Dose-related Decrease Research Articles

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24 h after a single pharyngeal aspiration of MWCNT-S (1–200 μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8 mg/m3) for 4 d, 4 h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200 μg/mouse) and in MNPCEs after inhalation exposure (17.5 mg/m3). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 μg/cm2), while MWCNT-T increased strand breakage only at 200 μg/cm2. Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.

Toxicological assessment of a prototype e-cigaret device and three flavor formulations: a 90-day inhalation study in rats

A prototype electronic cigaret device and three formulations were evaluated in a 90-day rat inhalation study followed by a 42-day recovery period. Animals were randomly assigned to groups for exposure to low-, mid- and high-dose levels of aerosols composed of vehicle (glycerin and propylene glycol mixture); vehicle and 2.0% nicotine; or vehicle, 2.0% nicotine and flavor mixture. Daily targeted aerosol total particulate matter (TPM) doses of 3.2, 9.6 and 32.0 mg/kg/day were achieved by exposure to 1 mg/L aerosol for 16, 48 and 160 min, respectively. Pre-study evaluations included indirect ophthalmoscopy, virology and bacteriological screening. Body weights, clinical observations and food consumption were monitored weekly. Plasma nicotine and cotinine and carboxyhemoglobin levels were measured at days 28 and 90. After days 28, 56 and 90, lung function measurements were obtained. Biological endpoints after 90-day exposure and 42-day recovery period included clinical pathology, urinalysis, bronchoalveolar fluid (BALF) analysis, necropsy and histopathology. Treatment-related effects following 90 days of exposure included changes in body weight, food consumption and respiratory rate. Dose-related decreases in thymus and spleen weights, and increased BALF lactate dehydrogenase, total protein, alveolar macrophages, neutrophils and lung weights were observed. Histopathology evaluations revealed sporadic increases in nasal section 1–4 epithelial hyperplasia and vacuolization. Following the recovery period, effects in the nose and BALF were persistent while other effects were resolved. The no observed effect level based upon body weight decreases is considered to be the mid-dose level for each formulation, equivalent to a daily TPM exposure dose of approximately 9.6 mg/kg/day.

Use of calcium phosphate- silver nanoparticles in chitosan coatings on titanium and for drug delivery

Event Abstract Back to Event Use of calcium phosphate- silver nanoparticles in chitosan coatings on titanium and for drug delivery Gillen Gonzales1, Joel D. Bumgardner1, Jegdish P. Babu2, Jessica A. Jennings1, Warren O. Haggard1, Hitesh Adhikari3, Diego V. Pulgarin1, Timothy B. Martin1, Vishnu P. Murali1 and Sanjay R. Mishra3 1 University of Memphis, Biomedical Engineering, United States 2 UTHCS, School of Dentistry, United States 3 University of Memphis, Department of Physics, United States Introduction: Calcium-phosphate (CaP) nanoparticles decorated with silver (Ag) have shown potential to kill infectious bacteria in vitro.[1] [2]A coating that localizes the antibacterial CaP- Ag nanoparticles using osteoconductive chitosan may provide a means to inhibit bacterial biofilm formation and subsequent infectious complications for these implants. This work evaluated antimicrobial, cytocompatibility, and protein delivery properties of CaP- Ag in chitosan coatings. Materials and Methods: Porous CaP nanospheres with 0, 15, 37, or 50% Ag were fabricated using a hydrothermal synthesis of CaP followed by microwave heating of CaP-AgNO3 for Ag decoration on CaP. [1] Chitosan (82 DDA) coatings with 30wt% CaP-Ag particles were made via silane-solution casting on to cpTi and sterilized using ethylene oxide gas. Coated and uncoated cpTi coupons (n=3/group) were incubated with 3ml of bacteria (5X106 colony forming units) under anaerobic conditions for common dental pathogens (P. gingivalis, ATC BAA-388; P. denticola, ATCC 35308) and aerobic conditions for orthopedic pathogens (P. aeruginosa: ATCC 15442; S. aureus, ATCC 25923, and S. epidermidis, ATCC 700576). Percent viability of bacteria was determined using MTT Bacterial Viability Assay (Roche Lab) using culture plastic controls. Coated and uncoated cpTi coupons were incubated with 2X104 NIH 3T3 cells for 24 hrs and viability determined using CellTitre-Glo Assay (Promega). For protein release, 20mg of CaP-Ag particles (n=4/group) were incubated in 3mL of 1mg/mL α-chymotrypsin (MW = 25kDa, pI ~ 9.1), used as an analogue to BMP-2 (MW = 26kDa, pI ~8.2), for 24 hrs at 37C under gentle rocking. After 24hrs, supernatant was collected for determining protein loading by subtraction. Samples were incubated in 3mL PBS at 37C and eluates were collected with complete PBS every 2 days for 3 weeks. Protein concentration was determined using Quant-iT Protein Kit (Mol. Probe). Size and Zeta potential of particles was determined using Delsa™ Nano Submicron Particle Size and Zeta Potential Particle Analyzer (Beckmann Coulter). Result:Uncoated cpTi, plain chitosan and chitosan coatings with CaP-0%Ag particles had minimal effect on viability of bacteria (Figure 1). There was a significant and dose-related decrease in viability of all bacteria with increasing Ag content on CaP nanoparticles with the CaP-50%Ag particles having the largest antibacterial properties (p<0.05). There were some differences in the sensitivity of bacteria to the coatings with S. epidermidis showing the highest sensitivity. Figure1: Viability of bacteria on chitosan with CaP- Ag coatings The total loading and percent release of protein was not different for the different CaP-Ag particles (Figure 2). This may be due to the similarly large negative Zeta potential of the particles (Table). Protein release pattern was also similar for the different CaP-Ag particles showing an initial burst release followed by a sustained release of approximately10 ng/µl from day 3 to 21. Figure2: Protein release from CaP- Ag particles in PBS Discussion: There was no difference in the viability of the fibroblasts on the uncoated cpTi, plain chitosan and chitosan coatings with CaP-0%Ag particles. However, cell viability was reduced 100-fold on all chitosan coatings incorporating CaP-Ag particles regardless of the %Ag in the particles (data not shown). Part of reduced viability of the fibroblasts may be due to early release of Ag from particles due to the acetic acid used in the coating process. Previous studies showed that CaP-Ag particles alone had little effect on cell viability.[1] Conclusion: Increasing concentration of Ag in CaP particles in chitosan coatings exhibited a dose dependent antibacterial effect on 5 implant pathogens in vitro. However, low cell viability to CaP-Ag particles in the coatings needs to be addressed. The CaP-Ag particles exhibited high protein loading and sustained protein release profile that may be advantageous for the local delivery of growth factors or other agents that require low and extended release profiles. FedEx Institute of Technology, Memphis USA

A Synthetic Biology Rheoswitch Therapeutic System® for the Controlled Local Expression of IL-12 as an Immunotherapy for the Treatment of Cancer

Tumors escape the immune system through the process of immunoediting. Restoration of the immune system’s ability to detect the tumor should result in improved therapeutic outcome. A gene delivery platform technology, RheoSwitch Therapeutic System® (RTS®), has been developed to enable the controlled, regulated expression of a target gene which locally delivers the desired therapy to patients. A replication-incompetent adenoviral vector, administered intratumoral, is engineered to express IL‑12 (Ad-RTS-IL-12) under control of the RheoSwitch Therapeutic System, where IL-12 expression is controlled via the administration of an oral activator ligand (veledimex). In the presented preclinical studies we have demonstrated that the oral administration of veledimex resulted in a dose-related increase in tumor veledimex levels. The increase in tumor veledimex levels in combination with Ad-RTS-mIL-12 resulted in a dose-related increase in the IL-12 mRNA (switch on) leading to dose-related increases in IL-12p70 in the tumor with minimal increase in serum IL-12. The increase in tumor IL-12 correlated with an increase in tumor CD8+ cytotoxic T cells and a concomitant decrease in regulatory T cells (Tregs) in the tumor microenvironment. Importantly, Ad-RTS-mIL-12 + veledimex elicited dose-related decreases in tumor growth rate with no significant change in body weight in both breast and melanoma syngeneic mouse models. When veledimex was discontinued, expression of IL-12 mRNA returned to baseline concomitant with tumor IL-12 levels. In summary, these results highlight the potential of a synthetic biology-based approach for cancer treatment, safely leveraging the full anticancer potential of some immunomodulators without the serious side-effects associated with systemic injection.

Continuous MPTP intoxication in the Göttingen minipig results in chronic parkinsonian deficits.

Parkinson's disease (PD) is a common neurodegenerative disorder, resulting from progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Neuroprotective therapies in PD are still not available, perhaps because animal models do not imitate the chronic and progressive nature of the clinical state of PD. To address this, we performed a feasibility study aimed at establishing a chronic non-primate large animal PD model in Göttingen minipigs based on continuous infusion of the neurotoxin 1-methyl-4-phenyl‑1,2,3,6-tetrahydropyridine (MPTP). Twelve female Göttingen minipigs were divided into groups of 2-4 animals and implanted with infusion pumps for continuous intramuscular MPTP delivery of 4-24 mg MPTP/day for 11 weeks. The animals showed parkinsonian symptoms with bradykinesia, rigidity, coordination and chewing difficulties. Symptoms were stable in the 12 and 18 mg MPTP/day groups, whereas the remaining groups showed partial or full behavioral recovery. Digital gait analysis, high performance liquid chromatography (HPLC) measurements and stereological counts of tyrosine hydroxylase-positive (TH+) neurons in the SNc revealed a dose-related decrease in gait velocity, striatal metabolite levels and neuron numbers with increasing doses of MPTP. No neuronal inclusions were observed, but alpha-synuclein staining intensified with increased cumulative MPTP dosages. We conclude that this large-animal model of chronic MPTP administration in Göttingen minipigs shows trends of stable parkinsonian deficits at 18 mg MPTP/day in all modalities examined. This PD model shares many of the characteristics seen in patients and, although preliminary, holds considerable promise for future pre-clinical trials of neuroprotective therapies.

The minipig as a new model for the evaluation of doxorubicin-induced chronic toxicity.

Doxorubicin can cause life-threatening toxic effects in several organs, with cardiotoxicity being the major concern. Although a large number of animal models have been utilized to study doxorubicin toxicity, several restrictions limit their use. Since the Göttingen minipig is an accepted species for non-clinical safety assessment and translation to man, we aimed at exploring its use as a non-rodent animal model for safety assessment and regulatory toxicity studies using doxorubicin. Three groups of three males and three females adult Göttingen minipigs received 1.5 mg kg(-1) , 3/2.3 mg kg(-1) or vehicle at intervals of 3 weeks for 7 cycles. Doxorubicin treatment resulted in a dose-related decrease in the erythrocytes, hemoglobin and hematocrit count, accompanied by leukopenia and thrombocytopenia. Bone marrow smears revealed dose-related hypocellularity. Urea and creatinine levels were elevated in treated animals, associated with proteinuria and hematuria. Histopathological evaluation detected nephropathy and atrophy of hematopoietic tissues/organs, mucosa of the intestinal tract and male genital tract. Cardiac lesions including chronic inflammation, endocardial hyperplasia, hemorrhage and myxomatous changes were evident in hematoxylin and eosin stains, and evaluation of semi-thin sections showed the presence of dose-related vacuolation in the atrial and ventricular cardiomyocytes. Cardiac troponin levels were increased in the high-dose group, but there was no direct correlation to the severity of the histopathological lesions. This study confirms that the Göttingen minipig has a comparable toxicity profile to humans and considering its anatomical, physiological, genetic and biochemical resemblance to humans, it should be considered as the non-rodent species of choice for studies on doxorubicin toxicity. Copyright © 2015 John Wiley & Sons, Ltd.

Integrin-linked kinase affects signaling pathways and migration in thyroid cancer cells and is a potential therapeutic target

Integrin-linked kinase (ILK) is a serine-threonine kinase that regulates interactions between the cell and the extracellular matrix. In many cancers, overexpression of ILK leads to increased cell proliferation, motility, and invasion. We hypothesized that ILK functions as a regulator of viability and migration in thyroid cancer cells. Eleven human thyroid cancer cell lines were screened for ILK protein expression. The cell lines with the greatest expression were treated with either ILK small interfering RNA (siRNA) or a novel ILK inhibitor, T315, and the effects were evaluated via Western blot and migration assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays were performed to assess cell viability. siRNA against ILK decreased phosphorylation of downstream effectors Akt and MLC, as well as decreased migration. Treatment with T315 showed a dose-related decrease in both Akt and MLC phosphorylation, as well as decreased migration. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays showed T315 to have an half maximal inhibitory concentration of less than 1μM in cell lines with high ILK expression. ILK is expressed differentially in thyroid cancer cell lines. Both ILK siRNA and T315 inhibit motility of thyroid cancer cell lines, and T315 is shown to be cytotoxic at low concentrations. Altogether, our study suggests that ILK may represent an important kinase in aggressive thyroid cancers.

Application of electroretinography (ERG) in early drug development for assessing retinal toxicity in rats

Retinal ocular toxicity is among the leading causes of drug development attrition in the pharmaceutical industry. Electroretinography (ERG) is a non-invasive functional assay used to assess neuro-retinal physiological integrity by measuring the electrical responses. To directly assess the utility of ERG, a series of studies was conducted following intravitreal and/or iv administration of pan-cyclin-dependent kinase inhibitors: AG-012,986 and AG-024,322 in rats. Both compounds have previously shown to induce retinal toxicity. Retinal injury was evaluated by ERG, histopathology and TUNEL staining. Intravitreal injection of AG-012,986 at ≥10μg/eye resulted in decreases (60%) in ERG b-wave and microscopic changes of mild to moderate retinal degeneration, and at 30μg/eye led to additional ophthalmic findings. Intravenous administration of AG-012,986 daily at ≥5mg/kg resulted in dose-related decreases (25 to 40%) in b-wave and sporadic to intense positive TUNEL staining. Intravitreal injection of AG-024,322 at 30μg/eye also resulted in decreases (50 to 60%) in b-wave, mild to marked retinal degeneration and mild vitreous debris. These experiments demonstrate that ERG can be used as a sensitive and reliable functional tool to evaluate retinal toxicity induced by test compounds in rats complementing other classical ocular safety measurements.

Etrolizumab Treatment Does Not Modify Levels of VCAM-1 in Ulcerative Colitis Patients

Introduction: Etrolizumab, an anti-β7 mAb, targets the integrins α4β7 and αEβ7 and inhibits the interaction of α4β7 with MAdCAM-1 and VCAM-1, and αEβ7 with E-cadherin. IBD patients have higher expression levels of MAdCAM-1 and ICAM-1 in the gut tissue compared to controls. Expression levels of VCAM-1 (Jones et al., 1995; Panes et al., 2003), an adhesion molecule involved in cell trafficking across the blood-brain barrier, are reported to be similar between IBD patients and controls. We demonstrated that etrolizumab treatment in patients with moderate-to-severe ulcerative colitis (UC) modulates MAd-CAM-1 and E-cadherin in serum and colonic tissue (Vermeire et al., 2012; Fuh et al., 2015; unpublished data) whereas natalizumab has been shown to decrease serum VCAM-1 in multiple sclerosis patients (Millonig et al., 2010). We explored the role of etrolizumab treatment on VCAM-1 and ICAM-1 serum levels and expression in colonic tissue among patients in the Phase 2 EUCALYPTUS study. Methods: Phase 2 UC patients were randomized into 1 of 3 different treatment arms during an induction period of 10 weeks: placebo (n=36), etrolizumab 100mg (n=28), and etrolizumab 300mg (n=34). Soluble VCAM-1 and ICAM-1 were measured at baseline and during the treatment period using commercially available ELISA assays and tissue expression was assessed in biopsy tissue by qPCR. Results: No differences were observed in serum soluble VCAM-1 levels in the etrolizumab- or placebotreated arms, nor was an association found with clinical remission or response. Similar results were observed in VCAM-1 expression in colonic tissue on day 43 and day 71. There was no dose-related decrease of ICAM-1 expression in serum or colonic tissue in the treated arms. However, levels of expression in colonic tissue of clinical responders, in both etrolizumab and placebo treated arms, had a median reduction of ≥50% at day 43 that was maintained through day 71. Conclusion: Lack of changes in VCAM-1 levels in both colonic tissue and in serum is consistent with the mechanism of action of etrolizumab, which does not block interaction of α4β1 with VCAM-1. The observed downmodulation of ICAM-1 expression is likely due to a decrease in tissue inflammation and not direct targeting of the pathway. These and prior data indicate that etrolizumab modulates expression of gut-specific MAdCAM-1 and suggests that it does not affect lymphocyte trafficking to the CNS via VCAM-1 modulation. Funded by Genentech.

NBCe1 expression is required for normal renal ammonia metabolism.

The mechanisms regulating proximal tubule ammonia metabolism are incompletely understood. The present study addressed the role of the proximal tubule basolateral electrogenic Na(+)-coupled bicarbonate cotransporter (NBCe1; Slc4a4) in renal ammonia metabolism. We used mice with heterozygous and homozygous NBCe1 gene deletion and compared these mice with their wild-type littermates. Because homozygous NBCe1 gene deletion causes 100% mortality before day 25, we studied mice at day 8 (±1 day). Both heterozygous and homozygous gene deletion caused a gene dose-related decrease in serum bicarbonate. The ability to lower urinary pH was intact, and even accentuated, with NBCe1 deletion. However, in contrast to the well-known effect of metabolic acidosis to increase urinary ammonia excretion, NBCe1 deletion caused a gene dose-related decrease in ammonia excretion. There was no identifiable change in proximal tubule structure by light microscopy. Examination of proteins involved in renal ammonia metabolism showed decreased expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase, key enzymes in proximal tubule ammonia generation, and increased expression of glutamine synthetase, which recycles intrarenal ammonia and regenerates glutamine. Expression of key proteins involved in ammonia transport outside of the proximal tubule (rhesus B glycoprotein and rhesus C glycoprotein) was not significantly changed by NBCe1 deletion. We conclude from these findings that NBCe1 expression is necessary for normal proximal tubule ammonia metabolism.

Impact of aflatoxin B1 on hypothalamic neuropeptides regulating feeding behavior

The presence of mycotoxins in food is a major problem of public health as they produce immunosuppressive, hepatotoxic and neurotoxic effects. Mycotoxins also induce mutagenic and carcinogenic effects after long exposure. Among mycotoxins that contaminate food are aflatoxins (AF) such as AFB1, which is the most powerful natural carcinogen. The AF poisoning results in symptoms of depression, anorexia, diarrhea, jaundice or anemia that can lead to death, but very few studies have explored the impact of AF on neuroendocrine regulations. To better understand the neurotoxic effects of AF related to anorexia, we explored in rat the impact of AFB1 on the major hypothalamic neuropeptides regulating feeding behavior, either orexigenic (NPY, Orexin, AgRP, MCH) or anorexigenic (α-MSH, CART, TRH). We also studied the effect of AFB1 on a novel neuropeptide, the secretogranin II (SgII)-derived peptide EM66, which has recently been linked to the control of food intake. For this, adult male rats were orally treated twice a week for 5 weeks with a low dose (150μg/kg) or a high dose (300μg/kg) of AFB1 dissolved in corn oil. Repeated exposure to AFB1 resulted in reduced body weight gain, which was highly significant for the high dose of AF. Immunocytochemical and quantitative PCR experiments revealed a dose-related decrease in the expression of all the hypothalamic neuropeptides studied in response to AFB1. Such orexigenic and anorexigenic alterations may underlie appetite disorders as they are correlated to a dose-dependent decrease in body weight gain of treated rats as compared to controls. We also found a decrease in the number of EM66-containing neurons in the arcuate nucleus of AFB1-treated animals, which was associated with a lower expression of its precursor SgII. These findings show for the first time that repeated consumption of AFB1 disrupts the hypothalamic regulation of neuropeptides involved in feeding behavior, which may contribute to the lower body weight gain associated to AF exposure.

Analysis of cardiovascular responses to the H2S donors Na2S and NaHS in the rat.

Hydrogen sulfide (H2S) is an endogenous gaseous molecule formed from L-cysteine in vascular tissue. In the present study, cardiovascular responses to the H2S donors Na2S and NaHS were investigated in the anesthetized rat. The intravenous injections of Na2S and NaHS 0.03-0.5 mg/kg produced dose-related decreases in systemic arterial pressure and heart rate, and at higher doses decreases in cardiac output, pulmonary arterial pressure, and systemic vascular resistance. H2S infusion studies show that decreases in systemic arterial pressure, heart rate, cardiac output, and systemic vascular resistance are well-maintained, and responses to Na2S are reversible. Decreases in heart rate were not blocked by atropine, suggesting that the bradycardia was independent of parasympathetic activation and was mediated by an effect on the sinus node. The decreases in systemic arterial pressure were not attenuated by hexamethonium, glybenclamide, N(w)-nitro-L-arginine methyl ester hydrochloride, sodium meclofenamate, ODQ, miconazole, 5-hydroxydecanoate, or tetraethylammonium, suggesting that ATP-sensitive potassium channels, nitric oxide, arachidonic acid metabolites, cyclic GMP, p450 epoxygenase metabolites, or large conductance calcium-activated potassium channels are not involved in mediating hypotensive responses to the H2S donors in the rat and that responses are not centrally mediated. The present data indicate that decreases in systemic arterial pressure in response to the H2S donors can be mediated by decreases in vascular resistance and cardiac output and that the donors have an effect on the sinus node independent of the parasympathetic system. The present data indicate that the mechanism of the peripherally mediated hypotensive response to the H2S donors is uncertain in the intact rat.

A CB2-Selective Cannabinoid Suppresses T-Cell Activities and Increases Tregs and IL-10.

We have previously shown that agonists selective for the cannabinoid receptor 2 (CB2), including O-1966, inhibit the Mixed Lymphocyte Reaction (MLR), an in vitro correlate of organ graft rejection, predominantly through effects on T-cells. Current studies explored the mechanism of this immunosuppression by O-1966 using mouse spleen cells. Treatment with O-1966 dose-relatedly decreased levels of the active nuclear forms of the transcription factors NF-κB and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures generated using a PCR T-cell activation array showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. Further, an increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures. Pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-related decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. These data support the potential of CB2-selective agonists as useful therapeutic agents to prolong graft survival in transplant patients, and strengthens their potential as a new class of immunosuppressive agents with broader applicability.

Hypoglycemic Effects of a Standardized Extract of Salvia miltiorrhiza Roots in Rats.

Background and Aims:Salvia miltiorrhiza Bunge (Labiatae) is a Chinese medicinal plant, the dried roots of which (known as Dan-Shen) have been used for hundreds of years in the treatment of a series of ailments, including hyperglycemia. This study was designed to evaluate the hypoglycemic effect of a new, standardized extract of S. miltiorrhiza.Materials and Methods:S. miltiorrhiza extract (containing 21% total tanshinones and 3.7% tanshinone IIA) was administered acutely and intragastrically at the doses of 0, 50, 100, and 200 mg/kg to male, healthy, fasted Wistar rats 60 min before the intragastric infusion of a bolus of starch (3 g/kg; a semi-naturalistic experimental condition) (Experiment 1) or glucose (2 g/kg) (Experiment 2).Results:In both experiments, treatment with S. miltiorrhiza extract produced a dose-related decrease in glycemia, evidenced in terms of reduction of peak value and/or area under the curve of the time-course of glycemia. The effect of S. miltiorrhiza extract occurred at doses devoid of any behavioral toxicity in rats.Conclusion:The results of this study suggest that the hypoglycemic effect of S. miltiorrhiza extract was likely secondary to an action on carbohydrate metabolism. These results are consistent with several preclinical and clinical data and add further support to the hypothesis that S. miltiorrhiza extracts may act as effective anti-hyperglycemic remedies.SUMMARY Acute treatment with Salvia miltiorrhiza extract reduced the glycemia rise induced by a bolus of starch in ratsAcute treatment with Salvia miltiorrhiza extract reduced the glycemia rise induced by a bolus of glucose in ratsThis effect relates to both glycemia peak value and area under the curve of the time-course of glycemia. Abbreviations used: Intragastric: i.g., Least significant difference: LSD test.

Phase 1 Study Results of a Novel Controlled-Release Formulation of Anagrelide (GALE-401) for the Treatment of Thrombocytosis

Introduction: Despite recent advances in the diagnosis and management of myeloproliferative neoplasms (MPNs), treatment of essential thrombocythemia (ET) has remained largely unchanged since the introduction of anagrelide in the US during 1997. Anagrelide is indicated for the treatment of thrombocythemia, to reduce the elevated platelet count and risk of thrombosis, and to ameliorate symptoms including thrombohemorrhagic events. The primary pharmacological effect of anagrelide is inhibition of megakaryocyte hypermaturation leading to reduced platelet production. Anagrelide also inhibits cyclic AMP phosphodiesterase III (PDE3), and common drug related adverse events (AEs; e.g., headache, palpitations, fluid retention, nausea and diarrhea) are believed to be due to this mechanism. Although initiating treatment at low doses and slowly increasing the dose to reach a target decreased platelet count may mitigate AEs, over 20% of patients still withdraw from treatment due to poor tolerability. As the currently marketed anagrelide product is an immediate release (IR) formulation with peak plasma concentrations (Cmax) that may exceed that needed for platelet reduction and cause unwanted PDE3 inhibition and AEs, an alternate formulation that modifies this pharmacokinetic (PK) profile may improve patient tolerability, adherence and treatment outcomes. This has led to the development and study of a controlled-release (CR) formulation of anagrelide (GALE-401).Methods: 98 healthy adult subjects were enrolled among 5 Phase 1 clinical trials of anagrelide CR, including 12 placebo-control subjects and 86 subjects who received single or multiple doses ranging from 0.2 to 0.6 mg twice daily (b.i.d.) for up to 41 days. The trials included an open-label, single dose developmental study; two placebo-controlled multiple dose ranging studies; a food effect study; and a comparative crossover PK study vs. IR reference product. Safety parameters included routine laboratory, ECG, and clinical evaluations. PK was assessed by measurements of plasma anagrelide and its active metabolite using a validated HPLC-MS/MS method. Pharmacodynamic activity was assessed by daily platelet count determinations in the multiple dosing studies.Results: Single doses of anagrelide CR were well tolerated, and the only drug-related AE reported in 2 or more subjects was headache. In the b.i.d. dose-ranging studies, the frequency and severity of AEs were similar between anagrelide CR and placebo groups, with the exception of decreased platelet counts in subjects receiving anagrelide CR. All AEs were transient, mild or moderate in severity, and no severe or serious AEs were reported.Anagrelide CR demonstrated dose proportional PK characteristics. Following a single 0.5 mg dose in the fasted state, the mean time to maximum plasma concentration (Tmax) and terminal elimination half-life (t1/2) were 2.0±1.5 hrs and 10.4±9.3 hrs (mean ± SD), respectively; in contrast, Tmax and t1/2 following IR was 1.0±0.9 hrs and 1.4±0.2 hrs, respectively. Cmax and total plasma exposure (AUC0-inf) with anagrelide CR were reduced to 26% and 59% of IR, respectively. However, steady-state PK following 6 daily 0.5 mg b.i.d. doses of anagrelide CR or IR showed similar AUC0-inf values, while Cmax with anagrelide CR remained nearly unchanged (29%). Plasma exposure was higher when anagrelide CR was administered in the fed state, as demonstrated by the ratio of least-squares mean values for Cmax and AUC0-t, which were increased by 100% and 60%, respectively.The platelet lowering effect of anagrelide CR was evident in the 2 multiple dose ranging studies. In a placebo-controlled study of 0.2 to 0.6 mg b.i.d. dosing for up to 21 days, a dose-related decrease in platelet counts was observed, and the 0.6 mg cohort was halted early due to excessive platelet reductions. Anagrelide CR did not have a relevant impact on platelet function as assessed by template bleeding time. [Display omitted] Conclusion: Anagrelide CR is a promising, novel formulation of anagrelide that exhibited the desired PK profile of a significantly reduced Cmax, while maintaining plasma exposure to induce platelet count reductions. The product was well tolerated with an AE profile that was not distinguishable from placebo. These data support the importance of an ongoing Phase 2 study in patients with MPN-related thrombocytosis, including ET. DisclosuresLaliberte:Galena Biopharma, Inc.: Employment, Equity Ownership. Glidden:Galena Biopharma, Inc.: Consultancy, Equity Ownership, Patents & Royalties. Hamilton:Galena Biopharma, Inc.: Employment, Equity Ownership.

Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay

The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds.

Abstract 3128: Efficacy of diethyldihydroxyhomospermine against human pancreatic adenocarcinoma using orthotopic implantation of human pancreatic L3.6pl cells into the pancreas of nude mice

Abstract Purpose: To determine the anti-neoplastic effects of (S,S)N1,N14-diethyl-3,12-dihydroxyhomospermine ([HO]2DEHSPM, SUN-101) after subcutaneous (s.c.) administration after orthotopic transplantation of human pancreatic cancer cells (L3.6pl) in the pancreas of nude mice. Methods: L3.6pl cells were injected into the pancreas of nude mice and seven to ten days later the treatment groups were: Study 1: mice (n=10/group) with saline (control), SUN-101 (25 mg/kg), SUN-101 (50 mg/kg), and SUN-101 (100 mg/kg) QD for 4 to 6 weeks. Study 2: mice (n=10/group) with saline (control), SUN-101 (25 mg/kg, QD), SUN-101 (25 mg/kg three times a week, QOD), SUN-101 (15 mg/kg, QOD), and SUN-101 (5 mg/kg, QOD) for 4 to 6 weeks Study 3: mice (n=10/group) with saline (control), Gemzar (100 mg/kg, intraperitoneal (i.p.), twice a week), SUN-101 (25 mg/kg, QOD), and Gemzar plus SUN-101 for 4 to 6 weeks. Results: In study 1, the optimal dose of SUN-101 was determined to be 25 mg/kg QD. This dose reduced up to 82.9% the weight of human pancreatic tumors in mice. Treatment with 50 and 100 mg/kg doses resulted in decreased body weight and proved to be toxic in mice. Histologic changes in the liver included hepatocyte reparative change and in the exocrine pancreas included a mild decrease of cytoplasmic granules in the epithelium of the pancreatic acini. In study 2, the optimal dose of 25 mg/kg administrated QOD was less toxic than daily 25 mg/kg administrations. The pancreatic weight and volume were decreased (47.8% and 66.6%, respectively) with 25 mg/kg administrated QOD and dose related decreases were observed at the 15 mg/kg QOD (20.1% and 52.6%, respectively). No decrease in tumor weight or volume was noted with 5 mg/kg administrated QOD. In study 3, the treatment of Gemzar, SUN-101, and Gemzar plus SUN-101 resulted in 18.7%, 35.6%, and 42.4% decreases in the body weight, respectively. Compared with tumor-bearing control, the treatment of Gemzar, SUN-101, and Gemzar plus SUN-101 resulted in 24.7%, 58.8%, and 67.2% decreases in the pancreas weight, respectively, and 37.8%, 58.4%, and 72.9% decreases in the tumor volumes, respectively. The incidence of liver metastasis was also decreased with SUN-101, Gemzar, and combination of Gemzar plus SUN-101. Conclusions: SUN-101 administered QD or QOD 25 mg/kg inhibited the growth of human pancreatic carcinoma in mice. SUN-101 demonstrated higher toxicity including disruption of the digestive process at daily 50 and 100 mg/kg doses. Co-administration of SUN-101 with gemcitabine appeared to have an additive or synergistic effect on reduction in the pancreatic tumor. Citation Format: Ajit K. Shah, Michael T. Cullen, Cheryl H. Baker. Efficacy of diethyldihydroxyhomospermine against human pancreatic adenocarcinoma using orthotopic implantation of human pancreatic L3.6pl cells into the pancreas of nude mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2014-3128

Comparative Study of Cigarette Smoke Cytotoxicity Using Two In Vitro Assay Systems

SUMMARY The aim of this study was to compare the results obtained from two in vitro cytotoxicity assays that depend upon different mechanisms/modes of action. The Neutral Red Uptake (NRU) assay is based on endocytotic activity whereas the Water Soluble Tetrazolium Salts (WST-1) assay is based on mitochondrial dehydrogenase activity. Both were investigated in light of their wide use and documented validation. The total particulate matter (TPM) and gas vapor phase (GVP) of main stream smoke derived from Kentucky reference cigarettes 3R4F and 10 test cigarettes made of 100% flue-cured or 100% Burley tobacco were individually applied to the two assays using CHO-K1 cells. In addition, cigarette smoke constituents and known cytotoxic agents, documented to affect specific endpoints, were evaluated within both assays. Although the NRU assay was primarily more sensitive than the WST-1 assay, both assays provided comparable results in terms of the rank order for the cytotoxicity of cigarette smoke samples. In addressing the cytotoxicity of constituents in cigarette smoke, acrolein, hydroquinone and catechol gave clear dose-related decreases in cell viability (an end point common in both assays). Moreover, enzyme inhibitors of the mitochondrial respiratory chain and chemicals causing membrane disruption also showed similar responses regardless of the specific endpoint addressed within the cytotoxicity assay. In conclusion, results from the NRU and WST-1 assay are comparable therefore indicating results were independent of the different assay detection mechanisms/modes of action. [Beitr. Tabakforsch. Int. 26 (2014) 98-108]

Reproductive responses of the earthworm (Eisenia fetida) to antiparasitic albendazole exposure

Albendazole (ABZ) is a veterinary drug with a high efficiency against helminths. Here reproductive responses of earthworms Eisenia fetida to ABZ exposure (0, 1, 3, 6, 9 and 12mgkg−1 soil dry weight) were investigated for 56d in chronic reproduction test, and deformed sperm were counted and morphological alterations in the seminal vesicles were qualitatively assessed by light and transmission electron microscopy. Results have showed that cocoon number of earthworms was more sensitive to low concentrations of ABZ than cocoon hatching success and hatching survival, showing a significant dose-related decrease in cocoon number at 3, 6, 9 and 12mgkg−1. In short-time exposure of 14d, the sperm deformity (%) of earthworms increased at 6, 9 and 12mgkg−1, and the microstructural alteration in seminal vesicles was also observed at these concentrations, whereas ultrastructural alteration of germ cells, particularly morphology of mitochondria, was observed at 3mgkg−1 and above, suggesting the high sensitivity of germ cell ultrastructure to low concentrations of ABZ in short-time exposure. The results can provide important information for prediction of ecologically significant toxic effects.

Blood pressure-lowering efficacy of monotherapy with thiazide diuretics for primary hypertension.

Blood pressure-lowering efficacy of monotherapy with thiazide diuretics for primary hypertension.