Invivo Assays Research Articles

Abstract B52: A phase I-IIA, dose-escalating study of intravenous melphalan-flufenamide (J1) in adult patients with advanced solid tumors (I), advanced ovarian cancer, or non-small cell lung cancer (IIA).

Abstract Introduction: Melphalan-flufenamide (previously designated J1) is an enzyme-activated (aminopeptidase N, APN) prodrug of melphalan. The mechanism of enzyme activation results in the selective intracellular trapping of melphalan in tumor cells. Treatment with melphalan-flufenamide results in a 10–20 fold higher intracellular concentration of melphalan in tumor cells than treatment with equal doses of melphalan, and has demonstrated enhanced cytotoxic activity in numerous in-vitro and in-vivo assays. Consistent with these observations, exposure to melphalan-flufenamide results in greater reductions in the rate of DNA synthesis and greater pro-apoptotic effects than direct treatment with the same doses of melphalan. Experimental: In the dose-escalating phase I part of this study patients with end-stage solid tumor malignancies were admitted. After definition of Recommended Phase Two Dose (RPTD) the trial shifted to a phase IIA trial and only patients with selected diagnoses could be included. Results: The dose limiting toxicity was bone marrow suppression, mainly reversible thrombocytopenia. This was particularly evident in heavily pretreated patients, and RPTD was set to an intravenous infusion of 50 mg (fixed dose) every third week. No cumulative toxicity was observed. In the phase IIA part of the study forty patients were included, twenty of which had ovarian cancer, and thirteen NSCLC. The ovarian cancer patients had received 3–8 previous lines of chemotherapy (mean 3.9, and 90 % had received both taxane and platinum drugs), the NSCLC patients 2–4 lines (mean 2.5, 100 % had received platinum drugs). Twenty-four patients could be radiologically evaluated after 3 cycles of melphalan-flufenamide. Out of twelve evaluable ovarian carcinoma patients two had PD, nine had SD and one had PR according to RECIST criteria. Two patients with SD had a CA125 response of −50 and −70 % respectively. Two patients with SD had clinical progression (ascites effusion) shortly after first evaluation. Eleven patients with NSCLC were evaluated; three had PD and eight SD, according to RECIST. Thus, in NSCLC disease stabilization was best response, one patient received nine cycles of melphalan-flufenamide, and progressed after seven months of therapy. Summary: The APN-activated prodrug melphalan-flufenamide can safely be given to cancer patients. Dose is limited by hematological toxicity. The study suggests clinical activity (biochemical and radiological) in ovarian cancer, even after failure to several lines of chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B52.

Asyn-anticonformational difference allows SRSF2 to recognize guanines and cytosines equally well

SRSF2 (SC35) is a key player in the regulation of alternative splicing events and binds degenerated RNA sequences with similar affinity in nanomolar range. We have determined the solution structure of the SRSF2 RRM bound to the 5'-UCCAGU-3' and 5'-UGGAGU-3' RNA, both identified as SRSF2 binding sites in the HIV-1 tat exon 2. RNA recognition is achieved through a novel sandwich-like structure with both termini forming a positively charged cavity to accommodate the four central nucleotides. To bind both RNA sequences equally well, SRSF2 forms a nearly identical network of intermolecular interactions by simply flipping the bases of the two consecutive C or G nucleotides into either anti or syn conformation. We validate this unusual mode of RNA recognition functionally by in-vitro and in-vivo splicing assays and propose a 5'-SSNG-3' (S=C/G) high-affinity binding consensus sequence for SRSF2. In conclusion, in addition to describe for the first time the RNA recognition mode of SRSF2, we provide the precise consensus sequence to identify new putative binding sites for this splicing factor.

CK2 phosphorylates AP-2α and increases its transcriptional activity

Transcription factor AP-2α involves in the process of mammalian embryonic development and tumorigenesis. Many studies have shown that AP-2α functions in association with other interacting proteins. In a two-hybrid screening, the regulatory subunit β of protein casein kinase 2 (CK2β) was identified as an interacting protein of AP-2α; we confirmed this interaction using in-vitro GST pull-down and in-vivo co-immunoprecipitation assays; in an endogenous co-immunoprecipitation experiment, we further found the catalytic subunit α of protein casein kinase 2 (CK2α) also exists in the complex. Phosphorylation analysis revealed that AP-2α was phosphorylated by CK2 kinase majorly at the site of Ser429, and such phosphorylation could be blocked by CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in a dose-dependent manner. Luciferase assays demonstrated that both CK2α and CK2β enhanced the transcription activity of AP-2α; moreover, CK2β increased the stability of AP-2α. Our data suggest a novel cellular function of CK-2 as a transcriptional co-activator of AP-2α.

The social recognition test of memory in the rat: A reliable primary in-vivo cognition assay for guiding drug discovery/development decision-making relevant to neuropsychiatric diseases

The social recognition test of memory in the rat: A reliable primary in-vivo cognition assay for guiding drug discovery/development decision-making relevant to neuropsychiatric diseases

Potential efficacy of a delta 5-aminolevulinic acid bioadhesive gel formulation for the photodynamic treatment of lesions of the gastrointestinal tract in mice.

A delta 5-aminolevulinic acid (ALA) bioadhesive gel has been developed and evaluated in an in-vivo mouse model for photodynamic treatment of gastric cancer or Barrett's oesophagus. Four gels were tested: noveon AA-1, keltrol T, lutrol and blanose. An initial in-vitro study of gel adhesion showed that noveon and keltrol had longer polyethylene transit times than lutrol and blanose. In-vivo assays indicated that protoporphyrin IX was synthesized by gastric mucosa when ALA-noveon and ALA-lutrol were used (preferable results for noveon). Keltrol was eliminated from the study after these investigations. Only ALA-noveon gel was retained for studies of the relationship between ALA dose and fluorescence. Fluorescence measurements in-vivo showed that ALA concentration and application time had an influence on protoporphyrin IX synthesis. Maximum intensity (2091 counts s-1) was found with 2 mg mL-1 ALA, and fluorescence intensities differed with application time, reaching 1805 counts s-1 after 240 min. ALA-noveon, showing good adhesion and enabling efficient diffusion of ALA at a pH < 6, was considered the best formulation for maintaining ALA stability.

A novel human microplasmin fold: new perspective to thrombosis treatment

The purpose of the present study was to obtain polypeptide chains with more specific fibrinolytic activity from normal human plasma. The isolation procedure was carried out in the presence of acetic acid and sodium borate (pH 9.9) purified with affinity and ionic interchange chromatography. Activity of microplasmin studied in vitro shows that the fibrin plate was used to measure the fibrinolytic activity. Rabbit thrombosis model was used to probe in-vivo fibrinolytic effects. Out of 13 male and female rabbits, seven (group A) were treated with microplasmin and six (group B) as placebo, weighing 2500-3200 g. Comparison of groups was made by analysis of variance with a statistical significance of 0.05%. In-vitro assay lysis (25 IU) was produced by microplasmin and tissue plasminogen activator. The fibrinolytic activity in rabbits showed 100% (7/7) reperfusion with microplasmin and 0% (0/7) with placebo (P = 0.002). The proposed scheme in this research for the fibrinolytic activity of microplasmin obtained by autolysis cleavage at new specific sites Lys-97-Val-98 and Ser-364-Thr-365 in the plasminogen involved in-vitro and in-vivo assays as a new specific fibrinolytic activity without haemorrhagic events. This microplasmin is different to the others and with more specific fibrinolysis.

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Antihyperalgesic activity of verbascoside in two models of neuropathic pain

This study reports on the rapid isolation of verbascoside from Lippia citriodora H.B.K. (Verbenaceae), an inexpensive and widespread source, and the evaluation of its antihyperalgesic activity. Isolation of verbascoside was achieved by size exclusion chromatography with Sephadex LH-20 eluting with 50% EtOH, which is proposed as a fast and efficient method of separation. The antihyperalgesic activity of verbascoside was tested by in-vivo assay using the paw-pressure test in two animal models of neuropathic pain: a peripheral mononeuropathy produced either by a chronic constriction injury of the sciatic nerve (CCI) or by an intra-articular injection of sodium monoiodoacetate (MIA). Verbascoside administered intraperitoneally at a dose of 100 mg/kg reverted the mechanical hyperalgesia in both CCI and MIA treated rats, as evaluated in the paw-pressure test. Verbascoside was also effective against mechanical hyperalgesia after oral administration at doses of 300 and 600 mg/kg.

Functional Characterization of Human Cancer-Derived TRKB Mutations

Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKBT695I and TRKBD751N). Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKBL138F). Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKBP507L) in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKBT695I and TRKBD751N, displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKBL138F and TRKBP507L) were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

Metabolites of the Ellagitannin Geraniin and Their Antioxidant Activities

Different types of ellagitannins are reported to have various biological activities, such as antioxidant, antiviral, and antitumor activities. However, there are few definitive studies on the absorption and metabolism of ellagitannins. This review compares the absorption and metabolism of ellagitannins, and the antioxidant properties of their metabolites in rats, with those of intact ellagitannins by means of IN VITRO and IN VIVO assays. We isolated 7 urinary and intestinal microbial metabolites in rats after the ingestion of geraniin, which is a typical ellagitannin isolated from GERANIUM THUNBERGII, an antidiarrheic remedy in Japan. The structures of these metabolites were determined to be dibenzopyran derivatives ( 1- 7), using NMR and mass spectroscopic data. Four major metabolites ( 1- 4) prepared by chemical synthesis were evaluated for their antioxidant activities by using 2,2-diphenyl-1-picrylhydrazyl radical scavenging and oxygen radical absorbance capacity (ORAC) methods. The metabolites exhibited more potent antioxidant activities in the ORAC assay than intact ellagitannins, such as geraniin and corilagin. Furthermore, plasma ORAC scores increased with increases in the plasma concentration of the metabolites after the oral administration of geraniin to rats. These findings suggest that these metabolites may contribute to the health benefits of ellagitannins as antioxidants in the body.

Development of anti-malaria herbal preparation / drugs from local medicinal plants

1. The screening for potential anti-malaria properties by bioassay guided fractionations. The biological properties were carried out using the plasmodium lactate dehydrogenase assay (pLDH) (Makler, 1993). 2. The harmful effect of the extract was determined by the MTT assay (Mossman, 1983). 3. The extracts were tested for its efficacy in animal model using rodent malaria parasite, P. berghei using the 4 Days Suppressive Test by Peters and Robinson, 1975. 4. The parasite and the host cell biology were investigated to determine the role of the drug on the biology of the host cells and the erythrocyte invasion pathways. 5. The probable mode of actions were also investigated through known mechanism of action such as the Heam Polymerization Assays (Basilico et al., 1997). The biologiccal screening was carried out using the pLDH assay, triplicate test were carried out at different time to ensure the reproducibility of the results. The data was analysed using probit analysis. The invivo assay was by 4 days suppressive test, For each parasite strain, 4 groups of mice with different doses given orally were used, each having 5 mice and another 4 groups of mice with the same doses given s/c and each group having as the earlier. The standard control used was chlorouine and the design was similar to the test group except that the oral and s/c doses were according to the standard dose from standard procedure by Peters and Robinson, 1975. Mode of actions, parasite erythorcyte invasion assay were according to known procedure. 30% of the local medicinal plants screened have potentials for anti-malarial. The herbal preparation of the E.longifolia standardized extracts were able to supressed the infection of the rodent malaria more than 80%. Detail results will be discuss in the presentation. E. longifolia herbal standardized extracts have potential for anti-malaria drug.

Maturation of the miR15a/miR16-1 Family Is Impaired In Chronic Lymphocytic Leukaemia

AbstractAbstract 53A role as tumor suppressor has been proposed for the miR15a/miR-16-1 family of miRNA genes localized in a critical region in 13q14 that is deleted in more than 50% of CLL patients. These two miRNA genes regulate cell cycle progression and apoptosis in cell lines and in vivo, and knockout of the syntenic region in mice results in the development of a CLL-like phenotype.We measured by qRT-PCR mir15a and miR-16 in CLL samples (n=38) and normal B-cells (n=14) and observed reduced levels in CLL independent of the 13q deletion status. We then assessed the levels of the primary precursors of the mature miRNAs (pri-miRNAs 15a, 16-1 and 16-2). No difference could be identified between patients with a deletion of 13q and patients with a retention of both copies of 13q14. However, CLL cells had increased levels of pri-miRNAs when compared to CD19-sorted normal B-cells. Moreover, in CLL cells levels of miR-16 and miR15a were inversely correlated to their pri-miRNA transcripts, while a miRNA used as a control (miR-155) had a positive correlation with its primary transcript as expected. In addition, also the levels of precursor miRNA molecules of the 13q14 miRNA genes were low in CLL patients. This strongly suggested a defect of miRNA maturation at the Drosha processing step that produces the pre-miRNA from the pri-miRNA molecule. Therefore, we grouped patients according to the observed levels of pri-miRNA and pre-miRNA: CLL patients with a ratio of these two processing intermediates (pri-/pre) above the average levels observed in non-malignant CD19-B-cells were included in the “high ratio” group (58% of our cohort CLL patients), while the rest was grouped in the “normal ratio” group (42%). Patients with high pri-/pre ratio had significantly lower mature miRNA levels compared to patients with normal ratio. In contrast, the respective ratio of the precursor molecules of miR-155 that were used as control did not differ in the two groups. These findings suggest that there is a processing defect that reduces maturation of the miR15a/miR-16 in a subset of CLL patients. In order to test the actual processing activity in CLL cells, we used a luciferase based in-vivo Drosha processing assay and could show a significant reduction of pri-16-1 processing in patients belonging to the “high pri-/pre ratio” group. These findings underline the role of miR15a/miR-16 in the pathomechanism of CLL and show a complementary route of inactivation that supplements genomic loss of the critical region in 13q14 and its transcriptional downregulation in CLL. Disclosures:Mertens:Mundipharma: Honoraria; Celgene: Honoraria. Stilgenbauer:Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding.

ALDH high adenoid cystic carcinoma cells display cancer stem cell properties and are responsible for mediating metastasis

The cancer stem cell (CSC) theory has been proposed to explain the tumor heterogeneity and carcinogenesis process. Recent studies indicate that aldehyde dehydrogenase (ALDH) activity represents a promising CSC marker. Here, we aimed to determine whether human adenoid cystic carcinoma (AdCC) also follows CSC model by exploring the CSC properties of AdCC cells expressing high level of ALDH activity. Utilizing in-vivo series transplantation assays, we found ALDH high AdCC cells were capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. Utilizing in-vitro assay, we found only ALDH high AdCC cells have tumorsphere-forming ability in anchorage-independent cultures. Finally, we showed ALDH high AdCC cells possess highly invasive capability and are responsible for mediating metastasis. These findings suggest the existence of a developmental hierarchy within human AdCC and further elucidation of the unique survival mechanism of AdCC derived CSC population may provide novel therapeutic strategies to treat AdCC.

A Protective Effect of Endomorphins on the Oxidative Injury of Islet

The antioxidative capacity of endomorphins (EMs), endogenous μ-opioid receptor agonists, has been demonstrated by IN VIVO assays. In this study, we attempt to evaluate the effects of endomorphin 1 (EM1) and endomorphin 2 (EM2) on pancreatic islet injuries induced by streptozotocin (STZ), alloxan (ALX) and H(2)O(2), respectively. Wistar rats' islets were isolated and purified. The function of the islet cells, the insulin response to glucose stimulation was examined by insulin Radio Immuno Assay and the cell viability was measured by MTT assay. DNA fragments were performed to evaluate the apoptosis, while the cell cycle distribution was analyzed by PI staining flow cytometric analysis. Furthermore, the islet were treated with EM1, EM2 or ALX for 24 h, and the expression of p53 and p21 protein were determined by Western blot. The results showed that STZ, ALX, and H(2)O(2) displayed clear concentration-dependent inhibitory effects on the pancreatic islet cells. While EMs improved the viability of islet induced by STZ, ALX or H(2)O(2), and EMs enhanced insulin accumulation of the cell supernatant after ALX and STZ stimulation. Our data also showed both that EMs inhibited cell apoptosis and cell cycle G1 arrest induced by STZ and ALX through down-regulaing p53 and p21 expression. Taken together, these results demonstrate that EMs can protect islet cells from STZ, ALX and H(2)O(2) induced injuries. Our observations imply that the endomorphins may have protective effects on islet cells oxidative injury.

Abstract 2314: Epithelial-mesenchymal transition derived cells exhibit multi-lineage differentiation potential similar to mesenchymal stem cells

Abstract The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows stem cell traits to breast epithelial cells. Mesenchymal stem cells (MSC) have the capacity to self-renew and differentiate into multiple tissue lineages. We hypothesized that the activation of EMT by ectopic expression of Twist, Snail or TGF-β in immortalized human mammary epithelial cells (HMEC) will result in the generation of cells with a phenotype and functionality similar to MSC. We found that the EMT-derived cells not only showed similar morphology but also displayed the typical MSC phenotype i.e. CD44+, CD24− and CD45−. Alternatively, MSC expressed EMT inducing genes such as Twist, Snail and FOXC2. Interestingly, CD140b (PDGFR-β), a marker for naive MSC, was exclusively expressed in EMT-derived cells compared to their epithelial counterparts. Moreover, functional analysis revealed that EMT-derived but not the control cells differentiate into Alizarin Red S-positive mature osteoblasts, Oil Red O-positive adipocytes and Alcian Blue-positive chondrocytes similar to MSC. We also observed that EMT-derived but not control cells invade and migrate towards MDA-MB-231 tumor cells in-vitro similar to MSC, displaying the characteristic tropism of MSC for tumor cells as previously reported by us. In-vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSC. In conclusion, we demonstrated that the EMT-derived cells are similar to MSC in gene-expression, multi-lineage differentiation, migration towards tumor cells and their ability to home to wounds. These results also suggest that EMT-derived MSC are active participants in cancer growth and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2314.

Gastroprotective activity of atractylenolide III from Atractylodes ovata on ethanol-induced gastric ulcer in vitro and in vivo

The rhizome of Atractylodes ovata De Candolle is popularly used in traditional Chinese medicine to treat gastrointestinal diseases. However, the major gastroprotective compounds of A. ovata have not been identified. This study reports on the principal gastro- protective component of A. ovata. Five sesquiterpenoids (atractylon, atractylenolides I, II, III and biatractylolide) were isolated from the extracts of A. ovata rhizome via silica gel column chromatography. The gastroprotective effects of these five sesquiterpenoids were measured in in-vitro ethanol-induced primary culture rat gastric mucosal (PRGM) cell damage and in-vivo ethanol-induced acute rat gastric ulcer models. Atractylon, atractylenolide I and biatractylolide were strongly toxic in PRGM cells, whilst atractylenolides II and III were not. Atractylenolide II did not show cytoprotective effects, but oral administration of atractylenolide III dose-dependently prevented ethanol-induced PRGM cell death and cell membrane damage. The EC50 values were 0.27 and 0.34 mm, respectively. In the in-vivo assay, atractylenolide III 10 mg/kg significantly reduced 70% ethanol-induced Wistar rat gastric ulcer. Atractylenolide III could inhibit matrix metalloproteinase (MMP)-2 and MMP-9 expression through upregulation of tissue inhibitors of metalloproteinase from the gastric ulcerated tissues. Atractylenolide III was the major gastroprotective component of A. ovata in ethanol-induced acute gastric ulcer. It is suggested that the gastroprotective mechanism of atractylenolide III was via inhibition of the MMP-2 and MMP-9 pathway.

An extract of Lannea microcarpa: composition, activity and evaluation of cutaneous irritation in cell cultures and reconstituted human epidermis

Lannea microcarpa (Anacardiaceae) is a tropical tree used in African folk medicine and commercial dermopharmaceutical formulations. Fractionation and analysis of its polar extract allowed the identification of 4'-methoxy-myricetin 3-O-alpha-L-rhamnopyranoside, myricetin 3-O-alpha-L-rhamnopyranoside, myricetin 3-O-beta-D-glucopyranoside, vitexin, isovitexin, gallic acid and epi-catechin, as the major constituents. In-vivo assay (the croton oil ear test in mice) showed that the extract had significant anti-inflammatory effect (ID50 = 900 microg cm(-2)) but ten times lower than that of indometacin (ID50 = 93 microg cm(-2)), the non-steroidal anti-inflammatory drug used as reference. Cytotoxicity and cutaneous irritation of the extract and its constituents were investigated. The crude extract and its major components did not affect cell viability in-vitro either in three different cultures (J774. A1, WEHI-164 and HEK-293) of cells grown in monolayers or in the reconstituted human epidermis (RHE, 3D model), nor did they cause release of pro-inflammatory mediators (IL-1alpha) or histomorphological modification of RHE.

Antitumour activity of 3-chlorodeoxylapachol, a naphthoquinone from Avicennia germinans collected from an experimental plot in southern Florida

As part of an ongoing collaborative effort to discover new anticancer agents from plants, an extract obtained from the leaves and twigs of Avicennia germinans, collected in a coastal area of southern Florida, was identified as possessing cytotoxic activity in a panel of human cancer cell lines. Fractionation of the petroleum ether partition, using cytotoxicity to guide the fractionation, led to the isolation of 3-chlorodeoxylapachol. The antitumour potential of 3-chlorodeoxylapachol was demonstrated with the in-vivo hollow fibre assay, a model of antitumour activity using human cancer cell-filled fibres implanted into mice. The possibility that this compound is an artefact of the isolation procedure was ruled out by liquid chromatography-mass spectrometry analysis of extracts prepared without the use of chlorinated solvent. In conclusion, 3-chlordeoxylapachol, a secondary metabolite obtained from the chloroform-soluble extract of a mangrove tree, was cytotoxic in a panel of human cancer cells, and active against KB human cancer cells in the murine hollow fibre antitumour model, with selectivity in KB cells for the intravenous site at lower doses, indicating possible metabolic activation.

Synthesis and evaluation of N1-substituted-3-propyl-1,4-benzodiazepine-2-ones as cholecystokinin (CCK2) receptor ligands.

A novel synthetic approach towards N1-alkylated 3-propyl-1,4-benzodiazepines was developed in five synthetic steps from 2-amino-4-chlorobenzophenone, in which the N-oxide 4 served as a key intermediate. The structure-activity relationship optimization of this 3-propyl-1,4-benzodiazepine template was carried out on the N1-position by selective alkylation reactions and resulted in a ligand with an improved affinity on the cholecystokinin (CCK2) receptor. The N-allyl-3-propyl-benzodiazepine 6d displayed an affinity towards the CCK2 (CCK-B) receptor of 170 nM in a radiolabelled receptor-binding assay. The anxiolytic activity of this allyl-3-propyl-1,4-benzodiazepine 6d was subsequently determined in in-vivo psychotropic assays. This novel ligand had ED50 values of 4.7 and 5.2 mg kg(-1) in the black and white box test and the x-maze, respectively, and no significant sedation/muscle relaxation was observed.

Cytotoxicity and antiangiogenic activity of grandisin

Abstract Objectives The antitumoural properties of grandisin, a tetrahydrofuran neolignan from Piper solmsianum, were investigated by in-vitro and in-vivo assays using the Ehrlich ascites tumoural (EAT) model. Methods Viability of the tumour cells was evaluated by Trypan blue exclusion and MTT methods, after incubation with grandisin (0.017-2.3 μM). The effects of grandisin on the activity of caspase-3, −6, −8, and −9 were also investigated using colorimetric protease kits. In-vivo studies were performed in EAT-bearing mice treated intraperitoneally with 2.5, 5 or 10 mg/kg grandisin for 10 days. Key findings Grandisin inhibited the growth of EAT cells, by both methods, with IC50 values less than 0.25 μM. The results showed that the activity of all the caspases studied increased in grandisin-treated cells, when compared with control, non-treated cells. Administering grandisin to EAT-bearing mice increased survival of the animals, in a dose-dependent manner. Simultaneously, we detected a 66.35% reduction of intraperitoneal tumour cell burden in the animals treated with 10 mg/kg grandisin. Additionally, in these animals, the marked increase of vascular endothelial growth factor (VEGF) levels, induced by EAT development, was decreased with treatment with grandisin, resulting in a reduction of 32.1% of VEGF levels in the peritoneal washing supernatant, when compared with the control. Conclusions The results demonstrated that grandisin induced in-vitro cytotoxicity and antiangiogenic effects in mice while it acted against tumour evolution, prolonging host survival.