Advancing Translational Drug Discovery

Pain


The formalin test has both an inflammatory and neuropathic pain component and thus serves as a useful initial screening test. Intra-plantar injection of formalin produces a biphasic incidence of licking the injected area, with an early (i.e. 0-10min) phase due to direct activation of TRPA1 channels localized on sensory neurons, and a late (10-60min) phase which is believed to reflect both central sensitization and local inflammatory response to formalin-induced TRPA1 channel activation and local tissue damage.

The withdrawal latency to a radiant heat source provides a very simple measure to an acute stimulus. This test is used to evaluate treatments for acute pain.

Intradermal injection of the mast cell degranulator Compound 48/80 produces a local release of histamine and intense scratching of the injected area. Since histamine elicits itch in humans this response is believed to reflect a similar sensation in mice.

Transection of the tibial and peroneal branches of the sciatic nerve produces a long lasting neuropathy of the denervated paw, characterized by tactile and cold allodynia. These effects may be maintained for several weeks post surgery enabling long term chronic studies, and/or within subject test designs to be undertaken.

Validation Data: Effect of Tegretol® (Carbamazepine) against allodynia and hyperalgesia following SNI

Cold allodynia chart Tactile allodynia chart

SNI and sham operated rats were surgically prepared. By 10 days all SNI lesioned animals developed tactile/cold allodynia relative to sham controls. Approximately 30 days post surgery the animals were treated with Carbamazepine (CBZ) 10-30mg/kg. Acute CBZ attenuated the thermal hyperalgesia and cold allodynia, but not the tactile allodynia

The intra-plantar injection of Freund’s Complete Adjuvant (FCA) produces an acute inflammatory response that peaks at approximately 24h, and is maintained for over 30 days post injection. Edema is measured by the threshold withdrawal response to Von Frey hairs. NCEs can be tested either acutely (i.e. 1 day post FCA), or longer term (i.e. 30 days post FCA). An advantage of testing longer term FCA injection is that this may more closely mimic the deleterious effects of long term inflammatory conditions such as rheumatoid arthritis.

Validation Data: Timecourse of paw oedema,tactile allodynia and histology following intra-planter injection of Freunds adjuvant.

  • FCA scans
  • Tactile allodynia
  • FCA time course

Rats were injected intra-plantar with Freunds adjuvant (0.5-1mg/ml, 50uL volume) and paw volume measured over 30 days. At 30 day (but not 1 day), some evidence of bone erosion (*) and cartilage loss (#) was evident in some animals.

A 1cm incision through the plantar surface of the hindpaw produces a sensitization to a tactile stimulus applied 1 to 2 days post surgery. NCE’s may be tested against the withdrawal response to this tactile response either 1 or 2 days post surgery. This model is used to evaluate treatments for post operative pain.

Epilepsy


Acute seizure tests include maximal electroshock (MES), the chemical convulsant pentylenetetrazol (s.c.PTZ), and the 6Hz model of psychomotor seizure. Similar to pain, epilepsy is a heterogeneous condition which responds differently to drugs. Assessment of an NCE across the MES, PTZ and 6Hz models provides a drug signature and insight as to which seizure type may be treatable. Together these 3 acute tests identify all known AED's, and the MES and PTZ tests are currently recognized as the gold standard early phase screening tests1.

Validation Data: Comparison of phenytoin, valproate and levetiracetam against MES, s.c.PTZ and 6Hz-induced seizures in the mouse.

  • Phenytoin
  • Valproate
  • Levetiracetam

Dose response curves for each drug against each seizure test. Note each drug has a unique profile across these tests, consistent with distinct mechanism of action and clinical profile.

Repeated subconvulsive electrical stimulation of discrete CNS structures (typically amygdala, hippocampus) will lead to induction (kindling) of overt seizures. As such this may model human partial seizures with secondary generalization. Typical endpoints are (a) behavioral assessment of seizure intensity (Racine scale), and (b) electrophysiological assessment of afterdischarge EEG. NCEs may be tested either as a pretreatment to examine a block of an already established kindled seizure, or administered during kindling development to assess antiepileptogenic potential. Today the kindling model is the only chronic model widely used in most AED screening programs1.

Validation Data: Effect of levetiracetam (Keppra ®) against amygdala kindled seizures

Amygdala seizures

Bialer & White (2010) Key factors in the discovery and development of new antiepileptic drugs. Nature Rev. Drug Discovery 9: 68-82.

Obesity


Both the Zucker fa/fa rat and Diet induced obesity models provide a means to study effect of an NCE on multiple endpoints important for determining its potential as an anti-obesity therapy. A brief overview of endpoints that InterVivo can measure and how these compare between the two models is presented below.

EndpointZucker modelDIO model
Body mass~50% > vs. lean controls~30% > vs. controls
Body composition (QMR)Fat mass ~35% vs. ~7% lean controls
Lean mass ~56% vs. 80% lean controls
Fat mass ~20% vs. ~10% controls
Lean mass ~59% vs. 75% controls
Food and water intakeFood and water intake elevated vs. lean controls.Food and water intake actuall reduced vs. lean controls (based on high fat diet).
Lipid profileTG's + Cholesterol elevated vs. lean controlsTG's + Cholesterol modestly elevated vs. lean controls
Blood insulin levelsSevere hyperinsulinaemiaHyperinsulinaemia
Blood glucose levelsHyperglycemiaNormal blood glucose levels
Glucose tolerance testElevated blood [glucose] following load - 2x AUC vs. lean controls.Elevated blood [glucose] following load - 1.5x AUC vs. controls.
Behavioural profileHypoactive, allodynia to a tactile stimulus vs. lean conrolsHypoactive vs. controls.

Acute feeding models provide a means to assess the effect of an NCE on specific factors that may contribute to anorectic efficacy or unwanted side effect of an NCE. Acute feeding tests may also assist with dose finding necessary for chronic studies.

Rats are trained either to consume their daily food ration over a specific time period each day, i.e. feeding induced by deprivation, or given access to a sweetened diet in a specific location and time each day without restriction to standard food, i.e feeding induced by palatability. Enables the administration of an NCE to be precisely timed to feeding.

Validation Data: Effect of lorcaserin (Belviq) on feeding induced by palatability or deprivation

% change in food intake

Percentage change in food intake following lorcaserin (0.3-3 mg/kg SC) compared to vehicle pretreatment in rats trained to consume food over a limited access period under conditions of 22h food deprivation, or palatability-induced feeding in non-deprived rats. Data modified from: Higgins et al (2012) Neuropsychopharmacology 37: 1177-1191.

Typically rats given access to food will display a sequence of behaviours as they feed to satiation, i.e feed>active/groom>rest. Provides the means to study effect of an NCE on this behavioural sequence to measure specificity of any anorectic effect.

Validation Data: Effect of lorcaserin (Belviq) on the behavioural satiety sequence

Effect of lorcaserin

Effect of lorcaserin (1 mg/kg) on the behavioural satiety sequence measured during the palatability-induced feeding experiment. Note in the vehicle controls and lorcaserin (1 mg/kg) groups, the predominant behaviours are feeding and active/grooming over the first 15-20min, before ‘inactive’ predominates from 20-25 min onwards. At this dose, lorcaserin is therefore reducing food intake with minimal effect on the behavioural satiety sequence. Data modified from: Higgins et al (2012) Neuropsychopharmacology 37: 1177-1191.

Rats may be trained to respond for food reward under a variety of schedules to study factors such as effect of an NCE on motivation for food. Because animals are typically well trained in these tasks, baseline performance can be tightly controlled and performance becomes predictable enabling robust baselines with which to study effects of an NCE.

Validation Data: Effect of lorcaserin (Belviq) on operant responding for food under a progressive ratio schedule of reinforcement

Effect of lorcaserin pretreatment on behaviours motivated by food

Effect of lorcaserin (0.3-1 mg/kg SC) pretreatment on behaviours motivated by food made available under a progressive ratio schedule of reinforcement. In this test the response requirement for food reward is increased with each successive reward. The critical measure is the break point, i.e at which response level will the animal cease to work for food. Lorcaserin reduced the break point for food under a progressive schedule of reinforcement consistent with a reduced motivation for food. The effect of lorcaserin wasreversed by the selective 5-HT2C receptor antagonist SB-242084 (SB). Data modified from: Higgins et al (2012) Neuropsychopharmacology 37: 1177-1191.

NCEs may reduce feeding behaviour by non-specific means such as malaise. InterVivo Solutions offer a variety of techniques to study malaise in rats, including measurement of pica, certain spontaneous behaviours and conditioned behaviours such as place aversion and conditioned disgust. Nausea and vomiting are one of the primary dose limiting side effects of CNS drugs in the clinic. Certain drug classes such as PDE4 inhibitors, e.g rolipram are associated with induction of nausea in humans.

Validation Data: Assessing the aversive properties of rolipram

Effect of rolipram

Effect of rolipram (0.3 – 1 mg/kg) pretreatment on various measures which may reflect malaise in rats. Rolipram produced a dose related incidence of checked signs such as chin rubbing, flat body posture and chewing. These were aggregated to give a ‘malaise score’. In a separate experiment, equivalent doses of rolipram increased the amount of clay eaten over a 4h time period. Rolipram (1 mg/kg) also produced a significant place aversion for a distinct compartment it was paired to (unbiased procedure).

Addiction

In addition to providing a platform to study the potential for an NCE to reduce a specific aspect of a drug, known to contribute to its liability for abuse, i.e. efficacy based study, these same tests may be applied to investigate abuse potential of an NCE, i.e safety based study. InterVivo Solutions has experience in either approaches to study an NCE.


Most drugs of abuse have locomotor stimulant effects which can be readily measured in open field activity chambers. The total distance travelled within unit time will give a reliable measure of any motor stimulant/depressant effect of an NCE. Alternatively an NCE may be tested for its ability to inhibit the stimulant property of a drug of abuse.

Validation Data: Motor stimulant effects of nicotine

Motor stimulant effects of nicotine

Rats were habituated to test apparatus and over a 1h time period the total distance travelled following treatment with either saline vehicle, or nicotine (0.05-0.4 mg/kg) was measured by an automated tracking system. Nicotine produced a robust increase in total distance travelled consistent with its known stimulant property.

All drugs of abuse, as well as certain non-abused drug classes, will produce an internal state that the rat can be trained to discriminate from a neutral stimulus such as saline injection. Drug discrimination procedures typically rely on a two-lever operant procedure. NCE’s may be tested for (a) their ability to either substitute (generalise) to a known drug cue (i.e suggestive of a similar interoceptive state) or (b) their ability to antagonise a known drug cue (i.e suggestive of an ability to block the interoceptive state of a known drug). Alternatively rats may be trained with an NCE to see if it can support a discrimination opening up opportunities to identify which known drugs may generalise. InterVivo Solutions has experience in each of these areas.

Validation Data: Cross generalisation between a nicotine and diazepam cue

Nicotine cue Diazepam cue

Separate groups of rats were trained to discriminate either nicotine (0.3 mg/kg) from saline (Nicotine), or Diazepam (2 mg/kg) from saline (DZP). As expected Nicotine trained rats showed a dose related generalisation to the nicotine lever, and DZP trained rats showed a dose related generalisation to the diazepam lever. However diazepam administered to Nicotine group, and nicotine administered to DZP group produced only at best a very weak (~25%) generalisation, thus showing the specificity of each cue type.

Many drugs of abuse following chronic treatment will produce adaptive changes in the animal which may lead to tolerance, i.e higher dose of drug to produce an equivalent pharmacological response, and/or dependence, i.e cessation of drug may lead to an aversive withdrawal state, which may also be precipitated by an acute pharmacological challenge (precipitated withdrawal). Avoidance of an aversive withdrawal state is a contributory factor in continued drug taking of certain abused drugs.

Validation Data: Nicotine dependence

Nicotine exposure Nicotine W/D

Adult, male rats were implanted with minipumps primed to deliver nicotine at 6 mg/kg/day or 9 mg/kg/day. Body weight gain was reduced in the animals receiving the 9 mg/kg/day dose. At day 7 post implant, rats injected with the nicotine antagonist mecamylamine (1 mg/kg SC). Somatic withdrawal signs, e.g ptosis, tremor, muscle jerks, chewing were manifest. Mecamylamine-induced W/D signs not evident in rats implanted with saline minipumps.

Many drugs of abuse will produce a preference for a specific location to which it has been paired, i.e the drug will produce a conditioned place preference based on the appetitive properties of that drug. Alternatively drugs which may elicit an aversive state, may produce an aversion for a specific location to which it has been paired, i.e the drug will produce a conditioned place aversion. This procedure may therefore be of value in determining an appetitive vs. an aversive interoceptive state generated by an NCE. Alternatively the procedure may be used to see if an NCE may modify a conditioned place preference/aversion produced by a known drug. InterVivo Solutions has experience with each approach.

The majority of abused drugs will support their self-administration, which defines their reinforcing property. InterVivo Solutions has access to intravenous self-administration procedures through sub-contract.

Parkinson’s Disease


In the rat, the systemic injection of rotenone results in a variety of behavioural, biochemical and histopathological changes which bear parallels with changes in Parkinson’s Disease. Together these measures provide multiple endpoints to assess the effect of an NCE in this model.

Validation Data: Effect of Rotenone dose on indices of neurological function

Rotenone effect on tasks of neurological function

A 10-12 daily regimen of rotenone (2.75 – 3 mg/kg IP) produces significant effects on tasks of neurological function The most robust changes are measured using rearing in open field, postural instability and beam walking. Tremor, hypolocomotion, catalepsy are inconsistently affected by rotenone treatment.

Alzheimer’s Disease


It is estimated that by 2050, 1 in 49 Americans will have Alzheimer’s disease. Given the epidemic like projections, Alzheimer’s disease represents an important therapeutic area in drug development. There are 2 classes of drugs currently approved for the treatment of Alzheimer’s disease, but neither can reverse or halt the progression of the disease. Over the last decade, several potential disease modifying drugs have been developed, but have consistently failed in clinical trials. One of our goals is to provide animal models for the development of therapeutics that will eradicate Alzheimer’s disease.

Amyloid (A) Amyloid (B) Amyloid (C) Amyloid (D) Amyloid (E) Amyloid (F)

We provide specialized and tailored studies in both rodents and dogs that can be used to screen potential therapeutics for symptomatic effects as well as disease modifying effects. Our translational approach across species can be used to effectively bring your therapeutic from early proof of concept to a characterized clinical candidate. With over 100 years combined of academic and industry experience within the Alzheimer’s field, our team of scientists can provide you with the scientific expertise to leverage your drug development program. Our team can also provide world-class expertise in cognitive assessment, in vivo imaging and CSF sampling. The combination of these services will allow you to assess domain specific effects of therapeutics, including effects on memory, attention, and executive function, as well as biomarkers either acutely or longitudinally. We can provide you with the information you need to better understand your development product.

Cognitive

InterVivo Solutions offers a capability for assessment of NCEs in rodent and canine tests of cognition, providing an opportunity to identify efficacy for therapeutic indications associated with a cognitive disorder, and to support safety assessment of novel NCEs. Multiple tests are selected to offer a broad analysis of drug effects against distinct cognitive domains. Various means to challenge cognitive function are available, from natural (aging, task manipulation) to pharmacological. Some of these tests have direct human equivalents offering translational approaches to studying NCEs.


The Morris Water Maze is a test of spatial learning and memory. Various test designs are available although a typical design may involve an initial cued learning phase (visible platform of variable location) followed by a hidden platform test phase (hidden platform of fixed location). Probe tests (i.e., platform removed) conducted at various stages of the hidden platform test phase serve to examine accuracy of spatial learning and retention of this information. The test is readily adaptable to both the rat and mouse, and virtual technology has enabled equivalent tests to be conducted in humans.

Validation Data: Age-dependent impairment in learning and memory assessed by the water maze task.

Place learning - mouse vs. rat

This test of recognition memory is based on the tendency of rodents to preferentially explore a novel compared to a familiar object. Test subjects are exposed to a sample stimulus (object), and after a delay of minutes to hours, are presented with the sample object together with a novel object. Memory of the familiar object is gauged by a time difference score spent exploring each object.

Validation Data: Age-dependent impairment in learning and memory assessed by the novel object recognition task.

Novel Object Recognition bar chart

Aged rats show a decline in novel object recognition at 3h post sample stage, unlike young controls. Both young and aged rats show decline at 24h post sample stage.

The 5-choice serial reaction time task (5-CSRTT) has become a widely used test to measure attentional perfermance in rodents, and having evolved from the continuous performance test in humans, the test has translational value. The basic test design involves training animals to respond to a brief visual stimulus presented unpredictably in one of five locations. One of the strengths of the 5-CSRTT is the ability to manipulate various test conditions to tax animals and provides a means of detecting bidirectional effects on performance within the same experiment.

Validation Data: Effect of nicotine (0.4mg/kg s.c.) against a vigilance decrement in aged rats

Aged rats (~20mos.) were trained to stable performance levels in the 5-CSRTT, and subjected to occasional tests of extended trials (250 trials per session, 0.5s SD, 5s ITI). Acute nicotine pretreatment (0.4mg/kg s.c.) prevented the within-session decline in accuracy and response speed seen in vehicle treated animals. Cumulative data are shown on the histobars. (Adapted from: Grottick and Higgins, 2002).

5-choice serial task figure

The 5-choice serial reaction time task (5-CSRTT) has become a widely used test to measure attentional perfermance in rodents, and having evolved from the continuous performance test in humans, the test has translational value. The basic test design involves training animals to respond to a brief visual stimulus presented unpredictably in one of five locations. One of the strengths of the 5-CSRTT is the ability to manipulate various test conditions to tax animals and provides a means of detecting bidirectional effects on performance within the same experiment.

Validation Data: Effect of donepezil (0.3-3mg/kg) on recognition memory assessed using the novel object recognition task. Relationship of dose response to cholinomimetic signs.

  • Motor activity bar chart
  • Novel object recognition bar chart
  • Cholinergic signs bar chart

Donepezil (Aricept®) (0.3-1mg/kg) produced a clear tendency to improve exploration of a novel object relative to a familiar object in a 24h retention test. No such effect was evident at a higher dose of 3mg/kg. In parallel studies, while donepezil doses of 0.3-1mg/kg had minimal effects on locomotor activity and signs reflective of cholinomimetic activity (secretory signs, tremor, hypothermia), the 3mg/kg dose of donepezil produced hypolocomotion and a clear incidence of cholinomimetic signs in 7/8 animals 0-2h post dose, which likely contributed to the decline in novel object recognition test performance.

Operant tests provide an opportunity to challenge rodents in very specific ways, including, but not limited to, tests to examine specific cognitive domains. Furthermore because animals are typically well trained in these tasks, baseline performance can be tightly controlled and performance becomes predictable. Thus, effects of NCEs can be evaluated against a robust baseline. InterVivo has considerable experience in multiple operant procedures including those to examine motivation (e.g. progressive ratio), behavioral flexibility (e.g., reversal learning) and response inhibition (e.g., Differential Low Rates of Reinforcement (DRL), Go-NoGo). Through the use of a flexible system and relevant proven research experience, InterVivo has the capability to conduct multiple schedules tailored to suit client needs.

Validation Data: Effect of dizocilpine (0.03-0.06 mg/kg s.c.) on two tests of response control in rats

Operant platform figures

Separate groups of adult rats were trained to stable performance levels in the DRL24s task and a signaled Go-NoGo successive discrimination task. In the DRL24s task, vehicle treated rats had peak responding around 24s consistent with accurate temporal discrimination. Dizocilpine (0.03mg/kg s.c.) produced a leftward shift in this response curve. In the Go-NoGo task, in vehicle treated animals, >90% responding was made during the S+ (i.e., Go) phase. Dizocilpine (0.03-0.06mg/kg s.c.) increased responding during the S- (i.e. NoGo) phase, such that only ~75% responding was during the S+ phase. Both tests highlight deficiencies in response control of rats following dizocilpine treatment.

InterVivo provides two tests of increasing difficulty that target this cognitive domain; the delayed non-matching to position test (DNMP) and the serial list learning task (SLL). Various test designs are available although a typical design may involve testing acute effects of an NCE on a variable-delay paradigm that allows assessment of rapid memory enhancing or impairing effects, or on long-term prevention on the progressive decline seen with age. The tests are readily adaptable to man and both MCI and Alzheimer’s patients show impairment on variations of these tasks compared to normal controls. These tasks are particularly sensitive to drugs targeting the cholinergic system, including the cholinesterase inhibitors.

To examine this cognitive domain, InterVivo uses a variable-object discrimination paradigm that reveals deficits related to both aging and difficulty. Impairment of selective attention occurs early in pathological aging and is impaired in various neuropsychiatric disorders such as schizophrenia. In this task, a dog is trained to respond to one object of two. Once learned, increasing the number of distracters (incorrect objects) impairs performance and increases response latency. This is augmented with aging, which may reflect impaired inhibitory control and processing speed, respectively, and by increasing task difficulty. This test has been effective in identifying therapeutics that enhance metabolic and mitochondrial function.

Discrimination and reversal learning are tests of simple learning and executive function, respectively, and the latter is severely impaired with age in multiple species and in Alzheimer’s patients. Fine analysis of reversal data can indicate whether NCEs affect perseverance (inhibitory control) or general learning, and this test has been effective in optimizing dose selection.

InterVivo encourages and provides tests to assess the effects of NCEs across several cognitive domains. This approach can be useful for dose optimization and to better understand the broader or selective effects of cognitive-modifying NCEs. Additional cognitive tests that have been validated in the dog include, but are not limited to:

  • Allocentric spatial learning and performance – the landmark task;
  • Egocentric learning and reversal – the egocentric task;
  • Complex learning – oddity discrimination learning; and
  • Psychomotor function – paw reaching task.

PK & Safety


A Functional observation test battery for either the rat or mouse adapted from the Irwin (1968) test screen, and incorporating additional objective measures of motor and autonomic function.

Animals can be chronically treated with an NCE under staggered designs such that 2nd/3rd level dosing can be determined by the effect at first dose. Daily observation, blood chemistry and cell counts are included. Additional options include: major organ histopathology; plasma sampling for toxicokinetic profiling; CSF sampling (in the dog); and incorporation of a functional CNS test battery into the study design. Our emphasis is on flexible designs to maximize the value of information to the customer to guide subsequent efficacy and GLP toxicology studies.

Frequently, drugs are administered in combination with another therapy. We offer the capability to test for potentially deleterious drug-drug interactions, both at the behavioural and pharmacokinetics level.

Animals can be treated with an NCE by either iv, sc, ip or oral routes of administration and plasma samples taken at multiple timepoints. Additional tissue samples, e.g. csf (rat) can be taken to determine CNS penetration of NCE. These can also be coordinated with efficacy studies to support PK-PD determinations.

Unique adverse effects may be suspected based on the pharmacological property or observations made during the drug characterization process. Additional concerns may arise during clinical trials. In such cases, follow-up studies are recommended (ICH S7A). We can provide the following supplemental services:

  1. Tests for dependence and abuse liability;
  2. Tests for cognitive liability; and
  3. Tests for pro-convulsant liability.

CNS Drug Disposition


  • Simultaneous collection of brain tissue and plasma from the same animal followed by LC-MS/MS bioanalysis of compound concentrations
  • Brain and plasma free-fraction determination using equilibrium dialysis
  • Useful for early-stage screening and lead selection programs, and can be combined with efficacy or POC studies
  • Non-survival sampling of cerebrospinal fluid from the cisterna magna of mice can be used to estimate free drug concentrations in the brain following drug administration 1
  • Can be combined with brain tissue harvesting and plasma collection to understand differences in drug exposure for CSF, brain and plasma
  • Also useful as an addition to efficacy or POC studies
1 1. Fan, J. et. al. Drug Metabolism Reviews.44(S1):138-139 (2012)
  • Collecting serial samples of CSF and plasma via surgically implanted catheters allows the determination of unbound drug delivery into the brain in awake rats
  • Reduced inter-animal variability improves quality and power of data over parallel sampling techniques
  • Studies can be performed in SAGE® ADME knockout rats to investigate how specific drug transporters affect the pharmacokinetics of drugs
  • Data rich study designs have demonstrated predictive validity in characterizing drug delivery into the brain 1
1 1. Fan, J. et. al. Drug Metabolism Reviews.44(S1):138-139 (2012)
  • ~150 beagle dogs of various ages are available for CSF and plasma sampling studies to evaluate drug delivery and exposure in a clinically relevant non-rodent species
  • Efficacy and safety profiles of new drugs can be further characterized by assessing drug or biomarker levels in CSF and plasma from the same animal over time
  • Aged dogs provide a natural and translational model for various age related conditions such as Alzheimer’s Disease progression, Osteoarthritis and Cancer
  • Industry accepted gold-standard model for determining unbound drug concentrations in specific brain regions over time
  • Combined drug and biomarker assessment enables PK-PD studies to establish robust dose-response profiles
  • In vitro and in vivo parameters are optimized for each compound to ensure accuracy and reliability of results which can be used to support development decisions.
Quinidine graph (n=4)
Figure 1. Mean + S.D. (n=4) unbound plasma and brain ECF concentrations of quinidine in Sprague-Dawley rats over time following i.v. administration of a loading dose and constant rate infusion of quinidine.
Quinidine graph (n=5)
Figure 2. Mean + S.D. (n=5) unbound plasma and CSF concentrations of quinidine in Sprague-Dawley rats over time following i.v. administration of a loading dose and constant rate infusion of quinidine.

Pathology


Canine amyloid-Aβ (Aβ) protein is identical to the human protein and the pattern and quantity of isoforms is analogous to that seen in humans.

Endogenous Aβ is naturally deposited into plaques of the diffuse subtype in aged dogs, which are vulnerable to post translational modification and are fibrillar at the ultrastructural level – cerebral amyloid angiopathy is also found in aged dogs. The pattern of Aβ deposition parallels that seen in humans and occurs over a 3 to 4 year window permitting the examination of interventions that may slow or halt deposition.

Cognitive deficits are positively correlated with Aβ deposition in Beagles. Oligomeric forms of Aβ, may correlate with cognitive decline occurring prior to Aβ deposition.

Drug Screening


A β-amyloid cleaving enzyme (BACE) inhibitor acutely reduces CSF and plasma levels of Aβ in a dose dependent manner.

The administration of a vaccine targeting fibrillar Aβ reduced Aβ deposition, but did not significantly modify longitudinal cognitive measures.

The effects of putative AD therapeutics on CSF biomarkers in aged dogs provides a rapid screen in a higher mammalian model that naturally develops AD-like pathology. We have experience in conducting relatively rapid screens for studying the effect of multiple NCEs on CSF biomarkers to support the lead optimization process which can be followed up with more extensive studies on selected compounds.

Imaging


  • MRI or CT to demonstrate structural changes in response to interventions
  • T1, T2. PD sequences
  • Diffusion Tensor Imaging to evaluate changes in myelination
  • Quantitative and qualitative assessment of morphological features or regions of interest by image processing
T1 weighted

Bar chart of diffusion tensor imaging data showing increasing fractional anisotropy
Diffusion Tensor Imaging data showing increasing fractional anisotropy in the frontal lobe, implying increasing myelination with increasing age in young beagle dogs.
Graph showing landmark allocentric learning improves with increased myelination in one year old beagle dogs
Data indicating performance on the landmark allocentric learning improves with increased myelination in one year old beagle dogs.
  • Dynamic contrast perfusion to assess blood brain barrier permeability and regional cerebral blood flow using either MRI or CT
  • PET/CT imaging with additional coregistration to MRI images if required to quantify glucose uptake
  • Imaging of brain tissues using standard PET radiopharmaceuticals eg F-18 DOPA
  • Imaging of brain tissues with custom developed radiopharmaceuticals linked to specific naturally occurring brain chemicals or novel compounds
F-18 DOPA

Dynamic perfusion CT (A)
Dynamic perfusion CT (B)

Dynamic perfusion CT images of a dog brain with cerebral blood flow (CBF) maps demonstrating an increase in CBF when end tidal CO2 is increased from 35mmHg (A) to 68mm Hg(B).

  • Chemical shift graph
    Quantification of changes in brain chemistry associated with the development and decline of neural function in response to therapeutics
  • MRS concentration bar chart
    Magnetic resonance spectroscopy data showing changes in brain metabolites associated with brain development in growing beagles, including markers of neuronal health. Effects of therapeutics can be assessed in attenuating or improving brain development
  • Percent change chart across metabolites
    Data showing change in concentration of brain metabolites in 10 year old compared to 2 year old beagle dogs

Quantitative magnetic resonance (QMR) is a non invasive method of determining body composition in awake animals. The principles of the method are similar to magnetic resonance imaging (MRI), except that the output is numerical rather than an image. Non sedated rodents are restrained in a Plexiglas tube that is placed within the bore of a low field magnet. Data collection takes two minutes, allowing a high throughput and repeated measurement for longitudinal study designs. During the data collection, hydrogen atoms in fat and lean tissue respond differently to radiofrequency energy, allowing the determination of the subject’s fat, lean and total body water mass.

Fat: Zucker vs. Lean control Rodents

Whole body imaging protocols are used for assessing the effects of therapeutics on metabolism and feeding behavior. Using dual energy x-ray absorptiometry (DEXA) body composition can be determined non invasively in longitudinal studies. These studies give body composition in terms of the percentage of fat, lean and bone tissue. Body composition is an important component in obesity studies.

DEXA image of a dog and MR images

From left to right: DEXA image of a dog; MR Image of rat abdomen; MR image segmented into visceral (yellow) and subcutaneous (blue) fat.

Several imaging protocols can be used to quantify the efficacy of new therapeutics. These can be conventional anatomic measures of tumor size using MRI, ultrasound or radiographs. Other functional measures that can be used on a quantitative basis pre and post treatment as a measure of the therapeutic effect of a new agent include:

  • FDG PET imaging of glucose metabolism in tumors. Reduced glucose metabolism is a measure of therapeutic success.
  • Dynamic contrast enhanced MRI to quantify effect of agents on angiogenesis. Reduced angiogenesis is a measure of therapeutic success

Depending on the research question to be answered several different imaging modalities can be used in musculoskeletal imaging. Magnetic Resonance Imaging provides images of the greatest range of tissues including bone marrow and changes in the internal structure of a joint, including articular cartilage changes, whereas CT can provide detailed images of bone structure.

Our range of magnets available allows MR imaging of beagles at up to 9.4T. Plain radiographs can illustrate basic morphometric changes with either contrast radiography or ultrasound adding details of the internal structure of joints. Nuclear medicine studies provide quantitative information on bone metabolic turnover and studies can be focused on a specific area or can include the entire skeletal system.

Dog knee radiograph

From left to right: Radiograph of a dog knee with osteoarthritis; MR image of a normal dog knee.

Our comprehensive imaging capabilities allow evaluation of any organ or body system. In addition to the sophisticated cross sectional imaging modalities we have extensive experience in plain and contrast radiography and ultrasound, including endocavitory, evaluation. These modalities continue to have applications in the development of new therapeutic applications and we can help you to select the most appropriate and cost effective imaging method for your study. Morphometric measures can be made from the images and some measures of echogencitiy can be made from ultrasound images.

Dog chest radiograph

From left to right: Dog chest radiograph; image of dog prostate with benign prostatic hypertrophy; dog kidney with chronic infection.

Interventional imaging provides minimally invasive methods of collecting tissue or depositing cells in virtually any organ system. We have extensive experience in interventional procedures with all imaging modalities. The complexity can range from simple collection of fluid or cells to implantation of cells within a fetus to create chimera or place a device within an organ or vessel using an endovascular approach. Biopsies can be collected from both bone and soft tissues. We can also develop instrumentation and methodology for unique procedures.

Ultrasound guided biopsy collection; catheter delivery of cells into the renal artery in a dog; 25 day canine fetus yolk sac injection.

From left to right: Dog chest radiograph; image of dog prostate with benign prostatic hypertrophy; dog kidney with chronic infection.

Segmented Brain
Dog brain segmented into grey matter, white matter and cerebrospinal fluid

InterVivo Solutions has experience in image processing of all modalities. Image processing extracts quantitative data from images. This can range from basic morphometric measurements made from images such as bone length or hippocampal volume to complex segmentation based on image signal intensities.

Project specific image processing routines are developed using scripts in standard software packages or can be custom developed according to the needs of the project. Interpretation of the data is routinely provided.

We are able to co-register images to enable low resolution quantitative images to be co-located with high resolution anatomical mages to allow accurate regions of interest to be drawn of the low resolution images. Brain images can be segmented into grey matter, white matter and cerebrospinal fluid. Regional blood flow and blood volume can be determined using either an MRI or a CT acquisition. Time activity curves can be generated from nuclear medicine studies to determine first pass kinetics.