ABSTRACT
Repulsive guidance molecule A (RGMa) is a potent inhibitor of axonal growth and a regulator of neuronal cell death. It is up-regulated following neuronal injury and accumulates in chronic neurodegenerative diseases. Neutralizing RGMa has the potential to promote neuroregeneration and neuroprotection. Previously we reported that a rat anti-N terminal RGMa (N-RGMa) antibody r5F9 and its humanized version h5F9 (ABT-207) promote neuroprotection and neuroregeneration in preclinical neurodegenerative disease models. However, due to its cross-reactivity to RGMc/hemojuvelin, ABT-207 causes iron accumulation in vivo, which could present a safety liability. Here we report the generation and characterization of a novel RGMa-selective anti-N-RGMa antibody elezanumab, which is currently under Phase 2 clinical evaluation in multiple disease indications. Elezanumab, a human monoclonal antibody generated by in vitro PROfusion mRNA display technology, competes with ABT-207 in binding to N-RGMa but lacks RGMc cross-reactivity with no impact on iron metabolism. It neutralizes repulsive activity of soluble RGMa in vitro and blocks membrane RGMa mediated BMP signaling. In the optic nerve crush and optic neuritis models, elezanumab promotes axonal regeneration and prevents retinal nerve fiber layer degeneration. In the spinal targeted experimental autoimmune encephalomyelitis (EAE) model, elezanumab promotes axonal regeneration and remyelination, decreases inflammatory lesion area and improves functional recovery. Finally, in the mouse cuprizone model, elezanumab reduces demyelination, which is consistent with its inhibitory effect on BMP signaling. Taken together, these preclinical data demonstrate that elezanumab has neuroregenerative and neuroprotective activities without impact on iron metabolism, thus providing a compelling rationale for its clinical development in neurodegenerative diseases.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , GPI-Linked Proteins , Nerve Regeneration , Nerve Tissue Proteins , Neuroprotection , Optic Nerve Injuries , Optic Nerve , Optic Neuritis , Recovery of Function , Retina , Animals , Mice , Cuprizone/toxicity , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/physiopathology , GPI-Linked Proteins/antagonists & inhibitors , Monoamine Oxidase Inhibitors/toxicity , Nerve Regeneration/drug effects , Nerve Regeneration/physiology , Nerve Tissue Proteins/antagonists & inhibitors , Neuroprotection/drug effects , Optic Nerve/drug effects , Optic Nerve/physiology , Optic Nerve Injuries/physiopathology , Optic Neuritis/physiopathology , Recovery of Function/drug effects , Recovery of Function/physiology , Retina/drug effects , Surface Plasmon ResonanceABSTRACT
Spinal cord injury (SCI) is a devastating condition characterized by loss of function, secondary to damaged spinal neurons, disrupted axonal connections, and myelin loss. Spontaneous recovery is limited, and there are no approved pharmaceutical treatments to reduce ongoing damage or promote repair. Repulsive guidance molecule A (RGMa) is upregulated following injury to the central nervous system (CNS), where it is believed to induce neuronal apoptosis and inhibit axonal growth and remyelination. We evaluated elezanumab, a human anti-RGMa monoclonal antibody, in a novel, newly characterized non-human primate (NHP) hemicompression model of thoracic SCI. Systemic intravenous (IV) administration of elezanumab over 6â¯months was well tolerated and associated with significant improvements in locomotor function. Treatment of animals for 16â¯weeks with a continuous intrathecal infusion of elezanumab below the lesion was not efficacious. IV elezanumab improved microstructural integrity of extralesional tissue as reflected by higher fractional anisotropy and magnetization transfer ratios in treated vs. untreated animals. IV elezanumab also reduced SCI-induced increases in soluble RGMa in cerebrospinal fluid, and membrane bound RGMa rostral and caudal to the lesion. Anterograde tracing of the corticospinal tract (CST) from the contralesional motor cortex following 20â¯weeks of IV elezanumab revealed a significant increase in the density of CST fibers emerging from the ipsilesional CST into the medial/ventral gray matter. There was a significant sprouting of serotonergic (5-HT) fibers rostral to the injury and in the ventral horn of lower thoracic regions. These data demonstrate that 6â¯months of intermittent IV administration of elezanumab, beginning within 24â¯h after a thoracic SCI, promotes neuroprotection and neuroplasticity of key descending pathways involved in locomotion. These findings emphasize the mechanisms leading to improved recovery of neuromotor functions with elezanumab in acute SCI in NHPs.
Subject(s)
Antibodies, Monoclonal/administration & dosage , GPI-Linked Proteins/antagonists & inhibitors , Nerve Tissue Proteins/antagonists & inhibitors , Neuronal Plasticity/drug effects , Neuroprotection/drug effects , Recovery of Function/drug effects , Spinal Cord Injuries/drug therapy , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Chlorocebus aethiops , Exercise Test/methods , Humans , Injections, Spinal , Male , Neuronal Plasticity/physiology , Neuroprotection/physiology , Primates , Recovery of Function/physiology , Spinal Cord Injuries/pathology , Spinal Cord Injuries/physiopathology , Thoracic Vertebrae/injuriesABSTRACT
The histopathology slide seminar "Classic Examples in Toxicologic Pathology XXVII" was held on February 21 and 22, 2020, at the Department of Pathology at the University of Veterinary Medicine in Hannover, Germany, with joint organization by the European Society of Toxicologic Pathology. The goal of this annual seminar is to present and discuss classical and actual cases of toxicologic pathology. This article summarizes the presentations given during the seminar, including images of representative lesions. Ten actual and classical cases of toxicologic pathology, mostly induced by a test article, were presented. These included small intestine pathology and transcriptomics induced by a γ-secretase modulator, liver findings in nonhuman primates induced by gene therapy, drug-induced neutropenia in dogs, device-induced growth plate lesions, polycystic lesions in CAR/PXR double knockout mice, inner ear lesions in transgenic mice, findings in Beagle dogs induced by an inhibitor of the myeloid leukemia cell differentiation protein MCL1, findings induced by a monovalent fibroblast growth factor receptor 1 antagonist, kidney lesions induced by a mammalian target of rapamycin inhibitor in combination therapy, and findings in mutation-specific drugs.
Subject(s)
Amyloid Precursor Protein Secretases , Pathology , Animals , Dogs , Fibroblast Growth Factor-23 , Genetic Therapy , Growth Plate , Mice , Mice, Knockout , Mice, TransgenicABSTRACT
The European Society of Toxicologic Pathology organized an expert workshop in May 2018 to address adversity considerations related to thyroid follicular cell hypertrophy and/or hyperplasia (FCHH), which is a common finding in nonclinical toxicity studies that can have important implications for risk assessment of pharmaceuticals, food additives, and environmental chemicals. The broad goal of the workshop was to facilitate better alignment in toxicologic pathology and regulatory sciences on how to determine adversity of FCHH. Key objectives were to describe common mechanisms leading to thyroid FCHH and potential functional consequences; provide working criteria to assess adversity of FCHH in context of associated findings; and describe additional methods and experimental data that may influence adversity determinations. The workshop panel was comprised of representatives from the European Union, Japan, and the United States. Participants shared case examples illustrating issues related to adversity assessments of thyroid changes. Provided here are summary discussions, key case presentations, and panel recommendations. This information should increase consistency in the interpretation of adverse changes in the thyroid based on pathology findings in nonclinical toxicity studies, help integrate new types of biomarker data into the review process, and facilitate a more systematic approach to communicating adversity determinations in toxicology reports.
Subject(s)
Thyroid Epithelial Cells , Biomarkers , Humans , Hyperplasia , Hypertrophy , Risk Assessment , United StatesABSTRACT
Hepcidin was originally detected as a liver peptide with antimicrobial activity and it functions as a central regulator in the systemic iron metabolism. Consequently suppression of hepcidin leads to iron accumulation in the liver. AbbVie developed a monoclonal antibody ([mAb]; repulsive guidance molecule [RGMa/c] mAb) that downregulates hepcidin expression by influencing the RGMc/bone morphogenetic protein (BMP)/neogenin receptor complex and causes iron deposition in the liver. In a dose range finding study with RGMa/c mAb, rats were treated with different dose levels for a total of 4 weekly doses. The results of this morphometric analysis in the liver showed that iron accumulation is not homogenous between liver lobes and the left lateral lobe was the most responsive lobe in the rat. Quantitative hepcidin messenger RNA analysis showed that the left lateral lobe was the most responsive lobe showing hepcidin downregulation with increasing antibody dose. In addition, the morphometric analysis had higher sensitivity than the chemical iron extraction and quantification using a colorimetric assay. In conclusion, the Prussian blue stain in combination with semi-quantitative and quantitative morphometric analysis is the most reliable method to demonstrate iron accumulation in the liver compared to direct measurement of iron in unfixed tissue using a colorimetric assay.
Subject(s)
Hepcidins/metabolism , Iron/analysis , Iron/metabolism , Liver/chemistry , Liver/metabolism , Animals , Antibodies, Monoclonal , Dose-Response Relationship, Drug , Female , GPI-Linked Proteins , Hepcidins/analysis , Hepcidins/genetics , Membrane Glycoproteins , Nerve Tissue Proteins , Rats , Rats, Sprague-DawleyABSTRACT
The use of natural pulmonary surfactants (PS) as a drug delivery vehicle for biologics is a more recent therapeutic modality. Herein, we tested different contents of PS regarding their physicochemical properties under stress conditions. The PS content of 12.25 mg/ml (Formulation B) showed desired properties such as an isotonic osmolality â¼300 mOsm/kg and an acceptable viscosity of 8.61 cSt, being lower than in commercially available PS solutions. Formulation B passed the specifications of surface lowering capacities of >80 % total lung capacity and physiologically desired formulation properties were independent of the antibody used in the composition. The identified formulation showed the capability of significantly increasing the oxygen saturation in ex vivo isolated perfused rat lungs, compared to a control and up to 30 min post lavage. In the in vivo setting, we showed that intratracheal administration of a human mAB with and without pulmonary surfactant led to higher amounts of delivered antibody within the alveolar tissue compared to intravenous administration. The antibody with the PS formulation remained longer in the alveolar tissues than the antibody without the PS formulation. Further, SARS-CoV-2 infected Golden Syrian hamsters showed that the intranasally applied antibody reached the site of infection in the alveoli and could be detected in the alveolar region 24 h after the last administration. With this work, we demonstrated that pulmonary surfactants can be used as a pulmonary drug delivery mechanism for antibodies and may subsequently improve the antibody efficacy by increasing the residence time at the desired site of action in the alveolar tissue.
ABSTRACT
The field of nasal drug delivery gained enormously on interest over the past decade. Performing nasal in vivo studies is expensive and time-consuming, but also unfeasible for an initial high-throughput compound and formulation screening. Therefore, the development of fast and high-throughput in vitro models to screen compounds for their permeability through the nasal epithelium and mucosa is constantly expanding. Yet, the protocols used for nasal in vitro permeability studies are varying, which limits the comparability and reproducibility of generated data. This project aimed to elucidate the influence of different culture and assay parameters of RPMI 2650 cells grown under air-liquid interface (ALI) conditions on the transepithelial electrical resistance (TEER) and apparent permeability (Papp) values of five selected reference compounds, covering the range of low to moderate to high permeability. The influence of the passage number, seeding density, and timepoint of airlift was minimal in our approach, while the substrate pore density had a significant influence on the Papp values of carbamazepine, propranolol, and metoprolol, classified as highly permeable compounds, but not on atenolol and aciclovir. Elevation of the experimental concentration of carbamazepine, propranolol, and metoprolol in the donor compartment had an increasing effect on the Papp values, while prolonging the assay time did not have a significant influence. Based on the results reported here, RPMI 2650 cells cultured under ALI conditions offer the possibility of a standardized high-throughput screening model for small molecules and their formulations for in vitro drug permeation studies to predict and select optimal conditions for their nasal delivery.
ABSTRACT
Even after more than two years of intensive research, not all of the pathophysiological processes of Coronavirus Disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, have been fully elucidated. The initial virus-host interaction at the respiratory epithelium plays a crucial role in the course and progression of the infection, and is highly dependent on the glycosylation pattern of the host cell and of the secreted mucins. Glycans are polysaccharides that can be attached to proteins and thereby add to their stability and functionality. Lectins are glycan-binding proteins that recognize specific glycan motifs, and lectin histochemistry is a suitable tool to visualize and examine glycosylation pattern changes in tissues. In this study we used lectins with different glycan-specificities for the visualization of glycosylation pattern changes in the respiratory tract of SARS-CoV-2 infected Golden Syrian hamsters. While some lectins (LEL, STL) enable the visualization of the damage to alveolar type 1 pneumocytes, other lectins, e.g., GSLI, visualized the loss and subsequent hyperplasia of type 2 pneumocytes. UEAI staining was co-localized with KI67, a proliferation marker. Double staining of lectins LEL, STL and WGA with specific immune cell markers (Iba1, CD68) showed co-localization and the dominant infiltration of monocyte-derived macrophages into infected alveolar tissue. The elucidation of the glycosylation pattern of the respiratory tract cells in uninfected and infected Golden Syrian hamsters revealed physiological and pathological aspects of the disease that may open new possibilities for therapeutic development.
ABSTRACT
Over the past 10 years, the interest in intranasal drug delivery in pharmaceutical R&D has increased. This review article summarises information on intranasal administration for local and systemic delivery, as well as for CNS indications. Nasal delivery offers many advantages over standard systemic delivery systems, such as its non-invasive character, a fast onset of action and in many cases reduced side effects due to a more targeted delivery. There are still formulation limitations and toxicological aspects to be optimised. Intranasal drug delivery in the field of drug development is an interesting delivery route for the treatment of neurological disorders. Systemic approaches often fail to efficiently supply the CNS with drugs. This review paper describes the anatomical, histological and physiological basis and summarises currently approved drugs for administration via intranasal delivery. Further, the review focuses on toxicological considerations of intranasally applied compounds and discusses formulation aspects that need to be considered for drug development.
Subject(s)
Drug Delivery Systems , Drug Development , Administration, Intranasal , Brain , Nasal Mucosa , Pharmaceutical PreparationsABSTRACT
Lectins are naturally occurring molecules which bind to specific carbohydrates of glycoconjugates. The binding specificity of lectins can therefore be used to specifically elucidate the glycosylation pattern in various tissues. While lectin histochemistry is usually carried out manually on single slides, a fully automated immunostaining system offers an easy, standardized, and high throughput system. In this study lectin histochemistry was implemented and optimized on a fully automated immunostaining system to investigate glycosylation patterns in the murine respiratory tract and the primary olfactory pathway. We tested 22 commercially available biotinylated lectins for their labelling-profiles to specifically identify morphologic structures. The results showed that lectin staining profiles using the implemented protocol on the automated system were constant and suitable for high throughput morphological studies. Further, the morphological evaluation of the stained slides revealed a complete characterization of the murine respiratory tract and primary olfactory pathway including the lectin binding profiles for the olfactory bulb, the vomeronasal organ and the nasal-associated lymphoid tissue.
Subject(s)
Lectins , Olfactory Pathways , Animals , Histocytochemistry , Lectins/metabolism , Mice , Olfactory Pathways/metabolism , Respiratory System/metabolism , Staining and LabelingABSTRACT
Here we demonstrate a dramatic improvement in Ti/Au ohmic contact performance by utilizing the anisotropic nature of ß-Ga2O3. Under a similar doping concentration, Ti/Au metallization on (100) Ga2O3 shows a specific contact resistivity 5.11 × 10-5 Ω·cm2, while that on (010) Ga2O3 is as high as 3.29 × 10-3 Ω·cm2. Temperature-dependent contact performance and analyses suggest that field emission or thermionic field emission is the dominant charge transport mechanism across the Ti/Au-(100) Ga2O3 junction, depending on whether reactive ion etching was used prior to metallization. Cross-sectional high-resolution microscopy and elemental mapping analysis show that the in situ-formed Ti-TiOx layer on (100) Ga2O3 is relatively thin (2-2.5 nm) and homogeneous, whereas that on (010) substrates is much thicker (3-5 nm) and shows nanoscale facet-like features at the interface. The anisotropic nature of monoclinic Ga2O3, including anisotropic surface energy and mass diffusivity, is likely to be the main cause of the differences observed under microscopy and in electrical properties. The findings here provide direct evidence and insights into the dependence of device performance on the atomic-scale structural anisotropy of ß-Ga2O3. Moreover, the investigative strategy hereâcombining comprehensive electrical and materials characterization of interfaces on different semiconductor orientationsâcan be applied to assess a variety of other anisotropic oxide junctions.
ABSTRACT
SARS-CoV-2 has been the cause of a global pandemic since 2019 and remains a medical urgency. siRNA-based therapies are a promising strategy to fight viral infections. By targeting a specific region of the viral genome, siRNAs can efficiently downregulate viral replication and suppress viral infection. However, to achieve the desired therapeutic activity, siRNA requires a suitable delivery system. The VIPER (virus-inspired polymer for endosomal release) block copolymer has been reported as promising delivery system for both plasmid DNA and siRNA in the past years. It is composed of a hydrophilic block for condensation of nucleic acids as well as a hydrophobic, pH-sensitive block that, at acidic pH, exposes the membrane lytic peptide melittin, which enhances endosomal escape. In this study, we aimed at developing a formulation for pulmonary administration of siRNA to suppress SARS-CoV-2 replication in lung epithelial cells. After characterizing siRNA/VIPER polyplexes, the activity and safety profile were confirmed in a lung epithelial cell line. To further investigate the activity of the polyplexes in a more sophisticated cell culture system, an air-liquid interface (ALI) culture was established. siRNA/VIPER polyplexes reached the cell monolayer and penetrated through the mucus layer secreted by the cells. Additionally, the activity against wild-type SARS-CoV-2 in the ALI model was confirmed by qRT-PCR. To investigate translatability of our findings, the activity against SARS-CoV-2 was tested ex vivo in human lung explants. Here, siRNA/VIPER polyplexes efficiently inhibited SARS-CoV-2 replication. Finally, we verified the delivery of siRNA/VIPER polyplexes to lung epithelial cells in vivo, which represent the main cellular target of viral infection in the lung. In conclusion, siRNA/VIPER polyplexes efficiently delivered siRNA to lung epithelial cells and mediated robust downregulation of viral replication both in vitro and ex vivo without toxic or immunogenic side effects in vivo, demonstrating the potential of local siRNA delivery as a promising antiviral therapy in the lung.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/therapy , Humans , Lung/metabolism , Polymers/chemistry , RNA, Small Interfering , SARS-CoV-2/genetics , Virus Replication/geneticsABSTRACT
The vast majority of the world population is infected with at least one member of the human herpesvirus family. Herpes simplex virus (HSV) infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. Since the milestone development in the late 1970s of acyclovir (Zovirax), a nucleosidic inhibitor of the herpes DNA polymerase, no new non-nucleosidic anti-herpes drugs have been introduced. Here we report new inhibitors of the HSV helicase-primase with potent in vitro anti-herpes activity, a novel mechanism of action, a low resistance rate and superior efficacy against HSV in animal models. BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide), a well-tolerated member of this class of compounds, significantly reduces time to healing, prevents rebound of disease after cessation of treatment and, most importantly, reduces frequency and severity of recurrent disease. Thus, this class of drugs has significant potential for the treatment of HSV disease in humans, including those resistant to current medications.
Subject(s)
Antiviral Agents/therapeutic use , DNA Helicases/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Herpes Simplex/drug therapy , Pyridines/therapeutic use , Thiazoles/therapeutic use , Acyclovir/therapeutic use , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , DNA Primase , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Female , Guinea Pigs , Herpes Simplex/enzymology , Herpes Simplex/pathology , Humans , Infant, Newborn , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Pregnancy , Pyridines/chemistry , Pyridines/pharmacokinetics , Safety , Sulfonamides , Thiazoles/chemistry , Thiazoles/pharmacokinetics , Viral ProteinsABSTRACT
ABT-736 is a humanized monoclonal antibody generated to target a specific conformation of the amyloid-beta (Aß) protein oligomer. Development of ABT-736 for Alzheimer's disease was discontinued due to severe adverse effects (AEs) observed in cynomolgus monkey toxicity studies. The acute nature of AEs observed only at the highest doses suggested potential binding of ABT-736 to an abundant plasma protein. Follow-up investigations indicated polyspecificity of ABT-736, including unintended high-affinity binding to monkey and human plasma protein platelet factor 4 (PF-4), known to be involved in heparin-induced thrombocytopenia (HIT) in humans. The chronic AEs observed at the lower doses after repeat administration in monkeys were consistent with HIT pathology. Screening for a backup antibody revealed that ABT-736 possessed additional unintended binding characteristics to other, unknown factors. A subsequently implemented screening funnel focused on nonspecific binding led to the identification of h4D10, a high-affinity Aß oligomer binding antibody that did not bind PF-4 or other unintended targets and had no AEs in vivo. This strengthened the hypothesis that ABT-736 toxicity was not Aß target-related, but instead was the consequence of polyspecificity including PF-4 binding, which likely mediated the acute and chronic AEs and the HIT-like pathology. In conclusion, thorough screening of antibody candidates for nonspecific interactions with unrelated molecules at early stages of discovery can eliminate candidates with polyspecificity and reduce potential for toxicity caused by off-target binding.
Subject(s)
Alzheimer Vaccines/immunology , Amyloid beta-Peptides/antagonists & inhibitors , Antibodies, Monoclonal, Humanized/toxicity , Blood Platelets/drug effects , Immunity, Heterologous , Platelet Factor 4/antagonists & inhibitors , Purpura, Thrombocytopenic, Idiopathic/chemically induced , Alzheimer Vaccines/pharmacokinetics , Alzheimer Vaccines/toxicity , Amyloid beta-Peptides/immunology , Animals , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/pharmacokinetics , Antibody Specificity , Blood Platelets/immunology , Blood Platelets/metabolism , Female , Humans , Macaca fascicularis , Male , Mice, Inbred BALB C , No-Observed-Adverse-Effect Level , Platelet Activation/drug effects , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/blood , Purpura, Thrombocytopenic, Idiopathic/immunology , Risk Assessment , Time Factors , Toxicity Tests, Acute , Toxicity Tests, ChronicABSTRACT
Endotoxins are frequent contaminants of recombinant proteins produced in Escherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg(-1) is, however, one of the most problematic steps during protein purification from E. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced in E. coli using a non-ionic detergent. Endotoxin content was as low as 5 to 9 EU mg(-1) with a recovery of antibody fragments of over 90%. Non-ionic detergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinant antibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.
Subject(s)
Chromatography, Affinity/methods , Endotoxins/chemistry , Immunoglobulin Fragments/chemistry , Metals , Antibodies, Monoclonal/chemistry , Binding Sites, Antibody , Cell Line, Tumor , Detergents/chemistry , Dimerization , Endothelial Cells/physiology , Endotoxins/isolation & purification , Escherichia coli , Humans , Intercellular Adhesion Molecule-1/immunology , Models, Molecular , Octoxynol , Polyethylene Glycols/chemistryABSTRACT
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.
Subject(s)
Clofibrate/toxicity , Hypolipidemic Agents/toxicity , Muscle, Skeletal/drug effects , Alanine Transaminase/blood , Alanine Transaminase/urine , Animals , Aspartate Aminotransferases/blood , Aspartate Aminotransferases/urine , Biomarkers/blood , Biomarkers/urine , Creatine Kinase/blood , Creatine Kinase/urine , Fatty Acid Binding Protein 3 , Fatty Acid-Binding Proteins/blood , Fatty Acid-Binding Proteins/urine , Gene Expression Profiling , Heart/drug effects , Male , Muscle, Skeletal/pathology , Myocardium/pathology , Myoglobin/blood , Myosin Light Chains/blood , Myosin Light Chains/urine , Parvalbumins/blood , Parvalbumins/urine , Rats , Rats, Sprague-Dawley , Troponin C/blood , Troponin C/urine , Troponin I/blood , Troponin I/urineABSTRACT
High levels of hepcidin, the main regulator of systemic iron metabolism, lead to various diseases. Targeting hepcidin and lowering its concentration is a possible form of intervention in order to treat these diseases. High turnover rate of hepcidin is a major drawback of therapies directly targeting this peptide. We developed two monoclonal antibodies ABT-207 and h5F9-AM8 which inhibit hemojuvelin/repulsive guidance molecule C (RGMc) and downregulate hepcidin. We conducted single-application and dose response studies to understand the antibodies' mechanism and subchronic toxicology studies to exclude safety-related concerns. Investigation was carried out at different biological levels through qPCR, Affymetrix, liquid chromatography coupled with mass spectrometry (LC-MS/MS), histopathology, serum iron, unsaturated iron binding capacity (UIBC), and drug concentration measurements. After a single application of these antibodies, hepcidin expression in liver and its serum protein levels were reduced. Serum iron increased for several weeks. The RGMc antibodies show a pronounced dose response relationship in rats with h5F9-AM8 having an IC50 (UIBC) of approximately 80-fold higher than ABT-207. When hepcidin levels were downregulated, iron deposition in the liver was visible histologically 1 week post application. These antibody-mediated iron depositions were not associated with any adverse toxicologically relevant effect at the doses and time points evaluated. Iron depositions seen after 14 weekly treatments with ABT-207 were reversible in rats and in cynomolgus monkeys. Due to their long-lasting effects and excellent safety profile, both RGMc-blocking antibodies ABT-207 and h5F9-AM8 are favorable clinical candidates for diseases characterized by high serum hepcidin levels like anemia of chronic disease.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Hepcidins/genetics , Iron/blood , Membrane Proteins/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Chromatography, Liquid , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Female , Hepcidins/blood , Inhibitory Concentration 50 , Macaca fascicularis , Male , Rats , Rats, Sprague-Dawley , Tandem Mass Spectrometry , Time FactorsABSTRACT
This article describes the design of HuCAL (human combinatorial antibody library) PLATINUM, an optimized, second-generation, synthetic human Fab antibody library with six trinucleotide-randomized complementarity-determining regions (CDRs). Major improvements regarding the optimized antibody library sequence space were implemented. Sequence space optimization is considered a multistep process that includes the analysis of unproductive antibody sequences in order to, for example, avoid motifs such as potential N-glycosylation sites, which are undesirable in antibody production. Gene optimization has been used to improve expression of the antibody master genes in the library context. As a result, full-length IgGs derived from the library show both significant improvements in expression levels and less undesirable glycosylation sites when compared to the previous HuCAL GOLD library. Additionally, in-depth analysis of sequences from public databases revealed that diversity of CDR-H3 is a function of loop length. Based upon this analysis, the relatively uniform diversification strategy used in the CDR-H3s of the previous HuCAL libraries was changed to a length-dependent design, which replicates the natural amino acid distribution of CDR-H3 in the human repertoire. In a side-by-side comparison of HuCAL GOLD and HuCAL PLATINUM, the new library concept led to isolation of about fourfold more unique sequences and to a higher number of high-affinity antibodies. In the majority of HuCAL PLATINUM projects, 100-300 antibodies each having different CDR-H3s are obtained against each antigen. This increased diversity pool has been shown to significantly benefit functional antibody profiling and screening for superior biophysical properties.
Subject(s)
Gene Library , Genetic Variation , Immunoglobulin Fab Fragments/genetics , Gene Expression , Genetic Vectors , Glycosylation , HumansABSTRACT
IL-22 is produced by activated T cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2. The aim of this study was to analyze IL-22 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal epithelial cells (IEC). Expression studies were performed by RT-PCR. Signal transduction was analyzed by Western blot experiments, cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and Fas-induced apoptosis by flow cytometry. IEC migration was studied in wounding assays. The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IL-22 receptor subunits IL-22R1 and IL-10R2. Stimulation with TNF-alpha, IL-1beta, and LPS significantly upregulated IL-22R1 without affecting IL-10R2 mRNA expression. IL-22 binding to its receptor complex activates STAT1/3, Akt, ERK1/2, and SAPK/JNK MAP kinases. IL-22 significantly increased cell proliferation (P = 0.002) and phosphatidylinsitol 3-kinase-dependent IEC cell migration (P < 0.00001) as well as mRNA expression of TNF-alpha, IL-8, and human beta-defensin-2. IL-22 had no effect on Fas-induced apoptosis. IL-22 mRNA expression was increased in inflamed colonic lesions of patients with Crohn's disease and correlated highly with the IL-8 expression in these lesions (r = 0.840). Moreover, IL-22 expression was increased in murine dextran sulfate sodium-induced colitis. IEC express functional receptors for IL-22, which increases the expression of proinflammatory cytokines and promotes the innate immune response by increased defensin expression. Moreover, our data indicate intestinal barrier functions for this cytokine-promoting IEC migration, which suggests an important function in intestinal inflammation and wound healing. IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and IEC migration.