David Krull

Applications of Laser Scanning Cytometry in Immunohistochemistry and Routine Histopathology

Laser scanning cytometry (LSC) is a powerful tool for qualitative and quantitative analysis of tissue sections in preclinical drug development. LSC combines the strengths of flow cytometry with tissue architecture retention. This technology has been used predominantly with immunofluorescent techniques on cell culture and tissue sections, but recently LSC has shown promise in evaluating chromogenic immunohistochemistry (IHC) and histochemical products in paraffin-embedded and/or frozen tissue sections. Inverted light scatter measurements or a combination of inverted scatter and fluorescence allows automated determination of cell/nuclear counts (e.g., proliferation labeling indices), cell area (e.g., cellular hypertrophy), stromal elements, and labeling intensity (e.g., cytoplasmic/organellar proteins) in chromogen-labeled IHC or histochemical stained sections that correlates well with standard manual quantification methods. Segmentation with autofluorescence or dual immunolabeling facilitates capture of labeling data from specific cell populations. LSC evaluation of HE-stained sections is accomplished using autofluorescence/eosin fluorescence and inverse scatter. A standardized fluorescent approach with archivability, a lack of fluorescence quenching (photobleaching), and amenability to evaluation of multiple markers in a section has been demonstrated using Qdot® nanocrystals. Examples of LSC use in chromogenic IHC, routine histopathology, and Qdot® labeling will be reviewed, and advantages and disadvantages of this technology will be discussed.

Safety, Pharmacokinetic, and Functional Effects of the Nogo-A Monoclonal Antibody in Amyotrophic Lateral Sclerosis: A Randomized, First-In-Human Clinical Trial

The neurite outgrowth inhibitor, Nogo-A, has been shown to be overexpressed in skeletal muscle in amyotrophic lateral sclerosis (ALS); it is both a potential biomarker and therapeutic target. We performed a double-blind, two-part, dose-escalation study, in subjects with ALS, assessing safety, pharmacokinetics (PK) and functional effects of ozanezumab, a humanized monoclonal antibody against Nogo-A. In Part 1, 40 subjects were randomized (3∶1) to receive single dose intravenous ozanezumab (0.01, 0.1, 1, 5, or 15 mg/kg) or placebo. In Part 2, 36 subjects were randomized (3∶1) to receive two repeat doses of intravenous ozanezumab (0.5, 2.5, or 15 mg/kg) or placebo, approximately 4 weeks apart. The primary endpoints were safety and tolerability (adverse events [AEs], vital signs, electrocardiogram (ECG), and clinical laboratory tests). Secondary endpoints included PK, immunogenicity, functional endpoints (clinical and electrophysiological), and biomarker parameters. Overall, ozanezumab treatment (0.01–15 mg/kg) was well tolerated. The overall incidence of AEs in the repeat dose 2.5 mg/kg and 15 mg/kg ozanezumab groups was higher than in the repeat dose placebo group and repeat dose 0.5 mg/kg ozanezumab group. The majority were considered not related to study drug by the investigators. Six serious AEs were reported in three subjects receiving ozanezumab; none were considered related to study drug. No study drug-related patterns were identified for ECG, laboratory, or vital signs parameters. One subject (repeat dose 15 mg/kg ozanezumab) showed a weak, positive anti-ozanezumab-antibody result. PK results were generally consistent with monoclonal antibody treatments. No apparent treatment effects were observed for functional endpoints or muscle biomarkers. Immunohistochemical staining showed dose-dependent co-localization of ozanezumab with Nogo-A in skeletal muscle. In conclusion, single and repeat dose ozanezumab treatment was well tolerated and demonstrated co-localization at the site of action. These findings support future studies with ozanezumab in ALS. Trial Registration ClinicalTrials.gov NCT00875446 GSK-ClinicalStudyRegister.com GSK ID 111330

Treatment with an antibody directed against Nogo-A delays disease progression in the SOD1G93A mouse model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder in which motor neurons in the spinal cord and motor cortex degenerate. Although the majority of ALS cases are sporadic, mutations in Cu-Zn superoxide dismutase-1 (SOD1) are causative for 10-20% of familial ALS (fALS), and recent findings show that a hexanucleotide repeat expansion in the C9ORF72 gene may account for >30% of fALS cases in Europe. SOD1(G93A) transgenic mice have a phenotype and pathology similar to human ALS. In both ALS patients and SOD1(G93A) mice, the first pathological features of disease manifest at the neuromuscular junction, where significant denervation occurs prior to motor neuron degeneration. Strategies aimed at preventing or delaying denervation may therefore be of benefit in ALS. In this study, we show that Nogo-A levels increase in muscle fibres of SOD1(G93A) mice along with the elevation of markers of neuromuscular dysfunction (CHRNA1/MUSK). Symptomatic treatment of SOD1(G93A) mice from 70 days of age with an anti-Nogo-A antibody (GSK577548) significantly improves hindlimb muscle innervation at 90 days, a late symptomatic stage of disease, resulting in increased muscle force and motor unit survival and a significant increase in motor neuron survival. However, not all aspects of this improvement in anti-Nogo-A antibody-treated SOD1(G93A) mice were maintained at end-stage disease. These results show that treatment with anti-Nogo-A antibody significantly improves neuromuscular function in the SOD1(G93A) mouse model of ALS, at least during the earlier stages of disease and suggest that pharmacological inhibition of Nogo-A may be a disease-modifying approach in ALS.

A Poorly Differentiated Germ Cell Tumor (Seminoma) in a Long Evans Rat

A large neoplasm that replaced 1 testis of a Long Evans Rat was noted at the final necropsy of a dietary 2-yr study. By light microscopy, the morphological features were consistent with a poorly differentiated seminoma. Ultrastructurally, the cells were polygonal, had a round nucleus, had straight cellular boundaries, and bore no resemblance to Sertoli cells. Although there was little evidence of spermatocytic differentiation, the presence of proacrosomal granules and vesicles, prominent Golgi apparatus, tight intercellular junctions, and a few centriolar pairs without axoneme development, in conjunction with the absence of lipid droplets or abundant smooth endoplasmic reticulum, supported the diagnosis of seminoma rather than Leydig cell tumor. The cells were S-100-and vimentin-positive, although cytokeratin- and a-fetoprotein-negative. Seminomas are extremely rare neoplasms in rats; this is the first report in this strain and the first extensive analysis of a rat seminoma without spermatocytic differentiation.

Drug-induced Liver Fibrosis: Testing Nevirapine in a Viral-like Liver Setting Using Histopathology, MALDI IMS, and Gene Expression.

Nevirapine (NVP) is associated with hepatotoxicity in 1–5% of patients. In rodent studies, NVP has been shown to cause hepatic enzyme induction, centrilobular hypertrophy, and skin rash in various rat strains but not liver toxicity. In an effort to understand whether NVP is metabolized differently in a transiently inflamed liver and whether a heightened immune response alters NVP-induced hepatic responses, female brown Norway rats were dosed with either vehicle or NVP alone (75 mg/kg/day for 15 days) or galactosamine alone (single intraperitoneal [ip] injection on day 7 to mimic viral hepatitis) or a combination of NVP (75/100/150 mg/kg/day for 15 days) and galactosamine (single 750 mg/kg ip on day 7). Livers were collected at necropsy for histopathology, matrix-assisted laser desorption/ionization imaging mass spectrometry and gene expression. Eight days after galactosamine, hepatic fibrosis was noted in rats dosed with the combination of NVP and galactosamine. No fibrosis occurred with NVP alone or galactosamine alone. Gene expression data suggested a viral-like response initiated by galactosamine via RNA sensors leading to apoptosis, toll-like receptor, and dendritic cell responses. These were exacerbated by NVP-induced growth factor, retinol, apoptosis, and periostin effects. This finding supports clinical reports warning against exacerbation of fibrosis by NVP in patients with hepatitis C.

Ozanezumab dose selection for amyotrophic lateral sclerosis by pharmacokinetic-pharmacodynamic modelling of immunohistochemistry data from patient muscle biopsies.

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disease with a high unmet medical need. In this context, a potential therapy should be brought to patients in the most expeditious way and early exploration of pharmacology is highly beneficial. Ozanezumab, a humanised IgG monoclonal antibody against Nogo-A protein which is an inhibitor of neurite outgrowth, is currently under development for the treatment of ALS and has been recently assessed in 76 patients in a first-in-human study. Inadequate target engagement has been recognised as a major contributing reason for drug trial failures. In this work, we describe the development of a pharmacokinetic-pharmacodynamic (PKPD) model using immunohistochemistry (IHC) data of co-localization of ozanezumab with Nogo-A in skeletal muscle as a surrogate measure of target engagement. The rich plasma concentration data and the sparse IHC data after one or two intravenous doses of ozanezumab were modelled simultaneously using a non-linear mixed-effect approach. The final PKPD model was a two-compartment PK model combined with an effect compartment PD model that accounted for the delay in ozanezumab concentrations to reach the site of action which is skeletal muscle. Diagnostic plots showed a satisfactory fit of both PK and IHC data. The model was used as a simulation tool to design a dose regimen for sustained drug-target co-localization in a phase II study.

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

ABSTRACT The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.

Morphologic Characterization of PhoenixBio® (uPA+/+/SCID) Humanized Liver Chimeric Mouse Model

Morphological evaluation of humanized chimeric mouse livers from the PhoenixBio (uPA(+/+)/SCID) mouse model show robust replacement and expansion with human hepatocytes, however areas of human hepatocytes had prominent steatosis and a variable lack of sinusoids which was consistent with decreased hepatocellular perfusion and lacked bile canalicular formation between human and mouse hepatocytes.

Histamine Receptor 3 negatively regulates oligodendrocyte differentiation and remyelination

Background Agents promoting oligodendrocyte precursor cell differentiation have the potential to restore halted and/or delayed remyelination in patients with multiple sclerosis. However, few therapeutic targets have been identified. The objective of this study was to identify novel targets for promotion of remyelination and characterize their activity in vitro and in vivo. Methods A high-content screening assay with differentiation of primary rat oligodendrocyte precursor cells was used to screen GSK-proprietary annotated libraries for remyelination-promoting compounds. Compounds were further validated in vitro and in vivo models; clinical relevance of target was confirmed in human post-mortem brain sections from patients with MS. Results Of ~1000 compounds screened, 36 promoted oligodendrocyte precursor cell differentiation in a concentration-dependent manner; seven were histamine receptor-3 (H3R) antagonists. Inverse agonists of H3R but not neutral antagonists promoted oligodendrocyte precursor cell (OPC) differentiation. H3R was expressed throughout OPC differentiation; H3R expression was transiently upregulated on Days 3–5 and subsequently downregulated. H3R gene knockdown in OPCs increased the expression of differentiation markers and the number of mature oligodendrocytes. Overexpression of full-length H3R reduced differentiation marker expression and the number of mature cells. H3R inverse agonist GSK247246 reduced intracellular cyclic AMP (cAMP) and downstream cAMP response element-binding protein (CREB) phosphorylation in a dose-dependent manner. Histone deacetylase (HDAC-1) and Hes-5 were identified as key downstream targets of H3R during OPC differentiation. In the mouse cuprizone/rapamycin model of demyelination, systemic administration of brain-penetrable GSK247246 enhanced remyelination and subsequently protected axons. Finally, we detected high H3R expression in oligodendroglial cells from demyelination lesions in human samples of patients with MS, and validated a genetic association between an exonic single nucleotide polymorphism in HRH3 and susceptibility to multiple sclerosis. Conclusions From phenotypic screening to human genetics, we provide evidence for H3R as a novel therapeutic target to promote remyelination in patients with multiple sclerosis.

Preclinical applications of quantitative imaging cytometry to support drug discovery.

Preclinical drug development is actively involved in testing compounds to find cures or to manage the effects of disease, such as diabetes. Animal models, such as the Zucker diabetic fatty (ZDF) rat, are used to measure efficacy of candidate drugs. This animal model was selected because of its clinical and pathological similarities to diabetic human patients. A method using immunofluorescence and laser scanning cytometry (LSC) technology has been used to measure the development of diabetic phenotype in the ZDF rat during a 17-week time course. The expression levels of insulin, glucagon, voltage-dependent anion channel (VDAC), and Ki67 were quantified. Insulin and VDAC expression were reduced in the ZDF animals in comparison to the lean control rats, while no significant change was seen in glucagon and Ki67 expression at week 17. This information is useful in the design of studies to test experimental compounds in this model. Screening drug targets or biomarkers in tissue sections is another important activity in drug development. Tissue microarrays (TMAs) are composed of 60 or more tissue cores from humans or animal models and may contain healthy and/or diseased tissues. Antibodies against target proteins are applied to TMAs using routine immunohistochemical reagents and protocols. The protein expression across the cores, as labeled by immunohistochemistry, is measured using LSC technology. The process provides an efficient and cost-effective method for evaluating multiple targets in a large number of tissue samples. More recently, IHC and LSC have been taken to the next level to quantify biopharmaceutical drug and target co-localization in tissue sections.