Abstract
Category: Methodology
100 Discovering Transcriptional Regulators of Cytochrome P450 1A Through Sirna High Throughput Screening
Parrisa Solaimani1 and Oliver Hankinson1
1University of California Los Angeles, Molecular Toxicology IDP, Los Angeles, CA, USA
Using RNAi high throughput technologies, we aim to identify novel proteins that modulate the Aryl Hydrocarbon Receptor (AHR)-dependent induction of the CYP1A1 gene in the Hepa-1 murine hepatic cancer cell line. Dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and certain polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene, are environmental contaminants and potent carcinogens. The mechanism of their toxicities lies in the ligand-mediated binding to AHR and subsequent activation of their target genes. After ligand binding, AHR dimerizes with the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) protein, and the AHR/ARNT dimer then activates transcription of CYP1A1 as well as a number of other genes. Metabolism of PAHs by CYP1A1 plays a major role in carcinogenesis by these compounds. TCDD is refractory to metabolism, but its carcinogenic effects also probably depend upon the activation of gene transcription by AHR/ARNT, although the responsible genes have not been fully defined. These cytochrome P450s then metabolize the PAHs to electrophilic derivatives that can mutate DNA. Further characterization of the mechanism(s) of AHR/ARNT-dependent induction of gene transcription is therefore a very important research objective. A siRNA library of 6000 proteins in the druggable genome has been used to transfect Hepa-1 cells, which were treated with TCDD for 24 hours, and then assayed for CYP1A1 activity using the EROD assay. Following RSA statistical analysis, we will follow up on promising hits and expect to identify novel chromatin proteins involved in dioxin-induced CYP1A1 expression.
Keywords: CYP1A1, high throughput screening, gene regulation
101 Withdrawn
102 Withdrawn
103 Integrated Strategy for Assessing Lung Irritation Potential From Inhaled Compounds
Reising A.1, Patel P.1, Cruz E.1, Sampath S.1, Danahay H.3, Wolf A.2, Pognan F.2, and Shangari N.1
1Novartis Institutes for Biomedical Research, East Hanover, NJ
2Novartis Institutes for Biomedical Research, Basel, Switzerland
3Novartis Institutes for Biomedical Research, Horsham, UK
Inhalation offers a noninvasive, highly targeted route for drug administration, particularly in the treatment of respiratory diseases. The major impediment to this dosing route is high local concentrations of the inhaled compounds in lung tissue immediately after exposure. Assessment of not only cytotoxicity, but irritation and inflammation in the exposed tissue is critical in aiding compound selection during drug discovery. We are developing an integrated in vitro and in vivo tool box starting with isolated rat macrophage cultures (both continuous and primary cultures), human bronchial epithelial cell (HBEC) cultures, precision-cut lung slices (PCLS) and ending with 4- to 10-day intertracheal (IT) dosing studies in rats to investigate irritancy potential. This stepwise approach will carry potential drugs through individual levels with decreasing throughput, but increasing sensitivity. The expected results are compounds with high irritancy potential being spotted early in the screen, while compounds with little or no irritancy being moved to the next stages. Currently, normal rat macrophage cell line NR8383 and PCLS have been characterized using LPS to test inflammation, paraquat to test cytotoxicity and ABC123 a Novartis compound shown to be irritant in lung tissue during 4-week rat inhalation studies. In both in vitro models, good correlation of cytotoxicity (ATP and LDH) and inflammation (TNF-α, IL-6, and Nitrite) were observed with these compounds. ABC123 has also been tested and shown to be a pulmonary irritant in 4-day rat IT studies. Development and characterization of primary rat macrophage and HBEC are currently under way. Preliminary results have show that this stepwise approach to pulmonary irritancy screening is effective and efficient. As the inhalation route becomes more widely used, this irritancy screening tool kit will be applied to enhance compound selection, hopefully reduce development times and late-stage complications in compound development.
Keywords: lung irritancy, inhalation dosing, in vitro toxicology
104 Refinement of the 3T3 Neutral Red Phototoxicity Assay
Sergey Ksenzenko1, Mark Schwartz1, Douglas B. Learn1, and Alan M. Hoberman1
1Charles River Laboratories, Horsham, PA, USA
The in vitro phototoxicity test in Balb/c 3T3 cells is an exceptionally sensitive assay to evaluate the phototoxic potential of a chemical. The relevance of this OECD compliant assay to human phototoxicity potential is an evolving story and open to some controversy. However, regardless of any potential shortcomings, this in vitro assay is an effective way to screen lead compounds for phototoxic potential when proper considerations and approaches are used and its limitations are taken into consideration. We have developed several screening techniques to perform OECD-compliant 3T3 assays to facilitate the most time- and cost-effective methodologies for phototoxicity assessments. Using the screening method, phototoxicity assessment of 24 test articles can be completed in 3 days using a single high-limit concentration, or at least 48 test articles per week, including the calculations of photo irritation factors (PIF) of each test article, the mean photo effects (MPE), and the IC50(+UVA) and IC50(–UVA) values, as in the standard OECD-compliant 3T3 assay. Depending on test article preparation procedures (whether solubility evaluations are needed), the vehicle used, etc from 20 micrograms to 200 milligrams of the test article is needed, an important consideration with limited availability. The turnaround time of GLP and OECD-compliant phototoxicity assessment can be reduced to 3 days, with 3 test articles tested, or 5 days for 6 test articles. The effective strategies and procedures of using these approaches are discussed with Sponsors and accomplished using relevant approaches in formulation solubility evaluations and dosing solution preparation, as well as the definitive testing and the dose formulation analysis. These approaches allow for customization of the assay to meet the needs of sponsors and allow for a rapid and cost-effective assay for phototoxic potential of multiple test articles.
Keywords: in vitro, phototoxicity, method refinement
105 Genetic Analysis of Formalin Fixed Tissues From Nonhuman Primates
Jean Dubach1, James H. Stolz2, and Donald Waller3
1Loyola University Medical Center, Maywood, IL, USA
2Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT, USA
3Prelabs, Oak Park IL, USA
Therapeutic development is increasingly taking advantage of the expanding field of human genetics. The genetic similarity between humans and nonhuman primates has increased the importance of the use of the nonhuman primate as a model to develop new therapeutic modalities. The cynomolgus monkey is the most frequently used nonhuman primate model for efficacy and safety studies. We have previously demonstrated the broad genetic diversity for this animal model. Pharmacologic and toxicologic responses to new agents can be greatly influenced by a number of factors, including background genetic variability. Retrospective genetic analysis of formalin fixed-tissues may provide some assistance in the interpretation of data where outliers cannot be easily explained. Blood, fresh frozen and formalin-fixed paraffin-embedded samples of heart and liver from cynomolgus monkeys were evaluated. A second set of formalin-fixed paraffin-embedded heart and liver samples from a previously performed study was also evaluated. DNA was extracted and all tissue/blood samples genotyped for twelve nuclear microsatellite loci using standard PCR protocols with products sized using a Beckman/Coulter CEQ8000 capillary electrophoresis system. All blood and frozen tissues could be easily genotyped. Genotyping of the formalin-fixed embedded tissues were also successful for both the new and the historical samples; however, an additional extraction was required to obtain adequate DNA for processing. The heart consistently provided better results for both the freshly prepared formalin-fixed embedded samples and the historical samples from a previous study when compared to the liver specimens. The ability to retrospectively evaluate the genetic profile of animals may provide an important tool to follow-up on studies with divergent responses leading to data which are difficult to interpret.
106 Withdrawn
107 Dried Blood Spot Comparison Study: Comparison of 2 Blood Collection Sites and 2 Blood Collection Methods After a Single Dose of Acetaminophen in Male Rats
Alan Stokes1, Tammy Moose1, Chhou Liv1, Simon Parry1, Matthew Barfield1, Cerys Lovatt1, Wesley Dopson1, David Melich1, Carol Overvold1, Sonya Gade1, Christopher Evans1, and Neil Spooner1
1Safety Assessment, GlaxoSmithKline, Research Triangle Park, NC, USA
Dried blood spot technology has been available for many decades but only in the last 5 years has it been considered for routine bioanalysis of blood samples collected on nonclinical and clinical studies as part of a drug development program. Advantages to using dried blood spots versus plasma samples include, but are not limited to, less blood volume required, less processing of the samples (eg, no centrifugation), potentially using less animals, and no storing or shipping frozen samples. The current study compared blood concentrations (AUC0-t and Cmax) of acetaminophen after a single 600 mg/kg dose using 2 different sampling sites (tail vein versus tail snip) and 2 different collection methods (3 separate 15 mL end-to-end EDTA-coated capillary tubes versus an integrated capillary tube/microcontainer) in male Crl:CD(SD) rats. The objective was to evaluate the reproducibility and identify differences in serial-microsampling of rats using these methods. After receiving a single oral gavage dose, each rat was sampled at multiple time points on the day of dosing and the resultant plasma analyzed for blood acetaminophen concentrations (AUC0-t and Cmax) using LC-MS/MS. The results showed that there were no meaningful differences (ie, twofold or greater) in blood concentrations of acetaminophen using the different sites or methods. Furthermore, to determine spot-to-spot variability, comparisons of the acetaminophen blood concentrations obtained after analysis of a duplicate blood spot from the same blood spot card were within 12% of the original concentration.
Keywords: dried blood spots, acetaminophen, tail snip
108 Non-Terminal Juvenile Rodent Repeat Blood Sampling Techniques for Dried Blood Spot Analysis—Opportunities for Reduction and Refinement?
E. S. Richmond1
1Sequani Limited, Bromyard Road, Ledbury, Herefordshire, HR8 1LH, UK
The utility of data identifying relationships between toxicological end points and drug exposure is well recognized and thus recommended by both the EU and FDA guidelines for nonclinical testing of pediatric pharmaceutical products. The difficulties in obtaining blood samples from juvenile rodents are, however, clearly acknowledged due to blood volume limitations and sampling feasibility. Consequently, terminal blood sample techniques are commonly employed, thereby increasing the number of animals required to generate data which is often limited to the assessment of proof of absorption or composite profiles. The more recent advances in the use of Dried Blood Spot technology (DBS) for bioanalysis of pharmaceutical products is therefore of great potential for the purposes of reduction and refinement on rodent juvenile studies, given the use of whole blood samples of drastically smaller volume. This provides opportunities for non-terminal serial blood sampling, thus improving the quality of toxicokinetic data and reducing the overall animal numbers required, in addition to those benefits already provided by the use of DBS technology.
Due to the current lack of methods for non-terminal blood sampling from the preweaning juvenile rat, this study investigated the suitability of different non-terminal blood sampling techniques in Sprague-Dawley rat pups of day 7 and day 13/14 of age, and the feasibility of repeat sampling. This study successfully identified a well-tolerated and a minimally invasive sampling route from the ventral tail artery. No anaesthesia was required, nor stimulation of peripheral vasodilation. Furthermore, samples were satisfactorily collected from the same animals on up to 2 occasions on day 7 of age and up to 6 occasions on day 13/14 of age. These findings therefore support the use of DBS analysis on nonclinical juvenile studies for safety assessment and present a superior approach for blood sampling in terms of ethics, cost, and quality of data.
Keywords: dried blood spot analysis, juvenile rodents, blood sampling
109 Dried Blood Spot (DBS) Sampling Methodology From Nonhuman Primates Following Dose Administration of Clonidine Hydrochloride
Carrie McMahon1, Fumin Li1, Stephen Ploch1, Andrew J. Attwood1, Brendan Stitcher1, and Tom W. Beck1
1Covance Laboratories, Madison, WI
Application of dried blood spot (DBS) methodology for bioanalysis in nonclinical toxicological study environments offers several advantages, including reduction of stress to study animals, long-term sample stability, decreased shipping costs, and elimination of freeze/thaw parameters in bioanalytical method validation. We assessed the feasibility of collecting small blood volumes from nonhuman primate distal tails for purposes of DBS bioanalysis following dose administration with a positive control article. Microvolume blood samples (~40 uL) were collected from twelve restrained cynomolgus monkeys during predose and 0.5, 1, 2, 4, 6, 12, and 24 hours after a single dose of 0, 0.01, or 0.05 mg/kg clonidine hydrochloride (n = 4 animals/group). Blood was collected from the distal tail using a single-use safety lancet and an anticoagulant-coated capillary tube. Samples were applied onto DBS cards (Whatman, WB129241). DBS cards were allowed to dry at room temperature and stored protected from light at room temperature in a sealed bag with desiccant prior to LC-MS/MS analysis. Animals recovered well with no observed signs of discomfort. Detection of clonidine in control animals was below the lower limit of detection (0.1 ng/mL), and the bioanalytical results agreed with the pharmacokinetics of clonidine previously reported in this species. In addition, exposure profiles correlated well with hemodynamic effects recorded for the same animals given the same dose levels of clonidine on a separate dosing day. While consideration of alternate collection sites is ongoing, these data demonstrate that the microvolume blood sampling technique from nonhuman primates is a reliable and practical approach for DBS bioanalysis in a nonclinical toxicological study environment.
Keywords: dried blood spot, bioanalysis, nonhuman primates
110 Impact of Serial Micro-Volume Blood Collection in Mice: Implications for Dried Blood Spot Methodology
Stephen Ploch1, Jodie Lasco1, Koren Brooks1, Pam Mielke1, Andrew Attwood1, and Niraj Tripathi1
1Covance Laboratories, Madison, WI
The use of dried blood spot methodology in nonclinical toxicology studies to establish toxicokinetic profiles offers numerous potential benefits, including a reduction in animal use, and exposure assessment in main study animals. In order to determine the impact of multiple serial sample collections in mice, microvolume blood samples (2 × 20 uL) were collected from mice 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after a single mock dose time. Blood was collected from the tip of the tail using a tail-tip method of collection into anticoagulant-coated capillary tubes. For comparison, another group of animals was handled similarly but animals were not bled, and a third (control) group was not handled or bled. Daily food consumption, body weights, and clinical observations were collected for 1 week. Clinical pathology samples were collected approximately 30 (day 2), 48 (day 3), and 168 (day 8) hours post mock dose time. Minor red skin discolorations were noted on the distal tail in a few mice that were bled for serial sampling, but also in some mice that were handled but not bled. A mild transient effect on body weight and food consumption was noted over the first 2 to 3 days post mock dose in mice that were handled, and those that were handled and bled. Transient clinical pathology effects were limited to mild decreases in red cell mass (ie, red blood cells, hemoglobin, and hematocrit) in mice that were bled for serial sampling. These hematology changes were observed on days 2 and 3, and recovered by day 8. A typical stress leukogram was not observed, indicating limited stress from handling or the blood collection procedure. These data support the use of the tail-tip method of microvolume sample collection in mice, and help guide appropriate study designs in nonclinical safety studies.
111 Reduction of Mice Required for Test Item Exposure Confirmation in Toxicology Studies Through Blood Microsampling Techniques
C. R. Mattis1, J. T. Miklulecky1, B. M. Bird1, M. Metchkarova1, K. X. Wan1, D. Zhao1, and L. A. Gallenberg1
1Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL
Assessment of systemic test article exposure in mouse toxicology studies is typically performed using terminal blood samples collected via cardiac puncture from satellite animals, resulting in large numbers of mice used in these studies. We have developed a study design that allows assessment of test article exposure from main study animals using a survival bleeding technique combined with a sparse sampling collection schedule. The pharmacokinetics of a test article and its metabolite were assessed in a series of experiments conducted in CD-1 and CByB6F1 (nontransgenic littermates of Tg.rasH2 model) mice. The pharmacokinetics were first determined from plasma, whole blood, and dried blood spots prepared from terminal blood samples from CD-1 mice following a single 10 mg/kg oral dose of the test article. Next, the suitability of non-terminal blood collection methods from the lateral tail vein and saphenous vein were assessed in a 7-day repeat-dose study in CD-1 mice. Finally, the feasibility of using the tail “nick” method with a sparse sampling collection schedule to assess systemic test article exposure was demonstrated in an experiment that compared the test article and metabolite pharmacokinetics in CByB6F1 and CD-1 mice. This alternate study design allowed assessment of test article exposure using approximately 75% fewer mice than would be used for a typical study design employing terminal bleeding techniques and satellite animals.
Keywords: blood microsampling, refinement, alternative
112 Comparison of Rapid Decapitation, Jugular, and Submandibular Blood Collection Methods From Conscious Rodents for the Measurement of Circulating Hormones
Musser N1, Colbeck B1, Robbins M2, Shoufler D2, and Kreckler L1
1Covance Laboratories Inc, Madison, WI, USA
2Covance Laboratories, Inc, Greenfield, IN, USA
Investigational analyses, such as hormone analyses, are increasingly included as part of preclinical studies to learn more about pharmacodynamic effects or additional toxic end points of a given drug candidate. However, evaluating test article-related effects on hormone levels can be complicated by a multitude of factors that impact hormones, including sex, age, exposure to anesthesia, circadian rhythm, and stress. Consequently, in rodents, rapid decapitation has been used as a blood collection technique when stress-sensitive hormones are to be assayed since it allows blood to be collected quickly from conscious rodents, thereby avoiding a confounding effect from systemic stress-induced hormone secretion. However, the use of blood collections from the jugular (rat) and submandibular (mice) of conscious animals would allow for serial collections from a single animal and an overall reduction in animal usage. The goal of this study was to compare hormone levels in blood collected by rapid decapitation in rats and mice with blood collected by the jugular method in rats or submandibular method in mice. Female rats and mice were acclimated at least overnight and left completely undisturbed the night prior to blood collection. Blood collections within a given set of animals were completed within 5 minutes of technicians entering the animal room. All samples collected were analyzed for corticosterone and prolactin levels. Blood collection by rapid decapitation was confirmed to be appropriate for hormone collection, but the jugular and submandibular collection techniques allow for the reduction in animal usage and serial collections from a single animal. Careful consideration of study goals is required when determining study design.
Keywords: hormone analyses, blood collection methods, rodents
113 Analytical Comparison of Bone Marrow Sample Collection Techniques and Immunophenotyping for Cynomolgus Monkeys
Pramod Joshi1, Joshua Arrington1, and Rick Haas1
1Covance Laboratories, Madison, WI, USA
The purpose of this study was to compare bone marrow sample collection techniques (aspiration and perfusion) for flow cytometric immunophenotyping and characterization of CD34+ cells from cynomolgus primates. These procedures have been developed to investigate perfusion bone marrow collection as a serial technique, to evaluate changes in bone marrow cells (in particular CD34+ cells) caused by new pharmaceutical products (either intentionally or unintentionally), and provide a basis for further investigation of stem cells or other precursor cell populations by flow cytometry. Bone marrow samples were collected from the humerus or femur by perfusion or aspiration and compared with whole blood T cells (total, helper, and cytotoxic), B cells, and natural killer cells to determine the degree of blood contamination in bone marrow samples and to quantify CD34+ cells. Additionally, 4 aspiration bone marrow sample processing techniques (peripheral blood mononuclear cell purification, lyse-wash-antibody staining-wash, antibody staining-lyse-wash, and antibody staining-lyse-no wash) were compared alongside whole blood samples and evaluated by flow cytometric immunophenotyping. The results show that bone marrow samples collected by perfusion had the least amount of whole blood contamination. Both perfusion and aspiration bone marrow samples measured much higher levels of CD34+ cells than peripheral blood. All 4 aspiration bone marrow sample processing techniques were able to detect CD34+ cells though PBMC isolation and wash methods were more effective for the cell populations investigated. In conclusion, bone marrow samples collected by perfusion yield a more accurate assessment of resident cell populations; bone marrow samples collected via aspiration or perfusion should be processed via peripheral blood mononuclear cell purification to eliminate sample particulates and provide cleaner samples for flow cytometry or cell culture applications. CD34+ cells can be evaluated with either perfusion or aspiration bone marrow samples and can be measured in peripheral blood.
Keywords: immunophenotyping, flow cytometry, bone marrow
114 An Improved System for Surgically Implanted In-Dwelling Catheterization for Long-Term Intravenous Infusion in Cynomolgus Monkeys, Using 2% Taurolidine-Citrate (Tc) to Maintain Catheter Patency
Robert Tavcar1, and Ken Draper1
1LAB Research, Inc, Laval, QC, Canada
Extended intravenous (IV) infusion with surgically implanted catheters is critical for nonclinical studies of many pharmaceuticals (eg, anticancer agents or anti-infectives). A “catheter-in-catheter” (CIC) system provides many advantages over the traditional surgically implanted indwelling catheter. The CIC system requires only a single surgical procedure to insert the catheter into an appropriate vessel and employs a subdermal port system and fluid lock. For infusion/dosing, a second catheter is inserted through the port/initial catheter and can be connected to a swivel and jacket system. If desired, the second catheter and/or jacket can be removed between dosing events. The CIC system allows permanent external port access to a dosing vein and easy replacement of the removable infusion catheter while eliminating the need for repair surgeries. CIC can be used in conjunction with telemetry equipment or other physiological monitoring to capture pharmacodynamic effects that can be obscured by animal handling for manipulation of an indwelling catheter. To evaluate the feasibility of using 2% TC (a broad spectrum antimicrobial agent) in the fluid lock of the CIC system, one group of cynomolgus monkeys was infused continuously with sterile saline for 6 months, and a second group was infused for 24-hour cycles, every 2 weeks for 6 months. In the group that received bimonthly infusions, TC from the catheter lock was flushed into the animals, and they were assessed for clinical pathology and/or toxicological effects at 24 hour postflush following each cycle. There were no TC-related clinical signs or clinical chemistry changes in the animals. Thus, 2% TC appears to be a safe, feasible option for use in the fluid lock of the CIC system during long-term nonclinical studies in monkeys.
Keywords: animal models, infusion, chronic toxicology
115 Optimization of Intravenous Dog Infusion via Vascular Access Port
Fritz W.1, Herman J.1, Brooks A.1, Taschwer M.1, and Alexander A.1
1Covance Laboratories Inc, Madison, WI
Intermittent intravenous infusion is becoming more common in preclinical toxicology studies. Vascular access port catheters (VAPs) can be locked with heparin/saline and animals disconnected from the infusion apparatus between doses, reducing opportunities for infection and increasing quality of life for the animals. In this study, 2 commercially available VAPs were evaluated in combination with different port locations and ambulatory or tethered infusion systems to optimize infusion study design in canines.
Male and female dogs were divided into 4 ambulatory (3 animals/group) and 2 tethered (6 animals/group) infusion groups. Animals were administered sterile saline at 5 mL/kg/h over approximately 3 hours, once daily for 11 days then once weekly for 2 weeks. A second set of 2 tethered infusion groups (5 animals/group) were administered D5W at 10 mL/kg/h for approximately 1 hour on days 1, 4, 8, 11, 22, 25, 29, and 32. Maintenance solution was infused between doses to maintain catheter patency and simulate continuous infusion. Infusion systems were assessed for external catheter breaks, needle dislodgements, leaks, occlusions, and disconnections; clinical signs, body weights, food consumption, and clinical and anatomical pathology findings were used to evaluate tolerability.
All port location/infusion system combinations allowed for successful intravenous dose administration. Needles bent with greater frequency with one of the port types, but marginally fewer needle pull outs occurred during ambulatory infusion. Accessing the catheter via the port was more labor intensive and sterility more difficult to maintain with one of the port types. No significant differences in clinical signs, body weight, food consumption, or clinical pathology were observed. Transient red skin and scabs were less frequent in the ambulatory group with lateral port location compared to cervical port location, but comparable in tethered groups regardless of port location. No discernable differences in the incidences or severity of microscopic observations were noted for infusion system or port location in dogs given sterile saline. The incidence or severity of fibrosis around the catheter was increased in tethered animals with ports in the cervical location compared to those with ports on the lateral thorax.
All combinations were acceptable for intravenous infusion in dogs, but the specific port/port location/infusion system used for a given study should be selected based on the study design, dose duration, and study end points.
Keywords: infusion, canine, VAP
116 Delayed-Type Hypersensitivity (DTH) in Cynomolgus Monkeys: Compared Induction Using Tetanus Toxoid or Keyhole Limpet Hemocyanin (KLH)
Caroline Bouchez1, Frederic Gervais1, Renaud Fleurance1, Bernard Palate1, Jean-Jacques Legrand1, and Jacques Descotes2
1CIT, Evreux, France
2Poison Center, Lyon, France
T-dependent antibody response (TDAR) is widely recommended for use as the first-line assay to measure immune function. However, a more focused assessment of cell-mediated immune response, eg, DTH, may be a helpful add-on. The aim of this study was to compare the induction of DTH in monkeys using either tetanus toxoid or KLH as the antigen. Male cynomolgus monkeys were injected intramuscularly either once or twice with 1 mg KLH, or twice with Pasteur tetanus vaccine® (40 IU tetanus toxoid + 0.06 mg aluminum hydroxide). Then, all animals were challenged by intradermal injections of the same antigen or aluminum hydroxide after 4, 6, and 8 weeks. Clinical reactions at the injection sites were scored 24, 48, and 72 hours postchallenge. Skin biopsies were taken on completion of the observation period after each challenge, and submitted to histopathologic examination. Tetanus toxoid induced stronger clinical reactions than KLH, whereas aluminum hydroxide induced no clinical reaction. Perivascular mononuclear cell infiltrates were histopathologic findings consistent with a DTH reaction in the animals injected with tetanus toxoid or KLH. These results suggest that tetanus toxoid adjuvanted with aluminum hydroxide can induce a more marked DTH response than KLH in cynomolgus monkeys.
117 A Preliminary Assessment of External ECG Telemetry Collections in Group-Housed Cynomolgus Monkeys
Ayo Olatunji1, Brian Maron1, C. Michael Foley1, and Steven M. Glaza1
1Covance Laboratories, Inc, Chandler, AZ
The collection of electrocardiogram (ECG) data via noninvasive external telemetry systems has gained recent popularity and utility for the assessment of potential cardiac effects during the course of experimental drug safety assessment. The major advantages of an external jacketed telemetry system in the assessments of ECGs are that they are noninvasive, are performed on nonsedated animal subjects, and allow for the continuous collection of ECG data over long periods of time. An external jacketed telemetry system (JET®, DSI, Inc) with leads placed in an approximate Lead II configuration was used in these trials. In typical toxicology study designs, these ECG assessments are made throughout the duration of a study while the animals are individually housed. There is also a recent trend to continuously group house primates as a means to achieve greater social enrichment for the animals and a reduction in stress-related disorders.
Recently, feasibility studies were undertaken to determine if external jacketed telemetry ECG collections can be successfully achieved in clinically healthy, naive Cynomolgus monkeys (Macaca fascicularis) while the animals are group housed. Two different jacket types were evaluated. Animal interference with the external telemetry apparatus was minimal with one jacket type and essentially nonexistent with another jacket type. The ECG data from the group-housed animals wearing both jacket types were of good quality and reflective of normal, healthy animals. Animals demonstrated good tolerance to the ECG collection procedures even after repeated data collection sessions in the group-housed environment. The collection of external telemetry ECG collections in Cynomolgus monkeys under group-housing conditions is deemed feasible and warrants additional investigation.
Keywords: external telemetry, primate, electrocardiogram
118 Rapid Visual Evaluation of ECG Changes in Ferrets, Dogs, and Monkeys After Moxifloxacin Treatment
B. W. Main1, C. A. Clawson1, R. H. Hunter1, M. Rosewarn1, J. Doyle1, and D. Leishman2
1Covance, Inc, Greenfield, IN
2Eli Lilly and Co. Indianapolis, IN
The QT interval is highly variable and statistical evaluation of rate-corrected QT interval can be complex and time-consuming. Spotfire® was used to visualize the QT changes from telemetered ferrets, dogs, and monkeys in response to moxifloxacin treatment. All animals were surgically instrumented with radio telemetry transmitters (Data Sciences International). Spotfire was capable of handling hundreds of thousands of data points allowing rapid visual evaluation of study results.
ECG data were collected continuously 24 hours prior to oral moxifloxacin treatment and continued for 48 hours after dose administration. Data were evaluated using beat-to-beat data and 1 minute averages. Moxifloxacin caused dose-related increases in QT intervals in all species. Maximal changes in QT values were approximately 85 msec in ferrets, 50 msec in dogs and 75 msec in monkeys. Using the features of Spotfire, we were able to move through the datasets using time after dose administration. The visualization process allows rapid examination of group means as well as individual animal effects. Individual evaluation is very effective since QT-HR clouds are often unique. Changes in QT intervals are easily visualized without having to use rate correction methods or/and can be used to visualize the effectiveness of any rate correction method. We have found visualization is a powerful and integral part of study results and study interpretation for any ECG analysis apart from any further analysis methods.
Keywords: ECG analysis, QT prolongation, cross-species translation
119 The Local Lymph Node Assay (LLNA) Using L92 Surfactant as Vehicle for Aqueous Mixtures
Tiwari V. K.1, Dalal V.1, Desai Y.1, Verma R.1, Pandey S.1, Gohel D.1, and Bagal A.1
1Jai Research Foundation, Valvada 396 108, Gujarat, India
We have conducted an in-house validation study using a vehicle (1% aqueous pluronic L92 surfactant) that is recommended by Boverhof et al (2008) for the testing of mixtures in the LLNA for assessing skin sensitization. We conducted the test according to the methods described by OECD 429 (Skin Sensitization: Local Lymph Node Assay). Two common pesticide formulations, oxyfluorfen EC (OEC) and trifluralin EC (TEC) and one known skin sensitizer α-Hexylcinnamaldehyde (HCA)*, were each applied to the dorsum of both ears (25 mL per ear) of groups of 5 female CBA/J mice. Each test material was applied at 3 separate concentrations for 3 consecutive days (days 0, 1, and 2). A further group was given the vehicle (1% aqueous pluronic L92 surfactant), alone. On day 5, all mice were injected intravenously (tail vein) with approximately 20 µCi of tritiated methyl thymidine. Five hours postadministration, the uptake of 3H-thymidine into the auricular (local) lymph nodes draining the site of chemical application was measured in order to assess the proliferative response of the lymph node. The DPM values were measured individually for each mouse. Stimulation Index (SI value) and EC3 values (the estimated concentration required for a threshold positive response) were calculated and are given below:
Values shown in bold have a SI 3 or more times greater than the control (Control SI = 1) and are considered to have the potential to cause skin sensitization.
The results obtained in the present study are comparable with the findings reported by Boverhof et al (2008), indicating good comparability between our laboratory and other international research centers. Hence, it is concluded that 1% aqueous pluronic L92 surfactant can be used as a vehicle for evaluation of mixtures in the Local Lymph Node Assay at Jai Research Foundation.
* with thanks to Dow Agro Science for supplying the test materials.
Keywords: Local Lymph Node Assay (LLNA), mixtures, vehicle
Category: Test Systems
200 Transcriptomic Analysis of Primary Canine Hepatocytes for Prediction of Dog Specific Hepatotoxicity
Michael J. Liguori1, Rita Ciurlionis1, Anthony J. Lee1, Xiaofeng Li1, Amy C. Ditewig1, Eric A. G. Blomme1, and Yi Yang1
1Abbott Laboratories
Hepatotoxicity in dogs is usually identified in the later stages of preclinical drug development. In vitro approaches to detect these toxicities at earlier stages could result in significant savings of resources and time. Traditional in vitro techniques, such as cell lines, are unable to predict certain changes and have limited utility because of their minimal metabolic capabilities. Primary hepatocytes are preferred to evaluate hepatotoxicity in vitro. The objective of this study was to evaluate primary canine hepatocytes compared to rat hepatocytes to predict and characterize hepatotoxicity specific to dogs. Primary hepatocytes isolated from the livers of male Beagle dogs or male Sprague-Dawley rats were cultured for 24 to 48 hours and treated with several prototypical toxic agents: acetaminophen (APAP; dog-preferential toxicity), zamifenacin (ZAM; dog-specific toxicity), or tacrine (TAC; mitochondrial toxicant with no species specificity). Cytotoxicity assays (LDH and MTT) and transcriptomics were performed. ZAM treatment resulted in enhanced cytotoxicity (TC20) in dog hepatocytes compared to rat while TAC was slightly more toxic to rat hepatocytes. For APAP, cytotoxicity was similar between species, but the number of gene expression changes was greater in dog hepatocytes relative to rat. Genes involved in oxidative stress response, glutathione (GSH) synthesis, and protein ubiquitination were highly upregulated in dog but to a lesser extent in rat. For ZAM, the overall gene expression profiles were markedly different for dog compared to rat with marked upregulation of the Nrf2 and GSH regulatory pathways. In contrast, TAC induced expression changes in genes associated with mitochondrial function and DNA damage response in both species. In conclusion, transcriptomic analysis of canine hepatocytes can be useful to predict species-specific hepatotoxicity and elucidate mechanisms for some compounds.
Keywords: hepatotoxicity, transcriptomics, hepatocytes
201 Standardization of the CFU Assay for Targeted Screening of Compounds on Hematopoietic Myeloid Progenitors
Elaine Lau1, Allen Eaves1, and Jackie Damen1
1STEMCELL Technologies, Inc, Vancouver, BC, Canada
A key feature of hematopoietic stem cells and their progeny is that their proliferation and differentiation can be exquisitely regulated by external stimulation from various cytokines. Cytokines differentially affect intracellular signaling pathways to direct lineage commitment and differentiation in hematopoietic progenitors which give rise to mature cell types such as granulocytes, monocyte/macrophages, and erythrocytes. The development of new compounds, such as the kinase inhibitors that specifically target key components of these hematopoietic signaling pathways, has increased dramatically. A crucial tool for the continued discovery and characterization of kinase inhibitors is the use and modification of the colony forming unit—granulocyte, monocyte (CFU-GM) assay which gives rise to isolated colonies of mature granulocytes, monocytes, or combinations of these. By manipulating the type and concentration of cytokines in the medium used, this assay can be customized to support the growth of specific myeloid progenitors. This then allows the differential assessment of the effects of test compounds on the progenitor cells of interest. To standardize this approach, we evaluated various media formulations to establish the ideal growth conditions for detecting inhibition of myeloid progenitors from human bone marrow. We then tested different tyrosine kinase inhibitors to determine IC50 and IC90 values for each progenitor population. Differential effects were observed in the IC50 values of Sunitinib and Imatinib on CFU-M and CFU-G. In addition, IC50 values obtained for these compounds on CFU-G inhibition were similar to what is reported in the literature and, more importantly, shown to be clinically relevant—with the more potent IC50 values correlating with an increased incidence of clinical neutropenia. This standardized assay produces biologically relevant results that are essential for the optimal determination of the differential effects of compounds on specific myeloid progenitors.
Keywords: hematopoietic stem cells, drug toxicity/screening, tyrosine kinase inhibitors
202 Cytotoxicity of 14 Oil Feedstocks on Cultured Alveolar Macrophages
Rigden M. D.1, Bouchard P. A.1, and Poon R.1
1Health Canada
Oils from plant and animal sources are the primary feedstock for biodiesels (monoalkyl esters of fatty acids). These sources include traditional oils derived from Palm, Coconut, and Canola, and emerging sources such as Jatropha, Castor, and fish oils. While many of the oils are nontoxic and in fact edible, some of the newer ones have little or no toxicity information. In this study, we characterized and compared the cellular effect of 14 oils using cultured alveolar macrophages cells. NR8383 cells, which are derived from rat bronchoalveolar macrophages and retained much of the antimicrobial and immune modulation functions, were used because inhalation is a major route of exposure for biodiesels due to aerosol generation and incomplete combustion. The cells were tested with oils from Canola, Castor, Coconut, Corn, Cottonseed, Jatropha, Lard, Mehaden fish, Olive, Palm, Rapeseed, Safflower, Soybean, and Sunflower oils at concentrations ranging from 0.02 to 5 mM. The cells were incubated for 18 to 20 hours and tested for LDH release (cell viability), secretion of TNF-alpha (a proinflammatory cytokine), and zymosan-induced chemiluminescence (phagocytosis-related oxidative burst). No significant increase in LDH release was observed indicating that at concentrations up to 5 mM the oils did not affect cell viability. All oils stimulated TNF-alpha secretion at the concentration range tested with maximum two- to sixfold increases at 0.05 to 0.2 mM. Jatropha showed a unique suppressive effect on zymosan-induced chemiluminescence with greater than 90% inhibition at 0.5 mM. All other oils caused mild to moderate stimulation of chemiluminescence at low (0.05-0.1 mM) concentrations followed by a gradual concentration-dependent suppression at 0.2 to 5 mM. Conclusion: oils used as feedstock for biodiesel production may interfere with the antimicrobial and immune activities of bronchoalveolar macrophages through their suppressive effect on phagocytic activity and modulation of cytokine-mediated inflammatory immune response. The toxicity and potential health effects of the emerging oils in particular require further study.
Keywords: oil toxicity, alveolar macrophage, phagocytic functions
203 Evaluating Porcora for Use in Consumer Products to Accurately Predict Ocular Injury and Reversibility
Eblin Kylee E.1, Jones Frank A.1, Rugen Pamela J.1, Steele Chris L.1, and Piehl Michelle2
1Global Safety and Regulatory Affairs, S.C. Johnson and Son, Inc, Racine, WI, USA
2MB Research Labs, Spinnerstown, PA, USA
Utilization of in vitro tests can provide rapid information that can greatly reduce live-animal use, and results that are potentially more relevant to human biology and human exposures. The current standard for ocular irritation is the Draize rabbit assay which predicts ocular injury while providing information on reversibility of the damage. In vitro assays to date have only been able to provide information regarding injury, not reversibility. A new assay developed by MB Research Labs, the Porcine Corneal Opacity Reversibility Assay (PorCORA) measures corneal damage and recovery in the same time frame as the Draize assay using porcine corneas (21 days); therefore, PorCORA results more closely resemble the standard regulatory classification methods of ocular irritancy which depend upon the time for an ocular injury to reverse.
Seven test formulas with a range of consumer product chemistries were screened through PorCORA to determine if it could accurately predict the hazard classifications determined by in vivo results as a stand alone assay or if it should be used in a tiered testing scheme to distinguish those formulas classified as moderate to severe/corrosive ocular irritants using the Bovine Corneal Opacity and Permeability (BCOP) Assay scoring classifications. For 4 of the 7 test substances, PorCORA predicted reversibility close to the time frame of the in vivo animal data thus resulting in the same eye irritancy classification of the product. Some variation between the PorCORA and in vivo results can be attributed to differences in protocol; aerosol method versus direct instillation of Test Article (TA). An important limitation to note is that similar to the Draize assay, the results are based on the last cornea to reverse rather than the average day of reversal. Variations in the prediction of irritancy classification for those TAs in the midrange of ocular irritation remain to be resolved; thus at this time, it is recommended that PorCORA be incorporated into the standard suite of in vitro/alternative assays used to predict eye irritation for formulations as a second tier assay to determine reversibility of TAs with BCOP in vitro scores (IVS) >75. Pairing PorCORA with other assays such as the BCOP and EpiOcular (assays under investigation for validation as regulatory accepted alternative methods), will enable toxicologists to more accurately predict the entire range of ocular irritancy without compromising the physiological accuracy of the Draize assay.
Keywords: in vitro/alternative test, ocular damage reversibility, porcine cornea
204 Porcine Corneal Ocular Reversibility Assay (Porcora) Predicts EU R41 and GHS Category 1
Piehl M.1, Soda R.1, Carathers M.1, DeGeorge G.1, and Cerven D.1
1MB Research Laboratories, Spinnerstown, PA
Currently, there is no alternative (non-“in vivo”) ocular irritation assay that can measure corneal tissue damage and reversibility. With the support of 2 Colgate-Palmolive Grants for Alternative Research, we have developed an alternative assay: Porcine Corneal Opacity Reversibility Assay (PorCORA). PorCORA measures corneal damage and recovery over extended time periods using porcine corneas excised from byproduct abattoir eyes. Test articles (liquid and solid) are dosed directly onto the corneal surface, and tissue damage and recovery are assessed by sodium fluorescein (NaFL) retention in the same corneas over time (up to 21 days). We have confirmed NaFL retention results and corneal recovery in the PorCORA system via several approaches. Both fluorescence and reflective confocal microscopy confirm damage repair indicated by fluorescein retention in the cultured corneas. In addition, we have shown histological evidence that also correlates well with NaFL staining in the PorCORA assay. Here, we report the results of a 32-reference chemical validation including chemicals from the following classes: acetates, acids, alcohols, alkalis, esters, hydrocarbons, ketones, inorganics, surfactants, and several solid compounds. To determine if the PorCORA system can predict R41 or GHS Category 1, we considered corneas that retained NaFL at 21 days postdose to be R41 and GHS Category 1. ECETOC historical rabbit eye data was used to classify EU and GHS eye irritation for the 32 compounds tested. PorCORA predicted 11/11 compounds classified as R41 and 12/13 compounds classified as GHS Category 1. Since PorCORA can predict these categories, then compounds that cause damage that is reversible in the PorCORA system may be considered R36 or Category 2. Thus, PorCORA is a highly predictive method to distinguish between ocular irritancy classifications R36 or R41 and Category 1 or 2 without the use of live animals.
Keywords: ocular toxicology, in vitro/alternative test, corneal damage reversibility
205 The Reproductive Performance of the Harlan Sprague-Dawley (HSD:SD) Rat
Mylchreest E.1 and Daniels K. K.1
1Southern Research, Birmingham, AL
As part of developing reproductive toxicology testing capabilities at our facility, a pilot study was conducted to collect reproductive data for the HSD:SD rat. Sexually mature male and female rats (30/sex) obtained from Harlan (Frederick, MD) were cohabited (for up to 2 weeks) on a 1:1 basis until evidence of mating was observed. Dams were allowed to deliver and rear their offspring until weaning. The number of live and dead, individual body weights, and clinical observations were determined for pups at postnatal day (PND) 0, 4, 7, 14, and 21. Litters were culled to 4/sex (litter size permitting) on PND 4. Reproduction data for the HSD:SD rat were compared to available historical control data for the Crl:CD rat, the most commonly used rat for reproductive toxicology studies. Mating was similar in both strains (>96%), however, fertility was lower in the HSD:SD rat (79%) compared to the Crl:CD rat (89%) and gestation was longer (22.9 vs 22.1 days, respectively). The HSD:SD rat had smaller litters than the Crl:CD rat (average of 11.8 vs 13.6 pups/litter on PND 0) despite similar numbers of implantations. Fewer litters had live pups at birth compared to the historical values for the Crl:CD rat (95.7% and 99.2%, respectively). Offspring viability and survival were lower in the HSD:SD rat compared to the Crl:CD rat. The percentage born alive, day 0 to 4 viability, and day 4-21 survival were 85%, 82%, and 86% in the HSD:SD rat, respectively, compared to >98% for all 3 indices in the Crl:CD rat. Only 78% of the HSD:SD litters survived to PND 21 compared to a historical mean of 100% for the Crl:CD rat. In conclusion, these preliminary data suggest that the reproductive performance of the HSD:SD rat is lower than the historically excellent reproduction of the Crl:CD rat. Studies will be conducted to further characterize the reproductive performance of both the HSD:SD and Crl:CD rats and generate facility historical control data for both rats under similar environmental conditions.
Keywords: reproductive performance, Sprague-Dawley rat, historical control data
206 Comparison of Toxicology Parameters in Pregnant and Nonpregnant Rats
Weaver E. V.1, McDowell E.1, Fisher B.2, Latimer K. S.1, and Suttie A. W.1
1Covance Laboratories, Vienna, VA, USA
2Division of Biology, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, U.S. Food and Drug Administration, White Oak, MD, USA
Pathology and toxicology parameters were compared between pregnant and nonpregnant rats. The object was to determine the similarities and differences between pregnant and nonpregnant rats. This would aid in identifying control data which could be used when no concurrent controls are available. Pregnant Sprague-Dawley and Wistar rats were sacrificed at gestation days 15 and 18 along with age-matched nonpregnant rats from the same source. Mild differences were noted in hematology, clinical chemistry, and nonreproductive organ weight parameters between pregnant and nonpregnant rats. Microscopic pathology showed cyclic changes in the ovary, alteration of the interfetal uterine wall, cervical and vaginal mucification, and mammary gland hyperplasia as well as minor effects in adrenal cortex. Changes in the developing fetus and placenta between days 15 and 18 included loss of red blood cell nucleation and skeletal mineralization. It is concluded that, with the exception of reproductive organs, pregnant rats are similar to nonpregnant rats with respect to clinical and anatomic pathology parameters. These data will aid in interpretation of findings from pregnant rats and help determination of whether findings are likely to be due to test compound toxicity or solely due to pregnancy.
Keywords: reproductive toxicology, clinical and anatomic pathology in pregnant rats, changes in rodent pregnancy
207 Stereosepecific Proconvulsant Activity of the Enantiomers of 7-Pyridine-3-YL-1, 7-Diazaspiro[4,4] Nonane (TC-2216) in the Pentylenetetrazol-Sensitized Seizure Model in Rats
Jianxun Xie1 and Steve M. Toler1
1Targacept Inc, 200 E 1st St., Suite 300, Winston-Salem, NC, USA
7-pyridine-3-yl-1,7-diazaspiro[4,4] nonane (TC-2216) is a racemic mixture comprised of the optical enantiomers TC-5684 and TC-5685. It is a competitive antagonist at the α4β2 neuronal nicotinic acetylcholine receptor subtype. The effect of TC-2216 and its enantiomers on proconvulsant activity was evaluated using the pentylenetetrazol (PTZ)-sensitized seizure model in rats. Eleven groups of 10 male rats/group were employed and treated by oral gavage with vehicle (0.5% methyl cellulose), 3 test articles (TC-2216, TC-5684, and TC-5685) at 300, 400, or 600 mg/kg, or positive control d-amphetamine sulfate at 35 mg/kg, respectively. PTZ was administered subcutaneously at 25 mg/kg to each group one hour post the administration of vehicle, test articles, or d-amphetamine. The rats were individually observed for up to 15 minutes and the onset time of a clonic or clonic–tonic seizure was recorded. The results showed that seizure onset time in the groups of TC-2216 and TC-5684 at 600 mg/kg was statistically significantly different from the vehicle control group, whereas the seizure onset time for TC-5685 at the same dose level was not significantly different from the vehicle control group. Thus the stereoisomers of TC-2216 showed stereospecific proconvulsant activity, with the S-(+) enantiomer TC-5684 being more potent in seizure induction in the PTZ rat model than R-(−) enantiomer TC-5685.
Keywords: TC-2216/TC-5684/TC-5685, proconvulsant activity, drug development
208 Impact of Strain, Supplier, and Facility Location on Rodent Historical Control Data in General Toxicology Studies
Hollie Skaggs1 and Rachel Avery1
1Covance Laboratories, Inc, Madison, Wisconsin
Historical control data (HCD), when developed carefully and used appropriately, can provide a useful follow-up assessment for unexpected or equivocal study-related findings. Although data derived from concurrent control animals are the most relevant comparator for assessing treatment-related effects, HCD provides a range of values for findings previously observed in control animals under similar conditions that can be used to better assess study-related findings.
The development of quality HCD is highly dependent upon the degree of homogeneity within the studies from which the HCD are derived. Ideally, studies used to develop HCD should be performed within the past 2 to 5 years and should be similar in terms of animal strain, supplier, age at initiation, dosing regimen, route of administration, control article, husbandry practices, facility location, and pathology practices.
In this work, we evaluated the impact of strain, supplier, and facility location on data/end points frequently evaluated in light of HCD. Two specific parameters/end points (convulsions and pancreatic necrosis) were investigated in 2 different rat strains (Sprague-Dawley and Han Wistar) from 2 suppliers (Charles River and Harlan) at 3 different CRO sites (Covance-Chandler, Madison, and Vienna). Data were selected from GLP-compliant studies completed within the past 5 years that exhibited a high degree of similarity. Results from this work help clarify the impact of these factors on HCD derived from a CRO setting.
Keywords: general toxicology, historical control data, rodent strains
209 THE Effects of Ifosfamide and Cypermethrin on the Male Reproductive Systems of New Zealand White (NZW) and Dutch Belted (DB) Rabbits
Cotton L. M.1, Burgess S.1, Crenshaw S.2, Stitcher B.1, and Fisher B. R.3
1Covance Laboratories, Vienna, VA, USA
2The Tauri Group, Alexandria, VA, USA
3Division of Biology, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, U.S. Food and Drug Administration, White Oak, MD
ICH guidelines for Developmental and Reproductive Toxicology Segment I Studies require the use of one rodent or nonrodent species. New Zealand White (NZW) and Dutch Belted (DB) rabbits are viable models for Segment I studies, when rodents cannot be utilized. NZW and DB rabbits, from Covance Research Products, were administered either; Ifosfamide, via a single intravenous injection on day 1 or Cypermethrin, via oral gavage every other day through day 36, both of which are compounds known to effect testicular development and semen quality in NZW rabbits [1,2], in an attempt to elicit biological responses for evaluation. Administration of Ifosfamide to NZW and DB rabbits resulted in significantly decreased testis weights and total sperm count for NZW rabbits and a significant increase in the total number of abnormal sperm in both NZW and DB rabbits. Administration of Cypermethrin to NZW and DB rabbits resulted in a significant decrease in total sperm count for NZW and DB rabbits and a significant increase in the total number of abnormal sperm in NZW rabbits only. There were no Ifosfamide- or Cypermethrin-related effects on sperm motility parameters for either strain. Our study demonstrated that administration of either positive control-induced biological responses in the male reproductive systems of both NZW and DB rabbits; however, NZW rabbits appeared to be slightly more sensitive than the DB rabbits.
1Yousef, M. I., et al., J. Environ Sci Health. 2003, B38: 463-478.
2Ypsilantis, P., et al., Reprod Toxicol. 2003, 17:237-245.
Keywords: male reproductive system, sperm analysis, New Zealand White and Dutch belted rabbits
210 Cutaneous, Ophthalmologic, and Histopathologic Phototoxicity Historical Control Data in the Long-Evans Rat
S. M. Jackman1, T. S. Coston1, J. F. Baker1, L. F. Rubin1, D. B. Learn1, and A. M. Hoberman1
1Charles River Laboratories, Horsham, PA, USA
Over the past 15 years, over 100 studies have been conducted using the Long-Evans rat model to assess the cutaneous and ocular phototoxic potential of test articles. In the study design, Long-Evan rats are administered the vehicle or test article formulations, lightly anesthetized using ketamine and xylazine, and the skin and eyes are exposed to solar-simulated ultraviolet radiation (UVR). The eyes are lubricated with a sterile ophthalmological solution immediately before, during, and after UVR exposure. A positive control group administered 8-methoxypsorlen (8-MOP) is included in the study to validate the test system and demonstrate positive phototoxic responses. Rats are examined after UVR exposure for general appearance and skin responses at the UVR exposure sites on the day of exposure and 1, 2, and 3 days after UVR exposure. Ophthalmologic examinations are performed on day 4 and the eyes are fixed with Davidson’s solution for histopathologic evaluation. The accumulated historical data from rats administered vehicle formulations demonstrate the lack of ocular and cutaneous reactions that can be confused with a positive phototoxic response and provides a basis for evaluating responses that are indicative of phototoxicity. The positive 8-MOP cutaneous and ocular responses define positive ocular phototoxic responses, either by clinical or histopathologic evaluation. Any effects of UVR exposure, anesthesia, and/or experimental manipulation are not consistent with responses indicative of phototoxicity. These background historical data support the uniformity of the phototoxic responses, the sensitivity of the model, and the ability to discern responses induced by methodology from those indicative of phototoxicity. These results support the consistency of the ocular and cutaneous responses and thus the model’s utility in evaluating the phototoxic potential of test articles.
Keywords: ocular and cutaneous phototoxicity, historical control data, Long-Evans rat
211 Characterization of Normal Skin Thickness for Various Body Regions, Ages, and Genders of Yucatan Miniature Swine
Brown L.1 , 2, Kim D. Y.3, Brocksmith D.2, Hodges M.2, Liu J.1, and Bouchard G. F.1 , 2
1Sinclair Research Center, LLC, Auxvasse, MO, USA
2Sinclair BioResources, LLC, Auxvasse, MO, USA
3Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO, USA
Dermatological studies of miniature swine provides a unique opportunity for risk assessment because of demonstrated similarities to human cutaneous anatomy, biochemistry, and physiology. The Yucatan is a popular miniature pig in use in biomedical research today. Yucatan skin is slate grey–black in color, slight-moderately pigmented, and contains a sparse haircoat (sometimes called the Mexican Hairless Minipig). The physical thickness of miniature pig skin can impact drug absorption during in vivo or ex vivo studies, or affect wound healing or phototoxicity studies, therefore a good understanding of the relative dimensions of each major skin application or collection site is indicated. This poster presents the results of a histologic skin characterization study in the Yucatan pig. Four skin samples [neck, dorsum or back (lumbar), flank, abdominal] were collected by 8-mm punch biopsy from various age animals following euthanasia and immediately fixed in 10% neutral buffered formalin. The number of hair follicles per surface area were first enumerated. The skin samples were then processed, embedded in paraffin, sectioned, and stained with haematoxylin and eosin for histologic evaluation. For site-specific skin quantitation, the layers were measured microscopically using an image analyzing system containing a verified standard micron scale. Each interfollicular measurement was taken at random and in triplicate using a 40x objective and averaged for each sample. Gender, age, and body region-specific means (µM), ± SDs, and ranges are presented for 5 interfollicular skin components (full thickness, stratum corneum thickness, epidermis thickness, number of epidermal cell layers, and dermis thickness). Relative ratios of stratum corneum, epidermis, and dermis to full thickness measurements were performed. Data are compared to published measurements of adult human skin illustrating similarities or differences. These cutaneous measurement data will aid investigators in contemplating a dermal study utilizing the Yucatan miniature swine model.
Keywords: dermal/skin, morphology, biometrics
212 Normal Physiological Ranges for Hanford Miniature Swine
Ross M.1, Brown L.1 , 2, Unterreiner D.1, Hanks C.1, Liu J.1, Hodges M.2, and Bouchard G. F.1 , 2
1Sinclair Research Center, LLC, Auxvasse, MO, USA
2Sinclair BioResources, LLC, Auxvasse, MO, USA
The miniature swine have been increasingly recognized as a nonrodent model in regulatory toxicity. Members of the FDA have even published on the use of miniature swine as an alternative to canine and nonhuman primates in regulatory toxicity. The similarities between the cardiovascular, renal, and digestive systems make the miniature swine a suitable animal to model the human counterpart. The miniature swine are also the most recognized species for dermal toxicology. The Hanford miniature swine (HMS) has other attractive traits that make them a good substitute to model humans. They are omnivorous, easy to handle, prone to obesity, and will develop atherosclerosis and dyslipidemia if fed a high-fat diet. With the advent of new techniques, all routes of compound administration can be used with miniature swine. The HMS should be considered as one of the nonrodent species in toxicity testing. In an effort to generate a database on baseline information about the normal physiological status of the Hanford miniature swine, we report expanded physiological data from normal intact and naive juvenile and young adult miniature swine of both genders. The normal physiological data gathered includes growth parameters, hematology, serum chemistry, coagulation profile, urinalysis, ECG rhythm and segment intervals, and organ weights.
Keywords: reference ranges, Hanford miniature swine, minipig
213 Book of Normal Data on Selected Lineages of Miniature Swine
Brown L.1 , 2, Liu J.1, Brocksmith D.2, Golightly A.2, Hodges M.2, and Bouchard G.F.1 , 2
1Sinclair Research Center, LLC, Auxvasse, MO, USA
2Sinclair BioResources, LLC, Auxvasse, MO, USA
Sinclair BioResources LLC has recently released a comprehensive dataset on normal data (reference intervals or ranges) for their 4 lineages of miniature swine. Included are Yucatan, Hanford, Sinclair S-1, and Micro-Yucatan lineages. This effort collates and summarizes normal biological and physiological data collected over many years. Parameters covered include: uses in biomedical research, clinical pathology (hematology, chemistry, coagulation, urinalysis), organ weights, growth, ECG, background histopathology lesions, blood glucose, ocular, diet/feeding, cardiovascular, dermal, reproduction data, and references. Over 80 tables of data are presented, along with most used citations and references. These data are offered to veterinarians, biomedical investigators, preclinical clients, and university staff to facilitate research, when using our miniature swine animal models. This poster will outline the contents of this “book” and present representative data tables. Copies of the full pdf on CDROM will be available on request.
Keywords: reference intervals or ranges, miniature swine, minipig
214 Cardiovascular and Neurobehavioral Preclinical Drug Safety End Points in a Göttingen Minipig Model
Monica R. Metea1, Angela S. Burke1, John J. Setser1, Teresa R. Gleason1, Kasey L. Landis1, Laurie J. Shellhammer1, Lyubomyr (Lou) Turchyn1, Amanda Allis1, Terence T. Enama1, and Philip R. Atterson1
1WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH, USA
Minipigs are increasingly used in biomedical research, especially due to their human-like cardiac physiology. The aim of this study was to validate cardiovascular (CV) and neurobehavioral (NB) end points for preclinical drug safety assessment in this model. Methods: The CV assessment was performed with 6 males, administered moxifloxacin and propanolol in a crossover design to elicit QT prolongation and hemodynamic changes, respectively. Telemetry data (heart rate, blood pressures, and ECG) were collected from unrestrained animals using both a surgically implanted telemetry device (TL11M2-D70-PCT, DSI) and a noninvasive Jacketed External Telemetry (JET™) device. Two surgical procedures for implanting telemetry probes were compared, using either epicardial or subcutaneous lead placement. Detection of arrhythmias, as well as test article effects were compared between the 2 acquisition methods. Additionally, a minipig-specific neurobehavioral assessment or functional observational battery (FOB) including home cage, open field, and tabletop observations was developed and subsequently validated using 9 animals/sex administered d-amphetamine or clonidine. Results: (1) The QT prolongation, hemodynamic effects, and waveform abnormalities were comparable between the 2 CV acquisition methods, confirming the suitability of this model for both toxicology and safety pharmacology studies in freely moving animals. Waveform quality collected from animals with epicardial and subcutaneous lead placement surgery was similar. Pig-specific ECG characteristics (especially short R-wave) were assessed, and an approach to data analysis developed. (2) The FOB was adequate for detecting the potential behavioral, motor, and/or neurological effects of the reference compounds administered, with interobserver reliability of ~95%. In conclusion, the results validated the use of the minipig as an alternate species for preclinical drug safety assessment.
Keywords: minipigs, cardiovascular, FOB
215 Ranges of Commonly Evaluated Hematology Parameters Using Siemens Advia 2120 Hematology Analyzer From Clinically Healthy Cynomolgus Monkeys of Chinese Origin
Mingyi W. Trimble1, Wendy A. Cherry1, Michael A. Heinitz1, Jay C. Albretsen1, and Steven M. Glaza1
1Covance Laboratories, Inc, Chandler, Arizona
Ranges of hematology parameters commonly evaluated in toxicology studies were summarized from samples of clinically healthy naive Cynomolgus monkeys (Macaca fascicularis) of Chinese origin in a recently established laboratory from 2009 to 2010. The range (minimum and maximum), median, mean, and standard deviation were reported by gender for red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean cell hemoglobin concentration, platelet count, reticulocyte count and percentage, white blood cell count, and white blood cell differentials (counts and percentages for neutrophils, lymphocytes, monocytes, eosinophils, basophils, and large unstained cells). A total of 1074 blood samples were collected from 486 naive monkeys almost exclusively from the femoral vein, after fasting overnight using potassium EDTA as an anticoagulant. Samples were analyzed utilizing a Siemens Advia 2120 (software v5.9.0-MS) on the same day of collection. Those samples were collected from 252 males and 234 females at the age range of 2.5 to 9.1 and 2.6 to 6.7 years old, respectively. Approximately 97% of the animals had multiple (2 to 4) measurements per parameter and no results were excluded. These historical hematology ranges are helpful in establishing typical or expected values from clinically healthy naive Cynomolgus monkeys and can serve as a baseline in newly established laboratories. It is important to note that there are many other variables that can impact the results of hematology testing. Hematology data interpretation on a toxicological study is most appropriately conducted by comparing test article treated animals versus control animals.
Keywords: hematology parameter, historical range, primate
216 Comparative Analysis of Clinical Pathology Data in Cynomolgus Monkeys of Chinese and Vietnamese Origin
U. Zühlke1 and G. F. Weinbauer1
1Covance Laboratories GmbH, Münster, Germany
Clinical pathology (CP) data—among other diagnostic parameters—are essential for the detection and interpretation of untoward findings in preclinical studies. Cynomolgus monkeys (Macaca fascicularis) are frequently used in toxicity studies and animals from various sources, such as Mauritius, China, and Vietnam, are available from established breeders. Clinical pathology parameters were found to be different among nonhuman primates (NHPs) of Mauritian and Asian origin previously. Hence, the aim of the present work was to compare CP data from group housed, untreated, and unsedated cynomolgus monkeys of Chinese and Vietnamese origin that were collected from 2006 to 2008. Individual data from 86 Chinese and 520 Vietnamese male and female animals were analyzed for haematological and clinical chemistry parameters.
Cynomolgus monkeys of Vietnamese origin had statistically significant higher (P < .05) percentages of eosinophils (42.9%), monocytes (31.0%), reticulocytes (29.1%), and lymphocytes (18.7%). For serum biochemistry parameters mean alkaline phosphatase (63.7%), lactate dehydrogenase (30.5%), free fatty acids (28.6%), HDL cholesterol (26.7%), and gamma GT (17.8%) were significantly higher in monkeys from China.
In summary, some statistically significant CP differences were seen among Asian NHPs of different origin. However, as reference ranges were overlapping, these changes were considered to be not biologically relevant.
Keywords: cynomolgus monkey, chinese/vietnamese origin, clinical pathology
217 Noninvasive Blood Pressure Monitoring in Ambulatory Beagle Dogs
M. Bétat1, J. G. Napoléoni2, A. Simonnard1, and R. Forster1
1CIT Technologies
2EMKA Technologies
The development of therapeutic biologics, for which Safety Pharmacology (SP) standard study designs are not well adapted, has led pharmaceutical companies to develop ways to integrate SP end points in toxicology studies. For this purpose, external telemetry, linked to a video camera, today allows continuous recording of ECG, respiration, and behavior in ambulatory nonrodent models. One of the major physiological signals missing from this panel until now was arterial blood pressure (ABP) for invasive approaches have been required. The aim of this study was to evaluate a novel technique for telemetered Non Invasive Blood Pressure (NIBP) recording developed by EMKA Technologies. The first step of the study was an evaluation of the habituation of dogs to the recording equipment. In a second step, the model was validated for evaluation of the potential of a drug to modify ABP. Finally a cross-validation study was performed comparing concomitant recordings of ABP signals from an implanted catheter (internal telemetry: IntT) and from the NIBP system (external telemetry: ExtT). Four beagle dogs implanted with telemetric devices were used. After habituation to the external telemetry equipment, they were treated with a reference drug, Prazosin. Decreases in systolic (IntT = –24%, ExtT = –16%) and mean ABP (IntT = –24%, ExtT = –25%) were demonstrated with both techniques. This study demonstrates that NIBP measured by external telemetry is an appropriate alternative method for recording ABP in dogs over long periods without the need for invasive surgery.
218 Significance of Clitoral Size in Cynomolgus Monkeys (Macaca fascicularis)
C. Marc Luetjens1, Belen Tornesi2, Antje Fuchs1, and Gerhard Weinbauer1
1Covance Laboratories GmbH, Münster, Germany
2Global Pharmaceutical Research & Development, Abbott Laboratories, Abbott Park, IL, USA
External examination of reproductive organs constitutes a major parameter in developmental toxicology. For primates, the critical steps of genital differentiation mostly occur prenatally, so that the shape of the external genitalia is complete at birth. Any alterations in size/shape of external genitalia may greatly impact the interpretation of study outcomes in nonhuman primates (NHP) as the number of animals used and the statistical power of studies is low. We recently evaluated the incidence of clitoris size variability in our female monkey colony. One hundred animals of Asian origin were selected randomly and clitoral size was assessed by imaging of the genitalia together with a scale bar. Animals were 1.5-8 years old (median age: 4.2 years) and weighed between 1.6 and 6.4 kg (median weight: 3.1). In 53 animals no clitoris was visible without opening the genital labia whilst the remaining 47 females exhibited a protruding clitoris. To evaluate the size of these protrusions the surface area in the images of the clitoris were determined from images. Clitoral surface area ranged from 3.4 mm2 to 19 mm2 with a median of 9.62 mm2. No significant correlation with age or weight of the females could be observed. Comparison of clitoral size in 15 primiparous monkeys with nine female offspring revealed, that 8 mothers and one infant exhibited clitoral enlargement. This infant was daughter to a mother with a nonprotruding clitoris. Hence, no association between maternal and offspring clitoral size was apparent. In conclusion, clitoral enlargement is considered a common variation among cynomolgus monkeys. Our limited mother–infant data suggest that clitoral enlargement represents a noninherited variation of the clitoris shape and in future pre/postnatal reproductive studies this observation has to be considered in the data interpretation.
Keywords: cynomolgus monkey, clitoris size, reproductive toxicology
219 A Review of Early Discovery Hepatotoxicity Screening Using the In Vivo Zebrafish Model
A. J. Hill1, M. Jones1, A. Dodd1, and H. Diekmann1
1Evotec AG, Hamburg, Germany
Toxicity remains one of the most prominent causes of candidate drug failure. Commonly identified during late stage animal testing, or even postmarketing in the case of idiosyncratic hepatotoxicity, there is an increasing drive in the pharmaceutical industry to reduce this expensive attrition by integrating new predictive assays to identify possible toxicity and safety liabilities much earlier in discovery, as well as to help prioritize better quality follow-up analogs.
Although well-established in the field of developmental and genetic research for the past 40 years, the zebrafish vertebrate model has only been used for drug testing over the past decade. However, due to numerous advantages over other in vivo screens, including the use of an in vitro-like assay format requiring only single milligrams of test compound to obtain a swift, full-dose response readout, the zebrafish has gained increasing popularity.
In particular, zebrafish larvae are now being utilized regularly as a model for the evaluation of hepatotoxicity early in discovery. This screen is primarily based upon a phenotypic readout for chemically induced alterations to hepatic tissue, hepatomegaly, loss of bile, and an assessment for yolk retention as a measure of hepatic function. Larvae are exposed postembryogenesis to each test compound for 48 hours using a standardized dose range after which they are anaesthetized and assessed for aberrant morphology.
As with all models there have been hurdles which must be overcome before achieving global acceptance, of which possible metabolism and issues with absorption have both benefited from the addition of bioanalysis. This bioanalytical measurement of the tissue and dosing medium allows correlation of any observed toxic phenotype with a level of actual compound uptake, versus having to rely on an assumed relationship with aqueous concentration. False negatives due to poor uptake as well as overt toxicity attributable to overdosing of compound have been demonstrated.
After reviewing the outcome of several studies consisting of over 70 compounds a high level of predictivity has been demonstrated for the zebrafish in vivo hepatotoxicity model and as such continues to show great potential as a screening tool to improve decision making in drug discovery.
220 A Review of Developmental Toxicity Screening Using Zebrafish Larvae
A. J. Hill1, M. Jones1, A. Dodd1, and H. Diekmann1
1Evotec AG, Hamburg, Germany
One of the most prominent and expensive causes of candidate drug failure is toxicity identified late in drug discovery. The pharmaceutical industry is therefore interested to reduce this attrition by integrating new predictive assays to help determine these liabilities. The zebrafish has gained increasing popularity for drug testing over the past decade due to numerous advantages over other vertebrate in vivo screens, including the use of an in vitro-like assay format requiring only single milligrams of test compound to obtain a swift, full dose response readout. In past years, Evotec has validated its zebrafish developmental toxicity assay with in excess of 70 compounds with known teratogenic potential in mammalian embryo foetal developmental toxicity studies.
Fertilized eggs (n = 14 per concentration) were exposed to a 6-point standard aqueous concentration range from 4 hours until 96 hours postfertilization, followed by morphological assessment (28 end points) using a dissecting stereomicroscope. Since the uptake into the zebrafish larva is compound dependent and can vary significantly even between similar compounds, the actual body burden was determined at the respective NOECs and LOECs.
The majority of drugs testing positive in traditional mammalian tests produced similar results in zebrafish larvae, although the specific phenotypes observed were different for some compounds. False negatives could be identified due to lack of compound getting into the larvae. Taking into account the interpretation from bioanalysis results, the overall predictivity for all compounds tested was 87%.
Based on these results, the zebrafish assay shows potential as a screening tool for developmental toxicity. Due to the higher throughput and lower costs compared to mammalian developmental toxicity assays, the larval zebrafish screen would be suitable for testing large numbers of compounds in early drug discovery.
Keywords: zebrafish, alternative toxicity model, developmental toxicity
221 Species Specific Clinical Pathology
Yamamoto B.1, Carter R.1, and Daniels, K. K.1
1Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205
The prevailing expectation from private and government sectors is repeatable, quality data from the pharmaceutical and contract research-based industries. Studies are performed in compliance with the US Food and Drug Administration’s Good Laboratory Practice (GLP) Regulations (21 CFR Part 58) to meet this criterion. Critically, no commercially available high throughput clinical pathology analyzers are manufactured for preclinical species’ assessment. Thus, it is paramount to establish on a per species basis, the utility and interpretability of animal data generated using such instruments. Validated analyzers are utilized by the Clinical Pathology Laboratory at Southern Research to generate hematology, clinical chemistry, and coagulation data according to industry expectation and GLP regulations. Species-specific qualifications have also been conducted on these analyzers to identify potential limitations in the analysis of each species.
Standard protocols were established for validation of the clinical pathology analyzers, the Advia 120, Cobas c501, and ACL Elite for hematological, clinical chemistry, and coagulation analyses, respectively. The validation process confirmed that the analyzers met manufacturer-based specifications for installation, operation, and performance qualifications. Subsequently, a standard validation plan was established for the qualification of individual species. Using untreated specimens procured from a commercial vendor, species-specific precision and suitability data for the analytes were generated. Data were then evaluated against predetermined acceptance criteria established by a Board Certified Veterinary Clinical Pathologist and compared to published reference interval ranges for the species.
Data from the Beagle dog, Sprague-Dawley rat, and the less commonly used Golden Syrian hamster will be presented to illustrate the lessons learned and the species-specific qualification process subsequently developed by Southern Research.
Keywords: validation, pathology, species-specific
General Toxicology
300 Safety and Immunogenicity of Measles Vaccine, Dry Powder in Rhesus Monkeys
Godin C. S.1, Krause E.1, Griffin D.2, Shermer C.3, Winston S.4, Sievers R.5, Lin W.-H.2, and Quinn B.4
1AVANZA Laboratories, Gaithersburg, MD
2Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD
3BD Technologies, RTP, NC
4Aktiv-Dry LLC, Boulder, CO
Needle-free aerosol delivery of a dry-powder measles vaccine may provide an effective and low-cost means of immunization of children in developing countries. The advantages over the freeze-dried marketed product are that the dry-powder vaccine requires no reconstitution or use of needles for clinical use. Aktiv-Dry has developed an alternative to freeze-drying that results in preservation of viral potency and particle sizes optimal for alveolar deposition. The purpose of this study was to determine the potential toxicity of Measles Vaccine, Dry Powder (Vaccine) when administered twice 3 weeks apart to rhesus monkeys by inhalation using either the PuffHaler® or SoloventTM dry-powder inhalers. Three groups (6/sex/group) received 50 mg of the control article via SoloventTM inhaler or 50 mg of the Vaccine via PuffHaler or SoloventTM inhalers. The first half of animals was euthanized 1 week following the first dose, and the remaining animals were euthanized 14 days following the second dose. Administration of Vaccine via PuffHaler or SoloventTM inhalers was well tolerated. There were no test article-related effects on mortality, clinical observations, body weights, food consumption, body temperatures, respiratory profile, breathing patterns, postdose evaluation of the nasal cavity, mouth, and pharynx, ocular examination, electrocardiogram evaluation, clinical pathology, gross pathology observations, organ weights, and histopathology. Administration of vaccine to all animals resulted in detection of measles virus RNA in either bronchoalveolar lavage or lung tissue samples after 1 dose. Serological analysis also confirmed that all animals in the study were measles seronegative prior to the first dose, and that the animals in the control group were not exposed to measles virus during the in-life phase of the study. Therefore, the no-observed-adverse effect level of the dry powder measles vaccine is 50 mg when administered once or twice via PuffHaler or SoloventTM inhalers. This work was funded by Aktiv-Dry LLC in support of its Grand Challenges in Global Health Initiative grant administered by the Foundation for the National Institutes of Health.
Keywords: vaccine, respiratory delivery, dry-powder
301 Safety and Immunogenicity of an Intranasal Anthrax Vaccine in New Zealand White Rabbits
Godin C. S.1 and Klas S.2
1AVANZA Laboratories, Gaithersburg, MD, USA
2LigoCyte Pharmaceuticals, Inc., Bozeman, MT, USA
LigoCyte's intranasal dry powder anthrax vaccine* (Vaccine), based on recombinant protective antigen (rPA), protects against both the anthrax toxin and the infectious disease. The Vaccine offers the potential for long-term stability and protects rabbits against inhaled anthrax. The purpose of this study was to evaluate the potential toxicity of the Vaccine following 4 repeated intranasal doses (N + 1 design based on a clinical regimen of 3 vaccinations) to New Zealand White rabbits. Sixty rabbits were assigned to one of 3 groups (10 animals/sex/group) and administered placebo, adjuvant-excipient, or Vaccine. Four days following the last dose, half were euthanized and necropsied, and the remaining half were euthanized and necropsied after a 1-month recovery interval. Parameters evaluated include mortality, clinical observations, body weights, food consumption, body temperature, nasal/pharyngeal examinations, ophthalmologic examinations, clinical pathology, C-reactive protein and serum protein electrophoresis, immunology, gross pathology, organ weights, and histopathology. There was no toxicity of the Vaccine; rPA-specific IgG and TNA responses were observed in vaccinated animals but not in animals treated with the adjuvant/excipient or placebo. In animals sacrificed 4 days after the final dose, minimal heterophilic infiltrates and apical blebbing of the respiratory epithelium lining the nasal cavity and minimal luminal exudate was present in animals treated with the adjuvant-excipient (alone or in combination with the Vaccine). Changes in nasal histopathology were not present in recovery animals, indicating full recovery of the nasal epithelium (ie reversibility of the effect) and the observation was therefore not considered adverse. There were several adjuvant-excipient-related increases but they were of no toxicological significance and were fully recoverable. In conclusion, there were no adverse effects on potential target organs and no evidence for delayed onset of toxicity. Based on the findings, the No-Observed-Adverse-Effect-Level (NOAEL) is a total dose of 1200 µg rPA contained in LigoCyte Anthrax Vaccine when dosed 4 times to both nostrils.
*The Vaccine incorporates chitosan. This application of chitosan (ChiSys®) has been licensed from Archimedes Development, Ltd.
Keywords: vaccine, anthrax, intranasal
302 Safety and Immunogenicity of Seasonal Influenza Trivalent Vlp Vaccine in New Zealand White Rabbits
Godin C. S.1, Song H.,2, and Connoly K2
1AVANZA Laboratories, Gaithersburg, MD, USA
2Novavax, Inc, Rockville, MD, USA
Virus-like particles (VLPs) are similar in structure to a virus but lack the genetic material required for viral replication. Because they more closely match an individual viral strain, VLPs can trigger a robust immune response without the need for adjuvant, and pose no risk of infection. The purpose of this study was to evaluate the potential toxicity and immunogenicity of Seasonal Trivalent Influenza Vaccine (Vaccine). Five rabbits/sex/group were assigned to one of 3 treatment groups, and 5 additional rabbits/sex/group were assigned as recovery animals. Animals were administered 0.5 mL of either control article or Vaccine at 30 or 85 µg HA (surface neutralizing antibody) twice by intramuscular injection 2 weeks apart. Three days following the second dose, the first 5 animals/sex/group were euthanized and necropsied, and the remaining animals were euthanized and necropsied one month following the second dose. Parameters evaluated included mortality, clinical observations, body weights, Draize observations, food consumption, body temperature, ocular examination, clinical pathology, hemagglutination inhibition (HAI), gross pathology, absolute and relative organ weights, and histopathology. Following the second dose of the vaccine, there were no dermal Draize observations in control animals of either sex or in treated females. However, several treated male animals had observations of slight edema and/or erythema at injection sites that were transient in nature but not adverse because of the low severity scores. Additional test article-related findings in treated males and females consisted of increased mean total white blood cell counts, increased absolute monocyte counts, and increased mean plasma fibrinogen. In addition, increased iliac lymph node and spleen weights and splenic lymphoid hyperplasia were noted in high-dose males, and subacute inflammation was present at the injection site of high-dose males and females. All of these effects were related to immune stimulation and nonadverse. Analysis of HAI for anti-Influenza (A/Brisbane 59/07 [H1], A/Brisbane 10/07 [H3], and B/Florida 4/06 [B]) antibodies indicated that treated animals mounted an HAI response to vaccination. Based on these findings, the No-Observed-Adverse-Effect Level for the vaccine is 85 µg HA.
Keywords: vaccine, virus-like particle, immunogenicity
303 Medi-528 Anti-Interleukin-9 Antibody: Use of a Murine Surrogate Antibody (MM9C1) for Nonclinical Safety Assessments
Manetz T. S.1, Leininger J.1, Robbie G.1, Ryan P. R.1, Kolbeck R.1, Humbles A.1, and Dixit R.1
1MedImmune, LLC, Gaithersburg, MD, USA
Interleukin-9 (IL-9) is a multifunctional cytokine that enhances the growth and/or activity of a variety of cells and inflammatory mediators that have been implicated in the pathogenesis of asthma and other inflammatory diseases. MEDI-528 is a fully humanized IgG1 kappa monoclonal antibody directed against human IL-9, currently in Phase 2b testing for treatment of patients with moderate-to-severe persistent asthma. In support of clinical development, nonclinical safety studies were conducted with MEDI-528 administration to cynomolgus monkeys. The nonclinical safety of MEDI-528 in monkeys was investigated in repeat subcutaneous (SC) dose studies of up to 26 weeks duration and showed no overt adverse findings or target organs of toxicity at the top dose level tested of 100 mg/kg/week. Since MEDI 528 binds to monkey IL-9 at a lower affinity than to human IL-9, additional nonclinical safety studies were performed in BALB/c mice using a surrogate murine anti-mouse IL-9 monoclonal antibody (referred to as MM9C1). This surrogate shows affinity for murine IL-9 similar to MEDI-528 affinity for human IL-9, exhibits strong neutralizing activity in vitro and in vivo, and allows evaluation of possible mechanism-based toxicity of an anti-IL-9 antibody in a pharmacologically relevant animal model. The nonclinical safety of the MM9C1 surrogate in BALB/c mice was investigated in repeat subcutaneous (SC) dose studies of up to 26 weeks duration. Similarly, there were no overt adverse findings or target organs of toxicity identified at the top dose level tested of 50 mg/kg/week. In conclusion, use of a surrogate antibody was useful for understanding the potential for adverse effects due to exaggerated pharmacology.
304 Hepatic and Vascular Changes Following Repeated Administration of an Anti-DLL4 Antibody
Krishna P. Allamneni1, Amrita V. Kamath2, Priyanka Gupta2, Heather Taylor1, Khiem Tran1, Shana Dalton4, Minhong Yan3, and Joseph C. Beyer1
1Safety Assessment, Genentech, Inc, South San Francisco, CA, USA
2Early Dev PKPD, Genentech, Inc, South San Francisco, CA, USA
3Tumor Biology & Angiogenesis, Genentech, Inc, South San Francisco, California, USA
4Covance Laboratories, Inc, Madison, Wisconsin, USA
Targeting Delta-like 4 (DLL4), a highly selective arterial endothelial cell-specific target, represents a novel therapeutic approach against tumor angiogenesis. In preclinical xenograft models, DLL4-blockade is associated with an increased density of functionally defective tumor vessels resulting in a growth inhibition of both VEGF-dependent and -independent tumors. We describe the safety assessment of an anti-DLL4 antibody administered intravenously for eight weeks followed by a dose-free recovery period, to Sprague-Dawley rats and cynomolgus macaques. Several unexpected drug-related findings were observed in both species in the context of dose-dependent systemic exposures maintained throughout the dosing period, with variable degrees of reversibility. Drug-related changes in clinical pathology included increases in reticulocytes associated with decreases in red cell mass in both species; and increases in serum ALT/AST observed primarily in rats. Most notable of the microscopic findings were marked histopathological changes in liver including bridging centrilobular sinusoidal dilatation in both rats and monkeys and proliferative vascular masses in rats. In addition, findings related to the expected pharmacology of blocking physiological angiogenesis i.e., physeal dysplasia and arrested ovarian function (rat only), and known effects of DLL4 in T cell survival (thymic atrophy) were noted. The diversity and character of the findings after anti-DLL4 administration suggests interplay between DLL4 and other angiogenic cytokines, possibly VEGF, in the maintenance of normal vasculature.
Keywords: therapeutic antibody, safety assessment, angiogenesis
305 Safety and Toxicokinetic Profile of Fidaxomicin Following Subchronic Toxicity Study in Beagle Dogs
C. K. Korgaonkar1,2, P. Sears1,2, Celeste R. MacElrevey1,2, and G. Gopakrishnan1,2
1WIL Research Laboratories, LLC, Ashland, OH
2Optimer Pharmaceuticals Inc, San Diego, CA
Fidaxomicin (OPT-80; PAR-101) is the first in a new class of narrow spectrum macrocyclic antibiotics that selectively eradicates pathogenic Clostridium difficile with minimal disruption of normal intestinal flora. Fidaxomicin clinical trial formulation (fidaxomicin 200 mg tablets) was administered in capsules once daily, for a minimum of 91 days to 3 groups (Groups 2-4) of Beagle dogs at dosage levels of 5, 16 and 48 tablets/day (1000, 3200 and 9600 mg/day) with a concurrent control group (Group 1) that received empty capsules, followed by a 28-day nondosing recovery period for the control and high dose groups. There were no fidaxomicin-related effects on survival, body weights, food consumption, clinical or anatomic pathology parameters, electrocardiography or ophthalmic examinations. The effects of fidaxomicin at dosage levels of 16 and 48 tablets/day were limited to mild gastrointestinal effects as evidenced by pale/yellow feces and sporadic emesis incidents. Systemic exposure to fidaxomicin and OP-1118 (main metabolite) generally increased more than proportionally to increasing fidaxomicin dosage for both genders over the 5 to 48 tablets/day dose group range, with the mean fidaxomicin Cmax in the high dose group ranging between 3010 and 4670 ng/mL across both genders and sampling days. Exposure to fidaxomicin was at least 5-fold greater than exposure to OP-1118 for all animals with the ratio of AUClast for the metabolite to AUClast for the parent ranging from 0.02 to 0.2 and from 0.01 to 0.18 for male and female dogs, respectively. The maximal fecal concentrations for fidaxomicin and OP-1118 were generally noted in the 48 tablets/day dose group with similar values for males and females at day 3 or day 86 intervals. The fecal concentrations of OP-1118 were approximately 0.5 to 4.3% of the fecal concentration of fidaxomicin. In conclusion, fidaxomicin was well tolerated even at the high dose of 48 tablets/day. Based on these results, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was 48 tablets/day, the highest dose administered (equivalent to a fidaxomicin dosage level of 9.6 g/animal/day).
Keywords: sub-chronic toxicity, fidaxomicin (PAR-101 or OPT-80), Clostridium difficile
306 Withdrawn
307 Withdrawn
308 Safety Evaluation of Algal Oil From Schizochytrium SP
I. Fedorova-Dahms1, P. A. Marone2, E. Bailey-Hall1, and A. S. Ryan1
1Martek Biosciences Corporation, Columbia, MD 21045
2Eurofins Product Safety Laboratories, Dayton, NJ 08810
The safety of Algal Oil from Schizochytrium sp. was evaluated by testing for gene mutations, clastogenicity and aneugenicity, and in a subchronic 90-day Sprague-Dawley rat dietary study. The results of all genotoxicity tests were negative. The 90-day study involved dietary exposure to 0.5, 1.5, and 5wt% of Algal Oil and 2 control diets: a standard low-fat basal diet and a basal diet supplemented with 5wt% menhaden oil (the fish oil control). There were no treatment-related effects of Algal Oil on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, coagulation, or urinalysis parameters. Increased mean liver weights and alveolar histiocytosis were observed in both the fish oil control and the high-dose Algal Oil-treated animals and were not considered to be adverse. Algal Oil was bioavailable as demonstrated by the dose-related increase of DHA and EPA levels in tissues and plasma. The no-observed-adverse-effect level (NOAEL) for Algal Oil under the conditions of this study was 5wt% in the diet, equivalent to an overall average Algal Oil intake of 3250 mg/kg bw/day for male and female rats. Based on the body surface area, the human equivalent dose is about 30 g Algal Oil/day for a 60 kg adult.
Keywords: DHA, algal oil, Omega-3 fatty acids
309 Withdrawn
Category: Special Studies
400 T-Dependent Antibody Response to Co- Administration of 2 Antigens (KLH AND TT) in Cynomolgus Monkey
C. Dumont1, J. N. Tichenor1, R. Bourgeois1, L. LeSauteur1, and C. M. Satterwhite
1Charles River, Preclinical Services
Monitoring the T-cell dependent antibody response (TDAR) to an antigen such as keyhole limpet hemocyanine (KLH) or tetanus toxoid (TT) represents routine humoral immune function evaluation within preclinical studies. The use of either KLH or TT in Cynomolgus monkeys has been previously reported, but their co-administration has been less documented. The option of using 2 neoantigens in a single study allows for better adaptation to various study designs such as: evaluating primary and secondary antibody responses within an IND-enabling study (ie ≤ 28 days) or evaluating TDAR of the same animals throughout the dosing and recovery periods. In this study, TDAR to immunization with both KLH (primary/secondary) and TT (primary) were evaluated via validated ELISA methods, in Cynomolgus monkeys untreated and treated with a known immunosuppressant, FK506. Animals were immunized with KLH 7 and 28 days after the FK506 dosing initiation (day 1) to measure primary and secondary humoral immune response, while TT was administered on day 28 to measure a primary humoral immune response. In FK506 untreated immunized animals, a primary and secondary immune response to KLH and a primary immune response to TT was detected for both antigens. In the presence of FK506, a dose-dependant reduction in the antibodies to KLH and TT was detected. These data indicate that both antigens are detectable after co-administration and the TDAR assay is sensitive enough to detect decreases in humoral immune function.
Keywords: immunotoxicology, immune function testing (preclinical), T-dependent antibody response (TDAR)
401 Integration of Pig-A, Micronucleus, Chromosome Aberration, and Comet Assays Into a 28-Day Rodent Toxicity Study With 4-Nitroquinoline-1-Oxide (4NQO)
Leon F. Stankowski Jr1, Daniel J. Roberts1, Hepei Chen1, Timothy E. Lawlor1, Marie McKeon1, Hemalatha Murli1, Ajit Thakur1, and Yong Xu1
1Covance Laboratories, Inc, Vienna, VA, USA
During a multi-lab validation, we examined induction of Pig-a mutant red blood cells (RBCs) and reticulocytes (RETs) by flow cytometry (FCM) during a 28-day study in male Sprague-Dawley rats using 4NQO (1.25, 2.50, 3.75, 5.00, and 7.50 mg/kg/day; high dose group sacrificed on day 15 due to excessive morbidity/mortality). Also evaluated were micronucleated RETs (mnRETs) by FCM, DNA damage using the comet assay (blood, liver, and stomach), and chromosome aberrations (CAbs) in peripheral blood lymphocytes (PBLs). All endpoints were analyzed at 2 or more timepoints where possible. Dose-dependent increases were observed, and they reached statistically significant levels (P < .05) for: Pig-a mutant RBCs at 7.50 mg/kg/day on day 15 and 5.00 mg/kg/day on day 29; Pig-a mutant RETs at 5.00 mg/kg/day on day 15 and 2.50 mg/kg/day on day 29; and mnRETs at 2.50 mg/kg/day on day 29. Generally, the responses were higher at the later timepoints, indicating accumulation of genetic damage with repeat dosing. The largest increases observed (fold-increase compared to control, where significant; all at 5.00 mg/kg/day on day 29) were: mutant RETs (21X) > mutant RBCs (9.0X) > mnRETs (2.0X). In contrast, no such increases were observed for CAbs in PBL (days 4 and 29), or DNA damage in liver (days 15 and 29), stomach (day 29), or PBL (days 1, 15, and 29). These results demonstrate the utility and relative sensitivity of the Pig-a endpoint in repeat dose studies under the conditions used herein. Additional work is in progress to evaluate responses of these endpoints using the same cumulative doses as evaluated here, but administered as a single dose, or fractionated into 3 daily doses as used in a typical Comet/micronucleus combination assay.
Keywords: genotoxicity, Pig-a, 4-nitroquinoline-1-oxide
402 Evaluation of the Carcinogenic Potential of FG-2216, a Small Molecule Inhibitor of HIF-PH in CD-1 Mice and Sprague-Dawley rats
Beck J.1, Meier-Davis S.1, Henschel C.1, Chou J.1, Lin A.1, and del Balzo U.1
1FibroGen, San Francisco, CA, USA
The carcinogenic potential of FG-2216, an orally active, heterocyclic small molecule inhibitor of the HIF prolyl hydroxylase (HIF-PH) enzymes being developed by FibroGen Inc for the treatment of anemia was evaluated in CD-1 mice and Sprague-Dawley rats. Inhibition of HIF-PH by FG-2216 leads to a rapid increase in the cytoplasmic concentration of HIF, its translocation to the nucleus and upregulation of HIF-responsive genes including erythropoietin (EPO). FG-2216 was evaluated following administration by oral gavage 3 times weekly for up to 104 weeks to mice at 0, 15, 30, and 80 mg/kg/dose and rats at doses of 0, 5, 12, and 25 mg/kg. Assessment of toxicity in both studies was based on survival, clinical observations, body weight and food consumption, hematology, clinical chemistry, macroscopic and microscopic pathology. Treatment with FG-2216 did not induce a significant increase in mortality compared to vehicle controls in either study. In both studies, test article-associated changes in hematology parameters were consistent with the pharmacologic activity of FG-2216 and included elevations in erythrocyte count, hemoglobin, hematocrit, absolute reticulocyte count, mean corpuscular volume, and mean corpuscular hemoglobin values in mice and rats at the highest dose tested. No test article-associated macroscopic, microscopic, or neoplastic effects were observed in either study. Relatively consistent AUC and Cmax values were found over the study duration suggesting that FG-2216 systemic exposure remained constant for both species. In conclusion, FG-2216 administered by oral gavage to mice and rats 3 times weekly for up to 104 weeks resulted in consistent exposure and hematologic effects at 80 mg/kg and 25 mg/kg, respectively. FG-2216 treatment had no test-article effect on survival or development of neoplastic lesions in CD-1 mice or Sprague-Dawley rats at doses of up to 80 mg/kg and 25 mg/kg, respectively.
Keywords: carcinogenicity, HIF propyl hydroxylase, anemia
403 Approaches for Reducing the Number of Animals Used for 26-Week Alternate Carcinogenicity Studies in Transgenic Mice (Tg.rasH2)
Sudhir A. Shah1, Madhav G. Paranjpe1, Martin. L. Wenk1, and Eias A. Zahalka1
1Toxicology, BioReliance, Rockville, MD, USA
The current lack of guidance and agreement on the adequate number of animals used for positive controls and toxicokinetics (TK) in the short-term (26-weeks) transgenic mouse carcinogenicity studies has resulted in unnecessary use of excessive animals. We compared our data from 15 studies using transgenic mice (+/− heterozygous c-Ha-ras; Tg.rasH2) and 28-day dose range studies using wild-type mice (CByB6F1-Tg(HRAS)2Jic (−/− homozygous c-Ha-ras; CByB6F1)). In each of the studies, the positive control group for validating the assay contained 25 animals/sex which were administered a total of 3 i.p. injections of Urethane (1000 mg/kg) on study days 1, 3, and 5. The animals were sacrificed on week 17 ± 1. The macroscopic, microscopic data and clinical observations showed that 95% of the animals exhibited a statistically significant increase in lung and spleen tumors as compared to the negative control group. We reanalyzed the same datasets by randomly excluding 10 animals/sex, resulting in an analysis of 15 animals/sex. Results showed that this number of animals was sufficient to produce a statistically significant response. The phase I and phase II enzymes are minimally changed by gene manipulation in Tg.rasH2 mice, and therefore the use of CByB6F1 wild-type mice is appropriate for TK evaluation in carcinogenicity studies. In the 28-day dose range studies, we evaluated toxicity and TK of the drugs with a separate cohort of 3 CByB6F1 mice/sex/dose/time-point, up to 6 time-points from 1 hour to 24 hours following drug administration on study day 1 and/or day 27 post daily dose. In the 26-week Tg.rasH2 carcinogenicity studies the collection of blood was at Tmax only, usually at weeks 13 and 26 of the study. The TK parameters were calculated for drug and/or metabolites in plasma using WinNonlin®. Based on acceptance of our protocols by the Carcinogenicity Assessment Committee (CAC) at the FDA, we recommend using 15 animals/sex for positive controls, full TK evaluation in 28-day study for calculating Tmax, Cmax and AUC, and a single time-point “exposure bleed” with 3 mice/sex/dose at the Tmax in 26-week Tg.rasH2 study to confirm the Cmax and to significantly reduce the number of animals used in carcinogenicity studies.
Keywords: transgenic mice, carcinogenicity, toxicokinetics
404 Developmental Toxicity Study of a Monoclonal Antibody When Administered Intravenously in New Zealand White Rabbits
Fleming-Weis S. L.1, Aleksandrowicz D.1, Hu X.1, Stevenson L.1, Donley J.1, Gharakhani E.1, Geanacou J.1, Leitermann K.1, Clarke J.1, and Hoberman A.2
1Biogen Idec, Cambridge, MA, USA
2Charles River Laboratories, Horsham, PA, USA
The purpose of this study was to evaluate effects of a fully-human IgG1 monoclonal antibody targeting a pro-inflammatory cytokine, when administered intravenously in presumed-pregnant New Zealand White rabbits and to evaluate effects on the development of the embryo and fetus following maternal dose administration from implantation to closure of the hard palate. Twenty-eight rabbits per group received the test article by IV slow bolus injection on days 7, 13, and 19 of presumed gestation at doses of 0, 10, 50, and 200 mg/kg/dose. There were no test article-related maternal effects observed. A reduction in fetal body weight and an increase in resorptions were observed in the 200 mg/kg dosage group. Although fetal body weights were also significantly reduced in the 50 mg/kg group, this result was not significant after accounting for litter-size as a covariate. There were no test article-related effects on any other reproductive or developmental parameter. Evidence of systemic maternal and fetal exposure was observed in all treated rabbits following both single and repeat dose administration. Maternal exposures were reasonably proportional to dose on DGs 7 and 19. Reduced exposure resulting from anti-drug antibody (ADA) was observed in some doses; however, given the small number of TK animals affected (29%), there was little impact on study interpretation. Transplacental exposure was observed during the period of organogenesis. Fetal exposure was low but measurable on DG19 and was generally greater than the respective maternal concentration on day 29. In conclusion, there was no maternal toxicity associated with test article administration up to the highest dose of 200 mg/kg/dose and therefore the NOAEL for maternal toxicity is 200 mg/kg. The decrease in fetal body weight, together with the increase in resorptions observed at the 200 mg/kg/dose level resulted in a developmental NOAEL of 50 mg/kg.
Keywords: reproductive toxicity, nonrodent, monoclonal antibody, pharmaceutical
405 The Effects of Continuous Infusion Rates on Pre- and Postnatal Development, Including Maternal Function in Rats
M. McKeon1,2, B. Fisher1,2, K. Latimer1,2, A. Suttie1,2, B. Makle1,2, and C. Williams1,2
1Covance Laboratories, Vienna, VA, USA
2Division of Biology, Office of Science and Engineering Laboratories, Center for Devices and Radiological health, US Food and Drug Administration, White Oak, MD
Continuous infusion is used for drugs with characteristics such as low pH, or high osmolarity that prevent administration via intravenous bolus injection. This study evaluated the maternal and embryo/fetal pre and postnatal effects of saline when administered at rates of 60, 100 and 150 mL/kg/day via continuous infusion to femorally cannulated, time-mated rats (Crl:CD(SD). One control group was not infused while a second control group received the maintenance rate (0.35 mL/hour). Dams (10/group) received infusions from GD6 to GD17 (teratology phase: Cesarean sections and fetal examination GD21) or GD6 to LD20 (delivery phase: necropsy of the F0 and evaluation of pups after weaning). In the teratology phase, there was no effect of infusion rate on maternal body weight or food consumption, no abnormal clinical signs, and no remarkable infusion-associated changes in clinical pathology, macroscopic findings, mean gravid uterine weights, corrected mean maternal body weights, or organ weights and no fetal anomalies attributed to changes in infusion flow rate. In the delivery phase, F0 females delivered and cared for pups while being infused and there were no developmental differences in the pups based upon the infusion rate. Dams all had increased urinary output of sodium and chloride consistent with maintenance of homeostasis. Microscopic observations incuded. Perivascular eosinophilc cuffing in the lungs and typical observations of intimal hyperplasia and fibrosis, inflammation, thrombus and abcesses at the infusion site and foreign body reaction at the catheterization site.
Keywords: developmental and reproductive toxicology, continuous infusion, time-mated rats
406 Trefoil Factor 3 as a Urinary Biomarker of Glomerular Alterations Induced by a VEGF-R2 Inhibitor in Rats
Jane A. Fagerland1,2, Wayne R. Buck1,2, Gerard D. Gagne1,2, Kenneth L. Kowalkowski1,2, Rita Ciurlionis1,2, Eric Blomme1,2, Hong Jin1,2, David L. Gerhold1,2, and Yi Yang1,2
1Abbott Laboratories, Abbott Park IL, USA
2Merck Research Laboratories, West Point PA, USA
Kidney injury is a common adverse effect observed during pharmaceutical development. Standard markers of renal function (ie, BUN and serum creatinine) are elevated only after severe renal damage, thus sensitive biomarkers for early detection would be very useful in both preclinical and clinical studies. Trefoil factor 3 (TFF3) is a known biomarker of renal tubule damage, and urinary levels decrease after administration of compounds that induce acute tubular injury. The purpose of this study was to determine whether TFF3 is an equally sensitive marker of glomerular injury, when compared to standard clinical chemical measures of kidney function and correlated with light and electron microscopic findings. ABT-123, an experimental VEGF-R2 inhibitor that affects glomeruli but not renal tubules, was administered orally to Sprague-Dawley rats for 7 days at 1, 3, and 10 mg/kg/day. Levels of TFF3 and other biomarkers were determined in urine, and expression of TFF3 mRNA was assessed in whole kidney and in glomeruli isolated using laser capture microdissection. BUN, urinary total protein, and creatinine (serum and urinary) were unchanged. Ultrastructural evaluation confirmed glomerular effects in all rats administered 10 mg/kg of ABT-123. Urinary levels of kidney injury biomarkers (albumin, lipocalin, osteopontin) were increased as early as day 4 in the 10 mg/kg dose group. Levels of a biomarker of tubular injury (KIM-1) were unchanged. While there was large inter-animal variability in both urinary levels and gene expression of TFF3, urinary TFF3 levels were decreased on day 4 (up to 10-fold decrease) and day 7 (up to 30-fold decrease) in the 10 mg/kg dose group. TFF3 gene expression in glomeruli was decreased by 10- to 16-fold in the 10 mg/kg group, and from 0- to 10-fold in the 3 mg/kg dose group. There was no change in TFF3 expression in whole kidney. Thus, decreased expression of TFF3 was demonstrated after administration of ABT-123, a compound known to preferentially affect glomeruli, suggesting that TFF3 is a useful biomarker of glomerular alterations.
Keywords: urinary biomarkers, kidney, VEGF
407 Evaluation of Renal Biomarkers in Urine of Rats Treated With Diuretics
Bogdan N.1, Procaccino J.1, Knight E.2, Johnson M.1, Sasaki J.1, and Sonee M.1
1Johnson and Johnson PRD, GPCD, Raritan, NJ 08869
2ECKnight Consulting Services, L.L.C., Bensalem, PA
Goals: Blood urea nitrogen and creatinine have been the traditional markers of renal injury. Recent literature shows the relevance of several novel urinary biomarkers for detection of renal injury. However, little is known about the performance of these markers under conditions of exaggerated pharmacology, which could cause nonspecific effects unrelated to toxicity. The primary goal of this study was to assess the effects of diuretics on urinary biomarkers, albumin, lipocalin-2, kidney injury molecule-1 (KIM-1), osteopontin, (glutathione-S-transferase) mu-GST, αGST and RPA-1 (renal papillary antigen-1). Methods: Male Sprague-Dawley rats were dosed daily for 14 days with erythritol (6000 mg/kg), acetazolamide (100 mg/kg), isosorbide (2000 mg/kg) and 5% sucrose (in drinking water). Urine was collected at 0-6 h post dose and 6-24 h post dose on days 0, 6, and 13. Urine volumes were measured and the samples were analyzed using the Meso Scale Discovery platform using the Kidney Injury kit and the Argutus AKI kit. Results: Acetazolamide treatment induced mild/moderate changes in some markers (albumin, mu-GST, and osteopontin) at earlier times but those markers returned to baseline by end of the treatment period. There was a trend in increased mu-GST levels and RPA-1 levels in the overnight urine collection time points in the 5% sucrose treated rats. In this study, Isosorbide and Erythritol treatments did not induce diuresis (based on the urinalysis) nor did they induce any notable changes in the 7 urinary biomarkers. Conclusions: The diuretic agent, Acetazolamide, induced very minimal changes in the urinary biomarkers at earlier times but most values returned to baseline by the end of the treatment. Further studies will be needed to understand the significance of the increase in mu-GST and RPA-1 in 5% sucrose treated rats.
Keywords: kidney, biomarkers, diuresis
Category: Environmental Toxicology
500 Withdrawn
501 Withdrawn
502 Withdrawn
503 Pubertal Effects of Postnatal Exposure to Sodium Arsenite in Rat Testis
Parul Kaushal1, Pushpa Dhar1, and Raj D. Mehra1
1Department of Anatomy, All India Institute of Medical Sciences, Delhi
In the present study, effects of exposure to sodium arsenite (NaAsO2) during early postnatal period were studied on rat testis. After Ethical clearance, pregnant Wistar rats were procured and day of delivery of pups was designated postnatal day (PND) 0. Pups of experimental and control animals received intraperitoneal injections of NaAsO2 (1.5 mg/kg body weight, bw) & distilled water respectively (PND 1-14). Further subgroups were made according to the day of obtaining the tissue ie PND 15 and 50 corresponding to cessation of exposure and onset of pubertal age respectively. After recording the body and testicular weights, testes were immersion fixed in Bouins fluid and processed for paraffin embedding. 7-µm thick sections were cut serially and stained with hematoxylin & eosin. The stained sections were used for determination of testicular volume (Cavalieri method) and parameters pertaining to morphology and morphometery (using NIS elements AR3.1 software attached to microscope). At PND 50, additional observations such as sperm count and sperm morphology along with electron microscopic features of the seminiferous tubules were carried out. Rate of weight gain and bw of experimental animals showed a significant (P < .05) decrease at PND 15 and 50. Weight and volume of testis in experimental animals showed a significant increase at PND 15 and a significant decrease at PND 50, thereby suggestive of initial inflammatory response of testes to NaAsO2 followed by impaired growth as a sequel to prolonged exposure to accumulated arsenic (As) in the testes. Light microscopic observations revealed decrease in the germinal cell lineage at PND 15 and increase in the seminferous epithelial height at PND 50 in the experimental animals as compared to the age matched controls. Vacuolization of the seminiferous epithelium was evident in the testis of the experimental animals both at PND 15 and 50. At PND 50, decreased sperm count and an increased percentage of abnormal sperms was noted in the experimental animals. Electron micrographs of testis of the experimental animals revealed apoptotic nuclei and abnormal mitochondria in seminiferous epithelial cells along with empty spaces in between the cells.
Keywords: sodium arsenite, postnatal period, seminiferous tubules
504 Hematotoxic Effects of Subchronic Exposure of MNX (Hexahydro-1-Nitroso-3, 5-Dinitro-1, 3, 5-Triazine), Environmental Degradation Product of Munitions Compound RDX (Hexahydro-1, 3, 5-Trinitro-1, 3, 5-Triazine)
Sindhura Ramasahayam1, Benny L. Blaylock1, and Sharon A. Meyer1
1Department of Toxicology, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA
Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) is an environmental degradation product of munitions compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Acute oral exposure of rats to MNX results in seizures, as observed for RDX in rats and humans. Our studies have additionally reported hematotoxicity with acute MNX exposure manifested as a delayed onset anemia. However, toxicity of longer term exposures to MNX has not been examined. The objective of this study was to determine whether hematotoxic effects continued with subchronic MNX exposure. Female Sprague-Dawley rats were gavaged daily for 4 or 6 weeks with 47 mg/kg/d MNX (1/4 LD50) or vehicle (5% DMSO in corn oil) and sacrificed 24 h after the last dose. Measured endpoints included body and organ weights, hematology, clinical chemistry, and tissue histopathology. The major toxicological effects observed were MNX-induced increases in blood granulocytes and platelets. In agreement, bone marrow exhibited an increased number of megakaryocyte number per area, but cellularity was unchanged. Fibrosis was absent as evidenced by no MNX-induced change in silver-stained reticulin fibers in iliac bone marrow. No changes in hemoglobin or hematocrit occurred with subchronic MNX. Clinical chemistry indicated an increase in serum potassium and decreases in sodium, chloride, glucose, and creatinine. Collectively, proliferation of megakaryocytes in bone marrow and thrombocytosis and granulocytosis after subchronic MNX exposure suggested continued hematotoxicity.
505 Role of DEB-Induced ROS Production During the Activation of Cell Survival Pathways in Prostate Cancer Cells
Lea Dixon2 and E. Martinez-Ceballos1,2
1Department of Biological Sciences, Southern University and A&M College, Baton Rouge, LA, USA
2Environmental Toxicology Program, Southern University and A&M College, Baton Rouge, LA, USA
BD has been deemed as an environmental pollutant and is also a volatile gas used in the production of rubber, plastics, insulation, acrylics, and other polymers. Its ubiquitous occurrence in environmental and industrial areas has been shown to cause an increased prevalence of respiratory illnesses. In addition, BD is a known mutagen and human carcinogen, and possesses multi-organ systems toxicity that includes bone marrow depletion, spleen, and thymus atrophy. Diepoxybutane (DEB) is one of the most toxic metabolites of 1,3-butadiene (BD). Previous investigations have shown that DEB causes the generation of reactive oxygen species (ROS) that may damage DNA or produce H2O2. This production in turn affects cell survival and cell death and may be responsible for the activation of the aforementioned pathways. N-acetyl l-cysteine (NAC) is an old mucolytic agent that has been proven to be an effective agent against oxidative stress. Thus, the objective of this study was to: (1) Determine whether ROS production occurs in DU145 prostate cancer cells as a consequence of low-dose (0.5 µM) DEB exposure. (2) Evaluate the effect of NAC on DEB-treated DU145 cells. (3) Investigate the involvement of DEB and ROS production during the activation of the PI3K/AKT cell survival pathway (CSP). We found that ROS production was prevalent in samples treated with 0.5 μM DEB after 12 h and that this occurrence increased with exposure. NAC was able to not only decrease ROS production, but totally eradicated the production of ROS after 12 h, when the samples were exposed to both NAC and DEB. The PI3K/AKT pathway was activated in response to DEB and NAC treatment. Our work indicates that genotoxic pollutants can stimulate the activation of CSPs in cancer cells through ROS production. The activation of CSPs in cancer cells may further increase their malignant status.
Keywords: environmental toxicology, diepoxybutane, prostate cancer
506 The Effect of Cigarette Smoke Extract (CSE) on the Production of Vascular Endothelial Growth Factor (VEGF) by Human Lung Fibroblasts: In Vitro Studies
Maha Farid1, Nobuhiro Kanaji1, Xiang De Liu1, Joel Michalski1, Masanori Nakanishi1, Tadashi Sato1, Xing Qi Wang1, Hesham Basma1, Amy J Nelson1, and Stephen Rennard1
1University of Nebraska Medical Center, Omaha, NE, USA
Chronic obstructive pulmonary disease (COPD) is characterized by progressive incompletely reversible expiratory airway limitation resulting from inflammation in the small airways and destruction of the alveoli. Cigarette smoke is the major cause of the disease, yet pathogenetic mechanisms are not fully understood. Vascular endothelial growth factor (VEGF) is one of the major regulators of angiogenesis and vascular permeability. Fibroblasts are a significant source of VEGF in the lungs; however the effect of cigarette smoke extract (CSE) stimulation on VEGF release by fibroblasts is not tested yet. We hypothesized that disturbed VEGF release by human lung fibroblasts in response to CSE is a potential pathogenetic mechanism that could contribute to COPD. We exposed human fetal lung fibroblast (HFL-1) to different concentrations of CSE (2.5, 5, 7.5, and 10% in serum-free media) and for different durations (24, 48 and 72 hours). The media were then collected and VEGF release into the media was measured using Enzyme-Linked Immunosorbent Assay (ELISA). CSE induced VEGF release by HFL-1 that was observed 48 hours after exposure and increased further after 72 hours. A clear dose response was observed with increasing concentrations of CSE. We also exposed lung fibroblasts from COPD patients and healthy smokers to the above mentioned CSE concentrations and measured VEGF release after 48 hours using ELISA. CSE stimulated release of VEGF from control but not from COPD fibroblasts. Finally, we investigated the role of Smad3 in CSE induced VEGF release by HFL-1. Transfection with siRNA to Smad3 eliminated the stimulatory effect of CSE on VEGF release by HFL-1. In conclusion our data indicates that CSE increases VEGF release by HFL-1. This response appears to involve the Smad3 pathway and fibroblasts from patients with COPD may have a deficient response. Knowing the essential angiogenic role of VEGF, our data shed light on a possible mechanism for the pathogenesis of cigarette smoke induced lung disease.
Keywords: cigarette smoke, VEGF, COPD
Molecular Mechanisms
600 Multiplexed High Content Screening and Proteomic Analysis Reveal That Cigarette Smoke Condensate Alters Stress-Activated Cell Signaling Pathways in Human Bronchial Cells
Charleata A. Carter1
1A. W. Spears Research Center, Lorillard Tobacco Company, Greensboro, NC, USA
Bronchial cells are one of the primary cell types affected by inhaled particulates. Previous work showed that treatment of the immortalized human bronchial epithelial cells (BEAS-2B) with cigarette smoke condensate (CSC) disrupted the F-actin cytoskeleton, and decreased cell spreading and motility (Carter and Hamm, 2009). In the current study, we sought to further elucidate the mechanisms by which these changes occurred. We hypothesized that CSC activated the tyrosine kinase protein focal adhesion kinase (FAK), mitogen-activated protein kinases (MAPKs) and heat shock proteins (Hsps) in BEAS-2B cells. Analysis was done using multiplexed high content screening and antibody-based proteomics. When BEAS-2B cells were treated with 20-50 mg/mL CSC for 1 hour, FAK staining and phosphorylation increased until a dose (100 µg/mL CSC) was reached whereby cells underwent anoikis. CSC treatment of BEAS-2B cells showed a dose-dependent increase in activation of the MAPKs c-Jun, c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK) and p38. At doses of 100 and 200 μg/mL CSC, increases in MAPKs correlated with a decrease in nuclear size. Hsp27 is responsive to toxins and involved in regulation of actin polymerization and cell migration. Hsp27 and phosphorylated Hsp27 were increased with increasing doses of CSC in BEAS-2B cells. CSC exerts changes in BEAS-2B human bronchial cells by altering morphology and concurrently activating pathways commonly responsive to stress stimuli. Protein analysis by antibody-based proteomic screening combined with high content screening not only further elucidate mechanisms of smoke-induced disease but also possess potential for use in in vitro toxicology screening.
Keywords: human bronchial cells, MAP kinases, focal adhesion kinase
601 Withdrawn
602 Regulation of CYP2S1 by Dexamethasone
Bebenek, Ilona G.1, Bui, Peter1, and Hankinson, Oliver1
1Department of Pathology, Molecular Toxicology Interdepartmental Program, UCLA, Los Angeles, CA, USA
Cytochrome P450s (CYPs) are monooxygenase proteins involved in the metabolism of both exogenous and endogenous compounds. Among the 57 or so human CYPs, CYP2S1 is among the 13 that are characterized as “orphan” CYPs due to the lack of information regarding their function. Unlike most CYPs, significant expression of CYP2S1 has been found in extrahepatic tissues such as the trachea, lung, stomach, small intestine, spleen, skin, breast, kidney, and placenta. CYP2S1 is upregulated by a variety of agents including UV radiation, coal-tar, all-trans retinoic acid (ATRA), and dioxin. We have previously demonstrated that that CYP2S1 is upregulated at least tenfold by TCDD and fivefold by hypoxia in mouse hepatoma cells. Differential expression of CYP2S1 has been associated with several tumors of epithelial origin and therefore, the characterization of its function and regulation may be useful in the treatment of disease. Upon a search for novel regulators of CYP2S1, we found that the synthetic glucorticoid receptor ligand dexamethasone (dex) repressed CYP2S1 expression. In this study, the transcriptional regulation of CYP2S1 by dex and the mechanism of this regulation were investigated.
Category: Data Analysis
700 The Next Challenge: Tailoring Statistics to the Specificities of a Study While Maintaining Productivity and Traceability
Fogel P.1, Suter H.1, Karakos A.2, Schuster M.2, and Suter D.2
1PDS Pathology Data Systems Ltd, Pratteln, Switzerland
2PDS Preclinical Data Systems Inc, Mount Arlington, NJ, USA
Regulatory agencies expect GLP-compliant statistical procedures to be planned and well-specified in order to ensure traceability, which helps assure unbiased analysis, reproducibility and reliability through validated calculations. As companies seek to respond to this challenge without adversely affecting productivity, they tend to “freeze” statistical procedures, which are then implemented in a software package and applied in a more or less systematical way. An obvious drawback of this approach is the lack of flexibility, which can lead to misuse of statistics that do not fit well with the experimental design of a study. For example, multiple comparison tests to a control must not be multiplicity adjusted when different treatments/endpoints are studied, by contrast with a dose-response study of a single treatment. The PDS solution developed to address this dilemma provides a number of different procedures that can be easily tailored to the specificities of each single measurement within a particular study, with respect to clearly identified characteristics such as test multiplicity strategy, type of multiple comparisons (vs control only, pairwise), trend type (2- or 1-sided), test type (automatic selection, parametric or non parametric), to cite just a few. Moreover, audit trail guarantees that all adjustments to existing procedures are defined and planned in advance of the study. Finally, productivity is enhanced, since all of the settings are stored in the database system—this allows easy reselection statistical procedures for each parameter studied.
Keywords: statistics, GLP compliance, software tools
701 Hierarchical Scaffold Classification of Repeated Dose Toxicity Databases
Glenn J. Myatt1, David Bower1, Kevin P. Cross1, Scott Miller1, and Donald P. Quigley1
1Leadscope, Inc, 1393 Dublin Rd., Columbus, OH, USA
High quality chemical databases with repeated dose toxicity information can be used to derive the chemical characteristics associated with positive organ toxicities. Multiple databases have been compiled from regulatory submissions, public databases, and the general literature. The underlying study information was carefully mapped to a controlled vocabulary, the tested chemicals verified and entered into the database. The database was analyzed using a new informatics approach based on a hierarchical scaffold tree. The method initially classifies the compounds on the basis of chemical scaffolds that are associated with organ toxicities. The hierarchical relationships between the scaffolds are used to create a tree, which can be used to guide the development of structural alerts. Branches of the resulting tree are carefully organized such that scaffolds mapping onto similar sets of chemical structures are positioned close together. This helps in explaining nonspecific promiscuous structural features, that is those that map onto unusually high numbers of positive compounds, but are merely co-occurring with other structural alerts.
Keywords: toxicity database, SAR, structural alerts
702 Withdrawn
Symposia Abstracts
Symp Ia Division of Scientific Investigations (DSI): Inspections of Nonclinical Testing Facilities
Jacqueline O’Shaughnessy1
1USFDA/CDER/Office of Compliance/Division of Scientific Investigations, Silver Spring, MD, USA
This session will introduce the FDA’s Good Laboratory Practice (GLP) Inspection Program within the Division of Scientific Investigations (DSI) in CDER’s Office of Compliance. The presentation will address directed and surveillance inspections of nonclinical testing facilities for test articles pertaining to CDER. Inspectional procedures, Establishment Inspection Reports (EIR), 483s, Warning Letters, and impact of inspections, along with case examples will be discussed. Interactions with Office of New Drugs and the reviewing divisions will also be included.
Symp Ib Introduction to Division of Anesthetic and Analgesia Products
Adam Wasserman
Pharmacology/Toxicology Supervisor, USFDA, CDER, Silver Spring, MD, USA
This session will introduce the FDA’s Office of Drug Evaluation II within the Office of New Drugs and its organizational structure, roles and responsibilities of the reviewing pharmacologist, and interactions of pharmacologists within and outside the Agency. ODE II is composed of Division of Anesthetic, Analgesia and Rheumatology Products, Divisions of Metabolism and Endocrinology Products, and Division of Pulmonary and Allergy Products. The main portion of the presentation will focus on the Division of Anesthetic and Analgesia Products. Nonclinical requirements and recommendations of the division in consideration of the target indications and the clinical phases of drug development will be discussed. The nonclinical differences and preferences that are unique and required or recommended by this division will be elaborated.
FDA
Regulatory
Pharmacology/Toxicology
Symp Ic Introduction to Division of Anti-Viral Products
Hanan Ghantous1
1Pharmacology/Toxicology Supervisor, USFDA, CDER, Silver Spring, MD, USA
This session will introduce the FDA’s Office of Antimicrobial Products within the Office of New Drugs and its organizational structure, roles and responsibilities of the reviewing pharmacologist, and interactions of pharmacologists within and outside the divisions. The Office of Antimicrobial Products is composed of Division of Anti-Viral Products, Division of Special Pathogen and Transplant Products, and Division of Anti-Infectives and Ophthalmology. The presentation will focus on the Division of Anti-Viral Products. Nonclinical requirements and recommendations that are unique to this division in consideration of the target indications and the clinical phases of drug development will be discussed.
Keywords: FDA, regulatory, pharmacology/toxicology
Symp Id Introduction to Division of Cardiovascular and Renal Products
Donald Nick Jensen1
1Pharmacology/Toxicology Supervisor, US FDA, CDER, Silver Spring, MD, USA
This presentation will introduce the FDA’s Office of Drug Evaluation I within the Office of New Drugs and its organizational structure, roles and responsibilities of the reviewing pharmacologist, and interactions of pharmacologists within and outside Agency. The ODE I is composed of Division of Cardiovascular and Renal Products, Division of Neurology Products, and Division of Psychiatry Products. The presentation will focus on the Division of Cardiovascular and Renal Products, and nonclinical requirements and recommendations of this division will be discussed. Examples will be provided in various disease indications within cardiovascular and renal areas that are covered by this division. The types of studies expected based on the different phases of the clinical program will be incorporated.
Keywords: FDA, regulatory, pharmacology/toxicology
Symp Ie Overview of Pharmacology/Toxicology at the Office of New Drugs
Adam Wasserman1
1Pharmacology/Toxicology Supervisor, US FDA, CDER, Silver Spring, MD, USA
This session will present an overview of the Office of New Drugs (OND) within the Center for Drug Evaluation and Research (CDER). The presentation will highlight key nonclinical issues in drug development under consideration in the OND and will include perspectives on administrative aspects within OND, interactions with other CDER offices and operational aspects of coordination and interaction among pharmacology/toxicology reviewers in various divisions. Additional topics such as how OND coordinates various committees/subcommittees and guidances/workshops within FDA and outside FDA, and the processes of the Executive Carcinogenicity Assessment Committee (ECAC) and the full Carcinogenicity Assessment Committee (CAC), as well as the differences between these committees, will be incorporated.
FDA
Regulatory
Pharmacology/Toxicology
Symp IIb Challenges in Developing Vaccines for Use in Pregnant Women
Martha E. I. Leibbrandt1
1Novartis Vaccines and Diagnostics
The benefit of vaccination for the prevention of serious childhood diseases is well established and globally accepted. However, primary infant vaccination schedules typically begin in the second month of life and, consequently, such immunization does not offer the opportunity to protect newborns from infectious disease. Newborns are protected from many diseases during the first 3 months of life by antibodies acquired from the mother through placental transfer. In the case of tetanus, the possibility of protecting newborns by vaccination of the mother during pregnancy has been realized through the widespread vaccination of pregnant women. Vaccination against other diseases including Group B Streptococcus and pertussis may offer similar benefit to the newborn. Because of the target population, extensive nonclinical evaluation of the vaccine’s safety should be completed prior to initiation of clinical trials in pregnant women using the vaccine formulation intended for clinical use. Issues in the reproductive and developmental toxicity testing of the vaccine include criteria for selection of one or 2 appropriate species (availability of a sufficient database, immunogenicity of the vaccine in the species, placental transfer comparable to humans, well-described endpoints for assessing the health of offspring) and a study design which assesses the toxicity of circulating vaccine-elicited antibodies and/or the vaccine itself. Finally, demonstration of the vaccine’s safety in pregnant animals may mitigate but not remove logistical, regulatory, societal and legal hurdles to vaccination of pregnant women for the aim of protecting the newborn.
Symp IIc The Nonclinical Safety Assessment of Gardasil®: A Human Papillomavirus Vaccine
Jayanthi Wolf1
1Safety Assessment, Merck
Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases in both men and women. A quadrivalent HPV vaccine, GARDASIL®, has been shown to be highly effective in the prevention of several HPV-mediated diseases. GARDASIL® is indicated for use in girls and women 9 through 26 for prevention of cervical, vulvar, and vaginal cancers caused by HPV types 16 and 18 and genital warts caused by HPV types 6 and 11. The vaccine indication has recently been extended for use in boys and men who are 9 through 26 years of age for the prevention of genital warts caused by HPV types 6 and 11. This presentation will describe the nonclinical safety assessment studies that were performed in support of clinical development and licensure of GARDASIL®. Since GARDASIL® was indicated for administration to girls and young women, particular focus will be given to the nonclinical developmental and reproductive toxicity evaluation that was required to support the development and licensure of this vaccine.
Keywords: HPV vaccine nonclinical safety assessment, Vaccine developmental and reproductive toxicity studies
Symp IId Medical Countermeasures: Developing Vaccines Against Biothreats and Emerging Infectious Diseases
Robert V. House1
1DVC LLC, Frederick, MD, USA
The term “medical countermeasure” has been colloquially used to refer to prophylactics and therapeutics against highly-hazardous biological, chemical, or radiological agents intended to be used as weapons or agents of terror. Increasingly, this term is also inclusive of emerging and re-emerging natural diseases such as pandemic influenza, Dengue, and other rare or zoonotic diseases. Regardless of the nature of the threat, they all share characteristics that create distinct challenges for advanced development and regulatory approval. For this presentation, we will be focusing on the challenges associated with developing vaccines, although other countermeasures face similar challenges. First and foremost, these vaccines can almost never be tested for efficacy directly in humans due to ethical considerations (direct challenge) or rarity in nature (epidemiological studies). Rather, a novel mechanism (the FDA “Animal Rule”) is in place to demonstrate efficacy. As a result, regulatory strategies for developing these products are somewhat different from other products. Another important difference is how these products will be used, specifically placed in strategic reserve and used only in emergency situations, which has significant ramifications on economies of production and desirability for long-term preservations. Finally, risk-benefit considerations are somewhat different from other products. This presentation will include several case studies which illustrate the similarities and differences encountered when developing these novel medical countermeasures.
Symp IIIa An Introduction to the “Animal Rule”—Approval of New Drug and Biological Products When Human Efficacy Studies are Not Ethical or Feasible
Andrea M. Powell1
1Office of Counter-Terrorism and Emergency Coordination, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA
In May 2002, the FDA amended its regulations for drugs (21 CFR 314.600) and biologics (21 CFR 601.90) to permit, under specific circumstances, the approval/licensure of medical countermeasures intended to reduce or prevent the serious toxicities that result from exposure to chemical, biological, radiological, or nuclear substances based on evidence of the effectiveness in animals. This change in the regulations is commonly referred to as the Animal Rule and its use as a regulatory pathway to approval/licensure is challenging. Adequate scientific data are necessary for the FDA to conclude that the efficacy demonstrated in animals is “reasonably likely” to be predictive of clinical benefit in humans. Thus, the selection of appropriate animal models in which the efficacy of the proposed medical countermeasure will be tested, and the subsequent design and conduct of the adequate and well-controlled efficacy studies in animals are of critical importance.
Keywords: animal rule, medical countermeasure, animal efficacy studies
Sypm IIIb The Nonclinical Safety Evaluation of Anthrax Anti-Toxins
Laura Kaufman1, Jeremy Middleton1, Karen Blodgett1, Elizabeth Posillico1, and Leslie Casey1
1Elusys Therapeutics, Inc, Pine Brook, NJ, USA
Anthrax attacks by mail in 2001 killed 5 of 11 individuals with documented inhalation anthrax despite aggressive antibiotic and supportive treatment. Mortality from inhalation anthrax is due to toxemia, which can be an early indicator of anthrax, and results from actions of the tripartite anthrax toxin: lethal factor and edema factor as the enzymatic moieties, protective antigen as the cell-binding moiety. Death can result from anthrax toxemia despite eradication of bacteremia. Since antibiotics do not target anthrax toxin, anti-toxins offer the prospect of life-saving intervention for patients who might otherwise not survive. Anti-toxin efficacy is also associated with reducing blood bacteremia to nondetectable levels.
The subject of this presentation will be nonclinical safety strategies used in the development of anthrax anti-toxins under the “Animal Rule.” Important considerations include pharmacokinetics and pharmaco-dynamics in animal models, the effect of anthrax on the pharmacokinetics and distribution of the anti-toxin in animal models, the need to characterize fully the effects of anthrax on target tissues. This presentation will address how all of these get factored into a nonclinical safety evaluation of an anthrax therapeutic. Efficacy data on ETI-204, a humanized, de-immunized monoclonal antibody with high affinity for anthrax protective antigen, will be presented in rabbit and cynomolgus monkey models of inhalation anthrax. ETI-204 development has been supported with federal funds from Biomedical Advanced Research and Development Authority (BARDA), DHHS, in conjunction with the National Institute of Allergy and Infectious Diseases (NIAID), NIH/HHS, under Contract No. HHSN272200700035C.
Keywords: animal rule, preclinical toxicology, medical countermeasure
Symp IIIc Aerobiology and Toxicology Exposure Studies With Nonhuman Primate Models
M. Louise1 and M. Pitt1
1United States Army Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD, USA
Aerobiology in biodefense is crucial to the nation’s goal to develop and license medical countermeasures to protect against aerosol biological threats. Vaccines or therapies developed to protect against biological warfare attack must be efficacious against an aerosol exposure. These products can be tested for safety in humans, but efficacy studies cannot be performed for both ethical and/or practical reasons, including following: aerosol exposure is not usually a natural route of exposure, the disease or intoxication is frequently deadly with no licensed available treatment, and the incidence of the naturally occurring disease is rare. Therefore, we have to rely on data from animal models as a substitute for human disease. The FDA amended its new drug and biological product regulations to allow appropriate studies in animals to provide substantial evidence of the effectiveness of new drug and biological products used to reduce or prevent the effects of these biological agents. The development of appropriate animal models depends on the ability to generate well-characterized aerosols of the biological agents and to consistently deliver specific inhaled doses to the animals. The disease outcome in the animal has to be predicted based on the delivered inhaled dose. Several nonhuman primate models have been developed and used to show efficacy of candidate medical countermeasures against relevant aerosol exposures. In this presentation, a selection of these models and efficacy data will be used to illustrate the challenges associated with this research.
Keywords: aerosol, nonhuman primate models, animal rule
Symp IIId Respiration Measures and Toxicology Affect Assessment in the Primate: Mechanisms of Injury and Physiological Responses From Selected Biological Agents
Paul A Dabisch1 and Jamie Kline1
1Aerosol Technology Department, Center for Aerobiological Sciences, USAMRIID, Fort Detrick, MD
Inhalation toxicology studies involving nonhuman primates within the biological safety laboratory environment entail unique methodologies to assure safety and to achieve accurate dosing results. The deposited dose is typically estimated as the product of airborne concentration, volume of air inhaled, and the efficiency of deposition of the airborne concentration. Traditionally, estimates of each of these parameters are made from historical data, or are determined prior to the inhalation exposure. However, this can result in the introduction of an unknown amount of error into the estimated deposited dose calculation due to inter-animal variability and potential unmeasured respiratory artifacts. Ideally, these parameters would be determined in situ during an inhalation exposure. This approach requires the integration of data from various technologies, but theoretically can result in increased accuracy of the estimated deposited dose. This case study details the use of technologies for the real-time measurement of airborne concentration and respiratory parameters in nonhuman primates, and compares the results to traditional methodologies which utilize historical or pre-exposure measurements
Symp IIIe Toxicology and Safety Assessments in Medical Countermeasure Development: An Overview of Research Trends and the Regulatory Framework
James E. Estep1 and Carol L. Sabourin1
Battelle, Columbus, OH
In 2002, the FDA amended its drug and biological product regulations to allow appropriate studies in animals to provide substantial evidence of the effectiveness of new drug and biological products that reduce or prevent the toxicity of chemical, biological, or radiological substances (CFR 21 Parts 314.610 and 601.91). The Animal Rule resulted in a focused effort to develop animal models to test medical products when well-controlled clinical efficacy studies cannot be ethically conducted in the intended patient population and field trials are not feasible. In addition to efficacy studies to satisfy licensure under the Animal Rule, the preclinical toxicology and safety studies are required to support an Investigative New Drug (IND) application and full licensure. These studies identify potential target organs toxicity, dose-limiting toxicities, Lowest Observed Adverse Effect Level dose, No-Observed Adverse Effect Levels dose, and associated plasma pharmacokinetic parameters of the candidate medical product. Studies demonstrating the reversible nature of toxicity are also usually performed. These studies are designed for understanding the toxicity, safety, bioavailability, toxicokinetics, and relationships between drug disposition and drug-induced toxicity in single-dose, repeated-dose, and continuous intravenous infusion study designs, as needed to support a clinical development plan. Beyond the IND stage of development, other commonly required studies include human metabolism characterization with safety assessment of the defined metabolites; chronic, reproductive, and juvenile toxicity studies; and carcinogenicity assessments.
Keywords: animal rule, preclinical toxicology, medical countermeasure
Symp IVa Current Challenges in NHP Infectious Diseases and Microbial Quality Control
Keith Mansfield1
1New England National Primate Research Center, Southborough, MA, USA
Nonhuman primates (NHP) are used extensively in biomedical research programs focused on infectious diseases, aging, neurosciences, reproductive biology, and drug development. Their close phylogenetic relationship to man and similarities in anatomy, physiology, and immunology place these species at an important crossroads for many types of translational research. While much has been learned concerning optimal animal husbandry and veterinary care for these species, infections with viral, bacterial, fungal, and parasitic agents still present challenges and obstacles in colony management and may impact NHP health and research in important ways. Although agents may still adversely impact animal and colony health causing overt morbidity and mortality, more problematic are infections of low virulence that may go unrecognized in colonies. These nonpathogenic agents may cause limited disease in the immunologically normal host but become virulent secondary to immunodysfunction induced through experimental manipulations. Moreover, pathogens may impact an animal’s immunologic and physiologic responses during study and serve as important confounders of such work. To assist with the control and reduction of infectious agents, specific pathogen free (SPF) colonies have been established for several species of NHPs. For macaques, the target agents for most programs include BV, STLV, SIV, SRV, MV, Shigella flexneri, and Mycobacterium tuberculosis. In general, derivation of such colonies has involved the use a test-and-remove strategy at the time of weaning of juvenile cohorts utilizing serologic and other assays to achieve agent eradication and control. While limitations exist with current assays, this approach coupled with extensive ongoing testing and has been highly successful in the development of SPF macaque colonies and is now being expanded to cover additional infectious agents as well as other NHP species. Even with these successful programs, it will be difficult to eliminate all infectious agents from NHP colonies and facilities should work to define the full spectrum of microbial agents present within a given population of animals and attempt to understand how these agents may impact pathobiology and experimental work. Based on a risk assessment of the species, source and experimental use, facilities should implement a rational microbial quality control program to abrogate the impact of infectious diseases. Such programs should include elements that address the pre-purchase evaluation of the source colony and animals, quarantine measures, preventative health and occupational health and safety programs, pathogen surveillance, and agent eradication and control. Because these agents may represent significant confounders of experimental work or cause diseases with overt morbidity and mortality, all facilities with NHPs should implement comprehensive microbial quality programs and we should seek methods to integrate and bridge disease surveillance across facilities.
Symp IVb Impact of Background Infection in NHP on Toxicology Studies
Ellen Evans1
1Pfizer, Inc, Groton, CT, USA
NHP carry a variety of background infections, depending on point of origin, source, husbandry, genetic variability, and exposure to humans. Most of the infections are subclinical, but clinical manifestations can occur during the course of a toxicology study due to stress, compromised condition, or immunomodulatory effects of a test article. The manifestation of background infections in NHP can adversely impact the conduct of toxicology studies in several ways: they can invalidate the study by compromising the model, result in early termination of individuals or an entire study for animal welfare reasons or to protect colony health, or confound data interpretation. Conversely, the manifestation of a background infection during the course of a study may be useful in identifying immunosuppressive effects of the test article. This presentation will discuss some of the issues regarding background infections in NHP related to the conduct of toxicology studies.
Symp IVc Herpesviruses, Retroviruses, Measles Virus, and Tuberculosis: Confounding Factors to Toxicology Study Design and Outcome
Joe Simmons1
1Charles River Labs, Houston, TX, USA
It is well recognized that adventitious infectious agents can interfere with biomedical research by increasing morbidity, mortality, and variability in scientific data, thus confounding research. Some infectious agents are acute and self-limiting, while others cause persistent or latent, life-long infections. Among the group of agents that cause life-long infections are the herpesviruses and retroviruses. Retroviruses can have direct immune suppressive effects and can thus confound studies by causing variability in data that is generated or loss of statistical power due to animals not surviving until study completion. Conversely, herpesviruses are typically self-limiting in immune competent animals; however, they can have a significant direct impact on the immune system and can cause a variety of study related problems from an increase in background lesions to clinically overt disease in animals that are immune suppressed as a result of test article treatment. While measles is a human disease it can infect a wide variety of nonhuman primates including macaques. Primary infection by virulent measles virus causes significant immune suppression for 6 to 12 months post infection, and vaccination with attenuated vaccine strains of measles virus causes a more limited immune suppression, especially in cell mediated immune responses, for a period of 6 to 8 weeks post inoculation. Thus, understanding the timing and implementation of a colony measles prevention strategy is extremely important for certain types of studies and for understanding the efficacy of testing for infection by Mycobacterium tuberculosis by the Mantoux tuberculin skin test. During this talk we will discuss the immunologic consequences and potential study-related impact of these viruses and of Mycobacterium tuberculosis on toxicology studies that utilize nonhuman primates.
Symp IVd A Clinical Veterinary Perspective on How to Prepare for Your NHP Study and Get a Good Nights Sleep as a Study Director
Joel J. Beren1
1FDA/CBER/DVS, Bethesda, MD , USA
Thorough planning and preparation is the foundation for a successful tox study. Avoiding problems is far better then having to deal with unexpected surprises once the study is underway. Controllable factors that influence a successful study include choosing an appropriate animal model and partnering with a trusted vendor/broker whose husbandry, housing, veterinary care, and transportation procedures are compatible with your specifications. Considerations include the NHP's country of origin and the endemic diseases found in that region. We will address health issues that go beyond the standard testing to avoid being blindsided by an unexpected problem.
Symp IVe Opportunistic Infections in Nonhuman Primates Exposed to Immunomodulatory Biopharmaceutics
David Hutto1
1Millennium Pharmaceuticals, Cambridge, MA, USA
Immunomodulatory biopharmaceutics may, through their intended effects on the immune system, render animals in nonclinical safety assessment studies more susceptible to various infections. Since biopharmaceuticals are usually studied in this way in nonhuman primates, the induced infections may be caused by primate specific agents similar to those that exist in humans. The relevance to human clinical safety of these specific infections, included factors associated with their onset and expression, will be discussed in the context of several case studies. A series of key questions that are felt to provide perspective regarding the clinical relevance of these infections will be considered.
Symp Va Immunology of Immunogenicity: Introduction
Daniel T. Mytych1
1Amgen, Inc, Thousand Oaks, CA
Immunogenicity toward biotechnology derived therapeutics can impact both the therapeutic activity and safety profile in human patients. The outcome of nonclinical studies designed to evaluate the safety and pharmacokinetics prior to clinical study can also be markedly affected by the development of anti-drug antibodies. Numerous factors have been identified that favor development of antidrug antibodies over tolerance. This presentation will review current understanding of the underlying immunology of immunogenicity toward foreign proteins. Particular emphasis will be placed on those factors that can be controlled to mitigate development of anti-drug antibodies.
Symp Vb Immunogenicity of Coagulation Proteins: What Can Animal Models Tell Us
Inge Ivens1
1Toxicology, Bayer Health Care, Richmond, CA, USA
Replacement therapy using exogenous FVIII is the first line of therapy for Hemophilia A in patients which lack FVIII. Immunogenicity, the development of binding (total) and neutralizing (inhibitory) antibodies against the protein is a clinical complication of this therapy The speaker will compare the immunogenicity of coagulation proteins in animal models to experience in humans where approximately one third of patients develop inhibitory antibodies. Animal models show a different pattern since most of the animal treated with FVIII develop inhibitory antibodies within a short time frame, abolishing exposure in longer term toxicity studies. Animal models of hemophilia A have been used to compare immunogenicity of proteins. The limitations of these models of will be discussed.
Symp Vc Immunogenicity and Animal Models During Vaccine Development - the Simple Life Isn’t Always So Simple
Leigh Ann Burns Naas1 and Deborah L. Novicki2
1Pfizer Global R&D, San Diego, CA, USA
2Global Head Toxicology, Novartis Vaccines, Cambridge, MA, USA
Immunogenicity is generally considered an unwanted and sometimes uncontrollable issue that toxicologists work through when conducting nonclinical testing of protein therapeutics intended for humans. But for vaccines, the goal is to generate an immune response to the immunogen that provides a sustained level of immunity over a period of time. When selecting the toxicology species for safety evaluation, one requirement is that the vaccine produce an immune response in the animal that mimics (to the extent possible) the immune response that is desired in humans. It sounds simple but it is not always the case, particularly for therapeutic vaccines which often target self-antigens. The goal there is to break natural tolerance. In some cases animal models either do not express the human antigen (or the altered for of the antigen) so while they can make an immune response to the injected vaccine, its translatability—and thus the overall species relevance—may be questionable. This presentation will discuss desired immunogenicity, animal models and the complexities surrounding the development of both prophylactic and therapeutic vaccines.
Symp Vd Immunogenicity Response in Gene Therapy Development
Timothy MacLachlan1
1Genzyme Corporation, Framingham, VA, USA
Gene therapy trials have relied most often on the use of viral vectors to deliver the therapeutic gene of interest. This has been a very effective way of introducing genetic material into cells, however the immune system is sometimes just as effective in responding to an agent that is obviously foreign. Both humoral and cellular immune responses have been detected in preclinical and clinical studies of gene therapy vectors and these have impacted the frequency of dosing, the serotype of vector used, the safety profile of the drug, and has even led to inclusion of immunosuppressive regimens concomitant with dosing. This discussion will focus on recent findings of humoral and cellular immune responses in preclinical and clinical safety studies and will evaluate how these events have led to evaluation of toxicity.
Symp Ve Animal Immune Responses Impact on Safety Studies of Human Cell Therapies
Lauren E. Black1
1Charles River Laboratories, Reno, NV, USA
Risk assessment of human cell therapies, prior to implantation in humans, is a key hurdle in clinical development and progress in the field of regenerative medicine. However, traditional animal toxicology approaches are not often feasible due to xenogeneic rejection of the human cells in animal models with competent immune systems. Alternatives include conducting toxicology in immunocompromised rodents or drug-immunosuppressed animals with otherwise normal immunity. Either approach introduces translational gaps. Approaches to bridging these gaps, as well as risk/benefit assessment, are described by a speaker with past CBER cell therapy review experience.
Symp VIa Translational Imaging in Preclinical Safety Assessment: Challenges and Opportunities
Brian R. Berridge1
1R&D Pathology, GlaxoSmithKline Safety Assessment, Research Triangle Park, NC, USA
Medical imaging capabilities are advancing at a rapid rate. Noninvasive imaging of organ structure, function, and even molecular events has become standard of care in many advanced clinical settings. Also, advances in the scalability of these imaging capabilities are facilitating their application to animal species commonly used in preclinical drug safety testing. Current societal, regulatory, and economic pressures on the pharmaceutical industry dictate a need for preclinical safety assessment to be more efficient, more translational, and use fewer animals. Translational imaging provides an opportunity to meet that challenge. This presentation will examine traditional nonclinical safety paradigms with the intent of identifying specific gaps and opportunities for imaging to fill those gaps. We will also explore challenges to integrating novel methods into very regimented and regulated paradigms. Finally, a few ideas about how to advance these novel approaches will be discussed.
Symp VIb Imaging Biomarkerrs for Safety Assessment
G. Allan Johnson1
1Duke Center for In Vivo Microscopy, Durham, NC, USA
The invention of digital imaging (CT, PET, and MRI) has revolutionized medicine. Over the last 25 years there has been considerable progress toward scaling these techniques from man to small animal. This talk will provide an overview of small animal imaging methods with examples of successful application in safety assessment.
Symp VIc A Survey on the Current Use of Imaging in Preclinical Safety Assessment
X. Ying1, K. McDorman1, S. D. Pettit1, and B. R. Berridge1
The HESI Project Committee on Translational Imaging
The HESI Project Committee on Translational Imaging has conducted a comprehensive survey to investigate the current practices and visions about the utilization of imaging technologies in preclinical safety assessment. The participants were from government agencies, the biopharmaceutical and chemical industries, preclinical CROs, and academic laboratories. Questions were asked on multiple translational imaging modalities such as MRI, CT, PET, SPECT, ultrasound, and optical imaging. This presentation will summarize the survey results, including what imaging techniques are currently being used in preclinical safety assessment, what are the imaging techniques used for, the animal species used on imaging studies and the biological systems targeted by imaging techniques. In addition, the survey results also include information about current preclinical imaging workflow and opinions from multidisciplinary experts, such as who is responsible for imaging data collection and interpretation, and what are the current imaging application opportunities and the areas for which there are remaining challenges and needs for improvement.
Symp VId Use of Ultrasound in Preclinical Hemodynamic and Safety Assessment of Investigational Drugs
Albert DeFelice1
1Office of Drug Evaluation-I, Office of New Drugs, Center for Drug Evaluation and Research, U. S. Food and Drug Administration, Silver Spring, MD
This presentation will address results of a study where both imaging and Doppler ultrasound were used to monitor chronic changes in left ventricular (LV) cardiac dimensions, LV systolic function, and regurgitation fraction in a rabbit model of aortic insufficiency and LV volume overload will be presented. The trajectory of these changes in 15-17 months following aortic vavulotomy will be presented. Acute beneficial hemodynamic effects of intervention with inotropic and vasodilating agents at 17 months, in compensated survivors will be shown. The speaker will offer perspective on the potential utility of these types of data in a regulatory safety context.
Symp VIIa Ecotoxicology—Individual Effects to Population Relevance
James Wheeler1
1Syngenta Ltd, Jealott's Hill International Research Centre, Bracknell Berkshire, United Kingdom
The field of ecotoxicology is focused around predicting population relevant effects from predominately laboratory studies on individual organisms. This is in contrast to human health assessments that use individual organisms to make judgments for the protection of individuals (man). With the advent of testing methodologies for endocrine disruption, for example the US-EPA’s Endocrine Disruptor Screening Program, more detailed individual endpoints are being incorporated into ecotoxicological test methods. These include endpoints such as histopathological evaluation that traditionally fall in the realm of toxicology. Consequently, there is a need to agree the value and use of such endpoints in ecotoxicological evaluations. This presentation will introduce the ecotoxicological test methods, discuss challenges presented by endocrine specific testing and highlight the learnings from toxicology practices that may be useful to move the debate forward.
Keywords: ecotoxicology, environmental risk assessment, histopathology
Symp VIIb 5-Legged Frogs and Other Croakers: The Use of Frogs in Environmental Monitoring and Toxicity Testing
Jeffrey C. Wolf1
1Experimental Pathology Laboratories, Inc, Sterling, VA, USA
Long gone are the days when scientific interest in frogs was limited to the anatomic dissection of these creatures by squeamish biology students. Triggered in part by the dramatic nature of frog limb abnormalities discovered serendipitously by Minnesota middle school children in 1995, anuran research has virtually exploded during the past ten years. This explosion has occurred in 3 major investigative directions, which comprise: (1) basic biological and biomedical research, (2) ecotoxicological testing, and (3) environmental monitoring. By virtue of many unique anatomical and physiological features, frogs are particularly suited to the exploration of certain basic research areas, diverse examples of which include the study of skin antimicrobial peptides, auditory functional anatomy, early vertebrate development, pheromone production, phylogeography, and tissue regeneration. In contrast, toxicological testing in frogs tends to be environmentally motivated; consequently, investigations often involve potential effects of anthropogenic substances found in surface waters or sediments. Whereas early studies focused on effects of metals or organochlorine compounds, subjects of recent emphasis have included thyroid or reproductive hormones, pharmaceutical agents, and human personal care products. Frog experiments are conducted in the laboratory, in the field, or in intermediate enclosed systems known as “mesocosms,” and the elaborate life cycles and well-characterized behavioral traits of frogs ensure that a wealth of endpoints are available for toxicologic bioassays. Lastly, the exquisite vulnerability of frogs to a host of aquatic and terrestrial predators, parasitic organisms, aqueous contaminants, and other types of exogenous stressors, makes them ideal sentinels for adverse environmental changes related to human or climatological impacts. Surveys monitor the ongoing declines in amphibian species and general biological diversity to determine if these might be bellwethers for ecosystem health and wildlife/human survival.
Keywords: frogs, environmental toxicity, endocrine disruption
Symp VIIc Slow Progress on Snail Sex
Klaus Weber1, Effrosini Kiporou2, Diana Mahnke1, Agnes Sieratowicz2, and Jörg Oehlmann2
1Harlan Laboratories Ltd., Itingen, Switzerland
2Goethe University Frankfurt am Main, Germany
Ecotoxicology deals with the impact of chemical compounds on the animate environment. Invertebrates play an important role in ecosystems as herbivores, carnivores, and destruents, thus contributing to the maintenance of energy flow and cycles of matter. With a number of approximately up to 130 000 living species, the diverse molluscan phylum can be found in almost all biotopes world-wide. The overwhelming majority of mollusc species belongs to the subphylum Conchifera (molluscs with shells). Approximately 80% of the molluscs are gastropods (snails), and from these about 35 000 species populate terrestrian, freshwater, or marine habitats.
The extent of possible effects of chemicals on molluscs came into public regard with the dramatic effects of tributyltin (TBT) compounds, which have broadly been used as antifouling agents for ships. The females of the Dog Whelk (Nucella lapillus) and of at least 160 further species developed male parts in addition to the female genital organs, a syndrome named imposex.
There were many discussions about the use of molluscs to control effects of chemicals to the environment. Consequently, there is a number of publications available dealing with the effects of chemicals on wild living molluscs or on molluscs under laboratory conditions. Strategies for the use of molluscs as multidisciplinary models in ecotoxicity studies are proposed, mainly due the fact that some metabolic pathways show homologies to vertebrates. The choice of the right model species is considerably affected by physiological conditions of the reproductive cycle.
Misinterpretation of endocrine effects in testing products on snails need to be elucidated by histopathology. Mainly irritative effects (inflammation, degeneration etc) and parasitic infections may be primary causes of changed physiological parameters (eg, fertility, fecundity).
Keywords: product safety for humans, product safety in environment, economical considerations
Symp VIId Finding Nemo in Muddy Waters
Mac Law1
1NC State University, College of Veterinary Medicine, Department of Population Health and Pathobiology, Raleigh, NC
Recent environmental catastrophes including large scale fish kills have heightened public awareness of fish as important indicators of the health of our waterways and as sentinels of human health. However, for every fish that is found washed ashore, there often are many more that either die undetected or suffer sublethal effects, leading to secondary disease conditions. More chronic conditions, such as the “epizootics” of fish tumors that occurred in the 1960s and 70s, helped usher in the Clean Water Act of 1977 and the Water Quality Act of 1987. This legislation has the goal of decreasing aquatic pollution and has necessitated increased testing of surface waters and the aquatic organisms within. This talk will discuss environmental monitoring of fish in surface waters, using specific examples ranging from acute toxicity to carcinogenesis and endocrine disruption.
The flip side of the coin involves the use of small fish models of human disease and the applications of these models in safety testing. Gone are the days when fish in the laboratory were considered a novelty. Indeed, the institution that fails to incorporate facilities for aquatic animals is in danger of falling behind in both basic and translational biomedical research. In addition, there is increased demand for alternative methods in safety testing which still incorporate the advantages of whole organisms. The fish embryo test (FET) is beginning to replace adult fish testing of effluent discharges in the European Union and is soon likely to see more widespread use in the United States.
Keywords: small fish models, medaka, fish embryo test
Symp VIIIa Introduction to Nanomaterials and Developing Green Products
David W. Hobson1
1LoneStar PharmTox LLC, Boerne, Texas
Green products are those that can be scientifically demonstrated as having either minimal to beneficial environmental impact ranging from minimal to beneficial. Growing recognition of the need to design products with “green” characteristics rather than attempt to deal with environmental impacts of products that have measurable adverse effects is creating consumer demand for such products. Nanotechnology is one path that many believe can substantially improve and advance current state of the art products and processes while also providing green solutions to current and emerging environmental concerns. Nanotechnology is not green by definition but can be by design, it technically is the manufacturing or engineering of materials that have at least one dimension of between 1 and 100 nm. Physically, materials with this dimensionality exhibit distinctive qualities that include chemical, particulate and physical interactions with their environment that are often unique. Since these dimensions are also those of important biomolecules and biomolecular structures, it should be no surprise that some nanotechnology products are being shown to have potential for both beneficial as well as adverse effects on living and environmental systems. The current nature of green product development and validation to include the development and use of nanotechnology to achieve green product objectives will be introduced using current and emerging practical examples in this talk along with relevant, toxicologic approaches toward demonstrating green characteristics.
Keywords: nanotechnology, green products, nanomaterials
Symp VIIIb Searching for Paradigms in Nanomaterial EHS-Insights From Science of Bionanointeractions
Kenneth Dawson1
1CBNI, University College Dublin, Ireland
A long-term and central question, with implications for scientists, regulators, and industry, is the degree to which nanomaterials can (in relation to their EHS properties) be treated as conventional chemicals. In this discussion we consider some general features of the interactions of nanomaterials with organisms that might give pointers to the future. This viewpoint cannot replace, and is complimentary to, the detailed consideration of specific materials, because individual nanomaterials may also have features requiring special attention.
At such small sizes we expect nanomaterials to be able to exploit energy dependant endogenous uptake and translocation processes at cellular, barrier, and higher levels. Also, clearance mechanisms may be muted for these smaller sizes. These observations suggest that conventional chemical thinking on biokinetics might be incomplete.
It is also striking to think that nanomaterials may present tens of square meters of surface area due to the very high surface to volume ratio at the nanometer scale. Finally, it is increasingly realized that a strongly bound and slowly exchanging protein (or biomolecular) corona surrounds nanomaterials in most biological environments, and this may well be a key part of the biological identity of the nanomaterials in situ.
Keywords: nanotechnology, protein corona, regulations in nanomaterials
Symp VIIIc Designing and Engineering Eco-Responsible Nanomaterials
Robert Hurt1
1Brown University, Providence, RI, USA
Lack of scientific data on the potential environmental and biological implications of nanomaterials has led to a climate of uncertainty for technology companies, regulatory agencies, and consumer advocacy groups. At the same time we have been given a unique “window of opportunity” to develop methods for managing EHS concerns before nanotechnology products become truly widespread in the marketplace. In contrast to some environmental toxicants, nanomaterials are emerging high-technology products under continual development and evolution. Nanomaterial toxicity often depends on specific material features, which are in principle under the control of nanomaterial developers, but can also undergo complex transformation in the environment or human body. The synthesis, surface modification, processing, release, environmental transformation, and biological impacts of nanomaterials form a cause-effect continuum that offer many opportunities for intelligent intervention on behalf of safety. This talk uses carbon nanotubes and nano-silver as example materials to discuss the role of individual material features on toxicity and on biological and environmental persistence. Data will be shown on the effects of metal impurities, hydrophobic surfaces, and chemical stability that influences biodegradation and the release of biological active ionic species. Mechanistic toxicity data will be used to suggest reformulation strategies to help manage EHS risks for these materials.
Keywords: nanotechnology, carbon nanotubes, nano-silver
Symp VIIId Getting the Good Stuff and Avoiding the Bad—How EPA is Addressing Industrial Nanoscale Materials to Achieve Environmental Benefits
Kristan Markey1
1Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency, Washington, DC, USA
The U. S. Environmental Protection Agency (EPA) regulates industrial chemicals under the Toxic Substances Control Act to control potential unreasonable risks to human health and the environment while also fostering new green technologies that have the potential to address environmental challenges including safer chemicals. Nanoscale materials (NMs) are a technological platform that can contribute to achieving these benefits, provided the implications are appropriately managed. However, avoiding unintended consequences is challenging because the models and metrics that have served to identify concerns in previous decades of chemicals management may not hold for NMs, and little valid data has been generated to develop new approaches. At the same time, there are tangible environmental benefits to be realized. Hence, maximizing the benefits while avoiding the down sides is tricky at best, and requires judicious choices to utilize the old approaches and develop new ones. Central to achieving this balance is developing a framework that attempts to define the implications and benefits of NMs throughout their life cycles and institutes policies to foster continuous improvements. To those ends, the Agency has multiple interacting initiatives and programs on nanotechnology to coordinate research, develop testing, collect data, and evaluate results. By considering the overall picture, the Agency is able to prioritize filling environmental, health, safety, and lifecycle data gaps. Specific case studies will examine how these different programs interact and how the EPA is contributing to the long-term foundations of nanotechnology sustainability.
Keywords: nanotechnology, green, regulation
Symp VIIIe Food Products and Nanotechnology: Toxicologic Approaches to Going Green and Unseen
Robin Guy1
1Robin Guy Consulting LLC
Nanotechnology applications for foods, food packaging, and agriculture are increasing. Technically, a nanomaterial has been defined as a material having at least one dimension between 1 and 100 nm; however, recent reviews oriented toward food nanotechnology are proposing wider limits between 1 and 1000 nm. Concerns over safety and environmental impacts of engineered nanoproducts in foods are a prime reason for increasing these limits as there is limited toxicologic information for food-oriented particles of this nature, but growing evidence that nanomaterials can be absorbed and distributed differentially to the chemical and molecular substances of which they are composed. Regulations to disclose the presence of engineered nanomaterials in foods are being considered and food companies generally do not publicize the presence of nanomaterials in their products, which adds to the confusion and raises even more questions regarding potential safety issues. How does using nanomaterials in food processing and packaging increase or decrease waste? Does “nano-sizing” components in dietary supplements and foods increase or decrease nutrient availability, increase safety or create a potential for toxicity? Can food-oriented nanomaterials enter the environment and wreak havoc? Demonstration of green product characteristics may help confirm the safety of a product through its lifecycle. Industry must be prepared to be subjected to worldwide regulations and guidances addressing the use of nanomaterials in foods, food ingredients, dietary supplements, packaging, and agriculture. The food industry is responding and developing green approaches to deal with nanomaterials. One way for a product line to be “green,” is for companies to include analytical and toxicologic testing to evaluate green product claims and thereby lowering toxicities to consumers and the environment.
Keywords: nanomaterials, food, green nano
Symp IXa Development of New in Vivo Models for Population-Based Risk Assessment
Alison Harrill1
1The Hamner-UNC Institute for Drug Safety Sciences, Research Triangle Park, NC, USA
Drug-induced liver injury (DILI) is the major adverse drug event that leads to regulatory actions on drugs. The most problematic and poorly predictable form of DILI is “idiosyncratic,” meaning the drug is safe for the vast majority of treated patients while causing liver injury in a few susceptible individuals, suggesting a possible genetic contribution. Genetic predisposition for complex traits results from the combined effects of variations within genes. Therefore, genetically heterogeneous animal models may be ideal for assessing human DILI responses and mechanisms. We previously demonstrated the utility of using a Mouse Diversity Panel (MDP) to model human toxicity responses due to acetaminophen toxicity. To demonstrate the ability of an MDP to predict clinical toxicities that have not previously been observed in preclinical models, the newly developed Collaborative Cross (CC) mouse population was utilized. The 8 founder strains of the CC were treated with 100 mg/kg isoniazid (i.g.) and sacrificed after regimens of sub-chronic once-daily dosing. We observed a time-dependent increase in the incidence and severity of microvesicular steatosis in some strains; however, other strains were resistant to the effect of the drug. Concurrent with the appearance of steatosis, an increase in the liver: body weight ratio was observed in susceptible strains. The data indicate that the mouse population-based approach may have utility to predict liver toxicity potential where other animal models have failed. A mouse population-based approach provides an attractive model for informing risk assessment of new drugs. The Collaborative Cross may provide a powerful tool that enables better risk assessment and better identification of susceptible subpopulations via biomarkers that can assess individual risk from these treatments. This work is supported by NIH ARRA award 1RC1DK087510-01.
Keywords: pharmacogenomics, drug-induced liver injury, personalized medicine
Symp IXb Preclinical and Translational Pharmacology of Nanoparticle Therapeutics
Whitney P. Caron1, Harvey Clewell2, Robert Dedrick3, Ramesh Ramanathan4, Ning Yu5, Margaret Tonda5, Jan H. Schellens6, Jos H. Beijnen6, and William C. Zamboni1,7,8,9,10
1Division of Pharmacotherapy and Experimental Therapeutics, University of North Carolina (UNC) Eshelman School of Pharmacy
2The Hamner Institute for Health Sciences, Research Triangle Park, NC, USA
3Biomedical Engineering and Instrumentation Branch, Division of Research Services, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, MD, USA
4Molecular Therapeutics and Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA
5ALZA Coporation, Mountain View CA, USA
6Department of Pharmacy and Pharmacology, Netherlands Cancer Institute/Slotervaart Hospital, Amsterdam, the Netherlands
7UNC Lineberger Comprehensive Cancer Center
8UNC Institute for Pharmacogenomics and Individualized Therapy
9Carolina Center of Cancer Nanotechnology Excellence
10North Carolina Bioanalytical Innovation Network
Carrier-mediated agents consist of nanoparticles, nanosomes (nanoparticle sized liposomes) and conjugates. The theoretical advantages for using carrier-mediated drugs, including increased drug solubility, prolonged duration of exposure, selective delivery of entrapped drug to the site of action, and improved therapeutic index. Pegylated nanosomal formulations contain lipid conjugated to polyethylene glycol. The disposition of encapsulated drug is dictated by the composition of the carrier, thus altering the pharmacokinetic (PK) profile of the drug. A proposed clearance pathway of carrier agents is the monocytes and macrophages of the reticuloendothelial system (RES). Our studies suggest there is a bi-directional interaction between nanosomal agents and the RES. However, potential factors associated with clearance of carrier agents in patients and preclinical animal models have not been extensively evaluated. Allometric scaling approaches for nanosomal agents did not scale across all species. Thus, new methods for allometric scaling and measures of RES function need to be developed for carrier-mediated agents. In addition, the most appropriate animal models for toxicology and pharmacology of carrier agents needs to be identified.
Keywords: nanoparticles, pharmacology/pharmacokinetics, allometric scaling
Symp IXc Local Vascular Effects of Drug Eluting Stents in Preclinical Models
Bradley S. Hubbard1
1DVM, SynecorLabs
Coronary Stent implantation was developed to overcome the acute recoil and high restenosis rate of balloon angioplasty. The introduction of this technology resulted in the development of a new clinical challenge, in-stent restenosis. The development of drug-eluting stents is one of the major revolutions in the field of Interventional Cardiology. Restenosis rates has been significantly reduced through the use of drug eluting stents. Local drug delivery from DES has been shown to inhibit intimal proliferation through various mechanisms. Preclinical and clinical trials have evaluated several pharmaceutical agents, including everolimus, sirolimus, and paclitaxel. The efficacy of these strategies has been proven, however a small percentage of patients are at risk of thrombosis in the area of the implant due to delayed re-endothelialization. Preclinical research results from multiple species will be discussed during this presentation.
Keywords: drug eluting stents, local tissue effect, impairment of normal vascular function
Symp IXd Application of Biosimulation Platforms to Safety Testing
Harvey J. Clewell1
1The Hamner Institutes for Health Sciences
The long-term objective of this joint Hamner/Entelos/USFDA research effort is to develop a dynamic, mechanistic computer model of liver homeostasis and drug-induced liver injury (DILI), using the Entelos Physiolab modeling platform. Initial model development has focused on acetaminophen (APAP); future efforts will include idiosyncratic liver toxicants such as isoniazid and valproate. The APAP model embodies a quantitative description of the key processes involved in APAP-induced liver injury: APAP metabolism, reactive metabolite formation, glutathione depletion and resynthesis, ROS formation, energy homeostasis, mitochondrial dysfunction, necrosis, apoptosis, and tissue regeneration. A whole-body physiologically based pharmacokinetic (PBPK) model for APAP and its metabolites in the mouse, rat, and human is linked to a pharmacodynamic (PD) model describing the reaction of APAP-derived N-acetyl-p-benzoquinone imine (NAPQI) with glutathione and the resulting depletion and resynthesis of glutathione. The formation of NAPQI-adducts with cellular proteins and increased oxidative stress resulting from glutathione depletion are linked to cellular damage and response. As compounds with other modalities of DILI are added to the platform, the resulting model will serve as a flexible biosimulation platform to enhance drug development by quickly identifying drug candidates that are likely to be associated with idiosyncratic hepatic toxicity, and to guide personalized medicine by determining patient characteristics associated with a greater likelihood of adverse liver responses. The DILI simulation model will also serve as a valuable knowledge management tool for organizing the available data on the elements of metabolism, cellular injury, and tissue response that are the determinants of susceptibility to DILI.
Keywords: hepatotoxicity, biological modeling, acetaminophen
Symp Xa Introduction to Minipigs: A Nonrodent Species in Nonclinical Safety Assessment
Niels-Christian Ganderup1
1Ellegaard Gottingen Minipigs, Dalmose, Denmark
This presentation shall set the stage for the subsequent presenters. It will do so by having 2 foci. (1) Covering the developmental history of minipigs and contrast that to the developmental history dogs and nonhuman primates, so as to convey a better understanding of the minipig as a laboratory animal species. (2) Outlining major areas of minipig use within biomedical research focusing on its use in the development of new medicines whereby giving the audience an appreciation of the diverse research applications this species is suitable for.
Symp Xb Minipigs in Safety Assessment of Pharmaceutical Drugs—With Examples From the Real Life
Jens Thing Mortensen1
1LAB Research Denmark, Hestehavevej 36A, Ejby, DK-4690 Lille Skensved, Denmark
Minipigs are used increasingly as the nonrodent species in regulatory preclinical safety programs for pharmaceutical drugs. Apart from being an obvious choice for testing of dermal drugs and devices, the minipig is actually also useful and relevant for testing of many other classes of drugs given by systemic or topical routes of administration.
The choice of nonrodent species for a pre-clinical testing program should be science driven. Among the aspects to be considered are the biological characteristics of the potential animal species, as well as the pharmacologic, immunogenic, metabolic and pharmacokinetic properties of the test drug. However, practical issues like the feasibility and ease of dosing the animal in a relevant manner and the life span of the animals should also be taken into consideration. With proper training, minipigs can be a good choice of species even for intranasal or other unusual routes of administration of drugs, and the length of pregnancy and the litter size etc make minipigs of interest for certain reproductive toxicology studies,.
The use of minipigs in safety assessment of pharmaceuticals will be illustrated by actual cases involving common classes of drugs, eg with anti-inflammatory, anti-diabetic, or anti-psoriatic properties. The rationale for species selection, typical toxicological findings, species-specific toxicities, as well as the clinical and regulatory relevance of the findings will be discussed, with reference to cross-species comparisons and clinical results, when possible. Also, practical experimental aspects will be discussed on background of casuistic observations.
Keywords: pharmaceuticals, safety assessment, minipigs
Symp Xc Use of Minipigs in Food Safety Assessment, Agrochemicals, Pesticides and Other Nonpharmaceuticals
Warren D. Harvey1
1General Toxicology, Charles River Laboratories, Edinburgh, Scotland
Swine are increasingly used as a test species in the nonpharmaceutical industry, particularly in the field of food safety assessments, food ingredients and food contaminants. When selecting an appropriate experimental research model, the porcine model has advantages over any other nonprimate test species because the anatomy and physiology of digestion and associated metabolic processes are very similar between humans and pigs. These important similarities to man make swine an ideal model in food and nutritional research. Despite the popularity of using swine in a regulatory setting they are rarely quoted in the regulatory guidelines as the nonrodent species in the testing guidelines. However, swine have extensively been used to elucidate the mechanisms associated with chronic over indulgence in certain foods (for example, olestra) as well as in the assessment of direct additives or residual contaminants introduced during the food production, processing, cooking, or storage conditions. Although the minipig appears not to have been used as the primary nonrodent species in standard regulatory toxicology studies for pesticide registration, their acceptability for regulatory testing is unquestioned. They have been used in organophosphorus research for decades and recently used to investigate the pathophysiology of dimethoate poisoning. Once practical hurdles are overcome then the minipig will be universally accepted by the agrochemical and chemical regulatory agencies as the most appropriate or more convenient species to employ.
Symp Xd The Use of Minipigs in FDA-Regulated Products: Experiences From the OND Division of Dermatology and Dental Products
Wafa A. Harrouk1
1US FDA, Silver Spring, MD, USA
The minipig has been used in recent years as an acceptable nonrodent model in the context of safety evaluation of investigational new drugs by regulatory agencies when scientifically justified. In particular, the minipig represents a good animal model to evaluate the safety profile of products intended to be used via the dermal route of exposure due to the similarity in stratum corneum thickness and density of hair follicles in human and minipig skin. Information presented in this talk will aim to convey experience with some FDA-regulated products where the minipig was used as the nonrodent species to test the systemic and dermal exposure in investigational products. Case studies of topical drug products will be used to illustrate the strengths and weaknesses of using this nonrodent species model for extrapolating risk assessment from minipigs to humans.
Symp XIa The History of ICH S6 and an Overview of the ICH Process for the Revision of ICH S6
James D. Green1
1Sudbury, MA, USA
The drafting, review, approval, and implementation of international guidance documents in the areas of preclinical safety assessment, clinical efficacy, and product quality are a complex process involving many scientific disciplines and regulatory authorities. For pharmaceutical and biopharmaceutical products, this process is overseen by a governance structure referred to as the International Conference of Harmonization (ICH). For a topic to be selected for the development of a new guidance document, a concept paper describing the topic and need for a new guidance in a particular area is first proposed to the ICH steering committee, which is comprised of senior members of worldwide health regulatory authorities and pharmaceutical trade organizations. Following approval of a new topic by this group, an expert working group is formed and the guidance document is developed through a prescribed path referred to as ‘Steps’ in which widespread critical scientific review and consensus is achieved. The ICH S6 guidance was the first worldwide attempt to achieve scientific consensus on what would be key considerations for the design of preclinical safety evaluation programs for biopharmaceuticals. This guidance document was released in final form in 1997 and represented a major advance in this area of biopharmaceutical drug development at that time. A key element of the ICH guidance process is referred to as “maintenance” in which current guidance documents are updated based upon new science and experience. After over a decade of use, the ICH steering committee determined that ICH S6 required clarification in several important topic areas and the process of maintenance was begun. This lecture will review the scientific and regulatory issues that led to the adoption of the principles described in the original ICH S6 and the maintenance process that produced the current addendum to ICH S6.
Keywords: ICH S6, safety assessment, biotechnology
Symp XIb Relevant Species Selection and General Toxicology Study Designs
Marque Todd1
1Drug Safety R&D, Pfizer, Inc, La Jolla, CA, USA
Selecting the relevant species and designing the general toxicology studies for the nonclinical development program for biotherapeutics remain foundational activities. Since the time when the original ICH S6 guideline was finalized, industry and health authorities have come to better understand best scientific practices around these activities. The selection of relevant species has evolved from more simple species homology searches and binding affinity assays to more sophisticated in vitro, ex vivo, and in vivo assays and biomarkers exploring the biological relevance and sensitivity of a species to the drug. The draft ICH S6 Addendum clarifies the role of tissue cross reactivity studies in species selection, when one or 2 species would be required and the use and limitation of alternative approaches such as homologous proteins, genetically modified animals, and disease models. Similarly, the design of the general toxicology studies has also evolved particularly in the areas of dose selection, study duration, and recovery periods. Dose selection has moved beyond the use maximum tolerated dose or maximum feasible dose and now encompasses exploration of PK/PD relationships including considerations around PAD and MABEL for selecting lower dose levels and maximum pharmacological responses and projected clinical dose levels when selecting the dose. Duration of the chronic toxicity studies and length or need for recovery periods are also discussed in the draft Addendum and will be covered in this presentation. Current case studies will be used to illustrate how best practices have evolved over the last decade and highlight the areas that the draft ICH S6 Addendum clarify or add to those practices today.
Keywords: biotherapeutic, relevant species, ICH S6, general toxicology
Symp XIc Evaluation of Immunogenicity of Biotherapeutics in Toxicology Studies—Impact and Update of ICH S6 Addendum
Stacie L. Wild1
1Amgen, Inc, Thousand Oaks, CA, USA
Over the years since the ICH S6 guidance document (Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals) was first issued, there has been a substantial gain of data and experience-based knowledge in the development of biotherapeutics. In addition, there has been further advancement in methods, models, and instrumentation used in the preclinical toxicology studies conducted for biotherapeutics. The experience gained and advancement of methods/models applied in toxicology studies has led to many topics being addressed in the addendum to ICH S6, including the assessment of immunogenicity in toxicology studies. There are many questions around the evaluation of immunogenicity responses in toxicology studies, with one important concern being how the immunogenicity data generated should be utilized (eg, assist in the interpretation of the toxicology study results or design of subsequent studies vs using the data to predict the immunogenic potential in humans). Additionally, there are several logistical questions regarding the assessment of immunogenicity in toxicology studies including the types of assays that should be used (eg, binding only or binding and neutralizing), when immunogenic responses should be measured during a toxicology study (eg, mid- and end of study or end of recovery period only), as well as which toxicology studies conducted in the preclinical toxicology program immunogenic responses should be measured. In the attempt to provide more updated/relevant guidance and clarification in the assessment of immunogenicity, all of these concerns are being addressed in the ICH S6 addendum. This presentation will provide a detailed review of these questions and challenges, as well as provide an update on the most recent recommendations being considered in the draft addendum (Step 3).
Keywords: biotherapeutic, immunogenicity, general toxicology, ICH S6
Symp XId Developmental and Reproductive Toxicity Testing for Biopharmaceuticals—Evolution and Impact of the Proposed ICH S6 Addendum
Pauline L. Martin1
1Centocor R&D Inc, Radnor, PA, USA
The ICH S6 guidance document states that “the need for reproductive/developmental toxicity studies is dependent upon the product, clinical indication, and intended patient population.” However, the guidance does not elaborate further on how these studies should be conducted. The presumption is that the ICH S5 (R2) document for the “Detection of Toxicity to Reproduction for Medicinal Products and Toxicity to Male Fertility” should be referred to for additional guidance. For biopharmaceuticals that have pharmacology activity in rodents and/or rabbits the study designs outlined in ICHS5 (R2) can be utilized. However, for those biopharmaceuticals that have pharmacological activity only in nonhuman primates (NHPs), experience has shown that attempts to adapt the rodent study designs for NHPs has not necessarily resulted in the optimal use of NHPs. Therefore, the ICH S6 addendum attempts to clarify the appropriate use of NHPs for DART testing and to clarify situations when alternate strategies may be acceptable. Proposed strategies for the optimization of NHP studies include: combining endpoints from the embryo/fetal development study and the pre and postnatal development study into a single enhanced PPND study, reducing the number of treatment groups and evaluating reproductive potential/“fertility” by histopathological examination of reproductive organs in sexually mature NHPs in place of conducting separate male and female “fertility” studies. Hormones, menstrual cycles, and semen analysis would only be necessary if there was a cause for concern. Although the testing of the clinical product is preferred over the use of surrogate molecules or genetically modified animals these alternate approaches can be used if scientifically justified. At the time of this writing the addendum was in draft (Step 3) and these recommendations are currently under negotiation.
Keywords: nonhuman primates, developmental and reproductive toxicity testing, ICH S6
Symp XIe Strategies for Carciongenicity Testing of Biotherapeutics
John L. Vahle1
1Toxicology and Pathology, Lilly Research Laboratories, Indianapolis, IN, USA
Assessing carcinogenic potential remains one of the most challenging areas of preclinical safety assessment. Biotherapeutics present unique challenges in this area as traditional rodent bioassays are often not scientifically appropriate. In addition, many of the therapeutic targets of biotherapeutics modulate immunologic or cell growth pathways further complicating an assessment of carcinogenic risk. The original ICH S6 guidance recognized the unique nature of these products and suggested a product-specific assessment that considered both the intended clinical use and biologic properties of the biotherapeutic. In the past several years, increased attention has been focused on carcinogenicity assessments of biotherapeutics leading to: (1) inclusion of carcinogenicity as a topic in the ICH S6 addendum and, (2) a collaborative industry effort to review best practices in this area. This presentation will review the carcinogenicity sections of the proposed ICH S6 addendum and highlights of the best practices review. Although a standardized set of assays or studies are not available to assess carcinogenic potential of biotherapeutics, there are several key principles that should be followed. These include carefully examining the mechanism of action to identify theoretical risks, interrogating existing data for evidence of proliferative or immunosuppressive potential, and, as needed, hypothesis-driven experiments to better characterize clinical risk. Case examples of approaches used for some recently approved biotherapeutics will highlight the challenges inherent in predicting human cancer risk and the impact on product labeling and risk management practices.
Keywords: biotherapeutic, carcinogenicity, ICH S6
Symp XIIa A Growing Challenge in the 21st Century: Drug Development for Elderly Patients
Greg Stevens1
1Drug Safety R&D, Pfizer, Inc, San Diego, CA, USA
A growing challenge for the pharmaceutical industry is the shift in demographics within the United States and other Western countries. The elderly population is growing at a logarithmic pace. Developing drugs for older patients is complicated by the physiological changes that occur with aging, the large number of concomitant medicines older patients are prescribed, and the complexity of the diseases being treated. This presentation will provide a brief overview of the physiological changes that occur with aging and the points to consider in developing drugs for the elderly.
Symp XIIb Mitochondrial Dysfunction and Aging/Chronic Disease—Differential Potential for Mitochondrial Toxicity in Young vs Aged Populations (or Animal Models)
Yvonne Will1
1Pfizer, Inc, Groton, CT
Mitochondria produce >95% of the body’s energy. Mitochondria are also the major source of ROS production. ROS production can lead to increase in mtDNA damage and impairment of normal function. Impairment of mitochondrial function can lead to disease. Mitochondrial function declines with age and many age related diseases have been linked to mitochondria dysfunction, such as diabetes and neurodegenerative diseases. The mitochondrial theory of aging was first proposed by Ames. In addition, mitochondria have been increasingly recognized as a target of drug toxicity resulting in disruption of bioenergetics and causing oxidant stress in sensitive organs including CNS, heart, and skeletal muscle. In addition, there is increasing awareness that mitochondria are key mediators of drug toxicity in a number of other organs including liver and kidney, often causing mitochondrial outer membrane permeabilization and release of cell death proteins. Underlying disease states like diabetes, infections, or neurodegenerative diseases can greatly alter mitochondrial function and make the mitochondria more vulnerable to drug toxicity. In recent years we have developed HTS applicable screens that can reveal mitochondrial dysfunction in vitro in isolated organelles and also cell models. Animal models rarely reveal mitochondrial toxicity, most likely due to the lack of genetic divergence in nuclear as well as mtDNA, and in general studies are performed using young male rats. Recently the heterozygous MnSOD mouse has been used to investigate drugs causing idiosyncratic liver toxicity in humans. It appears that compounds with the possibility to affect mitochondria can cause pathology in this model. Examination of other mitochondrial toxicants, targeting organs other than the liver, could be of value. In addition, several mouse models of mitochondrial disease are now available. None of these models have been utilized for the assessment of mitochondrial toxicity. In addition several models of premature aging are also available. Interestingly most of those are also related to mitochondrial dysfunction. Here, we will discuss the literature as it stands and propose a possible path forward in utilizing these animals as a more relevant representative for geriatric patients.
Symp XIIc Age-Related Changes in Skeletal Muscle Structure & Function—Correlations Between Data on Rodents and Humans
John A. Faulkner1
1Molecular & Integrative Physiology and Biomedical Engineering, University of Michigan Medical School, Ann Arbor, MI, USA
Mice and rats provide excellent models of the age-related changes that occur in the skeletal muscles of humans. Each of the 3 species, mice, rats and humans, shows age-related skeletal muscle atrophy, with linear decreases in total number of fibers and in the numbers of Type 2 (fast-twitch) motor units in a given skeletal muscle. The losses in the numbers of motor units and in muscle mass begin at approximately the same relative time in their respective life spans, ~50 years of age for humans and ~24 months of age for mice and rats. The loss in the number of motor units in rats results from loss of alpha-motorneurons. For humans after age 50, the losses in numbers of motor units and total number of fibers in the vastus lateralis muscle are linear out to age 90 and the losses appear to be inevitable. The rodent models are extremely useful since they not only provide valid models of aging, but also of obesity and hypertension. Rodents can be trained to run on a motor-driven treadmill and consequently a wide range of studies are possible on the interactions among: physical activity, diet, and aging.
Symp XIId Susceptibility to Influenza Virus in Aged Sprague-Dawley Rats Immunosuppressed With Dexamethasone
Gary R. Burleson1
1BRT-Burleson Research Technologies, Inc, Morrisville, NC, USA
Influenza virus is an infectious disease of public health significance for all age groups with special concerns for the elderly population. Dexamethasone suppresses many of the immunological functions necessary for viral clearance and differences in dexamethasone-treated versus vehicle-treated juvenile, adult, and aged rats are compared. Aged rats are more susceptible to influenza viral infection in dexamethasone-treated animals when compared to vehicle-treated rats. Aged rats treated with dexamethasone are also more susceptible to influenza virus infection than either juvenile or adult animals treated with dexamethasone and infected with virus
Symp XIIe Animal Models for Assessing Cardio-Pulmonary Adverse Effects in Geriatric Populations
Robert L. Hamlin1
1QTest; Stanton Youngberg Professor of Physiology/Pharmacology, Ohio State University
Regulatory agencies believe that a search for potential adverse cardiopulmonary effects of test articles in the ageing population should be conducted in surrogates for ageing humans possessing physiology and pathophysiology mimicking as closely as possible humans for whom the test article are intended. Because of the prevalence of diabetes, ventricular hypertrophy, heart failure, and degenerative pulmonary disease in the ageing human population, it is reasonable that the best surrogate for man would be ageing infrahuman mammals with these diseases propitious for the development of toxic events. Type I diabetes—unfortunately not type II—produced by either alloxan or streptozotocin, ventricular hypertrophy produced by banding of great arteries, heart failure produced by coronary ligation, rapid-pacing, or doxorubicin, and pulmonary injury produced by monocrataline, oleic acid, or paraquat have proven useful to predict both safety and efficacy. In addition to selecting the correct surrogate, it is equally important to interrogate the potential physiological targets (eg, heart rate, force development, hindrance and distribution of blood flow, energetics, venous return, receptor function, pulmonary compliance, resistance, and neural control) that if affected by test articles translate to adverse events. These topics appropriate for the pharmaceutical industry and regulatory agencies will be summarized using examples.
Symp XIIIa Dosimetry of Inhaled Chemicals: A Species Comparison
Fred J. Miller1
1Fred J. Miller and Associates LLC
Since the primary focus of this symposium is on pharmaceutical aerosols, this presentation will examine factors governing the dosimetry of inhaled particles in rats and humans and how complex interactions among these factors influence the inhalability, deposition, and clearance of particles. These factors can be broadly grouped into 2 categories, one dealing with the physicochemical properties of the particles and the other with species-specific factors such as airway structure, ventilatory level, and mucociliary and alveolar clearance rates. Particle transport and physical properties of the particles combine to yield the primary mechanisms by which particles are removed from the airstream: inertial impaction, sedimentation, and diffusion (Brownian). Electrostatic attraction can be an important mechanism for the deposition of particles if the aerosol delivery system generates charged particles and there is no method by which the aerosol is brought to Boltzman equilibrium. Due to differences in lung size and structure between rats and humans, the dominating deposition mechanism can vary among respiratory tract regions for rats compared to humans. Species-specific structure of the various respiratory tract regions in combination with the route and depth of breathing are critically important for determining where particles will deposit, but there are several other factors that affect dosimetry to differing degrees in laboratory rodents and humans, such as inhalability, route of breathing for humans, and heterogeneity in acinar deposition of particles. In addition, there are morphometric differences in alveolar parameters that may influence the choice of appropriate dose metrics for relating various types of effects. The above topics will be discussed in relationship to interspecies dosimetric comparisons.
Keywords: dosimetry of inhaled particles, rats versus humans, dose metrics
Symp XIIIb Pulmonary Histopathology Associated With Inhaled Particles
Fletcher F. Hahn1
1Lovelace Respiratory Research Institute, Albuquerque, NM, USA
Inhaled pharmaceuticals in an insoluble particulate form may be used to treat lung disease and in a soluble form to treat systemic disease. The lung has evolved with many mechanisms to protect against damage from inhaled particles. However, particles of various types are known to induce lesions in the lung. Many particles are toxic as a result of occupational or environmental exposures and include chemicals, metals, radioactive materials, fibers, and mixtures, such as cigarette smoke or diesel exhaust. The histopathologic reactions in the lung of rats exposed to these particles include inflammation, fibrosis, epithelial hyperplasia, squamous metaplasia, and emphysema. The basis histologic reactions seen in the lung of rats are similar to those seen in humans. In addition to the histopathologic reaction in the lung, important factors in the toxicity of particulates are site of deposition in the lung, concentration and biopersistence of the material in the lung, the shape and size of the particle and the chemical composition of the particle. Each of these factors needs to be evaluated in determining the relative toxicity of inhaled particles.
Keywords: inhaled particles, lung, histolpathology
Symp XIIIc Pathobiology of the Alveolar Macrophage Response to Inhaled Pharmaceuticals
Deon Hildebrand1
1Safety Assessment, GlaxoSmithKline, Ware, UK
In preclinical inhalation toxicology studies, the presence of enlarged macrophages with foamy cytoplasm in the terminal airways of the lung is a common finding in control and treated animals. In many studies, a treatment-related increase in the incidence and severity occurs, occasionally accompanied by macrophage pigmentation, local inflammation and hypertrophy/hyperplasia of terminal airway epithelium. The adversity of this spectrum of changes is assessed in each study and is mainly based on the microscopic appearance of the changes but other supporting study-related information is also taken into consideration. A simple increase in macrophage number would usually be considered a nonadverse, adaptive change in response to the physical presence of inhaled drug material. However, the presence of other secondary changes needs more careful interpretation in terms of their adversity. A greater understanding of the pathogenesis of the response will enhance our understanding of the adversity of these changes and their relevance to humans. This presentation will describe GSK’s recent experience with macrophage changes and current thinking about the underlying pathogenesis of the changes.
Keywords: foamy alveolar macrophages, adverse, pathogenesis
Symp XIIId Implications of the Pulmonary Alveolar Macrophage Response for Risk Assessment, Dose-Setting, and Monitoring in the Clinic
Timothy J. McGovern1
1SciLucent, LLC
Increases in alveolar macrophages are a commonly observed finding in nonclinical toxicology studies that utilize the inhalation route of administration. While this response is generally considered a normal adaptive response to inhaled particulate material, the response can have a significant impact on the regulatory safety evaluation of pharmaceuticals intended for clinical investigation, especially for trials including patient populations with compromised respiratory systems. This presentation will discuss the implications of the alveolar macrophage response in toxicology studies in regard to potential clinical holds, dose selection for clinical trials, and follow-up studies to further characterize the response as nonadverse versus those associated with secondary inflammation, necrosis and fibrosis. Various approaches to monitor the finding in the clinic will also be discussed although none are currently considered adequate from a regulatory perspective to offset safety concerns.
Keywords: alveolar macrophages, risk assessment, clinical monitoring
Symp XIVa Molecular Markers for Metal and Metalloid Toxicity
Bruce A. Fowler1
1Division of Toxicology and Environmental Medicine, Agency for Toxic Substances and Disease Registry, Atlanta, GA, USA
The ongoing development of molecular “omic” biomarkers for early detection of toxicity has provided new insights into both mechanisms of cell injury / cell death from agents such as toxic metals and metalloids. The continuum of genomic, proteomic and metabolomic / metabanomic biomarkers coupled with evolving computational tools have greatly increased the ability of scientists to make rapid and informed risk assessments. Combined in vivo and in vitro studies on gallium, indium and arsenic semiconductors, on an individual or binary mixture basis using proteomic endpoints (Fowler et al., 2005, 2008) demonstrated metal/metalloid—specific alterations in protein expression patterns in renal tubule cells which were associated with GaAs and InAs—specific proteinuria patterns in 2-D gel electrophoretograms visualized by silver staining. Other studies which compared differences in protein expression patterns between males and females showed gender—specific responses for renal cells from both hamsters and humans. Females generally produced a more robust protein expression response than males for an equivalent GaAs or InAs exposure. The results of these studies provide a useful demonstration of how proteomic biomarkers may be applied to both understanding cellular responses to toxic metallic agents alone or as mixtures and helping to identify sensitive subpopulations as function of gender.
Symp XIVb Elemental Contaminants and Dietary Exposure
Carrington C. D.1 and Bolger P. M.1
1Chemical Hazards Assessment Staff, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD, USA
The risks of dietary elemental contaminants are normally considered via a safety assessment which results in the identification of a level of exposure (ie, Tolerable Daily Intake [TDI]) deemed to represent negligible risk. The TDI is derived form a Point of Departure (POD) which is either a No Observed Adverse Effect Level (NOAEL) or Benchmark Dose Lower Confidence Limit (BMDL) from an animal or human study and the application of 10 fold safety/uncertainty factors which account for the uncertainty/variability in the sensitivity of members of a population and /or extrapolation between species. An example is the a prior position that children are more sensitive than adults. This is based on the generic pre- and post-natal sensitivity of the developing organism and higher exposure of children versus adults on a body weight basis. An example is the observed sensitivity of the fetus and children to lead. Sensitivity may also occur because of nutritional deficiencies, genetic polymorphisms, reduced clearance capacity, and preexisting health conditions. The safety assessment paradigm is useful for determining exposures which are deemed to be of no public health concern. The determination that an exposure is “unsafe” or a population is “at risk” should lead to a consideration of risk as a matter of degree. This will allow for the inclusion of other issues such as the avoidability of exposure and competing dietary risks. Dose-response information may be used quantitatively to describe the differential response of sensitive populations by deriving estimates that account for both variability and uncertainty of the risk. These methods can then be generalized to evaluate the merits of other public health and risk management programs that involve trade-offs between food borne risks. Four major toxic elements, arsenic, cadmium, lead and mercury, that can occur in food will be presented to illustrate these points.
Symp XIVc Elemental Impurity: USP's Perspective
K. Zaidi1
1Department of Standards Development, USP, 12601 Twinbrook Parkway, Rockville, MD, USA
Heavy metals tests have been available in the USP/NF for over 100 years. These tests include the general heavy metals screening tests contained in General Chapter Heavy Metals <231>, as well as specific tests for aluminum, arsenic, iron, lead, mercury, and selenium. The screening tests in <231> are nonelement-specific limit tests applicable to a relatively small number of elements. The element specific tests are also chemical tests, some of which can be difficult to perform. The current state of metal toxicology in humans now allows a better understanding of the health implications of metals with the potential to be present in pharmaceuticals. Metals with the highest potential toxicity, including lead, mercury, cadmium, and arsenic, are ubiquitous in the environment. These toxic metals provide no therapeutic benefit, thus should be controlled in all pharmaceuticals and dietary supplements. USP worked with toxicologists and measurement scientists to assess methodologies and limits that provide greater patient/consumer protection, and has published 3 new draft chapters this year. These chapters propose health-based acceptable levels (permissible daily exposures – PDE) for metal impurities, and methodology capable of measuring these metals at the appropriate levels. The proposed sample preparation and instrumental technology permit the quantification of a broad range of elements at low levels with excellent precision and accuracy. This presentation will provide the background and current status of these proposed general chapters and discuss the current efforts of the ICH Q3D working party to provide global acceptance of proposed PDE for selected metals in pharmaceuticals.
Symp XIVd Current Issues in Metal Toxicity With Specific References to As, Cd, Cr, and Se
Fan A. M.1 and Howd R.1
1Office of Environmental Health Hazard Assessment (OEHHA), California Environmental Protection Agency, Oakland, CA, USA
Current toxicological and risk assessment issues for As, Cd, Cr, and Se via the oral route assessed by OEHHA provide additional considerations for setting exposure limits.
Symp XIVe Toxicity of Heavy and Trace Metals: Regulatory Perspectives
Mamata De1
1US FDA, Silver Spring, MD, USA
Elemental impurities are unwanted chemicals which may affect efficacy and safety of the pharmaceutical products. It includes catalysts and environmental contaminants that may be present in APIs, excipients, or drug products. These impurities may occur naturally, be added intentionally, or be introduced inadvertently (eg, through interactions with processing equipment). The presence of elemental impurities in APIs and excipients must be controlled. The basis of setting limits for the elemental impurities is related to their toxicity and bioavailability. The toxicities associated with the major elemental impurities (heavy metals and trace metals) will be discussed in the presentation. The presentation will also include the rationales for the limits of the elemental impurities in the drug products.
Symp XVa Assessment of Safety Pharmacology Endpoints in General Toxicology Studies
Hugo M. Vargas1
1Department of Investigative Toxicology, Comparative Biology & Safety Sciences. Amgen, Inc, Thousand Oaks, CA, USA
Safety pharmacology (SP) studies are the in vivo/in vitro functional studies that investigate the potential undesirable effect of a new test substance on vital physiologic functions to characterize the potential risk to humans. Typically, SP studies are performed as stand-alone studies in conscious rats, dogs, pigs or nonhuman primates, however, regulatory guidance allows for the integration of SP endpoints into toxicology studies under certain conditions, eg, for biopharmaceuticals like monoclonal antibodies or recombinant proteins. The collection of SP endpoints in repeat-dose studies could benefit drug safety evaluation by refining data collection, animal use, and hazard identification. This session with provide an overview of the application of SP endpoints into general toxicology studies and describe techniques and approaches that can be employed to maximize risk identification and optimize animal use.
Symp XVc Established and Emerging Noninvasive Techniques for Functional Measurements in Repeat-Dose Toxicity Studies
Redfern W. S.1, Ewart L.1, Lainée P.1, Robinson S.1, Valentin J.-P.1
1Safety Assessment UK, AstraZeneca R&D, Alderley Park, Macclesfield, SK10 4TG, United Kingdom
In vivo safety pharmacology studies generally involve a single administration of a candidate drug, followed by functional measurements for up to 24 h. Whereas pharmacological responses tend to be fairly rapid in onset, and are therefore detectable after a single dose, some may diminish on repeated dosing (tolerance), whereas others increase in magnitude. Also, many toxic effects of drugs are either delayed in onset or require multiple exposures before they develop (eg, neuropathy). Therefore, single-dose safety pharmacology studies may sometimes miss these late-onset effects. Functional measurements can be incorporated into repeat-dose toxicity studies, either routinely (eg, ECG) or on an ad hoc basis. Drivers for this are both scientific (based on the reasons above), and regulatory, eg, testing of biotechnology products (ICHS6) and end-stage cancer therapies (ICHS9) does not necessarily require stand-alone safety pharmacology studies. There are inherent difficulties in achieving this: the availability of suitable technical and scientific expertise in the test facility; suboptimal laboratory conditions; use of simultaneous (as opposed to staggered) dosing; prioritisation of toxicokinetic sampling; unsuitability of certain techniques (eg, use of anaesthesia; surgical implantation); low portability of certain equipment. Nonetheless, “fit-for-purpose” data can still be acquired in rodents, dogs and monkeys, without requiring additional animals. Examples in our facility include assessment of behaviour, sensorimotor function, visual function, salivation, ambulatory ECG, echocardiography, and respiratory function. This is entirely achievable if measurements are relatively unobtrusive, both with respect to the animals and the toxicology study.
Keywords: safety pharmacology, repeat-dose toxicity studies, functional measurements
Symp XVIa Therapies for Type 2 Diabetes: Nonclinical Safety Assessments and Risk Evaluation
Jeri El-Hage1, Todd Bourcier2, David Carlson2, Brian Hummer2, and Mark Timerstein3
1Aclairo Pharmaceutical Development Group, Vienna, VA, USA
2Division of Metabolism and Endocrinology Products(DMEP), FDA
3Bristol Meyers Squibb, New Brunswick, NJ, USA
The incidence of Type 2 diabetes is reaching epidemic rates in the US and world-wide, resulting in a keen interest in developing new therapies acting at novel molecular targets. Product areas of active drug development include the incretins (dipeptidyl peptidase 4 inhibitors and glucagon-like peptide 1 agonists), glucokinase activators, sodium glucose transporter 2 inhibitors, and numerous others (PPAR agonists, FGF 19, GPR 40, GPR 119 agonists, 11B-HSD inhibitors, etc).
A general introduction to and overview of the development of compounds for type 2 diabetes will be presented. Standard animal models used to assess for anti-diabetic efficacy include ob/ob mice, db/db mice, and ZDF rats. Standard ICH M3 preclinical programs are needed with additional studies recommended dependent upon the drug class and the demonstrated toxicity profiles. The recent Diabetes Drugs Guidance outlines additional studies needed to characterize the chronic toxicity profile of peroxisome proliferator activated receptor (PPAR) agonists. Class specific preclinical and clinical toxicity profiles for the new molecular target products will be discussed. Examples include (1) skin lesions and anaphylactic reactions in monkeys treated with DPP-4 inhibitors, (2) thyroid C-cell tumors observed in carcinogenicity studies with long-acting GLP-1 agonists, (3) neuropathy secondary to prolonged hypoglycemia with GK activators, and (4) effects of SGLT 2 inhibitors on bone. Examples of recommendations for class specific preclinical safety studies include a toxicology study in monkeys for DPP-4 inhibitors or a toxicology study in a diabetic/hyperglycemic animal model for GK activators.
Symp XVIb Regulatory Preclinical Development of Dipeptidyl Peptidase 4 Inhibitors
Todd Bourcier1
1US FDA, CDER, Division of Metabolic & Endocrine Products, Silver Spring, MD, USA
Pursuit of dipeptidyl peptidase-4 inhibitors (DPP4i) as a treatment for Type 2 diabetes remains an active area of drug development. By inhibiting deactivation and prolonging the plasma half-life of incretins, DPP4 inhibitors enhance post-prandial insulin release and reduce peak plasma glucose and HbA1c in several hyperglycemic animal models and in patients with type 2 diabetes. Physiological substrates of DPP4 enzymatic activity may include several biologically active peptides with diverse functions, including neuropeptide Y, stromal derived factor-1, and growth hormone releasing factor, in addition to the intended incretin target, GLP1. Prolonged activity of off-target DPP4 substrates may contribute to toxicities observed in regulatory preclinical toxicology studies with DPP4 inhibitors, such as multi-organ inflammatory infiltrates. Off-target inhibition of structural homologues of DPP4 with overlapping enzymatic specificity, such as DPP8 and 9, may also contribute to toxicities observed in animals with some investigational compounds, including gastrointestinal bleeding in dogs and cutaneous ulcerations in nonhuman primates. The role of DPP4 in chemokine inactivation and of DPP4/CD26 in T-cell activation raises the potential for immunotoxicity with DPP4 inhibitors, yet severe adverse effects on responses to antigen have not been reported. The standard regulatory preclinical program for DPP4 inhibitors includes a 3-month assessment in nonhuman primates to address the potential for adverse cutaneous vascular toxicity prior to initiation of phase 3 clinical studies.
Keywords: type 2 diabetes, preclinical toxicology, dipeptidyl peptidase-4 inhibitors
Symp XVIc GLP-1 Receptor Agonists: Extrapolating Nonclinical Data to Human Risk
B. Timothy Hummer1
1US FDA, CDER, Division of Metabolic & Endocrine Products, Silver Spring, MD, USA
Glucagon like peptide-1 (GLP-1) is an endogenous incretin peptide that has glucoregulatory activities including stimulation of glucose-mediated insulin synthesis and secretion, enhancement of insulin sensitivity, suppression of glucagon release, and slowed gastric emptying. Endogenous GLP-1 is rapidly degraded by dipeptidyl peptidase 4 (DPP4). To extend the peptide’s half life and achieve therapeutic exposures, GLP-1 analogs that are resistant to DPP4 degradation have been created for the treatment of type 2 diabetes. However, enhanced exposure to GLP-1 analogs may elicit safety concerns that are not seen with the secretion pattern of endogenous GLP-1. First, an increase in thyroid c-cell tumors has been observed in 2 year studies in rodents, often at clinically relevant exposures. Tumor promoting activity appears more potent for GLP-1 receptor agonists that have a long half life or that are formulated to allow sustained release. Currently there is insufficient information on the mode of action for c cell tumorigenesis in rodents to conclude that the tumorigenic process is not relevant to humans. Therefore, additional nonclinical studies are needed to better understand the mode of action and relevance to human risk. Second, there are post-marketing reports of pancreatitis in patients administered GLP-1-based drugs. In general, nonclinical toxicology studies did not reveal pancreatic toxicity in normoglycemic, healthy animals during drug development. To further investigate the potential effect of GLP-1 analogs on pancreatitis in the diabetic population, companies developing GLP-1 analogs have been encouraged to conduct dedicated toxicity studies in animal models of diabetes.
Keywords: type 2 diabetes, glucagon like peptide-1 (GLP-1), thyroid c-cells
Symp XVId Preclinical Toxicology Of Glucokinase Activators - A Promising New Therapeutic Class for Diabetes?
Carlson, David B1
1US FDA, Silver Spring, MD, USA
Glucokinase activation is a promising therapeutic mechanism under development for the treatment of type 2 diabetes mellitus. Glucokinase catalyzes glucose phosphorylation and drives liver glycogen formation while also increasing pancreatic beta cell insulin secretion. The dual actions of increased insulin secretion and increased hepatic glucose processing are expected to improve blood glucose control in diabetic individuals. Several glucokinase activators (GKAs) are currently under development and efficacy has been established in nonclinical animal models. While GKA treatment improves blood glucose control in diabetic animals, euglycemic animals used in standard toxicology studies are subjected to both acute and prolonged hypoglycemia. Maximum tolerated doses in GKA toxicity studies are typically defined by clinical signs and physiologic effects of acute hypoglycemia, including tremors, convulsions, and mortality. Many GKAs also potently and severely reduce blood glucose for 12 to 24 hours after a single dose. Toxicity trends in GKA-treated euglycemic animals subjected to prolonged hypoglycemia include Wallerian degeneration of peripheral nerves and lens opacities. Supplementation with glucose or hyperglycemic agents has not typically been sufficient to counter the long term hypoglycemia and toxicity in euglycemic animals. Thus, a great challenge in nonclinical toxicity studies with chronic glucokinase activation seems to be balancing acute and prolonged hypoglycemia with the goal of pushing drug exposure to sufficient multiples of expected human therapeutic doses. GKAs have the potential to provide potent, prolonged glucose control in diabetics but neither standard euglycemic nor diabetic animal models alone are ideal for pharmacology and toxicology studies. Challenges in nonclinical safety assessment and potential strategies and regulatory issues for GKA drug development will be discussed.
Keywords: glucokinase activators, type 2 diabetes, animal models
Symp XVIe Nonclinical Bone Effects Of Sodium Glucose Transporter-2 Inhibitors Due to Off-Target Sodium Glucose Transporter-1 Inhibition
M. A. Tirmenstein1
1Drug Safety Evaluation, Department of Toxicology, Bristol-Myers Squibb Co. New Brunswick, NJ, USA
Sodium glucose transporter-2 (SGLT2) inhibitors are in development for the treatment of type 2 diabetes. They act by inhibiting SGLT2 in the kidney promoting the excretion of urinary glucose. In nonclinical toxicology studies, SGLT2 inhibitors induced pharmacology mediated loss of urinary glucose with associated decreases in body-weights gains despite increased food consumption. Increases in urinary volume and loss of urinary electrolytes (sodium, phosphorus, and calcium) due to osmotic diuresis were also observed without biologically significant decreases in serum electrolytes. At high doses, SGLT2 inhibitors inhibited the major sodium glucose transporter-1 (SGLT1) in the intestines resulting in decreased glucose absorption in the intestines. The resulting increased intestinal glucose would be expected to lead to microbial fermentation, decreased intestinal pH, and increased gas production. SGLT2 inhibitors also promoted increased serum calcium associated with tissue mineralization and increased trabecular bone formation but only at high doses and only in rats. An SGLT2 inhibitor was administered at high doses to 2 groups of rats. The first group was fed a standard glucose containing diet while the second group was fed a fructose-substituted diet lacking glucose. After 10 days, rats fed a standard diet had fecal effects and hypercalcemia with tissue mineralization but in rats fed the glucose-free diet, these effects were either entirely prevented or ameliorated. These results provide supportive evidence for an off-target SGLT1 effect of increased calcium absorption secondary to inhibition of intestinal glucose transport underlying the observed calcification of various tissues, including bone, at high doses in the rat. These effects were only seen at human exposure multiples > 2000× and therefore, are not relevant to human safety.
Symp XVIIa Epigenetics: Role in Toxicity, Disease Etiology and Risk Assessment
Jerrold J. Heindel (presenter)1 and Lisa Helbing Chadwick1
1Division of Extramural Research and Training, National Institute of Environmental Health Sciences
Epigenetic mechanisms play an important role in development, as they help the cells of an organism decide what set of instructions to follow—a specific subset of genes required for the cell to acquire its unique functions. A growing body of evidence indicates that exposure to certain environmental toxicants can disrupt epigenetic processes, which can result in disease. This talk will summarize the evidence linking environmental toxicants to epigenetic changes and discuss how exposures during critical periods of prenatal development can lead to epigenetic changes that lead to disease outcomes in the adult. Several studies have also suggested that these changes may be passed on through multiple generations, even in absence of a direct exposure. These findings have important implications for risk assessment and regulation.
Symp XVIIc Perspective of an FDA Pharmacology/Toxicology Reviewer on Change
Molly (Shea) Topper1
1Pharmacology/Toxicology Supervisor, FDA CDER Division of Pulmonary, Allergy, and Rheumatology Products
This discussion will surround the realignment from the Division of Pulmonary and Allergy Products to the Division of Pulmonary, Allergy and Rheumatology Products and its impact on reviewers and consequently on Sponsors. It will give a brief overview of what changes have been made and how toxicology data are viewed.
Symp XVIIe Genotoxic Impurities—An Overview of Planned ICH Updates
Sheila M. Galloway1
1Genetic Toxicology and Molecular Carcinogenesis, Merck Research Laboratories
This presentation will provide a brief review of the existing guidance on control of genotoxic impurities in pharmaceuticals, and an outline of the main issues under discussion for development of an ICH guideline on genotoxic impurities. Included in the discussion are: Acceptable levels of genotoxic impurities during drug development and for marketing; the suitability of the Threshold of Toxicological Concern (TTC) approach; the use of structure activity analyses; what sort of qualification testing should be followed for impurities that are metabolites; whether an allowable amount of an impurity should be applied to each impurity individually, or to a sum of “similar” impurities; and what information would be needed to justify higher acceptable daily intake than the TTC.
Symp XVIIf FDA Update—Pregnancy and Lactation Labeling Rule
Lynnda Reid
FDA CDER Division of Reproductive and Urologic Products
This presentation will discuss pending new regulations as they pertain to the “Pregnancy and Lactation Labeling Rule" for pharmaceutical agents (drugs and biologics). The presentation will focus on the revised format for labeling, formulation of a ‘risk statement' based on animal data, and presentation of animal reproductive and developmental data in new labels.
Symp XVIIg A Behind the Scenes Look at FDA Guidances
Hanan Ghantous1
1FDA CDER Division of Antiviral Products
Guidances are prepared to establish clarity and consistency in FDA policies, regulatory activities, and inspection and enforcement procedures. They reflect FDA’s current thinking on an issue. Guidances are intended to assist industry in carrying out its obligations under laws and regulations on subjects such as the processing, content, evaluation, and approval of drug product applications and the design, production, manufacturing, and testing of regulated products. This presentation will provide a behind the scenes look at how a guidance is developed, cleared, published, and used