In vivo evaluation of combination therapy targeting the isoprenoid biosynthetic pathway.

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.

Characterization of chitosan/alginate/lovastatin nanoparticles and investigation of their toxic effects in vitro and in vivo.

In this study, chitosan and alginate were selected to prepare alginate/chitosan nanoparticles to load the drug lovastatin by the ionic gelation method. The synthesized nanoparticles loaded with drug were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), laser scattering and differential scanning calorimetry (DSC) methods. The FTIR spectrum of the alginate/chitosan/lovastatin nanoparticles showed that chitosan and alginate interacted with lovastatin through hydrogen bonding and dipolar-dipolar interactions between the C-O, C=O, and OH groups in lovastatin, the C-O, NH, and OH groups in chitosan and the C-O, C=O, and OH groups in alginate. The laser scattering results and SEM images indicated that the alginate/chitosan/lovastatin nanoparticles have a spherical shape with a particle size in the range of 50-80 nm. The DSC diagrams displayed that the melting temperature of the alginate/chitosan/lovastatin nanoparticles was higher than that of chitosan and lower than that of alginate. This result means that the alginate and chitosan interact together, so that the nanoparticles have a larger crystal degree when compared with alginate and chitosan individually. Investigations of the in vitro lovastatin release from the alginate/chitosan/lovastatin nanoparticles under different conditions, including different alginate/chitosan ratios, different solution pH values and different lovastatin contents, were carried out by ultraviolet-visible spectroscopy. The rate of drug release from the nanoparticles is proportional to the increase in the solution pH and inversely proportional to the content of the loaded lovastatin. The drug release process is divided into two stages: a rapid stage over the first 10 hr, then the release becomes gradual and stable. The Korsmeyer-Peppas model is most suitable for the lovastatin release process from the alginate/chitosan/lovastatin nanoparticles in the first stage, and then the drug release complies with other models depending on solution pH in the slow release stage. In addition, the toxicity of alginate/chitosan/lovastatin (abbreviated ACL) nanoparticles was sufficiently low in mice in the acute toxicity test. The LD50 of the drug was higher than 5000 mg/kg, while in the subchronic toxicity test with treatments of 100 mg/kg and 300 mg/kg ACL nanoparticles, there were no abnormal signs, mortality, or toxicity in general to the function or structure of the crucial organs. The results show that the ACL nanoparticles are safe in mice and that these composite nanoparticles might be useful as a new drug carrier.

Biotechnological Production of Statins: Metabolic Aspects and Genetic Approaches.

Statins are drugs used for people with abnormal lipid levels (hyperlipidemia) and are among the best-selling medications in the United States. Thus, the aspects related to the production of these drugs are of extreme importance for the pharmaceutical industry. Herein, we provide a non-exhaustive review of fungal species used to produce statin and highlighted the major factors affecting the efficacy of this process. The current biotechnological approaches and the advances of a metabolic engineer to improve statins production are also emphasized. The biotechnological production of the main statins (lovastatin, pravastatin and simvastatin) uses different species of filamentous fungi, for example Aspergillus terreus. The statins production is influenced by different types of nutrients available in the medium such as the carbon and nitrogen sources, and several researches have focused their efforts to find the optimal cultivation conditions. Enzymes belonging to Lov class, play essential roles in statin production and have been targeted to genetic manipulations in order to improve the efficiency for Lovastatin and Simvastatin production. For instance, Escherichia coli strains expressing the LovD have been successfully used for lovastatin production. Other examples include the use of iRNA targeting LovF of A. terreus. Therefore, fungi are important allies in the fight against hyperlipidemias. Although many studies have been conducted, investigations on bioprocess optimization (using both native or genetic- modified strains) still necessary.

Analysis of a Skeletal Muscle Injury and Drug Interactions in Lovastatin- and Fenofibrate-Coadministered Dogs.

Because dogs are widely used in drug development as nonrodent experimental animals, using a dog model for drug-induced adverse reactions is considered to be relevant for an evaluation and investigation of a mechanism and a biomarker of clinical drug-induced adverse reactions. Skeletal muscle injury occurs by various drugs, including statins and fibrates, during drug development. However, there is almost no report of a dog model for drug-induced skeletal muscle injury. In the present study, we induced skeletal muscle injury in dogs by oral coadministration of lovastatin (LV) and fenofibrate (FF) for 4 weeks. Increases in plasma levels of creatine phosphokinase, myoglobin, miR-1, and miR-133a and degeneration/necrosis of myofibers in skeletal muscles but not in the heart were observed in LV- and FF-coadministered dogs. Plasma levels of lovastatin lactone and lovastatin acid were higher in LV- and FF-coadministered dogs than LV-administered dogs. Taken together, FF coadministration is considered to affect LV metabolism and result in skeletal muscle injury.

Establishment and characterization of a mouse model of rhabdomyolysis by coadministration of statin and fibrate.

Rhabdomyolysis is characterized by elevation of plasma creatine phosphokinase (CPK) level, and multiple organ disorders, especially renal failure, as well as approximately 50% of acquired rhabdomyolysis are caused by pharmaceuticals. Statins are known to cause rhabdomyolysis, and its incidence is ≥10 times higher with coadministration of statin and fibrate. The purpose of this study is to establish a mouse model of drug-induced rhabdomyolysis by coadministration of statin and fibrate to clarify the mechanisms of its myotoxicity. We administered lovastatin (LV) and gemfibrozil (GF) with a glutathione synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), to C57BL/6 J female mice once daily for 3 days. The plasma levels of CPK and aspartate aminotransferase (AST) were prominently increased, and the increase in plasma miR-206-3p and miR-133-3p levels, not the increase of miR-122-5p and miR-208-3p levels, suggested skeletal muscle-specific toxicity. The caspase 3/7 activity and mRNA levels of oxidative stress-related factors were elevated in skeletal muscle. Pharmacokinetic parameters showed that blood levels of statin were significantly increased by coadministered GF. The possibility of kidney injury was examined as in clinical rhabdomyolysis. In histological examination, vacuoles were observed in renal proximal tubules, and the plasma renal injury marker, lipocalin 2/neutrophil gelatinase-associated lipocalin (Lcn2/Ngal), was markedly increased in the mice coadministered LV and GF, suggesting mild complications of acute kidney injury. A quantitative comparison of the myotoxic potential of various statins was successfully performed using the present method. In this study, a rhabdomyolysis mouse model was established by coadministration of the clinically using statin and fibrate. This mouse model may be useful to identify drugs that have high risk for rhabdomyolysis.

Efficiency of hepatocyte pretreatment with coenzyme Q10 against statin toxicity.

Statins are potent cholesterol-lowering drugs that can have serious adverse effects on the muscles and liver. The aim of our in vitro study was to establish the protective effect of coenzyme Q10 (CoQ10, in its optimal dose of 200 µmol L⁻¹) against cytotoxicity induced by atorvastatin, simvastatin, and lovastatin in isolated rat hepatocytes by observing parameters such as cell death, reactive oxygen species formation, lipid peroxidation, mitochondrial membrane potential, and cellular reduced and oxidised glutathione content. Our findings have shown that pretreatment with CoQ10 was effective in reducing the toxic effects of statins in rat hepatocytes. This work demonstrates that the addition of CoQ10 to statin treatment regimens may protect hepatocytes (and also other types of cells) from statin-induced injuries and alleviate their side effects.

Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

Neurotoxic effect of statins on mouse neuroblastoma NB2a cell line.

OBJECTIVE: Evidences from cell culture experiments suggest a link between cholesterol and nervous system disease. Statins may have neurotoxic or neuroprotective effects, but these effects remain controversial. Therefore, the present study was aimed to investigate the possible toxicity of statins on a neurite outgrowth in mouse neuroblastoma NB2a cell line. MATERIALS AND METHODS: We have utilized d-cAMP-induced terminally differentiated NB2a cells in culture as an experimental model to study the effects of statins. The cell survival and proliferation were studied by MTT. Measurement of neurite outgrowth was done by neurotoxicity screening test. NB2a cell differentiation was achieved by serum free medium plus 0.5 mM dibutyryl cAMP. Cells were incubated for 24 hours at 37 degrees C. After this period, lovastatin, mevastatin and atorvastatin were added to wells at different concentrations (1, 3, 10, 100 microM). Approximately 100 cells were chosen for each sample and examined randomly 24 hours later, from 10 different fields. Total length of neurite was photographed microscopically and measured by image analyze software. Changes in neurite lengths were expressed as % inhibition compared to that of the control group. RESULTS: Results showed that three statins at high concentrations induced neurite inhibition, inhibited proliferation and reduced the viability of differentiated neuroblastoma NB2a cells. CONCLUSIONS: Our results suggest that statins could act as a neurotoxic agent at high doses depending upon their concentrations. These results require further investigation at ultra structural and molecular levels to understand long term side effects for clinical safety of statins.

Inhibition of neuronal cholesterol biosynthesis with lovastatin leads to impaired synaptic vesicle release even in the presence of lipoproteins or geranylgeraniol.

Cholesterol is highly enriched in the brain, and plays a key role in synapse formation and function. The brain does not derive cholesterol from the circulation; instead, the majority of cholesterol is made in glia and secreted in form of lipoproteins. Neurons can synthesize cholesterol, but the extent of neuronal cholesterol biosynthesis in the adult brain is unknown. Cholesterol biosynthesis inhibitors of the statin family are widely used to lower circulating cholesterol and cardiovascular risk. Lipophilic statins can cross the blood brain barrier and inhibit brain cholesterol biosynthesis with possible consequences for synaptic cholesterol homeostasis. We have investigated the effects of lovastatin on synapse maturation and synaptic vesicle release. Treatment of primary hippocampal neurons with low levels of lovastatin for one week reduced synapse density and impaired synaptic vesicle release. Neither lipoproteins nor geranylgeraniol fully counteracted the lovastatin-induced decrease of synaptic vesicle exocytosis, even when cholesterol depletion was prevented. In contrast, restoration of neuronal cholesterol synthesis with mevalonate prevented defects in vesicle exocytosis without fully normalizing neuronal cholesterol content. These results raise the possibility that chronic exposure of neurons to lipophilic statins may affect synaptic transmission, and indicate that hippocampal neurons need a certain level of endogenous cholesterol biosynthesis.

Statins inhibit iNOS-mediated microbicidal potential of activated monocyte-derived dendritic cells by an IFN-ß-dependent mechanism.

Statins are prescribed to 25 million people worldwide for treating hypercholesterolemia and reducing the risk of cardiovascular diseases. However, the side effects of statins on immunity, and particularly on DC immunobiology, have not been analyzed in-depth. Here, we have investigated the impact of lovastatin treatment during monocyte differentiation into DCs on the responsiveness of the resulting monocyte-derived DCs (moDCs) to TLR-mediated activation. Lovastatin positively regulated TLR4 signaling in LPS-stimulated moDCs, leading to strong activation of p38 MAP-kinase paralleled by increased proinflammatory cytokine and IFN-ß production. In contrast, lovastatin promoted negative regulation of IFN-ß-mediated autocrine signaling through the IFN-αß receptor, paralleled by low expression of the transcription factor IRF-1, leading to the inhibition of the enzymes iNOS and HO-1. Defective activation of iNOS/HO-1 resulted in limited cytoprotective capacity against ROS and reduced microbicidal potential. These data were validated using an in vivo model of Listeria monocytogenes infection, which revealed that iNOS activation by splenic inflammatory moDCs, specialized in NO and TNF-α production, was strongly reduced in lovastatin-treated, Listeria-infected mice. Statin treatment could have severe implications in immunity against pathogens due to defective iNOS/HO-1 metabolism activation in inflammatory moDCs that might lead to immune failure.

Juvenile toxicity assessment of open-acid lovastatin in rats.

BACKGROUND: Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, reduces de novo cholesterol biosynthesis primarily in the liver. Since cholesterol is a major component of brain myelin and peak periods of brain myelination occurs after birth, this study was designed to encompass this period in rats and evaluate the potential neurotoxic effects. METHODS: The pharmacologically active, open-acid form of lovastatin was administered to groups of 50 Sprague-Dawley rats per sex subcutaneously once daily at dose levels of 0 (vehicle), 2.5, 5, or 10 mg/kg/day beginning on postnatal day 4 and continuing until termination on postnatal day 41 to 51. Physical signs and body weights were monitored during the study. Animals were assessed in a battery of behavioral tests, and at termination a set of animals were examined for gross and histological changes. RESULTS: There were no test article-related deaths, physical signs, or effects on preweaning and postweaning body weights during the study. In the behavior tests there were no test article-related effects in the passive avoidance, auditory startle habituation, open-field motor activity, or FOB. No test article-related postmortem findings were observed, including brain weights and histomorphology of brain, spinal cord, eye, optic nerve, or peripheral nerve. CONCLUSION: Based on these results, the no-effect level for general and neurobehavioral toxicity in neonatal rats was ≥10 mg/kg/day for open-acid lovastatin.

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.

Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin.

Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell derived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development. Using the examples of warfarin and lovastatin, two substances with entirely different primary targets, the surrogate marker signature nevertheless indicates a common embryotoxic mode of action. We discuss these findings observed in in vitro toxicity tests, in a context of clinical validation and evidence-based toxicology.

Statins potentiate cytostatic/cytotoxic activity of sorafenib but not sunitinib against tumor cell lines in vitro.

The aim of this study was to investigate the potential cytostatic/cytotoxic effects of HMG-CoA reductase inhibitors and two orphan drugs registered for the treatment of advanced renal cell carcinoma, i.e. sorafenib and sunitinib against several different tumor cell lines. Cytostatic/cytotoxic effects were measured using crystal violet or MTT reduction assays. Cell cycle regulation was investigated using flow cytometry and Western blotting. The combination of lovastatin and sorafenib (but not sunitinib) produced synergistic cytostatic/cytotoxic effects against all tested tumor cell lines. In this study, an impairment of the protein prenylation, especially geranylgeranylation, resulted predominantly in the potentiation of the cytostatic/cytotoxic activity of sorafenib, in cell cycle arrest in G1 phase, and, in poor induction of apoptosis. Moreover, due to the fact that it has been well documented that sorafenib compromises the heart function, we studied the interaction of lovastatin and sorafenib using rat cardiomyoblast line H9c2. The combination showed strong synergistic cardiotoxic effects. Statins and tyrosine kinase inhibitors were used at doses that are achievable clinically, which makes the combination promising for future studies, especially in urooncology, bearing in mind possible cardiotoxic effects.

Expression, localization, and pharmacological role of Kv7 potassium channels in skeletal muscle proliferation, differentiation, and survival after myotoxic insults.

Changes in the expression of potassium channels regulate skeletal muscle development. The purpose of this study was to investigate the expression profile and pharmacological role of K(v)7 voltage-gated potassium channels in skeletal muscle differentiation, proliferation, and survival after myotoxic insults. Transcripts for all K(v)7 genes (K(v)7.1-K(v)7.5) were detected by polymerase chain reaction (PCR) and/or real-time PCR in murine C(2)C(12) myoblasts; K(v)7.1, K(v)7.3, and K(v)7.4 transcripts were up-regulated after myotube formation. Western blot experiments confirmed K(v)7.2, K(v)7.3, and K(v)7.4 subunit expression, and the up-regulation of K(v)7.3 and K(v)7.4 subunits during in vitro differentiation. In adult skeletal muscles from mice and humans, K(v)7.2 and K(v)7.3 immunoreactivity was mainly localized at the level of intracellular striations positioned between ankyrinG-positive triads, whereas that of K(v)7.4 subunits was largely restricted to the sarcolemmal membrane. In C(2)C(12) cells, retigabine (10 microM), a specific activator of neuronally expressed K(v)7.2 to K(v)7.5 subunits, reduced proliferation, accelerated myogenin expression, and inhibited the myotoxic effect of mevastatin (IC(50) approximately 7 microM); all these effects of retigabine were prevented by the K(v)7 channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 muM). These data collectively highlight neural K(v)7 channels as significant pharmacological targets to regulate skeletal muscle proliferation, differentiation, and myotoxic effects of drugs.

Geranylgeraniol prevents the cytotoxic effects of mevastatin in THP-1 cells, without decreasing the beneficial effects on cholesterol synthesis.

BACKGROUND AND PURPOSE: Statins, inhibitors of hydroxymethylglutaryl-CoA reductase, reduce the intracellular synthesis of cholesterol and prevent the onset of atherosclerosis. They also decrease the synthesis of isoprenoid molecules, such as the side chain of ubiquinone and geranylgeranyl pyrophosphate. As a consequence, statins impair mitochondrial metabolism and the activation of small monomeric GTPases (such as Rho and Ras), causing toxic effects. To date, a successful strategy to prevent statin toxicity is lacking. EXPERIMENTAL APPROACH: In human monocytic THP-1 cells, we measured the synthesis of cholesterol and isoprenoids, mitochondrial electron flow, the activity of RhoA and Rac, cell death and proliferation. KEY RESULTS: Mevastatin reduced the synthesis of cholesterol, geranylgeranyl pyrophosphate and ubiquinone, mitochondrial electron transport, activity of RhoA and Rac, and cell proliferation, accompanied by increased cell death. Geranylgeraniol, a cell-permeable analogue of geranylgeranyl pyrophosphate, reversed all these effects of mevastatin, without affecting its ability to reduce cholesterol synthesis. Notably, geranylgeraniol was more effective than the addition of exogenous ubiquinone, which rescued mitochondrial respiratory activity and reversed mevastatin cytotoxicity, but did not alter the decrease in cell proliferation. The same results were obtained in human liver HepG2 cells. CONCLUSIONS AND IMPLICATIONS: Geranylgeraniol had a broader protective effect against the cytotoxicity of statins than exogenous ubiquinone. Therefore, geranylgeraniol may be a more useful and practical means of limiting the toxicities of statins, without reducing their efficacy as cholesterol lowering agents.

Lovastatin enhances the genotoxicity of doxorubicin in Chinese hamster V79 cells via noncovalent DNA binding.

The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitor, lovastatin (lova), has been reported to both sensitize to, and protect against, the toxic effects of the antitumor anthracycline doxorubicin (dox) in cellular and in vivo systems. The mechanism by which these effects occur has not yet been determined. In the present study, lova is shown to enhance the genotoxicity of dox in the V79 cell in vitro micronucleus assay and to do so, most likely, via noncovalent interaction with DNA adjacent to sites of dox binding. These studies confirm and extend the experimental evidence strongly suggesting the importance of noncovalent drug/DNA interactions in cellular responses to genotoxic stimuli.

The Neuroprotection with Statin Therapy for Acute Recovery Trial (NeuSTART): an adaptive design phase I dose-escalation study of high-dose lovastatin in acute ischemic stroke.

There is growing experimental and clinical evidence that by reducing downstream products of the mevalonate pathway other than cholesterol, HMG-CoA reductase inhibitors ('statins') have beneficial effects on endothelial function, coronary and cerebral blood flow, inflammation, and hemostasis. Statins have been shown in rodent models of acute ischemic stroke to reduce neuronal injury and infarct size in a dose-dependent fashion. The objective of this early phase trial will be to determine the maximal-tolerated dose of lovastatin for short-term acute stroke therapy. In this multicenter phase 1B dose-escalation and dose-finding study, 33 patients with acute ischemic stroke will be administered lovastatin in increasing doses from one to 10 mg/kg daily for 3 days beginning within 24 hours after symptom onset. The primary safety outcome will be occurrence of myotoxicity or hepatotoxicity, defined by clinical and laboratory criteria, and the study is designed to determine the highest dose of lovastatin that can be administered with <10% risk of myotoxicity or hepatotoxicity. The statistical design of the study utilizes an adaptive design, the Continual Reassessment Method, which is novel to stroke trials, to find the optimal dosage. The dose-toxicity model is calibrated such that the method will eventually select a dose that causes 7-13% dose-limiting toxicity (within 3% of target). A sample size of 33 will ensure that estimates of any binary variables will have a 95% confidence interval of width

Zooplankton chitobiase activity as an endpoint of pharmaceutical effect.

Numerous human and veterinary pharmaceuticals are constantly entering surface waters, despite little understanding of their potential impacts on aquatic ecosystems. To address this concern, an attempt to create a simple, reproducible, inexpensive, and sublethal toxicity bioassay for freshwater zooplankton was initiated. The approach was centered on characterizing the response of a zooplankton enzyme, chitobiase, to the presence of a toxicant. The aim of the present research was to develop a reproducible laboratory-based assay for Daphnia magna chitobiase activity and to screen four commonly prescribed pharmaceuticals using that assay. The four pharmaceuticals tested for potential effects on D. magna chitobiase activity were atorvastatin, lovastatin, fluoxetine, and sertraline. We were able to detect exposure-associated differences in chitobiase activity at concentrations of 0.1 mug/L fluoxetine after 24 and 72 hours of exposure. Differences were also detected for the other compounds. The response of chitobiase was found to be promising as an assay to measure sublethal effects in D. magna and perhaps other zooplankton species.