Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

A DNAzyme based knockdown model for Fragile-X syndrome in zebrafish reveals a critical window for therapeutic intervention.

INTRODUCTION: FXS is the leading cause of intellectual disabilities in males and a major monogenic cause of ASD (Autism spectrum disorders). It occurs due to the loss of FMRP, whose role in early development is not well understood. In this study, we have used a novel DNAzyme based approach to create a larval model of FXS in zebrafish with specific focus on the early developmental window. METHODS: Fmr1specific DNAzymes were electroporated into embryos to create the knockdown. Changes in RNA and protein levels of FMRP and relevant biomarkers were measured in the 0-7dpf window. Behavioral tests to measure anxiety, cognitive impairments and irritability in the larvae were conducted at the 7dpf stage. Drug treatment was carried out at various time points in the 0-7dpf window to identify the critical window for pharmacological intervention. RESULTS: The DNAzyme based knockdown approach led to a significant knockdown of FMRP in the zebrafish embryos, accompanied by increased anxiety, irritability and cognitive impairments at 7dpf, thus creating a robust larval model of FXS. Treatment with the Mavoglurant was able to rescue the behavioral phenotypes in the FXS larvae, and found to be more efficacious in the 0-3dpf window. DISCUSSION: The results from this study have revealed that a) a DNAzyme based knockdown approach can be used to create robust larval zebrafish model of disease, in a high-throughput manner and b) optimal window for therapeutic intervention for FXS as well as other pediatric diseases with a monogenic cause can be identified using such a model.

Statins exacerbate glucose intolerance and hyperglycemia in a high sucrose fed rodent model.

Statins are first-line therapy drugs for cholesterol lowering. While they are highly effective at lowering cholesterol, they have propensity to induce hyperglycemia in patients. Only limited studies have been reported which studied the impact of statins on (a) whether they can worsen glucose tolerance in a high sucrose fed animal model and (b) if so, what could be the molecular mechanism. We designed studies using high sucrose fed animals to explore the above questions. The high sucrose fed animals were treated with atorvastatin and simvastatin, the two most prescribed statins. We examined the effects of statins on hyperglycemia, glucose tolerance, fatty acid accumulation and insulin signaling. We found that chronic treatment with atorvastatin made the animals hyperglycemic and glucose intolerant in comparison with diet alone. Treatment with both statins lead to fatty acid accumulation and inhibition of insulin signaling in the muscle tissue at multiple points in the pathway.

Magnetic nanoparticle formulation for targeted delivery of chemotherapeutic irinotecan to lungs.

Lung cancer is the single largest cause of cancer related deaths in the world. Current treatments include surgery, radiation therapy, chemotherapy using cytotoxic drugs, and monoclonal antibodies. Such treatments have limited efficacy due to diverse nature of lung cells involved and lack of tissue penetration. Cytotoxic drugs, while potent, have the enormous drawback of limited entry into the lung selectively, thus causing collateral damage to other tissues. To overcome these shortcomings, we report here the development of new magnetic irinotecan containing nanoparticles (NPs), which target the lung over other tissues by over 5-fold. Selective targeting of lungs is achieved by deliberately incorporating a facilitated transport mechanism into the NPs. The iron containing NPs can be further exploited to retain the drug into the lung for maximum efficacy using an external magnet. This irinotecan nanoformulation can be used as mono therapy or combination therapy and offers a cost-effective and efficacious therapy for lung cancers.

Correlation of pharmacokinetics and brain penetration data of adult zebrafish with higher mammals including humans.

INTRODUCTION: Adult zebrafish pharmacology is evolving rapidly for creating efficacy and safety models for drug discovery. However, there is very limited research in understanding pharmacokinetics (PK) in adult zebrafish. Methods for understanding PK will help in conducting pharmacokinetic - pharmacodynamic (PK-PD) correlations and improving the quality and applicability of data obtained using zebrafish. METHODS: We conducted adult zebrafish PK and brain penetration studies on two known compounds (irinotecan and lorcaserin) with distinct PK and brain penetration properties using validated LCMS/MS method. Irinotecan was studied at a dose of 100mg/kg i.p. and levels of the parent drug and active metabolite SN-38 were measured. Loracserin was studies at a dose of 10mg/kg by two routes i.p. and p.o. RESULTS: Zebrafish PK and brain penetration profiles for both compounds were very similar to that of higher mammals including humans. Irinotecan was metabolised to SN-38 in ratios similar to ratios seen in other species and the compound had long half life with very low brain penetration in our studies. Loracasin was highly permeable in brain as compared to the exposure in blood, with long half life and high relative bioavailability, similar to other mammalian species including humans. DISCUSSION: Adult zebrafish PK studies are relatively an unexplored area of zebrafish research. The zebrafish data for key parameters of irinotecan and loracserin shows a high correlation to the data from higher species, including human. This report explores and discusses the use of adult zebrafish as a predictive PK tool for higher animal studies.

Novel Zebrafish EAE model: A quick in vivo screen for multiple sclerosis.

INTRODUCTION: Pre-clinical drug discovery for multiple sclerosis (MS) is a labor intensive activity to perform in rodent models. This is owing to the long duration of disease induction and observation of treatment effects in an experimental autoimmune encephalomyelitis (EAE) model. We propose a novel adult zebrafish based model which offers a quick and simple protocol that can used to screen candidates as a step between in vitro experiments and rodent studies. The experiments conducted for this manuscript were to standardize a suitable model of EAE in adult zebrafish and validate it using known modulators. METHODS: The EAE model was developed by disease induction with myelin oligodendrocyte glycoprotein - 35-55 (MOG) and observation of survival, clinical signs and body weight changes. We have validated this model using fingolimod. We have further performed detailed validation using dimethyl fumarate, dexamethasone and SR1001, which are known modulators of rodent EAE. RESULTS: The immunization dose for the disease induction was observed to be 0.6mg/ml of MOG in CFA (Complete Freund's adjuvant), injected subcutaneously (s.c.) near spinal vertebrae. In the validation study with fingolimod, we have demonstrated the modulation of disease symptoms, which were also confirmed by histopathological evaluation. Furthermore, detailed validation with three other known drugs showed that our observations concur with those reported in conventional rodent models. DISCUSSION: We have standardized and validated the adult zebrafish EAE model. This model can help get a quick idea of in vivo activity of drugs in a week using very low quantities of candidate compounds. Further work will be required to define this model particularly as it is found that zebrafish may not express a MOG homologue.

Simvastatin may induce insulin resistance through a novel fatty acid mediated cholesterol independent mechanism.

Statins are a class of oral drugs that are widely used for treatment of hypercholesterolemia. Recent clinical data suggest that chronic use of these drugs increases the frequency of new onset diabetes. Studies to define the risks of statin-induced diabetes and its underlying mechanisms are clearly necessary. We explored the possible mechanism of statin induced insulin resistance using a well-established cell based model and simvastatin as a prototype statin. Our data show that simvastatin induces insulin resistance in a cholesterol biosynthesis inhibition independent fashion but does so by a fatty acid mediated effect on insulin signaling pathway. These data may help design strategies for prevention of statin induced insulin resistance and diabetes in patients with hypercholesterolemia.

Investigational drugs for the management of Huntington's disease: are we there yet?

INTRODUCTION: Huntington's disease is a hereditary neurodegenerative disease. It is designated as a rare disease in the US, which means there are < 200,000 patients in the country who suffer from it. The drugs that are currently used to treat this disease were not designed specifically for it but developed for other diseases. Presently, two classes of drugs are being developed; those that provide symptomatic relief and those that may modify course of the disease. AREAS COVERED: This review is focused on seven selected drugs currently in clinical testing and describes their progress. Five of the seven drugs that are reviewed here, can be categorized as 'symptomatic' drugs, and, selisistat and PBT-2 are amongst the ones that would qualify as 'disease modifying' drugs. EXPERT OPINION: The authors believe that the future treatment paradigm for this disease is best met by using a disease-modifying drug that can be administered together with symptomatic drugs. Towards that end, it is important for the industry to focus on disease-modifying drugs by targeting unique pathways and targets. Furthermore, they propose that neuroprotective drugs, that is, drugs that directly work by preserving neuronal health and function is an opportunity for such disease-modifying drugs.

Head and neck cancers, the neglected malignancies: present and future treatment strategies.

Head and neck cancers are the most prevalent cancers in Asia and result in 50% of deaths due to all cancers in that region. However, treatment regimen has not changed in the past two decades and there are no specific drugs for this cancer. As a result of this, treatment outcomes remain poor. Furthermore, there are no new breakthrough therapies on the horizon, in part, due to low commercial interest in these cancers. What is needed is new thinking, which combines targeted and risk mitigation approaches to develop novel drugs. This editorial will focus on summarising the present approaches to treatment and propose new targets for these cancers.

Bioenergetics failure in neurodegenerative diseases: back to the future.

Neurodegenerative disease such as Alzheimer's, Parkinson and Huntington's are all characterized by dysfunctional neurons and loss of cognitive/motor functions. Interestingly these three diseases involve overproduction, aggregation or abnormal degradation of a specific aberrant protein, which participates in disease pathogenesis. The aggregated proteins may induce disease causing pathways such as high oxidative stress and reduced neuronal metabolism. Several mechanisms are being considered as disease-causing and there is established and growing evidence that a breakdown in neuronal energy production may be an underlying cause in these diseases. The specific risk factors and molecular drivers for each disease vary, yet there are common defective bioenergetics pathways, which may drive neuronal dysfunction. While it has been appreciated that energy deficits can drive neuronal dysfunction and disease, it has for the most part been overlooked as a target pathway for designing novel disease modifying therapeutics. This editorial reviews selected evidence supporting energy deficits as disease-causing and proposes targets for design of new therapeutics.

Bioenergetics breakdown in Alzheimer's disease: targets for new therapies.

Alzheimer's disease is rapidly growing worldwide and yet there is no cure for it. Currently available drugs only provide symptomatic relief and do not intervene in disease process sufficiently enough to prevent or cure it. Characteristic features of this disease are decline in neuronal mass and cognitive functions. The most dominant hypothesis proposed for pathogenesis of this disease is called "amyloid hypothesis". It states that excessive production of amyloid peptides called abeta peptides (Aß) is the underlying cause of neuronal death and dysfunction. However, recent drugs designed based on amyloid hypothesis have failed in clinical trails, demanding fresh assessment. Early and persistent molecular events in this disease progression are energy deficiency and high oxidative stress in the neurons. Our review will put together a disease model based on known human and animal data with regards to breakdown in neuronal energy generation. The model will integrate energy deficits as the cause of neuronal dysfunction and abeta peptide production culminating in catastrophic loss of cognitive functions. Finally, based on this model, we will also suggest enzyme targets in neuronal bioenergetics pathway for design and development of new disease modifying therapies.

Alzheimer's disease amyloid hypothesis at crossroads: where do we go from here?

Alzheimer's disease has been the focus of several drug discovery approaches by the pharmaceutical industry. Four drug candidates coming out of such efforts have recently failed in late-stage clinical trials for lack of efficacy or safety concerns. These drugs were designed based on the presently dominant scientific hypothesis for Alzheimer's disease called the 'amyloid hypothesis'. This editorial will briefly review the failure of these drugs and the effect of this on the amyloid hypothesis. Rather than accept the status quo, this editorial suggests a revised version of this hypothesis to reconcile data from recent drug failures. We propose a two-phase disease process; a first phase that is independent of amyloid and a second robust phase dependent on the amyloid cascade. Further validation of this revised hypothesis could aid future drug discovery for this devastating disease.

Lipid metabolism and Alzheimer's disease: pathways and possibilities.

BACKGROUND: Alzheimer's disease (AD) is a growing condition in dire need of new therapeutics for its cure. Currently marketed drugs only provide symptomatic relief and do not intervene in disease-modifying pathways. Deposition of amyloid beta peptides in the brain is considered to be at the core of pathological events in AD. Studies indicate that lipid/lipoprotein metabolism has a role in several steps in the etiology of AD. OBJECTIVE/METHODS: Here we review the molecular pathways by which lipid metabolism may influence AD and could form the basis for design of new disease modifying therapeutics. RESULTS/CONCLUSIONS: Modulation of lipid metabolism has excellent potential to provide new avenues for disease modifying drugs for AD.

Lipoic acid synthase (LASY): a novel role in inflammation, mitochondrial function, and insulin resistance.

OBJECTIVE: Lipoic acid synthase (LASY) is the enzyme that is involved in the endogenous synthesis of lipoic acid, a potent mitochondrial antioxidant. The aim of this study was to study the role of LASY in type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied expression of LASY in animal models of type 2 diabetes. We also looked at regulation of LASY in vitro under conditions that exist in diabetes. Additionally, we looked at effects of LASY knockdown on cellular antioxidant status, inflammation, mitochondrial function, and insulin-stimulated glucose uptake. RESULTS: LASY expression is significantly reduced in tissues from animal models of diabetes and obesity compared with age- and sex-matched controls. In vitro, LASY mRNA levels were decreased by the proinflammatory cytokine tumor necrosis factor (TNF)-alpha and high glucose. Downregulation of the LASY gene by RNA interference (RNAi) reduced endogenous levels of lipoic acid, and the activities of critical components of the antioxidant defense network, increasing oxidative stress. Treatment with exogenous lipoic acid compensated for some of these defects. RNAi-mediated downregulation of LASY induced a significant loss of mitochondrial membrane potential and decreased insulin-stimulated glucose uptake in skeletal muscle cells. In endothelial cells, downregulation of LASY aggravated the inflammatory response that manifested as an increase in both basal and TNF-alpha-induced expression of the proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Overexpression of the LASY gene ameliorated the inflammatory response. CONCLUSIONS: Deficiency of LASY results in an overall disturbance in the antioxidant defense network, leading to increased inflammation, insulin resistance, and mitochondrial dysfunction.

Novel anti-inflammatory role for glycogen synthase kinase-3beta in the inhibition of tumor necrosis factor-alpha- and interleukin-1beta-induced inflammatory gene expression.

Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase with a broad array of cellular targets, such as cytoskeletal proteins and transcription factors. Recent studies with GSK-3beta-null mice showed impaired NFkappaB-mediated survival responses. Because NFkappaB serves a dual role as a key regulator of cytokine-induced inflammatory gene expression and apoptosis, we investigated whether modulation of GSK-3beta expression affects cytokine-induced and NFkappaB-mediated inflammatory gene expression. We observed that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) treatment of primary cultures of human microvascular cells reduced net endogenous active GSK-3beta protein levels while inducing inflammatory cytokine (IL-6 and monocyte chemoattractant protein-1 (MCP-1)) expression. Interestingly, inhibition of GSK-3beta by antisense oligonucleotides or pharmacological agent (10 mm lithium) potentiated TNF-induced expression of IL-6 and MCP-1 by 2-6-fold suggesting that inhibition of GSK-3beta under inflammatory conditions (exposure to TNF-alpha and IL-1beta) may contribute to enhanced cytokine expression. Overexpression of GSK-3beta in endothelial cells, in contrast, significantly inhibited (by 70%, p < 0.01) both TNF-alpha and IL-1beta-induced expression of IL-6, MCP-1, and vascular cell adhesion molecule-1. Using adenoviruses in lipopolysaccharide-stimulated mice, overexpression of GSK-3beta significantly decreased TNF-alpha expression in lung and heart tissues (38 and 15%, respectively), further confirming the anti-inflammatory role of GSK-3beta. Overexpression of GSK-3beta did not affect the TNF-alpha-induced nuclear translocation of NFkappaB but reduced the nuclear half-life of TNF-alpha-induced NFkappaB considerably (by as much as 9 h) and enhanced phosphorylation (by as much as 33%). Interestingly, neither endothelial cell survival nor NFkappaB-mediated expression of anti-apoptotic genes was affected by GSK-3beta overexpression. We conclude that GSK-3beta selectively regulates NFkappaB-mediated inflammatory gene expression by controlling the flow of NFkappaB activity between transcription of inflammatory and survival genes.

A new therapeutic target for atherosclerosis treatment: interview with Uday Saxena. Interviewed by Emma Quigley.

Uday Saxena was appointed Chief Scientific Officer at Dr Reddy's Laboratories in 2000. In this role he provides the leadership and general strategy for the company's drug discovery research into metabolic disorders, cardiovascular disorders, inflammation, cancer and anti-infectives. He is also a member of the company's Senior Management Council. He gained his PhD at the Memorial University of Newfoundland, his thesis covering biochemical and functional characterisation of rat C-reactive protein with respect to lipid, lipoprotein metabolism, atherosclerosis and inflammation. On completing his postdoctoral fellowship at Colombia University, he went onto work on various drug discovery projects as Senior Scientist and Principal Research Scientist at Parke-Davis Warner-Lambert and as Director and Vice-President for preclinical research at AtheroGenics, Inc., before undertaking his current position. Uday Saxena has written over 50 peer-reviewed articles and invited reviews. He is currently on the Editorial Board of two international drug discovery-related journals including Expert Opinion on Therapeutic Targets.

Discovery of novel phenolic antioxidants as inhibitors of vascular cell adhesion molecule-1 expression for use in chronic inflammatory diseases.

Vascular cell adhesion molecule-1 (VCAM-1) mediates recruitment of leukocytes to endothelial cells and is implicated in many inflammatory conditions. Since part of the signal transduction pathway that regulates the activation of VCAM-1 expression is redox-sensitive, compounds with antioxidant properties may have inhibitory effects on VCAM-1 expression. Novel phenolic compounds have been designed and synthesized starting from probucol (1). Many of these compounds demonstrated potent inhibitory effects on cytokine-induced VCAM-1 expression and displayed potent antioxidant effects in vitro. Some of these derivatives (4o, 4p, 4w, and 4x) inhibited lipopolysaccharide (LPS)-induced secretion of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 from human peripheral blood mononuclear cells (hPBMCs) in a concentration-dependent manner in vitro and showed antiinflammatory effects in an animal model. Compounds 4ad and 4ae are currently in clinical trials for the treatment of rheumatoid arthritis (RA) and prevention of chronic organ transplant rejection, respectively.

Role of oxidative stress and inflammation in the origin of Type 2 diabetes--a paradigm shift.

In Type 2 diabetes the body either produces too little insulin, or does not respond well to it. Current pharmacological treatments, which are less than optimal, either target defective insulin secretion (sulfonylureas, glinides) or insulin resistance (metformin, thiazolidinediones). Exciting new research is now helping us to understand novel pathways that may contribute to defective insulin secretion as well as decreased response to insulin. Such pathways may explain the development of diabetes and associated complications (atherosclerosis and diabetic nephropathy). Understanding the way a cell metabolises glucose may be the key to understanding how cells secrete insulin and respond to it.

Atherosclerosis -- new targets and therapeutics.

Atherosclerosis is well recognized as an inflammatory disease and circulating markers of inflammation such as C-reactive protein and soluble adhesion molecules are strong predictors of atherosclerotic lesion development and future cardiovascular events. Several cells (endothelial, smooth muscle and macrophages) and proteins (inflammatory cytokines and adhesion molecules) contribute to this inflammatory process and lesion development. Although lipid management with statins does reduce levels of circulating inflammatory markers, this appears to be unrelated LDL-lowering. Thus, the recent focus has been shifted to develop molecules that directly affect the atherosclerotic process without effects on plasma lipids. Much of this research was initially focused on cytokine antagonists and adhesion molecule expression inhibitors, which are now at different stages pre-clinical and clinical development. Additional targets have begun gaining prominence in the past few years -- modulation of proteins involved in reverse cholesterol transport and lipid metabolism in the vessel wall such as ApoA1/apoE/ABCA1, ACAT, and LpPLA2 and regulation of molecules involved in matrix remodeling and cell proliferation such as matrix metalloproteinases and heparan sulfate proteoglycans. The current approaches for the treatment of atherosclerosis are 1) reduction of risk factors for the disease -- e.g., lipids, hypertension and diabetes and 2) direct disease modifiers. The purpose of this review is to examine key scientific advances and the prospect of these approaches in the prevention of cardiovascular disease.

Inflammatory cytokines and fatty acids regulate endothelial cell heparanase expression.

Heparan sulfates, the carbohydrate chains of heparan sulfate proteoglycans, play an important role in basement membrane organization and endothelial barrier function. We explored whether endothelial cells secrete a heparan sulfate degrading heparanase under inflammatory conditions and what pathways were responsible for heparanase expression. Heparanase mRNA and protein by Western blot were induced when cultured endothelial cells were treated with cytokines, oxidized low-density lipoprotein (LDL) or fatty acids. Heparanase protein in the cell media was induced 2-10-fold when cells were treated with tumor necrosis factor alpha (TNFalpha) or interleukin 1beta (IL-1beta). Vascular endothelial growth factor (VEGF), in contrast, decreased heparanase secretion. Inhibitors to nuclear factor-kappaB (NFkappaB), PI3-kinase, MAP kinase, or c-jun kinase (JNK) did not affect TNFalpha-induced heparanase secretion. Interestingly, inhibition of caspase-8 completely abolished heparanase secretion induced by TNFalpha. Fatty acids also induced heparanase, and this required an Sp1 site in the heparanase promoter. Immunohistochemical analyses of cross sections of aorta showed intense staining for heparanase in the endothelium of apoE-null mice but not wild-type mice. Thus, heparanase is an inducible inflammatory gene product that may play an important role in vascular biology.