Melior Discovery Inc.

Non-alcoholic steatohepatitis (NASH) is a hepatic manifestation of metabolic syndrome that can progress to liver cirrhosis.

A rodent NASH model that is most commonly used (the STAM™ model) is relatively short in duration but lacks the obesity and insulin resistance features that are commonly associated with human NASH. Instead, we established a model with better face validity qualities that we believe is more clinically translatable with pronounced obesity, dyslipidemia, insulin resistance, accompanied by liver steatosis, inflammation, fibrosis and hepatic triglyceride content. In this way, we believe that this model closely mimics most features of human NASH and may represent the most relevant rodent model of NASH. Importantly, we have validated this model with the PPAR alpha agonist fenofibrate, and the clinical proven NASH candidate, obeticholic acid (OCA; an FXR agonist).

TheraTRACE® Phenotypic Screening Platform

Contact Hypersensitivity

• Contact hypersensitivity models mimic allergic contact dermatitis aspects of immune function. Many cutaneous disorders are characterized by inflammation. The immune reaction induced by the method described below causes swelling at the site of challenge. Melior has established models of allergic contact hypersensitivity using oxazolone, DNFB and other haptens. The localized inflammatory response can be attenuated by corticosteroids and topical agents used to treat atopic dermatitis.

theraTRACE®

Melior Discovery Inc (Exton, PA, USA) has pioneered a unique “high throughput” in vivo pharmacology platform (theraTRACE®) able to systematically identify novel indications for pre-clinical and development stage drugs.

The theraTRACE® indications discovery platform allows for known drug-like compounds to be comprehensively screened using a battery of in vivo assays spanning multiple therapeutic areas. Currently over 40 validated animal models are represented in the platform spanning a broad range of therapeutic areas including inflammation, immunology, diabetes & metabolic syndrome, dermatology, cardiovascular, gastrointestinal, psychiatric, neurological and neurodegenerative disorders. The in vivo models utilized in the theraTRACE® platform comprise those typically used by pharmaceutical companies to determine whether a drug candidate is suitable for further clinical evaluation, however, these models are assembled in a proprietary “multiplexed” format that allows drug discoverers to evaluate a compound for potential efficacy in a broad array of therapeutic areas rapidly and cost-effectively. In most cases, Melior’s theraTRACE® system is able to provide a complete pharmacological fingerprint/profile of a compound in 10 weeks. Melior’s theraTRACE® system has found particular utility in facilitating the crucial “go/no go” development decisions routinely faced by pharmaceutical and biopharmaceutical companies.

6-OHDA Rat Model of Parkinson's Disease:

The neurotoxin 6-hydroxydopamine (6-OHDA) is widely used to induce depletion of dopaminergic neurons in animal models of Parkinson's Disease (PD). Unilateral administration of 6-OHDA into the medial forebrain bundle can produce a 90-95% ipsilateral depletion of dopamine neurons in 80-90% of animals injected, leading to a PD - like motor dysfunction.

MPTP - Induced Parkinson's Disease:

MPTP is a potent and selective nigrostriatal dopaminergic neurotoxin that in mice produces many of the neuropathological features of idiopathic Parkinson’s Disease in humans. MPTP produces nigrostriatal dopaminergic degeneration and locomotor impairment. Pharmacological agents that increase dopaminergic function or that block the neurotoxicity of MPTP also attenuate MPTP-associated locomotor and dopaminergic dysfunction. Moreover, MPTP-mediated toxicity may have some relationship to the mechanisms associated with dopaminergic loss in the disease indicating that this model may also be potentially useful for identifying agents that slow or reduce nigrostriatal dopaminergic loss.

Pentylenetetrazole-Induced Seizure

Epileptic seizures are effectively modeled in experimental animals using several methods including maximal electroshock, kindling methods and chemically - induced seizures. Pentylenetetrazole (PTZ), a GABA receptor agonist, is a common chemically induced seizure model. When PTZ is administered to mice, it produces a myoclonic seizure that models absence (petit mal) seizures. Compounds that enhance GABAergic transmission are effective in ameliorating seizure activity induced by PTZ.

Pro- or anti-convulsant activity can be evaluated using EEG power analysis and can be more sensitive than models utilizing chemical or electrical stimuluation and behavioral outcome measures. For example, quantitative anti-convulsant activity can be evaluated using sub-convulsant doses of PTZ that produce characteristic EEG frequency changes that can be quantitatively analyzed for several hours.

Maximal electroshock (MES) is a widely accepted test that is considered to be an experimental model of tonic-clonic seizure. This model is designed to identify pharmacological agents that exhibit anti-epileptic activity.

Grip Strength:

Rats or mice are placed on a wire grid with either forelimbs alone or forelimb + hindlimb gripping the grid (A), and pulled backward by the tail. The maximal force the animal exerts at the time of release is recorded (B,C). This test is a simple and reliable measure of the function of the general motor capability of the animal, in particular function of the spinal motor neurons, efferent axonal conduction, neuromuscular junction, and the muscles themselves.

Locomotor and Open-Field Activity:

The locomotor and open-field activity tests are useful tools for assessing locomotor, psychological and other sensorimotor behavior in rodents. An automated system is in place that is capable of monitoring multiple locomotor and psychotherapeutic related parameters. Open-field assessments are useful for determining how a drug effects not only normal locomotion, but other activities including exploratory behavior, stereotypic activity and time spent in pre-defined zones within the chamber. The chamber can be modified to examine anxiety-related behaviors, learning and memory functions and place-preference assays for additiction.

Motor Evoked Potentials and Nerve Conduction:

Nerve conduction and/or neuromuscular function following drug treatment, injury, or as a function of genetic factors can be evaluated in a variety of ways. Stimulation of the sciatic nerve at the hip produces compound motor action potential (CMAP) responses in the plantar foot muscle that are delayed and reduced in amplitude in SOD (super-oxide dismutase) knock-out (SOD-/-) or transgenic (G93A) mice. Mice can be evaluated as early as 5 weeks of age, and can be tested weekly or biweekly.

Rotarod:

The rotarod test is a simple pharmacological assessment of motor coordination, balance, muscle strength and gait, as well as sensory. In addition to straightforward assessment of coordinated movement, data from rotarod performance experiments can also be beneficial for interpreting results from other behavior models. Specifically, many drugs that have either sedative or stimulant properties could interfere with assessment of behavioral tests that require a motoric response. Consequently, having an "a-piori" read on locomotor coordination effects of novel compounds is prudent, if not necessary for planning, executing and explaining behavioral results gathered during standard pre-clinical efficacy profiling.

EEG Sleep / Wake and Motor Activity:

Cortical activity recorded in rodents can be used to characterize basic behavioral states such as sleep/wake activity, as well as evaluating interactions between drugs and environmental factors such as fatigue. Rats or mice can be chronically implanted and, depending on treatments, can be tested repeatedly for several months. General motor activity and body temperature can also be monitored by implanted transmitters.

Open-Field Activity:

The open-field test is a useful tool for assessing locomotor, psychological and other sensorimotor behavior in rodents. An automated system is in place that is capable of monitoring multiple locomotor and psychotherapeutic related parameters. Open-field assessments are useful for determining how a drug affects not only normal locomotion but other activities including exploratory behavior, stereotypic activity and time spent in pre-defined zones within the chamber. The chamber can be modified to examine anxiety-related behaviors, learning and memory functions and place-preference assays for addiction.

db/db Mouse Model:

Type II diabetes is characterized by high blood glucose levels in the presence of normal or elevated serum insulin levels. There are many animal models of type II diabetes that involve administering high levels of glucose to otherwise normoglycemic mice (see OGTT mouse odel). db/db mice express mutations in leptin receptor that lead to decreased insulin receptor sensitivity and subsequent increased levels of blood glucose, decreased β-cell function, increased obesity and elevated HBA1c levels. Compounds from several structural classes can effectively regulate this hyperglycemic response including sulfonylureas, thiazoladinediones (PPARγ agonists; glitazones) and metformin (glucophage). These drug classes are also clinically approved for use in humans.

Streptozotocin-Induced Diabetes

Type I diabetes is a condition characterized by loss of insulin producing β-cells of the islets of Langerhans in the pancreas leading to a deficiency in insulin production and secretion. This decrease in insulin leads to a decrease cellular glucose utilization and elevated blood glucose levels. Diabetes can lead to a number of secondary pathologies including retinopathies, neuropathies, nephropathy and cardiomyopathy. Type I diabetes can be modeled by administration of streptozocin to experimental animals which results in selective destruction of β-cells of the pancreas reducing pancreatic insulin production, decreasing circulating insulin, elevating blood glucose levels and a number of secondary type I diabetes associated pathologies and neuropathies.

ob/ob Mouse Model:

Type II diabetes is characterized by high blood glucose levels in the presence of normal amounts of insulin. There are many animal models of type II diabetes that involve administering high levels of glucose to otherwise normoglycemic mice. ob/ob mice express mutations in leptin that lead to decreased leptin activity and decreased insulin receptor sensitivity. Subsequently these mice display increases in blood glucose levels, decreases in β-cell function, obesity and elevated HBA1c levels; consistent with the development of Type II diabetes. Using this model we can test the ability of a compound to effectively regulate this hyperglycemic response.

Diet-Induced Obesity:

Obesity is a condition of pandemic proportions in the modern world. A myriad of conditions ranging from cardiovascular disease, type-2 diabetes, cancer, and even depression have been linked to obesity. Research into the mechanisms and treatment of obesity is an ongoing area of interest to many in the pharmaceutical and academic communities. Animal models have proven useful in studying the effects of compounds on diet-induced obesity. At Melior we utilize a high fat diet platform to model a number of acute and chronic features of metabolic and obesity disorders consistent with diet induced obesity changes in humans.

Diet-Induced Obesity/ High Fat Diet:

High fat diets in rodents change multiple biochemical and physiological parameters that reflect the biochemical and physiological changes observed in obesity. Such diets induce dramatic changes in weight concomitant with elevations in serum cholesterol, lipids and tricglycerides. Moreover, these high fat diets can lead to atherosclerotic lesions as well as insulin resistance consistent with obesity-induced changes in humans.

At Melior, we have characterized a mouse model of induced-obesity by chronically feeding mice a modified high fat diet (Western Diet) that was designed to approximate the typical human diet of North America and Europe. This chronic assay also serves as a platform for a number of additional acute and subchronic assays designed to model various features of metabolic and obesity disorders consistent with diet-induced changes in humans.

Zymosan-A Induced Peritonitis:

Leukocyte infiltration into a site of injury or irritation is an integral part of an inflammatory process associated with the peritonitis response. The peritonitis model described in this report utilizes Zymosan-A to induce leukocyte accumulation in the peritoneum. This model can be can be utilized as a model of inflammation, acute peritonitis and polymorphonucleocyte migration. Importantly, this model is an effective model for detecting a test compounds ability to attenuate inflammatory processes associated with peritonitis. Anti-inflammatory agents including steroidal and non-steroidal anti-inflammatory compounds are effective in reducing leukocyte recruitment and/or infiltration in this model.

LPS-Pulmonary Inflammation:

Airway diseases such as chronic obstructive pulmonary disease and asthma exact an ever-increasing toll on human health as the population ages and environmental pollutants (including tobacco smoke) damage lung tissue of young and old alike. These conditions are induced and exacerbated by pro-inflammatory molecules that respond to acute infection, but often result in chronic immunological responses that induce acute crises and damage lung tissue over time.

An important goal in pharmaceutics is to break the inflammatory cascade by blocking or suppressing specific cytokines and chemokines known to induce and amplify lung inflammation. Two such proteins, monocyte chemoattractant protein (MCP-1), and tumor necrosis factor alpha (TNF-α), are found to be associated with early and chronic infection and inflammation in humans, and are mirrored in rodent models of airway inflammation. Intranasal administration of lipopolysaccharide (LPS), a bacterial endotoxin, has been shown to initiate a pro-inflammatory response in the lungs of mice. This model recapitulates aspects of inflammatory lung disease in humans, and is useful as a screen for compounds that might attenuate the disease process.

Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. There are many described types of fibrosis with pulmonary and hepatic fibrosis being the most common. Pulmonary fibrosis can be caused by chronic inflammatory processes, infections, environmental agents, exposure to ionizing radiation, chronic conditions and certain medications.

Pulmonary fibrosis is characterized by alveolar epithelial cell injury and hyperplasia, inflammatory cell accumulation, fibroblast hyperplasia, deposition of extracellular matrix, and scar formation. The end result of this process is the loss of lung elasticity and loss of alveolar surface area leading to impairment of gas exchange and pulmonary function. The disease is further characterized by the presence of chronic inflammatory infiltrates, myofibroblast hyperplasia, and disordered collagen deposition.

The bleomycin model of pulmonary fibrosis is the best characterized, and most widely used model. Direct delivery of bleomycin to the lung (e.g. intratracheal) induces rapidly developing fibrosis after a single administration. Direct pulmonary delivery of bleomycin causes direct damage to alveolar epithelial cells. This is followed by the development of neutrophilic and lymphocytic alveolitis within the first week. After the first week, alveolar inflammatory cells are cleared, fibroblast proliferation is initiated, with synthesis of extracellular matrix. The development of fibrosis in this model can be seen biochemically and histologically by day 14 with maximal responses observed between 21- 28 days.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. By administering a myelin basic protein peptide (PLP) fragment or myelin oligodendrocyte glycoprotein (MOG), an autoimmune response is directed to the myelin sheath surrounding the motor neurons. The demyelination of neurons leads to impaired locomotor function characterized by loss of nerve conduction and chronic progression of disability, mirroring the human disease.

Collagen-Induced Arthritis:

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by a chronic erosive inflammation in joints leading to the destruction of cartilage and bone. Immunization of mice with type II collagen leads to the development of arthritis in the collagen-induced arthritis (CIA) model for RA. A number of anti-inflammatory compounds are effective in reducing inflammation in this model of arthritis.

Cortical EEG activity can be recorded from chronically implanted rats and mice and evaluated as a function of frequency to characterize or compare effects of psychoactive drugs such as benzodiazepines, NMDA antagonists, etc. EEG activity (0.03 - 100 Hz) is recoreded for 24 hours or more, and evaluated over discrete frequency ranges and time periods depending on the observed drug effects.

Acetylcholine Writhing Test

Irritable Bowel Syndrome (IBS) is a disorder that is expressed as abdominal cramping, gastrointestial pain and disturbances in GI transit. These conditions are produced by colonic spasticity and irregular contractility. The cramping and pain component of IBS can be modeled by injecting acetylcholine (ACh) into the peritoneum of mice. Intraperitoneally administered ACh promotes smooth muscle contractility and can be monitored, in animals, by quantifying the writhing response.

Headache disorders consistently rank as one of the most prevalent neurological conditions globally, with migraine being one of the most disabling disorders. Though the mechanism of migraine is complex and unresolved, one hypothesis states repeated activation of the trigeminal vascular system leads to long-term physiological changes. Clinical symptoms include trigeminal allodynia and sensitivity to nitrates, both resulting in pain. After four weeks of prostaglandin infusions directly onto the dura, rats exhibit chronic periorbital allodynia and nitrate sensitivity measured by mechanical von Frey fibers.

C-fiber Pain Reflex

C-fibers are small unmyelinated fibers transmitting diffuse pain signals from the periphery to the spinal cord. Larger and faster myelinated Aδ and A β fibers transmit pain and touch information. C-fiber pain activity can be selectively evaluated by stimulating the foot-pad and recording reflex activity int he peroneal muscle between 150 and 400 msec following stimulus. Activity in this pathway can be recorded for several hours allowing within-animal determination of baseline, test article and drug interation effects.

The Hoffman (H) reflex is a proprioceptive spinal reflex produced by activation of Ia afferents originating in the plantar foot muscle that travel tot he spinal cord via the tibial nerve. Stimulation of the tibial n., which also contains plantar motor neuron efferents, first results in direct activation of the plantar muscle (M-response), followed by the reflex activation of the motoneurons and secondary muscle response (H-reflex). The ratio of H/M response amplitude has been used as a measure of spinal excitability.

Micturition : Diet-Induced Stress and Overactive Bladder
Micturition is a parasympathetic function of the nervous system and is achieved by complex neurological mechanisms involving the somatic, autonomic and central components of the urinary system. Micturition disorders are classified as disturbances in bladder storage or emptying. The failure to store urine leads to urgent and stress-related incontinence. This study validates a micturition assay in which latency to urinate and urination volume were produced by an oxybutynin challenge and measured using an automated collection chamber.

The tail immersion assay is a thermal test for evaluating the analgesic potential of compounds. A number of clinically approved pharmacological agents have been demonstrated to delay the onset of heat sensitivity upon tail exposure to heat including opiods such as morphine, and alpha adrenegic compounds. The tail immersion assay has been reputed to measure spinally driven aspects of pain and has the advantage of measurements not being affected by sedation as they would be in other assays such as the hot plate assay.

Anxiety is a psychological and physiological state characterized by learning and memory, emotional, and behavioral components. The Vogel water conflict assay is used to identify drugs that have anxiolytic (alleviate anxiety) potential. By punishing drinking behavior with a mild electric shock animal will decrease drinking behavior. Many different classes of antidepressant drugs will re-establish drinking behavior. As such, the Vogel water conflict can be used to screen test compounds for anxiolytic activity.

Stress and general emotional state can have a multitude of effects on the body as the two are inextricably linked via the hypothalamic–pituitary–adrenal axis (HPA axis). Thus, changes in physiology, such as temperature, can be indicative of changes in stress. When exposed to brief periods of stress rodents display an increase in body temperature (hyperthermia). Many classes of antidepressant drugs can attenuate stress-induced hyperthermia. Here at Melior Discovery, our stress-induced hyperthermia assay offers a quick way to screen for anxiolytic potential in test compounds.

There are a number of animal models of depression that are used to identify pharmacological agents that have clinical potential as anti-depressant agents. One such animal model is the mouse tail-suspension model. Mice, suspended by the tail, will rapidly develop behavioral despair that is characterized by immobility. This immobility is thought to reflect a state of lowered 'mood' (i.e. depression) in which animals have given up hope of escaping. Tail-suspension immobility is attenuated by a variety of clinically active antidepressants. Antidepressants increase the amount of struggle and the latency until the first extended immobility. This assay is considered to be a good predictor for antidepressant activity and identifies nearly all antidepressant classes including tricyclics, SSRIs, 5-HT1A receptor agonists, and MAOIs.

Novel object recognition (NOR) is a classic cognitive test used to determine the ability of a compound to increase or impair learning and memory. This model is based on the natural tendency of rodents to investigate a novel object instead of a familiar one. The choice to explore the novel object reflects the use of learning and recognition memory. Contrary to most classically-used learning and memory paradigms which require long periods of animal training, the object recognition task offers the ability to obtain rapid information on the cognitive impact of a new drug.

Hyponeophagia – the phenomenon in which exposure to a novel environment suppresses feeding behavior – can be used as an assessment of anxiety in animals. Here at Melior Discovery we have developed a novel environment induced feeding suppression assay (NEIFS) measuring the latency to eat. This test assesses stress-induced anxiety and as such can be used to screen compounds that may have anxiolytic or antidepressant efficacy.

The Morris Water Maze assessment is utilized to study spatial learning and memory in rodents. By studying and animal’s ability to find a hidden platform to escape from a pool of water, we can assess their ability to learn and remember the location of a platform. Given rodents’ size to volume ratio, they are highly motivated to seek escape and can be trained relatively quickly to locate a hidden platform. Using this assay we can assess test compounds effects of both learning and memory.

Anxiety is a psychological and physiological state characterized by cognitive, somatic, emotional, and behavioral components. When rodents are given the opportunity to explore novel environments, they display a typical exploration pattern generally preferring the perimeter of a dimly lit environment to a brightly lit environment.

By exploiting rodent’s innate spontaneous exploratory behavior and aversion to brightly lit areas, the light-dark transitions assay can be used to identify drugs that alter anxiety levels. In the light-dark transition assay, a rodent is placed in a chamber with the option to explore a brightly lit area or darkened area. By quantifying the amount of time, we can monitor exploratory as well as anxiogenic or anxiolytic behavior.

The forced swim test is a commonly used assay for detecting anti-depressant activity of pharmacological agents. When mice are required to swim in a container with no route of escape, they develop a behavioral despair that is characterized by immobility except for small movements needed to remain afloat. This immobility is thought to reflect a state of lowered 'mood' (i.e. depression) in which animals have given up "hope" of escaping.

Forced swim is attenuated by a variety of clinically active antidepressants. Antidepressants increase the amount of struggle and the latency until the first extended immobility. This assay is considered to be a good predictor for antidepressant activity and identifies nearly all antidepressant classes including tricyclics, SSRIs, 5-HT1A receptor agonists, and MAOIs.

The fear conditioning learning and memory model is a method for evaluating trial compounds ability to reverse a drug-induced deficit in memory. Fear conditioning involves pairing a context and/or cue (conditioning stimuli, CS) to a shock (unconditioned stimulus) and later measuring the 'fear' response to the CS in the absence of the US. A defensive freezing or immobility response serves as a reliable measure of fear. Contextual and auditory cue fear conditioning depend on different neural processes, whereas contextual conditioning is hippocampally driven and cued fear conditioning is not (Kim and Fanselow, 1992; Fendt and Fanselow, 1999). NMDA-type glutamate receptor antagonists and nicotinc cholinergic receptor antagonists cause amnestic effects in learning and memory tests such as fear conditioning (ref). These compounds are often used to produce deficits in these models. Several classes of drugs reverse learning and memory deficits in fear conditioning. These compounds include acetylcholinesterase inhibitors (Csernansky et al., 2005) and phosphodiesterase type 4 (PDE4) inhibitors (Gong et al. 2004).

The Elevated Plus Maze (EPM) is an anxiety assay for putative anxiolytic (alleviating anxiety) or anxiogenic (anxiety inducing) compounds. The test involves a plus-shaped maze with two open and two enclosed arms. The model relies on the rodent’s tendency to hide in enclosed spaces. Anxiety reduction in this model is measured by an increase in the amount of time spent in the open arms. Anxiolytic drugs that increase GABAergic transmission are reliably effective in this model.

Chronic Mild Stress- Corticosterone Levels
Chronic mild stress (CMS) is a paradigm developed in animals to model the relatively minor and unanticipated irritants that lead to a state of anhedonia (an inability to experience pleasure) in some individuals. Melior has developed a chronic mild stress paradigm that leads to increased behavioral depression and elevated corticosterone levels in rodents. In both instances, these changes are attenuated by treatment with an anti-depressant.

Chronic Mild Stress- Tail Suspension Test
The chronic unpredictable mild stress (CMS) is a paradigm developed in animals to model the relatively minor and unanticipated irritants that lead to a state of anhedonia (an inability to experience pleasure) in some individuals. Here at Melior Discovery, we have developed a chronic mild stress paradigm that leads to increased behavioral depression and elevated corticosterone levels in rodents. In both instances, these changes are attenuated by treatment with an anti-depressant.

Allodynia is an abnormal nociceptive response to a stimulus that does not provoke pain under normal conditions. Von Frey mechanical monofilaments, varying in diameter and corresponding force, are utilized to provoke a tactile pain response in mice.

The tail flick assay measures the latency to thermal pain response and effectiveness of analgesics. Thirty minutes prior to drug administration, rats are held on a pre-heated tail flick apparatus. Once a tail flick response is observed, latency (seconds) to pain response was recorded, with a 15-second time-out period.

The chronic constriction injury (CCI) model is induced by ligation to the sciatic nerve. Melior has experience in developing several versions, including partial and complete sciatic nerve ligation models. The CCI model induces a neuropathic pain response that can be detected by von Frey mechanical thresholds and Hargreaves thermal sensitivity, and can be attenuated by administering drugs such as gabapentin, a first-line analgesic for neuropathic pain.

Startle Prepulse Inhibition
Pre-pulse inhibition (PPI) is the ability to inhibit the reaction of a subject to a strong startling stimulus (pulse) with a weaker pre-stimulus (pre-pulse). This model is designed to identify pharmacological agents that disrupt sensorimotor gating in normal animals and improve it in affected animals. Neurological disorders associated with sensorimotor gating deficits include schizophrenia, post-traumatic stress disorder, obsessive compulsive disorder, Tourette’s syndrome and Alzheimer’s and Huntington’s Diseases.

Oral or intraperitoneal Glucose Tolerance Test
Type II diabetes is characterized by high blood glucose levels in the presence of normal amounts of insulin. There are many animal models of type II diabetes involve administering high levels of glucose and measuring blood glucose levels and the ability of the experimental animal to maintain glucose homeostasis over time. Several pharmacological structural classes can effectively regulate this hyperglycemic response including sulfonylureas, thiazoladinediones (PPARγ agonists; glitazones) and metformin (glucophage). These drug classes are also clinically approved for use in humans. In the current validation study, we established a mouse model of hyperglycemia by administering high levels of glucose to mice.

The Insulin Tolerance Test (ITT) is designed to determine the sensitivity of insulin receptors in tissue by measuring blood glucose levels before and after insulin administration. This is a standard test to determine the diabetic status in humans and experimental animals. This test is used to assess the efficacy of insulin-like compounds and pharmacological agents that can modify insulin responsiveness.

Food intake and the pattern of food intake over time is a useful means for determining the appetite suppressive effects of pharmacological agents. Antidepressants, specifically those that inhibit monoamine reuptake, have recently been found to be effective agents in reducing feeding behavior in mice and in the clinic.

Euglycemic/Hyperinsulinemic Clamp Study
The hyperinsulinemic/euglycemic clamp is a standard method for detecting insulin receptor sensitivity and glucose utilization in experimental animals and in the clinic. In this model, insulin is infused intravenously (IV) at a steady rate and blood glucose levels maintained by variable infusion of glucose. At steady-state, the glucose infusion rate (GIR) is equal to tissue glucose uptake. Drugs, such as the thiazolidinones (PPARγ agonists; e.g. rosiglitazone) increase GIR in animals subjected to the euglycemic clamp and this increase in glucose utilization is driven by increased insulin receptor sensitivity.

DEXA - Bone Mineral Density Scan
Obesity is a condition of pandemic proportions in the modern world. A myriad of conditions ranging from cardiovascular disease, type-2 diabetes, cancer, and even depression have been linked to obesity (Bloom SR, 2008). Research into the mechanisms and treatment of obesity is an ongoing area of interest to many in the pharmaceutical and academic communities. Animal models have proven useful in studying hte effects of compounds on diet-induced obesity (DIO) (Hildebrand AL, 2003; Levin BE, 2000).

In this study, we characterize a mouse model using a pharmacological approach at treating diet-induced obesity. At Melior, this assay serves as a platform for a number of additional acute and chronic disorders consistent with diet-induced obesity changes in humans.

Pulmonary Inflammation
Allergic asthma is a condition that is initiated by an immune response to a diverse array of allergens and is manifested by airway obstruction, hyper-reactivity, and lung inflammation. This asthmatic response can be mimicked in experimental animals by sensitizing and then exposing them to an array of antigens. Like the asthmatic response in humans, the animal model of pulmonary inflammation is characterized by an extensive inflammatory response including an influx of leukocytes including monocyte chemoattractant protein (MCP-1). This inflammatory response can be attenuated by administration of a number of known anti-inflammatory agents.

LPS Model of Systemic Inflammation
Bolus injection of lipopolysaccharide (LPS) the major component of bacterial cell wall, results in the rapid and transient rise in tumor necrosis factor (TNFα) levels in serum in mammals. This animal model was originally developed to mirror certain aspects of septic shock in humans. This model may be an effective first-line general inflammation model and may be used to determine the anti-inflammatory potential of test compounds. A variety of clinically approved anti-inflammatory compounds are extremely effective in this model including glucocorticoids, NSAIDS and cox-2 inhibitors.

Capsaicin Analgesia Assay:

• Direct injection of capsaicin to rodents induces a hyperalgesic response through activation of the vanilloid receptor. Capsaicin-mediated pain is responsive to a variety of analgesic compounds including morphine. We use the capsaicin hyperalgesic assay to evaluate efficacy of compounds for analgesic activity.

Formalin Analgesia Assay:

• The formalin chemical nociception assay is a commonly used analgesia assay that is typically included in a battery of analgesic tests. Formalin produces a painful irritation when injected into the skin producing measurable behaviors including licking, flicking, and guarding responses. Typically there are two phases: phase I which is an acute response to the formalin injection and occurs almost immediately after injection and lasts between 5 and 10 minutes, and phase II which lasts from approximately 20 to 40 minutes after injection and is caused by a central sensitization activity. Formalin-induced chemical nociception can be attenuated by classical anti-nociceptive agents including opioids and GABA enhancers.

Hot Plate Analgesia Assay:

• The thermal analgesia hot plate assay is a pharmacological test for evaluating the analgesic potential of test compounds. A number of clinically approved pharmacological agents delay the onset of sensitivity upon paw exposure to heat including opioids and GABA potentiators. Typically, the hot plate assay is included in a battery of assays evaluating the analgesic potential of test compounds.

Alopecia is a medical description of the loss of hair from the head or the body. There are many forms of alopecia with androgenic alopecia and alopecia areata being the most common. Although alopecia is not life-threatening, it can be life-altering and can produce profound psychological impact on those afflicted by the disease. There are several drugs that promote hair growth including Minoxidil. The data below show the effects of hair regrowth in our validated mouse model. After hair removal, mice were administered Minoxidil or vehicle for 25 days and hair re-growth monitored and visually scored.

Delayed Type Hypersensitivity

• Delayed-type hypersensitivity (DTH) reactions are antigen-specific cell-mediated immune responses that can invoke harmful (e.g., allergic dermatitis and autoimmunity) aspects of immune function. The immune reaction induced by the method described below is characterized by swelling at the site of challenge and by an infiltration of monocytes/macrophages and lymphocytes into the epidermis and dermis. This model for skin DTH reactions has been used widely to monitor cell-mediated immune responses in vivo. Melior has established a model of delayed type hypersensitivity using sheep red blood cells (SRBC) as the antigen. Dexamethasone, a steroid anti-inflammatory, was employed to pharmacologically validate the assay.

Acetylcholine Writhing Test:

• Irritable Bowel Syndrome (IBS) is a disorder that is expressed as abdominal cramping, gastrointestinal pain and disturbances in GI transit. These conditions are produced by colonic spasticity and irregular contractility. The cramping and pain component of IBS can be modeled by injecting acetylcholine (ACh) into the peritoneum of mice. Intraperitoneally administered ACh promotes smooth muscle contractility and can be monitored, in animals, by quantifying the writhing response.

Colonic Propulsion:

• Colonic propulsion is a method for evaluating a test compound’s ability to alter colonic motility. This model is designed to identify pharmacological agents that prolong gastrointestinal transit and is an excellent predictor of drugs that may be useful for the therapy of irritable bowel syndrome (IBS). This assay can also be modified to identify constipation reversal agents or diarrhea producing drugs.
• A glass bead is inserted into the distal colon of a mouse and the expulsion time is measured.

Gastrointestinal Transit Assay:

• The gastrointestinal (GI) transit model is a method for evaluating the ability of a test compound to alter intestinal transit and fecal output. A number of studies have shown various drugs can alter bowl function and GI motility. Melior's GI Transit Assay offers a way to monitor and quantify these changes.
• Following compound administration fecal output is monitored and quantified. This assay can be altered to measure stomach contents and GI motility of a bolus of orally dosed charcoal solution.

Morphine - Induced Constipation:

• This gastrointestinal transit model is a method for evaluating a test compound's ability to alter colonic propulsive motility. This model is designed to identify pharmacological agents that can functionally antagonize the constipative activities produced by morphine as measured by an increase in colonic propulsive activity. This model can be used to identify drugs that directly affect morphine receptors or that functionally affect morphine-induced colonic activity.

The two most commonly used models of experimental hepatic fibrosis are induced chemically (carbon tetrachloride; CCl4) or by bile duct ligation (BDL). CCl4 causes hepatocyte damage, necrosis, inflammation, and fibrosis after 4 weeks of treatment and over 8 weeks causes cirrhosis. In contrast, BDL stimulates the proliferation of biliary epithelial cells and oval cells causing proliferating bile ductules with an accompanying portal inflammation and fibrosis.

Primary hypertension is a chronic medical condition in which the arteriole blood pressure is constantly elevated with no apparent medical cause such as kidney diseases, tumors or medication side effects. Spontaneous hypertensive rats (SHR) are one of the most commonly used animal models for primary hypertension. These rats exhibit elevated blood pressure beginning at 6 weeks of age. Telemetry methods allow for the continuous and stress free measurement of blood pressure parameters including diastolic, systolic, mean arterial pressure, pulse pressure and pulse.

Thrombosis is defined as the formation or presence of a blood clot inside a blood vessel. This clotting can be deleterious and evoke events such as stroke and heart attacks. Anti-coagulants and anti-thrombotic agents are used to prevent this unwanted blood clotting and are commonly used prophylactically in prothrombotic situations. Bleeding time assays in rodents can be used to predict the anti-thrombotic/anti-coagulant potential of test agents.

Bleeding time was assessed by a tail transection method. After warming, mice tails were sliced with a razor and re-immersed into the water flask. The endpoint was calculated as bleeding cessation lasting for 30 seconds.