Preclinical Safety Studies Research Articles

Role of Phytochemicals in the Prevention of Menopausal Bone Loss: Evidence from In Vitro and In Vivo, Human Interventional and Pharmacokinetic Studies

Substantial body of data generated from cultured bone cells and rat models of osteoporosis supports a significant bone-conserving effect of phytochemicals. Flavonoids including isoflavones, stilbenes and lignans with variable efficacy have shown promising therapeutic application in osteoporosis. Majority of the phytochemicals assessed for their effects on bone cells revealed multiple beneficial actions such as promoting osteoblast functions, and inhibiting osteoclast and adipocyte functions. A variety of molecular targets mediate multiple effects of phytochemicals in bone cells. In vivo, quite a few phytochemicals have been found to afford bone-sparing effect and in some cases even bone restoring effect. However, important pharmacokinetic and bioavailaibility studies associated with these phytochemicals are mostly lacking. As a result, translating these findings to the clinic has been challenging, and so far only a few clinical studies have attempted to evaluate the effect of phytochemicals in menopausal osteoporosis. Clinical studies so far performed are with dietary supplements rather than pure phytochemicals. Clinical trials with pure molecules necessitate preclinical regulatory and safety studies that are not available with the phytochemicals except ipriflavone with bone-conserving properties. Ipriflavone is the only marketed anti-osteoporosis agent that was obtained following a lead from natural substance. As phytochemicals have multiple beneficial influences on bone cells, making analogues of the most potent molecule for developing synthetic series with rational drug design approach could pay rich dividends in menopausal osteoporosis therapy.

Safety Assessment and Dose Selection for First-in-Human Clinical Trials With Immunomodulatory Monoclonal Antibodies

Modulating immune responses with monoclonal antibodies (mAbs) that target immune molecules has become a promising therapeutic strategy and is under investigation for the treatment of cancer and (auto)-immune diseases. A major hurdle to the development and early clinical investigation of many immunomodulatory mAbs is the inherent risk of adverse immune-mediated drug reactions in humans, such as cytokine storms, autoimmunity, and immunosuppression. Dose selection for first-in-human (FIH) clinical trials involving immunomodulatory mAbs, and mAbs in general, is based on specifically designed preclinical safety studies, primarily in nonhuman primates (NHPs), and on mechanistic ex vivo investigations. Dose selection in such trials is challenging for a number of reasons related to safety. In this context, safety-relevant differences between NHP and human immune systems, species selection/qualification and preclinical study design considerations, the receptor occupancy model and its calculation, the minimal anticipated biological effect level (MABEL) and its use in the selection of a safe starting dose in humans, microdosing and the impact of immunogenicity on safety assessment of mAbs, and safety-relevant formulation properties of therapeutic mAbs are critically reviewed. In addition, the current regulatory requirements are presented and discussed to demonstrate how the TeGenero TGN1412 case is leading to increased regulatory scrutiny regarding dose selection for FIH clinical trials.

Translational strategies for development of monoclonal antibodies from discovery to the clinic

Successful strategies for the development of monoclonal antibodies require integration of knowledge with respect to target antigen properties, antibody design criteria such as affinity, isotype selection, Fc domain engineering, PK/PD properties and antibody cross-reactivity across species from the early stages of antibody development. Biophysical measurements are one of the critical components necessary for the design of effective translational strategies for lead selection and evaluation of relevant animal species for preclinical safety and efficacy studies. Incorporation of effective translational strategies from the early stages of the antibody development process is a necessity; when considered it not only reduces development time and cost, but also fosters implementation of rational decision-making throughout all phases of antibody development.

A novel biodegradable pancreatic stent for human pancreatic applications: a preclinical safety study in a large animal model

A novel biodegradable pancreatic stent for human pancreatic applications: a preclinical safety study in a large animal model

Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro

Human adipose stem cells (ASC) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue. ASC have been shown to have therapeutic applicability in pre-clinical studies, but a standardized expansion method for clinical cell therapy has yet to be established. Isolated ASC are typically expanded in medium containing fetal bovine serum (FBS); however, sera and other culturing reagents of animal origin in clinical therapy pose numerous safety issues, including possible infections and severe immune reactions. To identify optimal conditions for ex vivo expansion of ASC, the effects of seven serum-free (SF) and xeno-free (XF) media were investigated with both FBS and allogeneic human serum (alloHS; as a control media). Surface marker expression, proliferation, morphology and differentiation analyzes were utilized for investigating the effects of media on ASC. The proliferation and morphology analysis demonstrated significant differences between ASC cultured in SF/XF culture media compared with serum-containing culture media, with medium prototype StemPro MSC SFM XenoFree providing significantly higher proliferation rates than ASC cultured in media containing serum, while still maintaining the differentiation potential and surface marker expression profile characteristic of ASC. Looking forward, fully defined XF media formulations will provide the means for the development and approval of safer clinical cell therapy treatments. However, to fully recognize the capacity of these XF culture media, further pre-clinical safety and efficacy studies must be performed.

The basics of preclinical drug development for neurodegenerative disease indications

Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up; select the best formulation; determine the route, frequency, and duration of exposure; and ultimately support the intended clinical trial design. The details of each preclinical development package can vary, but all have some common features. Rodent and nonrodent mammalian models are used to delineate the pharmacokinetic profile and general safety, as well as to identify toxicity patterns. One or more species may be used to determine the drug's mean residence time in the body, which depends on inherent absorption, distribution, metabolism, and excretion properties. For drugs intended to treat Alzheimer's disease or other brain-targeted diseases, the ability of a drug to cross the blood brain barrier may be a key issue. Toxicology and safety studies identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Pivotal preclinical safety studies generally require regulatory oversight as defined by US Food and Drug Administration (FDA) Good Laboratory Practices and international guidelines, including the International Conference on Harmonisation. Concurrent preclinical development activities include developing the Clinical Plan and preparing the new drug product, including the associated documentation to meet stringent FDA Good Manufacturing Practices regulatory guidelines. A wide range of commercial and government contract options are available for investigators seeking to advance their candidate(s). Government programs such as the Small Business Innovative Research and Small Business Technology Transfer grants and the National Institutes of Health Rapid Access to Interventional Development Pilot Program provide funding and services to assist applicants in preparing the preclinical programs and documentation for their drugs. Increasingly, private foundations are also funding preclinical work. Close interaction with the FDA, including a meeting to prepare for submission of an Investigational New Drug application, is critical to ensure that the preclinical development package properly supports the planned phase I clinical trial.

Pulmozyme® Treatment of Vectors Produced by Transient Transfection Reduces Residual Plasmid DNA on Human CD34+ Hematopoietic Progenitor Cells without Loss of Transduction or Engraftment Efficiency

A significant percentage of clinical grade safety-enhanced gamma-retrovirus and lentivirus vector supernatants are currently produced by transient transfection. These products contain high concentrations of plasmid DNA that pose the risk of transfer into subjects, and preclude accurate estimation of transduction efficiency. Most envelope pseudotyped vectors are too labile for DNAse treatment or other purification steps and adding non-clinical grade reagents into any production step further complicates product qualification. Here we present development of a method to remove residual plasmid as a part of the transduction procedure of human CD34+ cells or mouse bone marrow progenitors, using the DNAse Pulmozyme®. Using a standard clinical transduction protocol, CD34+ were transduced with SRS11.EFS.IL2RG.pre* or SRS11.EFS.EGFP. pre* gamma-retroviral vectors pseudotyped with GALV envelope or a mock control. Retronectin coated flasks preloaded with vector were either treated with Pulmozyme® or no Pulmozyme®. Cells were transduced on two consecutive days and collected 4 –24 hrs after the 2nd transduction (day1). CFU were plated and enumerated and proportion of transduced progenitors quantified by real time PCR (qPCR) using wPRE primers. For the EGFP containing vector, gene transfer was determined by flow cytometry in cultures that were continued for up to 14 days. DNA was extracted and tested by qPCR on days 1, 7, and 14. Residual plasmid DNA was quantified using GALV primers. Transduction was also estimated on the bulk cultures using qPCR. In all qPCR reactions human ApoB was quantified concurrently to asses the cell number. Pulmozyme® treatment reduced GALV copy numbers in cells one day following transduction by up to 20-fold. After 7 days in culture, there was no residual plasmid detectable in all groups. More importantly, Pulmozyme® treatment did not alter the transduction efficiency of the progenitors in any of the experiments, as evaluated by either wPRE qPCR on Day-7 DNA (4.3 vs. 4.2, 6.8 vs. 7.6 and 2.0 vs. 1.9 vector copies per cell for Pulmozyme® treated and non-treated respectively), colony PCR (69.4 vs. 66.7% vector positive CFUs) or flow cytometry (81.8 vs. 85.7% EGFP positive cells). We also analyzed the engraftment potential of cells transduced using Pulmozyme®-treated versus non-treated vector after transplanting cells in NOD/SCID and NOD/SCID-IL2Rg null (NOG) mice. Analysis of mice at 6 weeks post transplantation showed no significant reduction in human cell engraftment in the Pulmozyme® and the average copy number per human cell in NOG bone marrow was 0.65 vs. 0.52 and in NOD/SCID bone marrow 1.03 vs. 0.67 (Pulmozyme® treated versus non-treated, respectively). We also tested Pulmozyme® treatment in mouse experiments using gamma-retroviral vector pseudotyped with ecotropoc envelope with a similar result. We conclude that treatment of gamma-retroviral vector supernatant with Pulmozyme® after vector preload on Retronectin® coated plates reduces vector and packaging plasmids and does not inhibit transduction, clonogenic potential, or engraftment. The removal of excess plasmid from transiently produced virus supernatant without affecting the virus transducibility allows accurate estimation of transduction efficiencies prior to transplant and will conceivably reduce the toxicity to primary cells from excess plasmid. These studies can be directly translated to preclinical animal safety studies and clinical trials.

Inhibition of Nuclear Translocation of Apoptosis-Inducing Factor Is an Essential Mechanism of the Neuroprotective Activity of Pigment Epithelium-Derived Factor in a Rat Model of Retinal Degeneration

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Histologic Processing of the Eye for Obtaining the Macular Region of the Retina of the Cynomolgus Monkey

Microscopic evaluation of the macular region of the retina is essential in preclinical drug safety studies in cynomolgus monkeys. A number of therapeutic agents are suspected of causing damage to the macula in humans; therefore, the macular region of the retina should be evaluated in preclinical drug safety studies in monkeys. Histopathologic examination of the fovea may be required for preclinical drug safety evaluation of certain ocular drugs for human risk assessment. Using the inferior oblique muscle as a landmark, the macula including the fovea was obtained in 100% of 20 eye sections. It is easier to obtain just the macular region of the retina than the fovea in the section. Measurements for the inferior oblique muscle (point of insertion), fovea, macula, and the optic disc for 20 eyes of untreated cynomolgus monkeys were taken. In the cynomolgus monkey, the macula was 2.01 mm in diameter and the fovea was 0.53 mm in diameter. The distance from the center of the optic disc to the center of the fovea was 3.23 mm. In the fixed eyes, the point of insertion of the inferior oblique muscle to the sclera was 5.96 mm. (The J Histotechnol 31:113, 2008)Submitted January 7, 2008; accepted with revisions June 22, 2008

Translation of New Technologies: From Basic Research to Drug Discovery and Development

Despite increasing investment in drug discovery and development, only around one in every ten new medicinal products that progresses to clinical testing ever reach from the registration stage. Approximately half of all drug failures are attributed to problems with efficacy and toxicity not anticipated from preclinical studies. As a consequence, the pharmaceutical industry is adopting a much more flexible and multi-disciplinary approach to drug discovery and development. Indeed, the line between basic and applied science is constantly being eroded, not least because of the increasing sophistication of therapeutic procedures and the complexity of the diseases that they aim to treat. Here, we look at the new technologies that are being explored as a way of reducing drug attrition rates and the development of chemical drugs and biotherapeutics. Specifically, we will consider the ways in which genomics and related disciplines, engineered cell-based and microfluidics systems, and nanotechnologies are being developed and used alongside in silico platforms during early drug pharmacokinetics and toxicity studies. The way in which information from such systems biology-oriented approaches can be integrated with information from animal based preclinical safety, toxicological and pharmacological studies on investigative medicinal products is considered, in view of its current and possible impact on clinical trial design.

End‐stage Liver Disease and Liver Transplant: Current Situation and Key Issues

The aim of this article is to identify the current challenges in pediatric OLT. It is directed to all physicians who care for pediatric patients from the early prospect of liver transplant to posttransplant life. Besides assessing current information on transplant results, the coauthors of this article have identified a number of issues that they consider as the most important that need to be addressed in 2008. Some are only an update of old questions, and others are newly identified problems. The recommendations from the group are summarized at the end of each specific issue and in the consensus guidelines.

Ligand-Based Targeting of Apoptosis in Cancer: The Potential of Recombinant Human Apoptosis Ligand 2/Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (rhApo2L/TRAIL)

Cancer is a leading cause of premature human death and commands considerable research attention. Apoptosis (type 1 programmed cell death) is critical in maintaining tissue homeostasis in metazoan organisms, and its dysregulation underpins the initiation and progression of cancer. Conventional chemotherapy and radiotherapy can induce apoptosis as a secondary consequence of inflicting cell damage. However, more direct and selective strategies to manipulate the apoptotic process in cancer cells are emerging as potential therapeutic tools. Genetic and biochemical understanding of the cellular signaling mechanisms that control apoptosis has increased substantially during the last decade. These advances provide a strong scientific framework for developing several types of targeted proapoptotic anticancer therapies. One promising class of agents is the proapoptotic receptor agonists. Of these, recombinant human apoptosis ligand 2/tumor necrosis factor-related apoptosis-inducing ligand (rhApo2L/TRAIL)-an optimized soluble form of an endogenous apoptosis-inducing ligand-is unique in that it activates two related proapoptotic receptors, DR4 and DR5. Preclinical data indicate that rhApo2L/TRAIL can induce apoptosis in a broad range of human cancer cell lines while sparing most normal cell types. In vitro, and in various in vivo tumor xenograft models, rhApo2L/TRAIL exhibits single-agent antitumor activity and/or cooperation with certain conventional and targeted therapies. Preclinical safety studies in nonhuman primates show rhApo2L/TRAIL to be well tolerated. Moreover, early clinical trial data suggest that rhApo2L/TRAIL is generally safe and provide preliminary evidence for potential antitumor activity. Clinical studies are ongoing to assess the safety and efficacy of this novel agent in combination with established anticancer therapies.

Drug‐induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies

Mitochondrial dysfunction is a major mechanism whereby drugs can induce liver injury and other serious side effects such as lactic acidosis and rhabdomyolysis in some patients. By severely altering mitochondrial function in the liver, drugs can induce microvesicular steatosis, a potentially severe lesion that can be associated with profound hypoglycaemia and encephalopathy. They can also trigger hepatic necrosis and/or apoptosis, causing cytolytic hepatitis, which can evolve into liver failure. Milder mitochondrial dysfunction, sometimes combined with an inhibition of triglyceride egress from the liver, can induce macrovacuolar steatosis, a benign lesion in the short term. However, in the long term this lesion can evolve in some individuals towards steatohepatitis, which itself can progress to extensive fibrosis and cirrhosis. As liver injury caused by mitochondrial dysfunction can induce the premature end of clinical trials, or drug withdrawal after marketing, it should be detected during the preclinical safety studies. Several in vitro and in vivo investigations can be performed to determine if newly developed drugs disturb mitochondrial fatty acid oxidation (FAO) and the oxidative phosphorylation (OXPHOS) process, deplete hepatic mitochondrial DNA (mtDNA), or trigger the opening of the mitochondrial permeability transition (MPT) pore. As drugs can be deleterious for hepatic mitochondria in some individuals but not in others, it may also be important to use novel animal models with underlying mitochondrial and/or metabolic abnormalities. This could help us to better predict idiosyncratic liver injury caused by drug-induced mitochondrial dysfunction.

Species selection considerations for preclinical toxicology studies for biotherapeutics

Background: Preclinical efficacy and safety studies, especially chronic studies, can be difficult to perform when the candidate therapeutic agent is a human protein due to the specificity of these molecules for the human target. The main issues are: i) the human protein or target may not be pharmacologically active in rodents or dogs, the standard toxicology species; or ii) the therapeutic agent may be so immunogenic in these species, that longer duration studies are not possible due to the formation of neutralizing antibodies. Thus, preclinical safety testing of biotherapeutics poses a particular challenge in selecting a relevant animal species for use in toxicology studies. Objective: This article will discuss the considerations that are unique to safety assessment of biotherapeutics and will provide alternatives to the standard toxicity testing which is conducted for small molecules. Methods: This article is based on published information with regards to species selection considerations as well as information from the FDA website on several marketed compounds. In addition, discussions of this topic that have occurred in public forums as well as the experience of the author are considered. Conclusion: The most important consideration in species selection for a biotherapeutic is that the drug is pharmacologically active in the preclinical species. This is a key consideration as biotherapeutics are highly targeted and rarely, if ever, demonstrate off-target toxicity. Because of this species specificity, nonhuman primates are often the only relevant species that can be used to assess the safety of a biotherapeutic. Other alternatives such as use of a homologous protein in rodents or the use of transgenic or knockout mice can also be used to assess safety although the caveats to these approaches must be considered.

On the effects of topical synthetic pseudoceramides: Comparison of possible keratinocyte toxicities provoked by the pseudoceramides, PC104 and BIO391, and natural ceramides

Ceramides (Cer) in the stratum corneum are essential for epidermal permeability barrier function. Thus, topical Cer replacement therapy has been employed to improve barrier function in clinical situations associated with Cer deficiency, e.g., atopic dermatitis. Because of the disadvantages of both natural- and skin identical-Cer (central nervous system origins and cost, respectively), synthetic chemical mimics, or pseudoceramides (pseudo-Cer), have been utilized as Cer substitutes. Whereas increased levels of intracellular Cer trigger cell growth inhibition and apoptosis, Cer levels are maintained by metabolic/catabolic pathways protecting cells from Cer-induced apoptosis. However, since the metabolic fates of each pseudo-Cer remain unknown, their widespread deployment in topical agents has raised concern about potential toxicities. We compared the effects of two chemically unrelated commercially available pseudo-Cer to exogenous cell-permeant (C2)- or natural (C18)-Cer on cell growth and apoptosis thresholds in cultured human keratinocytes (CHK). Cell growth and cell toxicity of CHK exposed to either C2-Cer or pseudo-Cer were assessed by MTT and lactate dehydrogenase release assays. Mitochondrial membrane potential, an indicator of apoptosis, was measured using membrane permeabilized semi-intact keratinocytes exposed C2-Cer, natural-Cer or pseudo-Cer. While the cell-permeant-Cer inhibits keratinocyte growth and increases cell toxicity, neither of the pseudo-Cer showed these effects. Decreased mitochondrial membrane potential occurred in CHK incubated with cell-permeant- and natural-Cer, but not pseudo-Cer. Taken together with preclinical safety studies of these pseudo-Cer and their widespread use over the counter without evidence of toxicity, these studies provide further assurance about the safety of these pseudo-Cer for topical use.

A novel biodegradable pancreatic stent for human pancreatic applications: a preclinical safety study in a large animal model

Endoscopic stenting is one treatment method for pancreatic strictures or pseudocysts in patients with symptomatic chronic pancreatitis. With a biodegradable stent, the later removal of the stent could be avoided. We investigated the degradation, patency, and toxicity of a novel biodegradable, self-expanding radiopaque polylactide-barium sulfate pancreatic stent in a large animal model. Animal study. Five swine had a biodegradable pancreatic stent placed into the pancreatic duct (PD) and were followed-up for 6 months. Repeated blood tests and radiographs were studied during the follow-up. The animals were euthanized at 6 months, at which time, the PD inner diameter was measured, and histology was analyzed and graded. For comparison, histology from 5 nonstented animals was also analyzed. The stent was correctly inserted into the PD in 4 of 5 animals. All the animals remained healthy, gained weight, and showed no signs of pancreatitis. A radiograph showed that the stent was in its original place in all animals at 1 month but had disappeared in all animals by 3 months. At 6 months, the autopsied pancreatic tissue was soft, and the PD was patent in all of the animals. The PD was slightly dilated at the site of the stent in the head of the pancreas compared with the preoperative diameter (5 mm [range 3-6 mm] vs 2 mm [range 1-3 mm], P < .05), but, in the body of the pancreas, no significant dilatation was seen. In the histology of the pancreata, there was no difference between the samples from the exposed parts of the biodegradable pancreatic stent compared with the distal nonexposed parts, or to the samples from the nonstented animals. This novel biodegradable pancreatic stent, studied in these swine, appeared to be safe for use in the PD. These encouraging results warrant further clinical trials with this biodegradable pancreatic stent in pancreatic applications in human beings.

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Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs

Introduction Seizure liability is an adverse property of new candidate drugs typically detected only in later stage pre-clinical safety studies. Consequently, pharmaceutical discovery needs small scale (microplate-based), rapid throughput screens to ‘front-load’ such adverse endpoints in order to reduce associated attrition rates. Of the potential methods available, previously published studies have shown that the quantification of seizure-associated locomotion in the larval zebrafish ( Danio rerio) offers high potential for development into such a screen. Here, we present methodology and validation data (on 25 compounds) from a larval zebrafish (Zf) convulsant assay, based on the quantification of high speed locomotion after exposure to a range of test compounds. Methods All assays were undertaken in 7 days post fertilization (dpf), WIK-strain Zf larvae, at 27 ± 1 °C. The blinded validation test set consisted of 17 positive and 8 negative controls, based on literature evidence for seizure liability. Initially, a Maximum Tolerated Concentration (MTC) assay was undertaken on each compound to identify the maximum concentration not causing general toxicity, sedation or overt neuromuscular effects. Next, the convulsant assay was undertaken on 5 concentrations from the MTC down, plus a dilution water control. Exposed larvae were videotracked for 1 h, using the Viewpoint Videotrack for Zebrafish™ system, and high speed movements, typically associated with seizure-like locomotor activity, were quantified. Results According to classification criteria proposed by the European Centre for the Validation of Alternative Methods (ECVAM), the data generated appeared to offer “sufficient” predictivity (72% overall), particularly considering the potential for throughput and likely positioning within a safety pharmacology front-loading screening cascade. Discussion Possible reasons for the misclassifications are discussed, and potential improvements to increase sensitivity and specificity outlined. In all, these initial validation data suggest that this assay offers potential as a medium throughput screen aimed at the early drug discovery detection of this complex safety pharmacological endpoint.

Human health risk assessments for three neuropharmaceutical compounds in surface waters

Enhanced sensitivity of analytical chemistry methods has enabled the detection of low-levels of pharmaceuticals in the environment, resulting in questions about the safety of surface waters used for drinking supplies. Human health risk assessments were performed to evaluate the risks from residues of atomoxetine, duloxetine, and olanzapine, which might be found in surface waters. Preclinical safety studies and human clinical data were used to determine an acceptable daily intake (ADI) for each compound: atomoxetine, 1.4 μg/kg/day; duloxetine, 1.8 μg/kg/day; and olanzapine, 1.4 μg/kg/day. The calculated predicted no-effect concentrations (PNECs) for children were 25.7, 19.1, and 35.9 μg/L for atomoxetine, duloxetine, and olanzapine, respectively. Estimated exposure concentrations determined using United States Food and Drug Administration guidelines and predicted exposure concentrations from the P hATE™ model were compared with each PNEC to determine margins of safety, which ranged from 147 to 642. Based on currently available data used in this assessment, no appreciable human health risks exist from exposure to the highest 99th percentile of predicted residue levels of atomoxetine, duloxetine or olanzapine in surface waters under low-flow conditions.

Impact of Infections and Normal Flora in Nonhuman Primates on Drug Development

Preclinical safety studies that are required for the marketing approval of a pharmaceutical include single and repeat dose studies in rodent and nonrodent species. The use of nonhuman primates (NHPs), primarily macaques, as the nonrodent species has increased in recent years, in part due to the increase in development of biopharmaceuticals and immunomodulatory agents. Depending on the source of the macaques, they may vary in genetic background, normal flora, and/or the incidence of preexisting pathogens and inflammatory conditions. As the use of alternative sources of macaques rises to meet the increased demand for these animals in biomedical research, the toxicologic pathologist should be well versed in NHP pathology to adequately assess potential drug-related effects in the context of these variations. Such knowledge is particularly important in studies involving immunomodulatory drugs as the toxicologic pathologist should anticipate which type(s) of infections are most likely to arise depending on which arm of the immune system is modulated. The purpose of this review is to discuss the immunosuppressive (e.g., simian type D retrovirus, simian immunodeficiency virus) and opportunistic viruses (e.g., cytomegalovirus, adenovirus, simian virus 40, rhesus rhadinovirus, and lymphocryptovirus), primary and opportunistic bacteria (e.g., Campylobacter spp., Shigella flexneri, Yersinia enterocolitica, Moraxella catarrhalis, Mycobacterium avium complex, enteropathogenic Escherichia coli), and parasites (e.g., Plasmodium spp., Schistosoma spp., Strongyloides fulleborni) that have had the most profound impact on the interpretation of drug safety studies and/or that may reemerge as alternative sources of NHPs are used for drug safety studies.