Neutralizing RGMa with Elezanumab Promotes Cerebroprotection and Recovery in Rabbit Middle Cerebral Artery Occlusion.

Repulsive guidance molecule A (RGMa) is an inhibitor of neuronal growth and survival which is upregulated in the damaged central nervous system following acute spinal cord injury (SCI), traumatic brain injury, acute ischemic stroke (AIS), and other neuropathological conditions. Neutralization of RGMa is neuroprotective and promotes neuroplasticity in several preclinical models of neurodegeneration and injury including multiple sclerosis, AIS, and SCI. Given the limitations of current treatments for AIS due to narrow time windows to intervention (TTI), and restrictive patient selection criteria, there is significant unmet need for therapeutic agents that enable tissue survival and repair following acute ischemic damage for a broader population of stroke patients. In this preclinical study, we evaluated whether elezanumab, a human anti-RGMa monoclonal antibody, could improve neuromotor function and modulate neuroinflammatory cell activation following AIS with delayed intervention times up to 24 h using a rabbit embolic permanent middle cerebral artery occlusion model (pMCAO). In two replicate 28-day pMCAO studies, weekly intravenous infusions of elezanumab, over a range of doses and TTIs of 6 and 24 h after stroke, significantly improved neuromotor function in both pMCAO studies when first administered 6 h after stroke. All elezanumab treatment groups, including the 24 h TTI group, had significantly less neuroinflammation as assessed by microglial and astrocyte activation. The novel mechanism of action and potential for expanding TTI in human AIS make elezanumab distinct from current acute reperfusion therapies, and support evaluation in clinical trials of acute CNS damage to determine optimal dose and TTI in humans. A: Ramified/resting astrocytes and microglia in a normal, uninjured rabbit brain. B: Rabbit pMCAO brain illustrating lesion on right side of brain (red), surrounded by penumbra (pink) during acute phase post stroke, with minimal injury to left brain hemisphere. Penumbra characterized by activated astrocytes and microglia (region in crosshair within circle), with upregulation of free and bound RGMa. C: Elezanumab binds to both free and bound RGMa, preventing full activation of astrocytes and microglia. D: Elezanumab is efficacious in rabbit pMCAO with a 4 × larger TTI window vs. tPA (6 vs. 1.5 h, respectively). In human AIS, tPA is approved for a TTI of 3-4.5 h. Elezanumab is currently being evaluated in a clinical Ph2 study of AIS to determine the optimal dose and TTI (NCT04309474).

In-vitro modeling of intravenous drug precipitation by the optical spatial precipitation analyzer (OSPREY).

Intravenous (IV) administration of poorly water-soluble small molecule therapeutics can lead to precipitation during mixing with blood. This can limit characterization of pharmacological and safety endpoints in preclinical models. Most often, tests of kinetic and thermodynamic solubility are used to optimize the formulation for solubility prior to infusion in animals, but these do not capture the dynamic precipitation processes that take place during in-vivo administration. To better capture the fluid dynamic processes that occur during IV administration, we developed the Optical Spatial PREcipitation analYzer (OSPREY) as a method to quantify the amount and size of compound precipitates in whole blood using a flow-through system that mimics IV administration. Here, we describe the OSPREY device and its underlying imaging processing methods. We then validate the ability to accurately segment particles according to their size using monodisperse suspensions of microspheres (diameter 50 to 425 µm). Next, we use a tool compound, ABT-737, to study the effects of compound concentration, vessel flow rate, compound infusion rate and vessel diameter on precipitation. Finally, we use the physiological diameter and flow rate of rat femoral vein and dog saphenous vein to demonstrate the potential of OSPREY to model in-vivo precipitation in a controlled, dynamic in-vitro assay.

Evaluation of Quantitative Platforms for Single Target Mass Spectrometry Imaging.

(1) Imaging of pharmaceutical compounds in tissue is an increasingly important subsection of Mass Spectrometry Imaging (MSI). Identifying proper target engagement requires MS platforms with high sensitivity and spatial resolution. Three prominent categories of drugs are small molecule drugs, antibody-drug conjugate payloads, and protein degraders. (2) We tested six common MSI platforms for their limit of detection (LoD) on a representative compound for each category: a Matrix-Assisted Laser Desorption/Ionization (MALDI) Fourier Transform Ion Cyclotron, a MALDI-2 Time-of-Flight (ToF), a MALDI-2 Trapped Ion Mobility Spectrometry ToF, a Desorption Electrospray Ionization Orbitrap, and 2 Atmospheric Pressure-MALDI Triple Quadrupoles. Samples were homogenized tissue mimetic models of rat liver spiked with known concentrations of analytes. (3) We found that the AP-MALDI-QQQ platform outperformed all 4 competing platforms by a minimum of 2- to 52-fold increase in LoD for representative compounds from each category of pharmaceutical. (4) AP-MALDI-QQQ platforms are effective, cost-efficient mass spectrometers for the identification of targeted analytes of interest.

Identification of VEGF Signaling Inhibition-Induced Glomerular Injury in Rats through Site-Specific Urinary Biomarkers.

Cancer therapies targeting the vascular endothelial growth factor (VEGF) signaling pathway can lead to renal damage by disrupting the glomerular ultrafiltration apparatus. The objective of the current study was to identify sensitive biomarkers for VEGF inhibition-induced glomerular changes in rats. Male Sprague-Dawley rats were administered an experimental VEGF receptor (VEGFR) inhibitor, ABT-123, for seven days to investigate the correlation of several biomarkers with microscopic and ultrastructural changes. Glomeruli obtained by laser capture microdissection were also subjected to gene expression analysis to investigate the underlying molecular events of VEGFR inhibition in glomerulus. ABT-123 induced characteristic glomerular ultrastructural changes in rats, including fusion of podocyte foot processes, the presence of subendothelial electron-dense deposits, and swelling and loss of fenestrations in glomerular endothelium. The subtle morphological changes cannot be detected with light microscopy or by changes in standard clinical chemistry and urinalysis. However, urinary albumin increased 44-fold as early as Day three. Urinary ß2-microglobulin levels were also increased. Other urinary biomarkers that are typically associated with tubular injury were not significantly impacted. Such patterns in urinary biomarkers can provide valuable diagnostic insight to VEGF inhibition therapy-induced glomeruli injuries.

Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs.

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

Comparison of RNA-Seq and Microarray Gene Expression Platforms for the Toxicogenomic Evaluation of Liver From Short-Term Rat Toxicity Studies.

Gene expression profiling is a useful tool to predict and interrogate mechanisms of toxicity. RNA-Seq technology has emerged as an attractive alternative to traditional microarray platforms for conducting transcriptional profiling. The objective of this work was to compare both transcriptomic platforms to determine whether RNA-Seq offered significant advantages over microarrays for toxicogenomic studies. RNA samples from the livers of rats treated for 5 days with five tool hepatotoxicants (α-naphthylisothiocyanate/ANIT, carbon tetrachloride/CCl4, methylenedianiline/MDA, acetaminophen/APAP, and diclofenac/DCLF) were analyzed with both gene expression platforms (RNA-Seq and microarray). Data were compared to determine any potential added scientific (i.e., better biological or toxicological insight) value offered by RNA-Seq compared to microarrays. RNA-Seq identified more differentially expressed protein-coding genes and provided a wider quantitative range of expression level changes when compared to microarrays. Both platforms identified a larger number of differentially expressed genes (DEGs) in livers of rats treated with ANIT, MDA, and CCl4 compared to APAP and DCLF, in agreement with the severity of histopathological findings. Approximately 78% of DEGs identified with microarrays overlapped with RNA-Seq data, with a Spearman's correlation of 0.7 to 0.83. Consistent with the mechanisms of toxicity of ANIT, APAP, MDA and CCl4, both platforms identified dysregulation of liver relevant pathways such as Nrf2, cholesterol biosynthesis, eiF2, hepatic cholestasis, glutathione and LPS/IL-1 mediated RXR inhibition. RNA-Seq data showed additional DEGs that not only significantly enriched these pathways, but also suggested modulation of additional liver relevant pathways. In addition, RNA-Seq enabled the identification of non-coding DEGs that offer a potential for improved mechanistic clarity. Overall, these results indicate that RNA-Seq is an acceptable alternative platform to microarrays for rat toxicogenomic studies with several advantages. Because of its wider dynamic range as well as its ability to identify a larger number of DEGs, RNA-Seq may generate more insight into mechanisms of toxicity. However, more extensive reference data will be necessary to fully leverage these additional RNA-Seq data, especially for non-coding sequences.

Immunomodulatory effects of tick saliva on dermal cells exposed to Borrelia burgdorferi, the agent of Lyme disease.

BACKGROUND: The prolonged feeding process of ixodid ticks, in combination with bacterial transmission, should lead to a robust inflammatory response at the blood-feeding site. Yet, factors present in tick saliva may down-regulate such responses, which may be beneficial to spirochete transmission. The primary goal of this study was to test the hypothesis that tick saliva, in the context of Borrelia burgdorferi, can have widespread effects on the production of immune mediators in skin. METHODS: A cross-section of tick feeding on skin was examined histologically. Human THP-1 cells stimulated with B. burgdorferi and grown in the presence or absence of tick saliva were examined by human DNA microarray, cytokine bead array, sandwich ELISA, and qRT-PCR. Similar experiments were also conducted using dermal fibroblasts. RESULTS: Tick feeding on skin showed dermal infiltration of histiocytes and granulocytes at the bite location. Changes in monocytic transcript levels during co-culture with B. burgdorferi and saliva indicated that tick saliva had a suppressive effect on the expression of certain pro-inflammatory mediators, such as IL-8 (CXCL8) and TLR2, but had a stimulatory effect on specific molecules such as the Interleukin 10 receptor, alpha subunit (IL-10RA), a known mediator of the immunosuppressive signal of IL-10. Stimulated cell culture supernatants were analyzed via antigen-capture ELISA and cytokine bead array for inflammatory mediator production. Treatment of monocytes with saliva significantly reduced the expression of several key mediators including IL-6, IL-8 and TNF-alpha. Tick saliva had an opposite effect on dermal fibroblasts. Rather than inhibiting, saliva enhanced production of pro-inflammatory mediators, including IL-8 and IL-6 from these sentinel skin cells. CONCLUSIONS: The effects of ixodid tick saliva on resident skin cells is cell type-dependent. The response to both tick and pathogen at the site of feeding favors pathogen transmission, but may not be wholly suppressed by tick saliva.

Biomarker evaluation of skeletal muscle toxicity following clofibrate administration in rats.

The use of sensitive biomarkers to monitor skeletal muscle toxicity in preclinical toxicity studies is important for the risk assessment in humans during the development of a novel compound. Skeletal muscle toxicity in Sprague Dawley Rats was induced with clofibrate at different dose levels for 7 days to compare standard clinical pathology assays with novel skeletal muscle and cardiac muscle biomarkers, gene expression and histopathological changes. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) enzyme activity were compared to novel biomarkers fatty acid binding protein 3 (Fabp3), myosin light chain 3 (Myl3), muscular isoform of CK immunoreactivity (three isoforms CKBB, CKMM, CKMB), parvalbumin (Prv), skeletal troponin I (sTnI), cardiac troponin T (cTnT), cardiac troponin I (cTnI), CKMM, and myoglobin (Myo). The biomarker elevations were correlated to histopathological findings detected in several muscles and gene expression changes. Clofibrate predominantly induced skeletal muscle toxicity of type I fibers of low magnitude. Useful biomarkers for skeletal muscle toxicity were AST, Fabp3, Myl3, (CKMB) and sTnI. Measurements of CK enzyme activity by a standard clinical assay were not useful for monitoring clofibrate-induced skeletal muscle toxicity in the rat at the doses used in this study.

Cross-laboratory analytical validation of the cardiac biomarker NT-proANP in rat.

INTRODUCTION: Natriuretic peptides, including N-terminal-proatrial natriuretic peptide (NT-proANP) are cardiac hormones that are produced in response to myocardial stretch and have been used in rats and humans as blood based functional cardiac biomarkers. There are limited validation data of these assays in rats and therefore the Predictive Safety Testing Consortium, Cardiac Hypertrophy Working Group (PSTC-CHWG) performed a cross-laboratory (5 laboratories) analytical evaluation of a commercially available NT-proANP ELISA for use with rat samples. METHODS: Serum samples were collected from normal Sprague Dawley (SD) rats and were spiked with kit calibrator material or rat heart tissue extracts to provide specimens for the validation. In addition, the cardiotoxicant, isoproterenol, was used to induce elevated endogenous NT-proANP levels in a subgroup of rats for additional validation specimens. The Biomedica™ (BI-20892, Vienna, Austria) proANP (1-98) enzyme-linked immunoabsorbent assay (ELISA) kit was used to measure NT-proANP. Intra-assay and inter-assay precisions, accuracy, sample linearity, recovery, limit of detection, upper and lower limits of quantitation (ULOQ and LLOQ, respectively), sample-freeze/thaw stability and stored sample stability were assessed and compared to pre-determined acceptance criteria. RESULTS: The majority of the experimental assessments met the established validation criteria, however there were individual results that did not meet these standards. Overall, acceptable intra- and inter-assay precisions and accuracies as well as inter-laboratory precision and accuracy were demonstrated. Linearity and recovery values fell within the pre-determined acceptance criteria, samples remained stable for up to three freeze-thaw cycles and frozen samples were stable at ~-70 °C for 12 months. The limit of detection (LOD) and LLOQ and ULOQ were similar to those specified by the manufacturer. DISCUSSION: Overall, the assay was demonstrated to be technically adequate for the detection of NT-proANP serum levels in SD rats.

Enrichment with wood blocks does not affect toxicity assessment in an exploratory toxicology model using Sprague-Dawley rats.

Environmental enrichment in rodents may improve animal well-being but can affect neurologic development, immune system function, and aging. We tested the hypothesis that wood block enrichment affects the interpretation of traditional and transcriptomic endpoints in an exploratory toxicology testing model using a well-characterized reference compound, cyclophosphamide. ANOVA was performed to distinguish effects of wood block enrichment separate from effects of 40 mg/kg cyclophosphamide treatment. Biologically relevant and statistically significant effects of wood block enrichment occurred only for body weight gain. ANOVA demonstrated the expected effects of cyclophosphamide on food consumption, spleen weight, and hematology. According to transcriptomic endpoints, cyclophosphamide induced fewer changes in gene expression in liver than in spleen. Splenic transcriptomic pathways affected by cyclophosphamide included: iron hemostasis; vascular tissue angiotensin system; hepatic stellate cell activation and fibrosis; complement activation; TGFß-induced hypertrophy and fibrosis; monocytes, macrophages, and atherosclerosis; and platelet activation. Changes in these pathways due to cyclophosphamide treatment were consistent with bone marrow toxicity regardless of enrichment. In a second study, neither enrichment nor type of cage flooring altered body weight or food consumption over a 28-d period after the first week. In conclusion, wood block enrichment did not interfere with a typical exploratory toxicology study; the effects of ingested wood on drug level kinetics may require further consideration.

Selective extraction and effective separation of galactosylsphingosine (psychosine) and glucosylsphingosine from other glycosphingolipids in pathological tissue samples.

To facilitate the study of the chemical pathology of galactosylsphingosine (psychosine, GalSph) in Krabbe disease and glucosylsphingosine (GlcSph) in Gaucher disease, we have devised a facile method for the effective separation of these two glycosylsphingosines from other glycosphingolipids (GSLs) in Krabbe brain and Gaucher spleen samples. The procedure involves the use of acetone to selectively extract GalSph and GlcSph, respectively, from Krabbe brain and Gaucher spleen samples. Since acetone does not extract other GSLs except modest amounts of galactosylceramide, sulfatide, and glucosylceramide, the positively charged GalSph or GlcSph in the acetone extract can be readily separated from other GSLs by batchwise cation-exchange chromatography using a Waters Accell Plus CM Cartridge. GalSph or GlcSph enriched by this simple procedure can be readily analyzed by thin-layer chromatography or high-performance liquid chromatography.

Comparing levels of biochemical markers in CSF from cannulated and non-cannulated rats.

Cerebrospinal fluid (CSF) is commonly used for assessing biomarkers of drug efficacy or disease progression in the central nervous system. Studies of CSF from pre-clinical species can characterize biomarkers for use in clinical trials. However, obtaining CSF from pre-clinical species, particularly rodents, can be challenging due to small body sizes, and consequently, low volumes of CSF. Surgical cannulation of rats is commonly used to allow for CSF withdrawal from the cisterna magna. However, cannulae do not remain patent over multiple days, making chronic studies on the same rats difficult. Moreover, CSF biomarkers may be affected by cannulation. Thus cannulation may contribute confounding factors to the understanding of CSF biomarkers. To determine the potential impact on biomarkers, CSF was analyzed from cannulated rats, surgically implanted with catheters as well as from non-cannulated rats. Brain protein biomarkers (αII-spectrin SBDP150 and total tau) and albumin, were measured in the CSF using ELISA assays. Overall, cannulated rat CSF had elevated levels of the biomarkers examined compared to non-cannulated rat CSF. Additionally, the variation in biomarker levels observed among CSF from cannulated rats was greater than that observed for non-cannulated rat CSF. These results demonstrate that in some cases, biomarker assessment using CSF from cannulated rats may differ from that of non-cannulated animals and may contribute confounding factors to biomarker measurements and assay development for clinical use.

Purification of drugs from biological fluids by counter-current chromatography.

Experiments were performed to demonstrate the potential of counter-current chromatography (CCC) for the isolation of drugs and their metabolites from biological matrices relevant to the metabolism studies of pharmaceutical research. Examples of typical drugs are spiked into biological media ex vivo to provide test samples for analysis. A mass spectrometer hyphenated to a CCC allows for the detection of small molecule drugs within the matrix through selected ion monitoring, and fraction collection can provide material for further structural elucidation by NMR.

Use of traditional end points and gene dysregulation to understand mechanisms of toxicity: toxicogenomics in mechanistic toxicology.

Microarray technologies can be used to generate massive amounts of gene expression information as an initial step to decipher the molecular mechanisms of toxicologic changes. Identifying genes whose expression is associated with specific toxic end points is an initial step in predicting, characterizing, and understanding toxicity. Analysis of gene function and the chronology of gene expression changes represent additional methods to generate hypotheses of the mechanisms of toxicity. Follow-up experiments are typically required to confirm or refute hypotheses derived from toxicogenomic data. Understanding the mechanism of toxicity for a compound is a critical step in forming a rational plan for developing counterscreens for toxicity and for increasing productivity of research and development while decreasing the risk of late-stage failure in pharmaceutical development.

Molecular beacon genotyping for globoid cell leukodystrophy from hair roots in the twitcher mouse and rhesus macaque.

Rapid and accurate genotype determination is ideal for the maintenance of breeding colonies of laboratory animal models of genetic disease. The rhesus macaque and murine (twitcher) models of globoid cell leukodystrophy have a dinucleotide deletion or single nucleotide substitution, respectively, which abolish ceramide beta-galactosidase activity and are authentic models of Krabbe disease. We report a molecular beacon PCR assay for each species which allows unambiguous determination of the genotype in under 4h. The assay works reliably with DNA extracted from hair roots using Chelex-100 in a 20 min, 100 degrees C incubation. We demonstrate that genotyping from hair roots is a preferred alternative to collecting blood or tissue for DNA extraction because it reduces animal distress, uses an inexpensive reagent, and is simpler and faster. Following amplification on a standard thermocycler with a 96-well plate format, these molecular beacon assays can be read on a standard laboratory fluorescent plate reader, eliminating the need to use a real-time thermocycler or to open the plate for subsequent restriction enzyme digestion and gel electrophoresis. The multiplexed ratio of fluorescence from wild-type- and mutant-specific beacons reporting at 560 nm and 535 nm wavelengths is distinct for each genotype.

Experimental infection of rhesus macaques with Streptococcus pneumoniae: a possible model for vaccine assessment.

BACKGROUND: We explored the possibility of using normal adult rhesus macaques for the preclinical assessment of safety, immunogenicity, and efficacy of newly developed vaccines against Streptococcus pneumoniae infection of the lung. METHODS: Our primary objective was to determine whether an intra-bronchial inoculum of at least 10(6)S. pneumoniae colony-forming units, or one as high as 10(8)-10(9) organisms, could detectably survive in rhesus macaques for a period longer than 1-2 weeks. If so, we hypothesized, it would be possible to observe signs of pneumonia commonly observed in humans, and discriminate between vaccinated/protected animals and controls. Infection was detectable in bronchoalveolar lavage fluids 3-5 weeks post-inoculation. RESULTS: The clinical course of disease mimicked aspects of that of human pneumococcal pneumonia. Signs of inflammation typical of the disease in humans, such as elevated concentrations of neutrophils and of pro-inflammatory cytokines in bronchoalveolar lavage fluids were also observed. CONCLUSIONS: These findings underscore the utility of this model to assess the safety, immunogenicity, and efficacy of newly developed S. pneumoniae vaccines.

Lentivirus-specific cytotoxic T-lymphocyte responses are rapidly lost in thymectomized cats infected with feline immunodeficiency virus.

To what extent the thymus is needed to preserve the virus-specific cytotoxic T-lymphocyte (CTL) response of lentivirus-infected adults is unclear. Presented here is the first definitive study using thymectomized (ThX) animals to directly evaluate the contribution of thymic function to lentivirus-specific CTL response and the control of lentivirus infections. ThX and mock-ThX cats were inoculated with feline immunodeficiency virus (FIV) and monitored for their FIV-specific CTL responses. Early in infection, both FIV-ThX and FIV-mock-ThX cats produced similar CTL responses, but surprisingly, after 20 weeks, the FIV-ThX cats showed a statistically significant loss of FIV-specific CTL activity, while FIV-infected cats with intact thymuses continued to maintain FIV-specific CTL. The loss of CTL did not affect plasma virus load, which remained elevated for both groups. These results emphasize the importance of thymic integrity in maintaining immunity to lentiviruses, but also bring into question the notion that virus load is regulated predominantly by the virus-specific CTL response.

Animal models of retroviral encephalopathies: feline model.

Human immunodeficiency virus infection in children and adults results in a progressive neurodegenerative disease consistent with a predominant subcortical mediated dementia. Techniques for developing a feline model of the early stages of lentiviral-associated neurodegeneration are presented. The behavioral, neurophysiologic, immunologic, virologic, and neuropathologic aspects of this model are also described.