RATIONAL OF ANIMALS USE IN DRUG DISCOVERY

 

ABOUT AUTHORS:
Aruni Kumari, Ajit Kumar Thakur, Vikas Kumar*
Neuropharmacology Research Laboratory, Department of Pharmaceutics,
Indian Institute of Technology (Banaras Hindu University),
Varanasi-221 005, India
*vikas.phe@iitbhu.ac.in

ABSTRACT
Animals play a pivotal role in science and scientific technology. All the animals used in experiments are treated ethically following legislative rules. Small animal such as mice, turtles, frogs, rats etc. are used which has short span of reproductive cycle. Their population can be multiplied within six months. The use of animals in research is analyzed from the viewpoint of three distinct ethical approaches: contractarianism, utilitarianism and animal rights view. On contractarian point of view, research on animal is ethical to the extent that human as a social party should care about its well being. According to utilitarian point of view, harm caused to the sentient animal during research should be compensated with benefit to human society. From the animal rights view, use of animals in science is not in animal’s best interest. In this review, comprehensive information is provided on contribution of animal research in drug discovery and development.

REFERENCE ID: PHARMATUTOR-ART-1814

1. INTRODUCTION
Animal experimentation is the backbone of the medical and scientific research. But the main focus should be on input and output (Rowan, 1997). The use of animals in scientific investigation has been traced back to ancient history. For instance, the writings of Aristotle (384-322 B.C.) and Erasitratus (304-258 B.C.) indicate that they had studied the anatomy of various animals. Use of animals in scientific investigation is very important and ineluctable in the interest of human welfare(Baumans, 2004). Various alternatives have been designed like in-vitro methods, but these only acts as an adjunct and reduce the number of animals used. Also the rate of biotransformation is rapid in small laboratory animals than in human, which lead to species difference in response to drugs(Kumar et al., 2000).Certain life processes can only be investigated by using whole animal system asin-vitroalternatives provide limited information. At the same time, scientists are required to follow the legislative rules for ethical treatment of animals (Sahni,2000). In this review, through a systematic search, contributions made by animal research in drug discovery are compiled.

2. MATERIAL AND METHODS
We have highlighted the rational use of animals in drug discovery via scientific search engines such as PubMed, ScienceDirect, Google Scholar, Scopus etc. using key words animal, drug discovery, animal pharmacology and animal physiology.

3. MAJOR ADVANCES IN BASIC RESEARCH THAT DEPENDED ON ANIMALS
The animals were frequently used as a model organism in the early 19th century for drug discovery. Vaccines for various diseases like smallpox, anthrax, rabies, typhoid were discovered using animal experimentation. In early 90s, treatment for rickets, Vitamin C, local anaesthetic, vaccine for tetanus, polio, diptheria, antibiotic(penicillin, streptomycin) was discovered which served the purpose of human well being. With the advent of 20th century various modern developments were made which revolutionized the scientific era. Mechanism and treatment of disease like cancer, AIDS, influenza, and several other fatal diseases were discovered. This contribution of animal life in experiments saved millions of human life across the world (Table 1).

4. CONTRIBUTION OF ANIMALS USE IN DRUG DISCOVERY

4.1. CNS Disorders:Animalexperimentation has made a significant contribution in this field in understanding various disorders and invention of its cure.Monkeys are used to understand and cure Parkinson’s disease (Redmond, 2012). Now several drugs are available for its treatment.With the findings in mouse model of neuro-developmental disorders such asfragile X,Rett syndrome, Down syndrome, and neurofibromatosis type I suggest that it is possible to reverse certain molecular, electrophysiological, and behavioral deficits associated with these disorders in adults by genetic or pharmacological manipulations.Earlier brain developmentduring neuronal disorder and any abnormalities were considered irreversible in adulthood (Castren,2012).P7C3,an aminopropylcarbazolehasproneurogenic and neuroprotective properties in newborn neural precursor cells of the hippocampal dentate gyrus. This has been found that this chemical is efficaciousinprotecting dopaminergic neurons of the substantianigra following exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,in a mouse model of Parkinson’s disease. An active analog of P7C3, known as P7C3A20, protects ventral horn spinal cord motor neurons from cell death in the G93A-SOD1 mutant mouse model of amyotrophic lateral sclerosis (ALS).It has been proposed that the chemical scaffold represented by P7C3 and P7C3A20 may provide a basis for the discovery and optimization of pharmacologic agents for the treatment of ALS (Teslaetal., 2012). A multifunctional DNA/RNA-binding protein TDP-43 has been identified as the major component of the cytoplasmic ubiquitin (+) inclusions (UBIs) in diseased cells of frontotemporal lobar dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) and three other autophagy activators (spermidine, carbamazepine and tamoxifen) have been tested in a FTLD-U mouse model with TDP-43 proteinopathies.Some behavioral improvements has been reported upon rapamycin treatment are accompanied by a decreased level of caspase-3 and a reduction of neuron loss in the forebrain of FTLD-U mice,as well as increases in the autophagic marker LC3. Finally, rapamycin as well as spermidine, carbamazepine, and tamoxifen could also rescue the motor dysfunction of 7-mo-old FTLD-U mice. These data suggest that autophagy activation is a potentially useful route for the therapy of neurodegenerative diseases with TDP-43 proteinopathies(Wang etal., 2012). Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons.By screening a zebrafish model of ALS Epha4, a receptor in the Ephrin axonal repellent system, as a modifier of the disease phenotype in fish, rodents and humans has been identified. Genetic as well as pharmacological inhibition of Epha4 signaling rescues the mutant SOD1 phenotype in zebrafish and increases survival in mouse and rat models of ALS. Motor neurons that are most vulnerable to degeneration in ALS express higher levels of Epha4, and neuromuscular re-innervation by axotomized motor neurons is inhibited by the presence of Epha4. In humans with ALS, EPHA4 expression inversely correlates with disease onset and survival, and loss-of-function mutations in EPHA4 are associated with long survival. This suggests that Epha4 generically modulates the vulnerability of (motor) neurons to axonal degeneration and may represent a new target for therapeutic intervention (Van Hoeckeet al., 2012). Middle cerebral artery occlusion(MCAO) is a widely used experimental technique in rodents to model both the short-term pathological events and longer term neuroanatomical and functional damage associated with focal ischemia. Various neurobehavioral tasks have been developed to assess the motor and cognitive dysfunctions associated with MCAO in rodents, and these studies have revealed deficits related to long-term sensorimotor function, as well as retention of spatial memory. Combined behavioral results from post-MCAO mice provide evidence of a RAP deficit as well as deficits in spatial, non-spatial, and motor learning. Overall results support a fuller characterization of behavioral deficits in auditory processing after MCAO (Troungetal., 2012). Repulsive guidance molecule a (RGMa) is a neurite growth inhibitor. It is of great interest in the study of CNS neuronal regeneration. RNA interference (RNAi) as a means of suppressing the expression of RGMa has been adopted and observed the improvement in axonal regeneration and neurological function of rats after cerebral ischemic injury. RGMa in rat brains after middle cerebral artery occlusion (MCAO) can be down-regulated by RNAi successfully, which may lead to improved axonal growth and neural anatomy plasticity, as well as neuron functional recovery(Fengetal., 2012). High mobility group box-1 (HMGB1) plays an important role in triggering inflammatory responses in many types of diseases. The involvement of HMGB1 in traumatic brain injury (TBI) has been examined and the ability of intravenously administered neutralizing anti-HMGB1 monoclonal antibody (mAb) to attenuate brain injury is evaluated by using rat or mice model(Okuma etal., 2012). Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. This disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. MB(methylene blue) increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in-vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD(Sotang et al., 2012). Comparative neuroprotective potential of silymarin, piracetam and protocatechuic acid ethyl ester (PCA) was evaluated in focal ischemic rats. Various pharmacological, biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite content, brain water content) and behavioural (memory impairment, motor control, neurological score) including infarct size and histopathological alterations were evaluated.Silymarin was found to be more effective than other(Muley etal., 2012). Incorporation of adeno-associated virus and short-hairpin RNA against superoxide dismutase-1 bypassed the blood-brain barrier to target the spinal cord and dorsal root ganglia. The therapeutic vector effectively suppressed mRNA and protein expression of endogenous superoxide dismutase-1 in the lumbar spinal cord and dorsal root ganglia. Moreover, neither neurological side effects nor toxicity due to the incorporated short-hairpin RNA occurred after the injection. Thus it was proposed that this approach could be developed into novel therapies for motor neuron diseases and chronic pain conditions, such as complex regional pain syndrome, through silencing of the genes responsible for pathologies in the spinal cord and dorsal root ganglia(Hirai etal., 2012). The pharmacological activity of the essential oil obtained from Spirantheraodoratissimaleaves and its major component on the murine CNS has been evaluated. It showedpossibleanxiolytic-like effect and the underlying mechanisms involved were also investigated. β-caryophyllene, the main component of this essential oil, andhasanti inflammatory activity. Spirantheraodoratissimais used in folk medicine to treat renal and hepatic diseases, stomachache, headaches and rheumatism(Galdino etal., 2012).

4.2. GIT Disorders: Due to the ever-present threat of a radiological or nuclear accident or attack, the National Institute of Allergy and Infectious Diseases, Radiation Medical Countermeasures Program was initiated in 2004. Since that time, the Program has funded research to establish small and large animal models for radiation damage, as well as the development of approaches to mitigate/treat normal tissue damage following radiation exposure(DiCarlo etal., 2012). Approximately 25% of the patients have established colorectal liver metastasis. Optimal management of disseminated disease requires therapies targeting multiple stages in hepatic colorectal cancer metastasis development. To facilitate this, biologically accurate in-vivo models are required. Early colonic cancer liver metastases development was studied using BDIX and Sprague-Dawley rat strains with human HT29 and rat DHDK12 colonic cancer cell lines(Robertson etal., 2008). The protective effect of electro-acupuncture (EA) pretreatment on acetylsalicylic acid (ASA)-induced ulceration in rats was investigated. Protection was thought to be through gastric defense component(Hwang et al., 2009). The incretin effect of the gut peptide hormone glucagon-like peptide-1 (GLP-1) is a combined result of inhibition of gastric emptying and stimulation of insulin secretion via an incretin mechanism. Animal experimentation in the rat clearly showed that not only gastric emptying, but also small bowel motility with the migrating myoelectric complex was profoundly inhibited by GLP-1 at low doses.Thus GLP-1 analogue ROSE-010 is believed to cause relaxation of the gut and can thereby relieve an acute pain attack of IBS, even though its precise mechanism is yet to be defined(Hellstrom,2009). The expression of substance P (SP) in the airway mucosa of guinea pigs was observed with repetitive esophageal stimulation by hydrochloric acid (HCL). Airway neurogenic inflammation was found in guinea pigs with repetitive esophageal stimulation by HCL, which maybe closely related to the pathogenesis of gastro-esophageal reflux disease (Liet al.,2009). Fluoro-D-glucose position emission tomography (FDG-PET) would allow for the introduction of metabolic imaging into intraoperative and minimally invasive settings. Through in-vitro and in-vivoexperimentation an evaluation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescently labeled glucose molecule has been presented as a molecular beacon of glucose utilization. The competitive inhibition of 2-NBDG uptake by excess free glucose is directly compared against FDG uptake inhibition in cultured cells. 2-NBDG uptake in the brain of a mouse experiencing a generalized seizure was measured, as well as in subcutaneously implanted tumors in mice during fed and fasting states. Localization of 2-NBDG into malignant tissues was studied by laser scanning microscopy. The clinical relevance of 2-NBDG imaging is examined by performing fluorescence colonoscopy, and by correlating preoperative FDG-PET with intraoperative fluorescence imaging. 2-NBDG exhibits a similar uptake inhibition to FDG by excess glucose in the growth media. Uptake was significantly increased in the brain of an animal experiencing seizure versus control, and in subcutaneous tumors after the animals are kept nil per os (NPO) for 24 h versus ad libitum feeding. The clinical utility of 2-NBDG is confirmed by the demonstration of very high target-to-background ratios in minimally invasive and intraoperative imaging of malignant lesions. We present an optical analog of FDG-PET to extend the applicability of metabolic imaging to minimally invasive and intraoperative settings (Sheth et al., 2009). The preventive effect of sodium butyrate in the appearance of aberrant crypt foci (ACF) in rats was evaluated after induction with the carcinogen 1,2-dimethylhydrazine (DMH). The use of sodium butyrate together with the induction of colorectal cancer was not effective in the prevention of the disease progression (Silva et al., 2010). The experimental infection of newborn calves with bovine norovirus was used as a homologous large animal model to study the pathogenesis of norovirus infection and to determine target cells for viral replication(Otto et al., 2011). Experiment done on Sprague Dawley male rats showed that a significant alteration in the enzyme activity as well as changes in the structure and function of the intestine in the presence of the pro-carcinogen, DMH(1,2 Dimethylhydrazine), which suggests the possible chemopreventive efficacy of NSAIDs against the intestinal cancer(Mittalet al., 2008).

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