, which netazepide prevented. Thus, netazepide is a potential treatment for the trophic effects of hypergastrinaemia and, with or without a PPI, is a potential treatment for acid-related conditions.

BMS-871: a novel orally active pan-Notch inhibitor as an anticancer agent.

This Letter describes synthesis, SAR, and biological activity of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides as inhibitors of γ-secretase mediated signaling of Notch receptors. Optimization of this series led to the identification of BMS-871 (compound 30) which displayed robust in vivo efficacy in Notch-dependent leukemia and solid tumor xenograft models.

ERK5 pathway regulates transcription factors important for monocytic differentiation of human myeloid leukemia cells.

Mitogen-activated protein kinases (MAPKs) are important transducers of external signals for cell growth, survival, and other cellular responses including cell differentiation. Several MAPK cascades are known with the MEK1/2-ERK1/2, JNK, and p38MAPKs receiving most attention, but the role of MEK5-ERK5 in intracellular signaling deserves more scrutiny, as this pathway transmits signals that can complement ERK/2 signaling. We hypothesized that the ERK5 pathway plays a role in the control of monocytic differentiation, which is disturbed in myeloid leukemia. We therefore examined the cellular phenotype and key molecular events which occur when human myeloid leukemia cells, acute (AML) or chronic (CML), are forced to differentiate by vitamin D derivatives (VDDs). This study was performed using established cell lines HL60 and U937, and primary cultures of blasts from 10 patients with ML. We found that ERK5 and its direct downstream target transcription factor MEF2C are upregulated by 1,25D in parallel with monocytic differentiation. Further, inhibition of ERK5 activity by specific pharmacological agents BIX02189 and XMD8-92 alters the phenotype of these cells by reducing the abundance of the VDD-induced surface monocytic marker CD14, and concomitantly increasing surface expression of the general myeloid marker CD11b. Similar results were obtained when the expression of ERK5 was reduced by siRNA or short hairpin (sh) RNA. ERK5 inhibition resulted in an expected decrease in MEF2C activation. We also found that in AML cells the transcription factor C/EBPß is positively regulated, while C/EBPα is negatively regulated by ERK5. These findings provide new understanding of dysregulated differentiation in human myeloid leukemia.

Effect of repeated doses of netazepide, a gastrin receptor antagonist, omeprazole and placebo on 24 h gastric acidity and gastrin in healthy subjects.

AIM: To administer repeated oral doses of netazepide to healthy subjects for the first time, to assess safety, tolerability, pharmacokinetics and effect on 24 h gastric pH and plasma gastrin. METHOD: We did two randomized, double-blind, parallel group studies. The first compared netazepide 25 and 100 mg 12 hourly, omeprazole 20 mg once daily and placebo for 7 days. On day 7 only, we measured pH and assayed plasma gastrin. The second study compared netazepide 5, 10 and 25 mg and placebo once daily for 14 days. We measured pH on days 1, 7 and 14 and assayed plasma gastrin on days 1 and 14. We compared treatments by time gastric pH ≥ 4 during 0-4, 4-9, 9-13 and 13-24 h after the morning dose, and by plasma gastrin. P < 0.05 was significant. RESULTS: Netazepide was well tolerated. On day 7 of the first study, netazepide increased pH significantly only during 9-13 h after the 100 mg dose, whereas omeprazole raised pH significantly during all periods. Both netazepide and omeprazole increased plasma gastrin significantly. Netazepide had linear pharmacokinetics. In the second study, netazepide caused dose-dependent, sustained increases in pH on day 1, but as in the first study, netazepide had little effect on pH on days 7 and 14. Again, netazepide increased plasma gastrin significantly. CONCLUSION: Although repeated doses of netazepide led to tolerance to its effect on pH, the accompanying increase in plasma gastrin is consistent with continued inhibition of acid secretion, via gastrin receptor antagonism and gene up-regulation.

Netazepide, a gastrin/CCK2 receptor antagonist, causes dose-dependent, persistent inhibition of the responses to pentagastrin in healthy subjects.

AIMS: To confirm by means of pentagastrin, a synthetic gastrin agonist, that netazepide is a gastrin/CCK2 receptor antagonist in healthy subjects, and that antagonism persists during repeated dosing. METHODS: We did two studies in which we infused pentagastrin (0.6 µg kg(-1) h(-1) intravenously), aspirated gastric secretion and measured the volume, pH and H(+) secretion rate of the gastric aspirate. First, we did a double-blind, five-way crossover study (n = 10) to assess the effect of single oral doses of netazepide (1, 5, 25 and 100 mg) and placebo on the response to pentagastrin. Then, we did a single-blind, placebo-controlled study (n = 8) to assess the effect of the first and last oral doses of netazepide (100 mg) twice daily for 13 doses on the response to pentagastrin. RESULTS: Netazepide was well tolerated. After placebo, pentagastrin increased the volume and H(+) secretion rate and reduced the pH of gastric aspirate. Compared with placebo, single doses of netazepide caused dose-dependent inhibition of the pentagastrin response (P < 0.02); netazepide (100 mg) abolished the response. After 13 doses, the reduction in volume and H(+) secretion rate persisted (P < 0.001), but the pH effect was mostly lost. CONCLUSIONS: Netazepide is an orally active, potent, competitive antagonist of human gastrin/CCK2 receptors. Antagonism is dose dependent and persists during repeated dosing, despite tolerance to the effect on pH. Further studies are required to explain that tolerance. Netazepide is a tool to study the physiology and pharmacology of gastrin, and merits studies in patients to assess its potential to treat gastric acid-related conditions and the trophic effects of hypergastrinaemia.

The gastrin receptor antagonist netazepide (YF476) prevents oxyntic mucosal inflammation induced by Helicobacter pylori infection in Mongolian gerbils.

OBJECTIVE: Long-term Helicobacter pylori infection causes gastritis leading to hypergastrinemia and predisposes to gastric cancer. Our aim was to assess the role of gastrin in oxyntic mucosal inflammation in H. pylori-infected Mongolian gerbils by means of the gastrin receptor antagonist netazepide (YF476). DESIGN: We studied 60 gerbils for 18 months and left five animals uninfected (control group), inoculated 55 with H. pylori, and treated 28 of the infected animals with netazepide (Hp+YF476 group). Twenty-seven infected animals were given no treatment (Hp group). We measured plasma gastrin and intraluminal pH. H. pylori detection and histologic evaluations of the stomach were carried out. RESULTS: All 55 inoculated animals were H. pylori positive at termination. Eighteen animals in the Hp group had gastritis. There was a threefold increase in mucosal thickness in the Hp group compared to the Hp+YF476 group, and a threefold increase in oxyntic neuroendocrine cells in the Hp group compared to the Hp+YF476 group (p < .05). All animals in the Hp+YF476 group had macro- and microscopically normal findings in the stomach. Plasma gastrin was higher in the Hp group than in the control group (172 ± 16 pmol/L vs 124 ± 5 pmol/L, p < .05) and highest in the Hp+YF476 group (530 ± 36 pmol/L). Intraluminal pH was higher in the Hp group than in the Hp+YF476 group (2.51 vs 2.30, p < .05). CONCLUSION: The gastrin antagonist netazepide prevents H. pylori-induced gastritis in Mongolian gerbils. Thus, gastrin has a key role in the inflammatory reaction of the gastric mucosa to H. pylori infection in this species.

JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide]: a novel, potent, and selective cholecystokinin 2 receptor antagonist with good oral bioavailability.

JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide] is a representative of a new chemical class of competitive antagonists of cholecystokinin 2 (CCK2) receptors. In this study, the primary in vitro pharmacology of JNJ-26070109 was evaluated along with the pharmacokinetic and pharmacodynamic properties of this compound in rat and canine models of gastric acid secretion. JNJ-26070109 expressed high affinity for human (pK(I) = 8.49 ± 0.13), rat (pK(I) = 7.99 ± 0.08), and dog (pK(I) = 7.70 ± 0.14) CCK2 receptors. The selectivity of JNJ-26070109 at the CCK2 receptor versus the CCK1 receptor was species-dependent, with the greatest degree of selectivity (>1200-fold) measured at the human isoforms of the CCK1 receptor (selectivity at CCK2 versus CCK1 receptors: human, ∼1222-fold; rat, ∼324-fold; dog ∼336-fold). JNJ-26070109 behaved as a surmountable, competitive, antagonist of human CCK2 receptors in a calcium mobilization assay (pK(B) = 8.53 ± 0.05) and in pentagastrin-stimulated gastric acid secretion in the isolated, lumen-perfused, mouse stomach assay (pK(B) = 8.19 ± 0.13). The pharmacokinetic profile of this compound was determined in vivo in rats and dogs. JNJ-26070109 was shown to have high oral bioavailability (%F rat = 73 ± 16; %F dog = 92 ± 12) with half lives of 1.8 ± 0.3 and 1.2 ± 0.1 h in rat and dog, respectively. The pharmacodynamic properties of this compound were investigated using two in vivo models. In conscious rat and dog chronic gastric fistula models of pentagastrin-stimulated acid secretion, JNJ-26070109 had oral EC(50) values of 1.5 and 0.26 µM, respectively. Overall, we have demonstrated that JNJ-26070109 is a high-affinity, selective CCK2 receptor antagonist with good pharmacokinetic properties.

Randomized Controlled Trial of the Gastrin/CCK2 Receptor Antagonist Netazepide in Patients with Barrett's Esophagus.

Hypergastrinemia has been associated with high-grade dysplasia and adenocarcinoma in patients with Barrett's esophagus, and experimental studies suggest proinflammatory and proneoplastic effects of gastrin on Barrett's esophagus. This is of potential concern, as patients with Barrett's esophagus are treated with medications that suppress gastric acid production, resulting in increased physiologic levels of gastrin. We aimed to determine whether treatment with the novel gastrin/CCK2 receptor antagonist netazepide reduces expression of markers associated with inflammation and neoplasia in Barrett's esophagus. This was a randomized, double-blind, placebo-controlled trial of netazepide in patients with Barrett's esophagus without dysplasia. Subjects were treated for 12 weeks, with endoscopic assessment at baseline and at end of treatment. The primary outcome was within-individual change in cellular proliferation as assessed by Ki67. Secondary analyses included changes in gene expression, assessed by RNA-sequencing, and safety and tolerability. A total of 20 subjects completed the study and were included in the analyses. There was no difference between arms in mean change in cellular proliferation (netazepide: +35.6 Ki67+ cells/mm2, SD 620.7; placebo: +307.8 Ki67+ cells/mm2, SD 640.3; P = 0.35). Netazepide treatment resulted in increased expression of genes related to gastric phenotype (TFF2, MUC5B) and certain cancer-associated markers (REG3A, PAX9, MUC1), and decreased expression of intestinal markers MUC2, FABP1, FABP2, and CDX1 No serious adverse events related to study drug occurred. The gastrin/CCK2 receptor antagonist netazepide did not reduce cellular proliferation in patients with nondysplastic Barrett's esophagus. Further research should focus on the biological effects of gastrin in Barrett's esophagus.Prevention Relevance: Treatment of patients with Barrett's esophagus with a gastrin/CCK2 receptor antagonist did not have obvious chemopreventive effects.

Effects of Rovalpituzumab Tesirine on Ventricular Repolarization in Patients With Small-Cell Lung Cancer.

Small cell lung cancer (SCLC) is a leading cause of cancer death worldwide, with few treatment options. Rovalpituzumab tesirine (Rova-T) is an antibody-drug conjugate that targets delta-like 3 on SCLC cells to deliver a cytotoxic payload directly to tumor cells. In this study, the cardiac safety profile of Rova-T was assessed by evaluating changes in QT interval, electrocardiogram (ECG) waveform, heart rate, and proarrhythmic adverse events (AEs) after treatment with Rova-T in patients with previously treated extensive-stage SCLC. Patients underwent ECG monitoring for 2 weeks after each of 2 i.v. infusions of 0.3 mg/kg Rova-T over 30 minutes, administered 6 weeks apart. Forty-six patients received at least one dose of Rova-T. At the geometric mean Rova-T maximum serum concentration of 7,940 ng/mL, ECG monitoring showed no significant changes in the Fridericia-corrected QT (QTcF) interval; the upper limit of the 2-sided 90% confidence interval did not exceed 10 msec for any time point. There were no clinically significant changes in QRS or PR intervals, ECG waveforms, or heart rate after Rova-T administration. All patients experienced a treatment-emergent AE (TEAE); 78% had a grade ≥ 3 TEAE, 59% had a serious TEAE, and 41% had a cardiac-related TEAE. The TEAEs that might signal proarrhythmia tendencies were uncommon. Confirmed partial responses were observed in 24% of patients. Based on the evaluation of ECG data collected in this study from patients treated with Rova-T at 0.3 mg/kg i.v. administered every 6 weeks, a QTcF effect of clinical concern can be excluded.

Phase I safety and pharmacokinetics study of rovalpituzumab tesirine in Japanese patients with advanced, recurrent small cell lung cancer.

OBJECTIVES: Rovalpituzumab tesirine (Rova-T™) is an antibody-drug conjugate that targets delta-like protein 3 (DLL3) on small cell lung cancer (SCLC) tumors, is internalized and releases the toxin pyrrolobenzodiazepine to induce cell death. This open label phase I study was the first study of Rova-T in Japanese patients. The aim of this study was to evaluate, safety, pharmacokinetics, and preliminary efficacy of Rova-T in Japanese patients with advanced recurrent SCLC. MATERIALS AND METHODS: Patients received Rova-T (0.2 or 0.3 mg/kg) by intravenous infusion on Day (D) 1 of each 6-week cycle for 2 doses and dexamethasone (8 mg BID oral) on D-1, D1, and D2 of each 6-week cycle. Retreatment with Rova-T was permitted for patients who tolerated their initial doses and then progressed after disease control (defined as stable disease or better) was observed for at least 12 weeks after their last dose of Rova-T. RESULTS: Rova-T exhibited toxicity that was generally manageable in Japanese patients (N = 29). No dose-limiting toxicities were experienced. The most common treatment-related adverse events (≥25% of patients, all grades) were platelet count decreased, pleural effusion, peripheral edema, aspartate aminotransferase increased, white blood cell count decreased, neutrophil count decreased, alanine aminotransferase increased, hypoalbuminaemia, anemia and decreased appetite. Safety and pharmacokinetics exposures were similar to previous observations in non-Japanese populations. Per investigator assessment of DLL3 high patients, 17% (3/18) had confirmed partial responses, and the disease control rate was 56%, mPFS was 2.9 months, and mOS was 7.4 months. CONCLUSIONS: These preliminary data support further exploration of Rova-T treatment in Japanese patients with SCLC in global studies. This trial was registered with ClinicalTrials.gov as NCT03086239.

Novel targeted strategies to overcome resistance in small-cell lung cancer: focus on PARP inhibitors and rovalpituzumab tesirine.

Introduction: Small-cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumour, and its outcome is strongly conditioned by the rapid onset of resistance to conventional chemotherapeutics. First-line treatment with a combination of platinum agents and topoisomerase inhibitors has been the standard of care for over 30 years, with disappointing clinical outcome caused by early-acquired chemoresistance. In this disheartening scenario, novel treatment strategies are being implemented in order to either revert or bypass resistance mechanisms. Areas covered: The general mechanism of action of the standard frontline treatment regimens for SCLC, as well as the known resistance mechanisms to these drugs, is reviewed. Moreover, we focus on the current preclinical and clinical evidence on the potential role of PARP inhibitors and rovalpituzumab tesirine (Rova-T) to tackle chemoresistance in SCLC. Expert opinion: Preliminary evidence supports PARP inhibitors and Rova-T as two promising approaches to either revert or bypass chemoresistance in SCLC, respectively. The identification of potential predictive biomarkers of response to these innovative treatments (SLFN11 and DLL3) has shortened the gap between SCLC and personalized targeted therapy. Further large-scale clinical studies are urgently needed for a better designation of PARP inhibitors and Rova-T in the therapeutic algorithm of SCLC patients.

Effect of Z-360, a novel orally active CCK-2/gastrin receptor antagonist on tumor growth in human pancreatic adenocarcinoma cell lines in vivo and mode of action determinations in vitro.

PURPOSE: Gastrin is known to enhance the growth of pancreatic carcinoma via the cholecystokinin (CCK)-2/gastrin receptor. We investigated the anti-tumor effect of Z-360 (calcium bis [(R)-(-)-3-[3-{5-cyclohexyl-1-(3,3-dimethyl-2-oxo-butyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-yl}ureido]benzoate]), a novel orally active CCK-2 receptor antagonist alone or combined with the chemotherapeutic agent, gemcitabine in human pancreatic adenocarcinoma cell lines. RESULTS: Z-360 potently inhibited specific binding of [3H]CCK-8 to the human CCK-2 receptor, with a Ki value of 0.47 nmol/l, and showed antagonistic activity for this receptor. The anti-tumor effect of Z-360 alone or combined with gemcitabine was assessed using subcutaneous xenografts of MiaPaCa2 and PANC-1 and an orthotopic xenograft model (PANC-1). Oral administration of Z-360 significantly inhibited the growth of MiaPaCa2 (41.7% inhibition at 100 mg/kg, P<0.01). Combined administration of Z-360 and gemcitabine significantly inhibited subcutaneous PANC-1 tumor growth compared with either agent alone (27.1% inhibition compared to effect with gemcitabine, P<0.05), and significantly prolonged survival compared with the vehicle control (median survival of 49 days in vehicle compared to 57 days in the combination group, P<0.05). In vitro studies showed that Z-360 significantly inhibited gastrin-induced proliferation of human CCK-2 receptor-expressing cells, and also significantly reduced gastrin-induced PKB/Akt phosphorylation to the level of untreated controls. CONCLUSION: In the present study, we have shown that Z-360 combined with gemcitabine can inhibit pancreatic tumor growth and prolong survival in a pancreatic carcinoma xenograft model, on a possible mode of action being the inhibition of gastrin-induced PKB/Akt phosphorylation through blockade of the CCK-2 receptor. Our results suggest that Z-360 may be a useful adjunct to gemcitabine for the treatment of pancreatic carcinoma and a therapeutic option for patients with advanced pancreatic cancer.

A locus and mechanism of action for associative morphine tolerance.

Repeated administration of an opioid in the presence of specific environmental cues can induce tolerance specific to that setting (associative tolerance). Prolonged or repeated administration of an opioid without consistent contextual pairing yields non-associative tolerance. Here we demonstrate that cholecystokinin acting at the cholecystokinin-B receptor is required for associative but not non-associative morphine tolerance. Morphine given in the morphine-associated context increased Fos-like immunoreactivity in the lateral amygdala and hippocampal area CA1. Microinjection of the cholecystokinin B antagonist L-365,260 into the amygdala blocked associative tolerance. These results indicate that cholecystokinin acting in the amygdala is necessary for associative tolerance to morphine's analgesic effect.

Mediators of glucagon-like peptide 2-induced blood flow: responses in different vascular sites.

The aims of the present study were: to characterize the mechanisms of hemodynamic alterations induced by GLP-2, and, to compare the responses elicited in the superior mesenteric artery (SMA) to other vascular beds. Anesthetized rats were infused at the doses of 0.9, 2.3, 4.6 and 9.3 nmol/kg into the jugular vein for 60 min. Blood flow in the various arteries was measured by the ultrasonic transit time technique. Some animals were pretreated with indomethacin (5 mg/kg, ip), L-NAME (9, 18, 36 and 72 micromol/kg, iv), atropine sulfate (1-2 mg/kg, iv), CCK-1 and CCK-2 receptor antagonists (L-364,718 and L-365,260, 1 mg/kg, iv), exendin (9-39) amide (35 nmol/kg, iv) and lidocaine (74 micromol/kg, iv) prior to the infusion of GLP-2 (4.6 nmol/kg). In another group, capsaicin was applied either systematically (125 mg/kg, sc) or vagally (1 mg/rat). GLP-2 administration at all doses significantly increased the SMA blood flow throughout the experiments. GLP-2 (4.6 nmol/kg) infusion significantly increased blood flow of inferior mesenteric artery and carotid artery but not in any other vessel measured. Only the pretreatments with L-NAME and lidocaine were ineffective in preventing the GLP-2-induced responses. These results implicate that GLP-2-induced blood flow alterations are most significant in the SMA and are not mediated by prostaglandins, muscarinic, GLP-1 or CCK receptors. Our results also suggest that the stimulatory effect of GLP-2 on SMA blood flow is NO-dependent and mediated via intrinsic, non-cholinergic enteric neurons.

Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo.

The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC(50)s of 14 and 0.7 micromol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage-independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21(waf1/cip1) levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors.

A randomized phase II study of gemcitabine plus Z-360, a CCK2 receptor-selective antagonist, in patients with metastatic pancreatic cancer as compared with gemcitabine plus placebo.

BACKGROUND: We investigated the efficacy and safety of 60, 120, or 240 mg of Z-360, which is a highly potent cholecystokinin2-receptor-selective antagonist, combined with gemcitabine in patients with metastatic pancreatic cancer. METHODS: Patients were randomly assigned in a 1:1:1:1 ratio to one of four treatment groups. Patients received 1000 mg/m2 gemcitabine for each cycle and Z-360 tablets of 60 mg (GZ 60 mg group), 120, 240 mg or placebo tablets (Gem group) orally twice daily. The primary endpoint was overall survival (OS). RESULTS: The median OS was 1.3 months longer in the GZ 60 mg group compared with the Gem group (8.5 vs. 7.2 months) and the risk of death was reduced by 19% compared with the Gem group, although there were no statistically significant differences. The study treatments were well tolerated. CONCLUSIONS: In this Phase II study, no statistically significant differences between the GZ groups and Gem group were detected in any analysis. However, Z-360 in dose of 60 mg tends to improve OS in patients with metastatic pancreatic cancer with low toxic effect. Further exploratory trials with other agents such as gemcitabine plus nab-paclitaxel might be beneficial.