ABSTRACT
In October 2019, Novartis launched brolucizumab, a single-chain variable fragment molecule targeting vascular endothelial growth factor A, for the treatment of neovascular age-related macular degeneration. In 2020, rare cases of retinal vasculitis and/or retinal vascular occlusion (RV/RO) were reported, often during the first few months after treatment initiation, consistent with a possible immunologic pathobiology. This finding was inconsistent with preclinical studies in cynomolgus monkeys that demonstrated no drug-related intraocular inflammation, or RV/RO, despite the presence of preexisting and treatment-emergent antidrug antibodies (ADAs) in some animals. In this study, the immune response against brolucizumab in humans was assessed using samples from clinical trials and clinical practice. In the brolucizumab-naïve population, anti-brolucizumab ADA responses were detected before any treatment, which was supported by the finding that healthy donors can harbor brolucizumab-specific B cells. This suggested prior exposure of the immune system to proteins with structural similarity. Experiments on samples showed that naïve and brolucizumab-treated ADA-positive patients developed a class-switched, high-affinity immune response, with several linear epitopes being recognized by ADAs. Only patients with RV/RO showed a meaningful T cell response upon recall with brolucizumab. Further studies in cynomolgus monkeys preimmunized against brolucizumab with adjuvant followed by intravitreal brolucizumab challenge demonstrated that high ADA titers were required to generate ocular inflammation and vasculitis/vascular thrombosis, comparable to RV/RO in humans. Immunogenicity therefore seems to be a prerequisite to develop RV/RO. However, because only 2.1% of patients with ADA develop RV/RO, additional factors must play a role in the development of RV/RO.
Subject(s)
Retinal Vasculitis , Animals , Humans , Adjuvants, Immunologic , Angiogenesis Inhibitors , Inflammation , Intravitreal Injections , Macaca fascicularis , Vascular Endothelial Growth Factor AABSTRACT
Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.
Subject(s)
Antigen-Antibody Complex , Root Cause Analysis , Humans , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , Inflammation , Angiogenesis Inhibitors , Intravitreal InjectionsABSTRACT
Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.
Subject(s)
Dengue Virus , Dengue , Animals , Antiviral Agents/therapeutic use , Dengue/drug therapy , Dogs , Mice , Models, Animal , Rats , SerogroupABSTRACT
The eye is a complex organ with a series of anatomic barriers that provide protection from physical and chemical injury while maintaining homeostasis and function. The physiology of the eye is multifaceted, with dynamic flows and clearance mechanisms. This review highlights that in vitro ocular transport and metabolism models are confined by the availability of clinically relevant absorption, distribution, metabolism, and excretion (ADME) data. In vitro ocular transport models used for pharmacology and toxicity poorly predict ocular exposure. Although ocular cell lines cannot replicate in vivo conditions, these models can help rank-order new chemical entities in discovery. Historic ocular metabolism of small molecules was assumed to be inconsequential or assessed using authentic standards. While various in vitro models have been cited, no single system is perfect, and many must be used in combination. Several studies document the use of laboratory animals for the prediction of ocular pharmacokinetics in humans. This review focuses on the use of human-relevant and human-derived models which can be utilized in discovery and development to understand ocular disposition of new chemical entities. The benefits and caveats of each model are discussed. Furthermore, ADME case studies are summarized retrospectively and capture the ADME data collected for health authorities in the absence of definitive guidelines. Finally, we discuss the novel technologies and a hypothesis-driven ocular drug classification system to provide a holistic perspective on the ADME properties of drugs administered by the ocular route.
Subject(s)
Eye/drug effects , Eye/metabolism , Pharmaceutical Preparations/administration & dosage , Pharmaceutical Preparations/metabolism , Small Molecule Libraries/administration & dosage , Small Molecule Libraries/metabolism , Administration, Ophthalmic , Animals , Drug Discovery/methods , Humans , Small Molecule Libraries/adverse effectsABSTRACT
The nonclinical safety evaluation of therapeutic drug candidates is commonly conducted in two species (rodent and non-rodent) in keeping with international health authority guidance. Biologic drugs typically have restricted species cross-reactivity, necessitating the evaluation of safety in non-human primates and thus limiting the utility of lower order species. Safety studies of cross-reactive ocular biologic drug candidates have been conducted in rabbits as a second toxicology species, despite the fact that rabbits are not a rodent species. Such studies are often confounded by the development of anti-drug antibodies and severe ocular inflammation, the latter requiring studies to be terminated prematurely for animal welfare reasons. Notably, these confounding factors preclude the interpretation of safety. Nonclinical toxicology programs should be designed with consideration of ethical animal use and 3Rs principles (Replacement, Reduction and Refinement). The experience of several pharmaceutical sponsors, demonstrating that toxicology studies of ocular (intravitreal and topical ocular) biologic drug candidates in the rabbit are of limited interpretive value, calls into question the utility of such studies in this species and indicates that such studies should not be conducted.
Subject(s)
Biological Products/adverse effects , Drug Evaluation, Preclinical/methods , Eye Diseases/immunology , Rabbits , Animals , Eye/immunology , Inflammation/immunology , Species SpecificityABSTRACT
Equine herpesvirus 9 (EHV-9), the newest member of the equine herpesvirus family, is a highly neurotropic herpesvirus that induces encephalitis in a variety of animals. To access transmission of EHV-9 in the nasal cavity and brain, a suckling hamster model was developed so that precise sagittal sections of nasal and cranial cavities including the brain could be processed, which proved useful in detecting viral transmission as well as extension of pathological lesions. Suckling hamsters were inoculated intranasally with EHV-9, and were sacrificed at 6, 12, 18, 24, 36, 48, and 60 h post inoculation (PI). Sagittal sections of the entire head, including nasal and cranial cavities including the brain, were made to assess viral kinetics and identify the progress of the neuropathological lesions. At 12 to 24 h PI the virus attached to and propagated in the olfactory epithelium, and infected adjacent epithelial cells. At 48 h PI, immunohistochemistry for EHV-9 viral antigen showed that virus had extended from the site of infection into the olfactory bulb and olfactory nerve. These results indicate that EHV-9 rapidly invades the brain via the olfactory route after experimental intranasal infection.
Subject(s)
Herpesviridae Infections/immunology , Herpesviridae Infections/transmission , Nasal Cavity/virology , Olfactory Bulb/virology , Varicellovirus , Acute Disease , Administration, Intranasal , Animals , Animals, Suckling , Antigens, Viral/metabolism , Cricetinae , Disease Models, Animal , Encephalitis, Herpes Simplex/immunology , Encephalitis, Herpes Simplex/pathology , Encephalitis, Herpes Simplex/transmission , Female , Herpesviridae Infections/pathology , Horses , Immunohistochemistry , Mesocricetus , Nasal Cavity/pathology , Olfactory Bulb/pathology , Olfactory Pathways/pathology , Olfactory Pathways/virology , PregnancyABSTRACT
The increased demand for non-human primates (NHPs) in biomedical research has resulted in alternative sources of animals being used, which has allowed for importation of animals with varying background incidences of bacterial, viral, parasitic, and fungal pathogens. This can be of minimal consequence when animals from different sources are kept isolated. However, when NHPs from different sources with varying incidences of primary and opportunistic pathogens are mixed, there can be a rapid spread of these pathogens and an increase in the seroconversion of susceptible animals. If this process occurs during the conduct of a study, interpretation of that study can be confounded. Furthermore, NHPs imported from areas enzootic for pathogens such as Plasmodium or with high incidences of human diseases such as measles and tuberculosis can introduce diseases that can be a threat to colony health, have zoonotic risk, and can severely impact study outcome. Thus, knowledge of the common primary and opportunistic NHP infections, as well as reemerging pathogens, enables the toxicologist to use information on disease status for pre-study animal selection and intelligent study design. This is particularly important when immunomodulatory compounds are being investigated. Moreover, the toxicologic pathologist well versed in the common spontaneous infections, opportunistic pathogens, and background lesions in NHPs is able to assess possible drug-related effects in drug safety studies. This review identifies the common primary and opportunistic pathogens, as well as newly emerging infections of NHPs, that can directly or indirectly affect colony health and the interpretation of drug safety studies.
Subject(s)
Communicable Diseases/veterinary , Macaca fascicularis/microbiology , Monkey Diseases/microbiology , Opportunistic Infections/veterinary , Zoonoses/microbiology , Animals , Bacterial Infections/microbiology , Bacterial Infections/veterinary , Blood-Borne Pathogens , Communicable Diseases/microbiology , Mycoses/microbiology , Mycoses/veterinary , Opportunistic Infections/microbiology , Parasitic Diseases, Animal/microbiology , Toxicity Tests , Virus Diseases/veterinaryABSTRACT
Preclinical safety studies that are required for the marketing approval of a pharmaceutical include single and repeat dose studies in rodent and nonrodent species. The use of nonhuman primates (NHPs), primarily macaques, as the nonrodent species has increased in recent years, in part due to the increase in development of biopharmaceuticals and immunomodulatory agents. Depending on the source of the macaques, they may vary in genetic background, normal flora, and/or the incidence of preexisting pathogens and inflammatory conditions. As the use of alternative sources of macaques rises to meet the increased demand for these animals in biomedical research, the toxicologic pathologist should be well versed in NHP pathology to adequately assess potential drug-related effects in the context of these variations. Such knowledge is particularly important in studies involving immunomodulatory drugs as the toxicologic pathologist should anticipate which type(s) of infections are most likely to arise depending on which arm of the immune system is modulated. The purpose of this review is to discuss the immunosuppressive (e.g., simian type D retrovirus, simian immunodeficiency virus) and opportunistic viruses (e.g., cytomegalovirus, adenovirus, simian virus 40, rhesus rhadinovirus, and lymphocryptovirus), primary and opportunistic bacteria (e.g., Campylobacter spp., Shigella flexneri, Yersinia enterocolitica, Moraxella catarrhalis, Mycobacterium avium complex, enteropathogenic Escherichia coli), and parasites (e.g., Plasmodium spp., Schistosoma spp., Strongyloides fulleborni) that have had the most profound impact on the interpretation of drug safety studies and/or that may reemerge as alternative sources of NHPs are used for drug safety studies.
Subject(s)
Communicable Diseases/drug therapy , Drug Design , Animals , Communicable Diseases/microbiology , Communicable Diseases/parasitology , Macaca/microbiology , Macaca/parasitology , Mycobacterium/drug effects , Mycobacterium/growth & development , Opportunistic Infections/drug therapy , Plasmodium/drug effects , Plasmodium/growth & development , Primates , Retroviridae Infections/drug therapy , Retroviridae Infections/virologyABSTRACT
We noted naturally occurring infection with Clostridium piliforme (Tyzzer's disease) in 2 captive-reared cotton-top tamarins (Saguinus oedipus). Spontaneous Tyzzer's disease has been reported in multiple species of laboratory, domestic, and wild animals but is extremely rare in humans and nonhuman primates. Distinct from idiopathic colitis, which is common in cotton-top tamarins, these 2 tamarins had severe, transmural, necrotizing typhlocolitis accompanied by myocarditis and hepatitis. Abundant bacteria compatible with C. piliforme, the etiologic agent of Tyzzer's disease, were present adjacent to lesions in the cecum-colon, liver, and heart. Therefore, colitis caused by C. piliforme, although rare, should be included as a differential diagnosis in cotton-top tamarins and as a cause of postnatal mortality in this species.
Subject(s)
Animals, Laboratory/microbiology , Clostridium Infections/veterinary , Clostridium , Colitis/veterinary , Monkey Diseases/microbiology , Monkey Diseases/pathology , Saguinus , Animals , Cecum/microbiology , Cecum/pathology , Clostridium Infections/pathology , Colitis/pathology , Colon/microbiology , Colon/pathology , Heart/microbiology , Liver/microbiology , Liver/pathologyABSTRACT
A novel series of 2-hydroxy-N-arylbenzenesulfonamides were identified to be ATP-citrate lyase (ACL) inhibitors with compound 9 displaying potent in vitro activity (IC(50)=0.13 microM). Chronic oral dosing of compound 9 in high-fat fed mice lowered plasma cholesterol, triglyceride, and glucose, as well as inhibited weight gain.
Subject(s)
ATP Citrate (pro-S)-Lyase/antagonists & inhibitors , Anti-Obesity Agents/chemistry , Anti-Obesity Agents/pharmacology , Benzene/chemistry , Sulfonamides/chemistry , Sulfonamides/pharmacology , Adipose Tissue/drug effects , Animals , Anti-Obesity Agents/chemical synthesis , Blood Glucose/analysis , Body Weight/drug effects , Cells, Cultured , Cholesterol/blood , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Mice , Rats , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Triglycerides/bloodABSTRACT
The transcription factor NF-kappaB plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKKbeta, an Ikappa-B kinase that regulates NF-kappaB. IKKbeta and NF-kappaB are required in vivo for prevention of TNFalpha-mediated apoptosis. ML120B sensitized mouse bone marrow progenitors and granulocytes, but not mature B cells to TNFalpha killing in vitro, and induced apoptosis in vivo in the bone marrow and spleen within 6 hours of a single oral dose. In vivo inhibition of IKKbeta with ML120B resulted in depletion of thymocytes and B cells in all stages of development in the bone marrow but did not deplete granulocytes. TNF receptor-deficient mouse thymocytes and B cells were resistant to ML120B-induced depletion in vivo. Surprisingly, surviving bone marrow granulocytes expressed TNFR1 and TNFR2 after dosing in vivo with ML120B. Our results show that inhibition of IKKbeta with a small molecule in vivo leads to rapid TNF-dependent depletion of T and B cells. This observation has several implications for potential use of IKKbeta inhibitors for the treatment of inflammatory disease and cancer.
Subject(s)
I-kappa B Kinase/antagonists & inhibitors , Lymphocyte Depletion/methods , Receptors, Tumor Necrosis Factor, Type I/physiology , Animals , Apoptosis/drug effects , B-Lymphocytes/drug effects , Bone Marrow Cells/drug effects , Enzyme Inhibitors/pharmacology , Granulocytes/drug effects , Granulocytes/metabolism , Hematopoietic Stem Cells/drug effects , Mice , Mice, Knockout , Receptors, Tumor Necrosis Factor , Receptors, Tumor Necrosis Factor, Type I/deficiency , Receptors, Tumor Necrosis Factor, Type II/deficiency , Receptors, Tumor Necrosis Factor, Type II/physiology , Tumor Necrosis Factor Decoy Receptors , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Muraglitazar/BMS-298585 (2) has been identified as a non-thiazolidinedione PPAR alpha/gamma dual agonist that shows potent activity in vitro at human PPARalpha (EC(50) = 320 nM) and PPARgamma(EC(50) = 110 nM). Compound 2 shows excellent efficacy for lowering glucose, insulin, triglycerides, and free fatty acids in genetically obese, severely diabetic db/db mice and has a favorable ADME profile. Compound 2 is currently in clinical development for the treatment of type 2 diabetes and dyslipidemia.
Subject(s)
Glycine/analogs & derivatives , Glycine/chemical synthesis , Hypoglycemic Agents/chemical synthesis , Hypolipidemic Agents/chemical synthesis , Oxazoles/chemical synthesis , PPAR alpha/agonists , PPAR gamma/agonists , Adipocytes/cytology , Animals , Blood Glucose/drug effects , Cell Line , Diabetes Mellitus, Type 2/drug therapy , Fatty Acids/blood , Glycine/chemistry , Glycine/pharmacology , Humans , Hyperlipidemias/drug therapy , Hypoglycemic Agents/pharmacokinetics , Hypoglycemic Agents/pharmacology , Hypolipidemic Agents/pharmacokinetics , Hypolipidemic Agents/pharmacology , Insulin/blood , Male , Mice , Mice, Obese , Oxazoles/chemistry , Oxazoles/pharmacology , Transcriptional Activation , Triglycerides/bloodABSTRACT
Inflammatory cytokines are believed to play an important role in the pathogenesis of human immunodeficiency virus type 1-associated encephalitis. To examine this in the simian immunodeficiency virus (SIV)-infected macaque model of neuroAIDS, inflammatory cytokine gene expression was evaluated in the brains of macaques infected with pathogenic SIV(mac251) by reverse transcriptase PCR. Interleukin-1 beta was readily detected in the brains of all animals evaluated, regardless of infection status or duration of infection. Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) transcripts were undetectable in the brains of uninfected control animals but were upregulated at 7 and 14 days postinoculation. At the terminal stage of infection, TNF-alpha and IFN-gamma transcripts were coexpressed in the brains of four of five animals with SIV encephalitis (SIVE). Within an encephalitic brain, TNF-alpha and IFN-gamma transcripts were detected in six of seven regions with histologic evidence of SIVE, suggesting a direct relationship between neuropathology and altered cytokine gene expression. With combined fluorescent in situ hybridization and immunofluorescence, TNF-alpha-expressing cells were frequently identified as CD68-positive macrophages within perivascular lesions. These observations provide evidence that cytokines produced by activated inflammatory macrophages are an important element in the pathogenesis of SIVE.