Synthesis of a dual functional anti-MDR tumor agent PH II-7 with elucidations of anti-tumor effects and mechanisms

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux.     

Discriminative stimulus effects of caffeine: tolerance and cross-tolerance with methylphenidate

Twice daily injection of caffeine (30 mg/kg) for 3-1/2 days shifted the caffeine stimulus generalization curve to the right by 3-fold in rats trained to discriminate saline from 30 mg/kg of caffeine, and by 4-fold in rats trained to discriminate saline from 10 mg/kg of caffeine. The latter group was also tested for cross-tolerance with methylphenidate, a drug that generalizes completely with caffeine. Twice daily injection of caffeine (30 mg/kg) for 3-1/2 days increased the ED50 of methylphenidate for caffeine-appropriate responding from 1.5 to 5.5 mg/kg. Conversely, injections of methylphenidate (3.0 mg/kg) increased the ED50 of caffeine from 5.2 to 15 mg/kg. The development of symmetrical cross-tolerance to the discriminative effects of caffeine and to the caffeine-like discriminative effects of methylphenidate supports previous observations suggesting commonalities in the cellular bases of the stimulus properties of these drugs.     

Dasatinib as a bone-modifying agent: anabolic and anti-resorptive effects

Background

Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function.

Methods

For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model.

Results

Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2–5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1–2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression.

Conclusions

Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease.     

On the effects of a constant sub-threshold conditioning stimulus upon the response to a constant current test stimulus

The strength of a test stimulus, which is just adequate to produce a response after a sub-threshold conditioning stimulus, is calculated on the basis of Rashevsky's two-factor theory as a function of the strength and duration of the conditioning stimulus. The results are compared with available data for one of a family of curves and found to be in satisfactory agreement.     

Evaluating a dual microbial agent biopesticide with Bacillus thuringiensis var. kurstaki and Beauveria bassiana blastospores

A biopesticide with a mixture of entomopathogenic microbial agents was studied for improvements in efficacy. Recently developed liquid fermentation techniques were used to produce blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb), which were mixed with traditional fermentation of Bacillus thuringiensis Berliner kurstaki (Bt) spores and crystals. Based on a dosage response bioassay using Trichoplusia ni (Hübner) neonates exposed to treated leaf disks, the LC₅₀ values were determined to be 3.85?×?10⁷ spores mL^?1 for the unformulated Bt and 3.58?×?10⁷ blastospores mL^?1 for the unformulated Bb. Using the same bioassay conditions, mixtures of Bt:Bb at ratios of 1:0, 0.75:0.25. 0.5:0.5, 0.25:0.75, and 0:1 indicated that the greatest mortality was caused by the 0.5:0.5 mixture ratio of the microbial agents. Based on these results, the 0.5:0.5 mixture was formulated as a dry powder by spray drying and applied to field-grown cabbage for comparison with commercial Bt and Bb products. Overall, the mixed agent treatment caused equal or greater mortality of exposed insects when compared with commercial products. The insecticidal interaction was determined to be synergistic when combining these two microbial agents into a single product, providing greater mortality of target insects. Assuming similar fermentation costs, the combined treatment could provide improved efficacy without increasing production costs. These benefits support the concept of formulating mixed microbial agents as an environmentally friendly pest control tool.     

BF066, a novel dual target antiplatelet agent without significant bleeding

In this study, we report BF066, a novel adenine derivative, inhibits platelet activation and thrombosis via the adenosine receptor (A(2A)) activation and phosphodiesterase (PDE) inhibition. BF066 inhibits platelet aggregation and ATP releasing induced by multiple platelet agonists in a dose-dependent manner. The inhibition of BF066 on ADP-induced aggregation is potentiated by adenosine and can be dramatically antagonized by the A(2A) antagonist SCH58261. BF066 also inhibits the PDE activity and platelet spreading on fibrinogen. In FeCl(3)-injured mouse mesenteric arterial thrombosis model, BF066 prevents thrombus formation effectively, similar to clopidogrel. Intriguingly, at dose achieving similar antithrombotic effect compared to clopidogrel, BF066 does not increase bleeding significantly. Taken together, these results suggest that BF066 may be an effective and safe antiplatelet agent targeting both PDE and A(2A). Considering the successful use of combined antiplatelet therapy, BF066 may be further developed as a novel dual target antiplatelet agent.     

Investigations using immunization to attenuate the psychoactive effects of nicotine

Despite the enormous health risks, people continue to smoke and use tobacco primarily as a result of nicotine addiction. As part of our immunopharmacotherapy research, the effects of active and passive immunizations on acute nicotine-induced locomotor activity in rats were investigated. To this end, rats were immunized with either a NIC-KLH immunoconjugate vaccine designed to elicit an antinicotine immune response, or were administered an antinicotine monoclonal antibody, NIC9D9, prior to a series of nicotine challenges and testing sessions. Vaccinated rats showed a 45% decrease in locomotor activity compared to a 16% decrease in controls. Passive immunization with NIC9D9 resulted in a 66.9% decrease in locomotor activity versus a 3.4% decrease in controls. Consistent with the behavioral data, much less nicotine was found in the brains of immunized rats. The results support the potential clinical value of immunopharmacotherapy for nicotine addiction in the context of tobacco cessation programs.     

Discriminative control of variability: effects of successive stimulus reversals

A growing body of evidence suggests that behavioral variability can come under control of discriminative stimuli. The present experiment further examined discriminative control of variability in a novel way by using the discrimination-reversal paradigm. Eight pigeons responded under a multiple schedule of Vary and Yoke components signaled by different-colored keylights. In the Vary component, 4-response sequences that differed from the previous 10 produced food, while in the Yoke component, any 4-response sequence had a fixed probability of producing food, yoked to the prior Vary component. Following stability in this procedure, the key colors signaling the Vary and Yoke components were reversed across four successive conditions. Across the experiment, variability of keypeck sequences was higher in the Vary than in the Yoke component. Across successive reversals, the level of variability in the Vary component adapted more rapidly to the reversed contingencies, while the rate of adaptation in the Yoke component did not change systematically. These results are interpreted in terms of the different contingencies in the Vary and Yoke components. In addition, the improvement in the rate of adaptation across successive reversals in the Vary component appears consistent with a proactive interference account of discrimination-reversal performance. These results join others in suggesting that variability may be an operant dimension of behavior.     

Host-genotype and agent effects in scrapie incubation: Change in allelic interaction with different strains of agent

Summary Incubation period in mice of ME7 scrapie agent is known to be controlled by the gene sinc. Evidence is presented that the same gene probably also determines incubation period for the 22A scrapie agent which has very different biological properties. Not only is there a reversal of the direction of gene effect when these agents are compared in mice homozygous for two different alleles of sinc, but there is also a different type of allelic interaction—absence of dominance with ME7 and overdominance with 22A. Other agents are mentioned which extend this range of allelic interaction even further. It is suggested that replication of scrapie agent depends on a multimeric host site and that the two alleles of sinc contribute different types of subunits to these sites.     

Off-target effects of psychoactive drugs revealed by genome-wide assays in yeast

To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes.     

Selective effects of serotonergic psychoactive agents on gastrointestinal functions in health

This study evaluated the effects of serotonergic psychoactive agents on gastrointestinal functions in healthy human subjects. Participants received one of four regimens in a randomized, double-blind manner: buspirone, a 5-HT(1A) receptor agonist (10 mg twice daily); paroxetine, a selective serotonin reuptake inhibitor (20 mg daily); venlafaxine-XR, a selective serotonin and norepinephrine reuptake inhibitor (75 mg daily); or placebo for 11 days. Physiological testing performed on days 8-11 included scintigraphic assessment of gastrointestinal and colonic transit, the nutrient drink test, and assessment of the postprandial change in gastric volume. Fifty-one healthy adults (40 females, 11 males) participated in this study. No effects on gastric emptying or colonic transit were identified with any agent. Small bowel transit of a solid meal was accelerated by paroxetine. Buspirone decreased postprandial aggregate symptom and nausea scores. Venlafaxine-XR increased the postprandial change in gastric volume. Buspirone, paroxetine, and venlafaxine-XR affect upper gastrointestinal functions in healthy humans. These data support the need for clinical and physiological studies of these agents in functional gastrointestinal disorders.     

Plasmodium falciparum falcilysin: a metalloprotease with dual specificity

The malaria parasite Plasmodium falciparum degrades host cell hemoglobin within an acidic food vacuole. The metalloprotease falcilysin has previously been identified as an important component of this catabolic process. Using random peptide substrate analysis, we confirm that recombinant falcilysin is highly active at acidic pH, consistent with its role in hemoglobin degradation. Unexpectedly, the enzyme is also robustly active at neutral pH, but with a substantially different substrate specificity. Imaging studies confirm the location of falcilysin in the food vacuole and reveal association with vesicular structures elsewhere within the parasite. These data suggest that falcilysin may have an expanded role beyond globin catabolism and may function as two different proteases in its two locations.     

Chlorimipramine: a novel anticancer agent with a mitochondrial target

Mitochondria have been suggested to be a potential intracellular target for cancer chemotherapy. In this report, we demonstrate the ability of the tricyclic antidepressant chlorimipramine to kill human glioma cells in vitro by a molecular mechanism resulting in an increase in caspase 3 activity following inhibition of glioma oxygen consumption. Studies with isolated rat mitochondria showed that chlorimipramine specifically inhibited mitochondrial complex III activity, which causes decreased mitochondrial membrane potential as well as mitochondrial swelling and vacuolation. The use of chlorimipramine in human as an effective, non-toxic cancer therapeutic having a strong selectivity between cancer cells and normal cells on the basis of their mitochondrial function is discussed.