ACUTE AND INFLAMMATORY PAIN
In terms of ease of execution and reasonable throughput, which inflammatory and neuropathic pain models would you recommend starting with (assuming acute pain models have already been studied)?
A study using the carrageenan model can be run in a single day. However, chronicity is debatable as it lasts 8–12 hours. The 24-hour CFA model is another quick option. For neuropathic pain models, there are differences in chronicity. For example, the partial sciatic nerve ligation lasts for 2−3 weeks while the SNL model and SNI model last 8–10 weeks after surgery. Therefore, the MOA of a drug candidate should be considered—inflammatory vs. neuropathic.
How sensitive is the formalin test? How many stages of pain response can be distinguished?
When measuring sensitivity as intensity of response, the formalin concentration determines response level to a small extent. 1%, 3% or 5% formalin can be used— 5% formalin induces a very intense response in phase 1. Pain responses are in two phases with an interphase with no or very low pain response.
Can topical drugs (e.g., creams) be tested in this model? Can topical drugs be tested on the tail flick model?
Capsaicin studies are possible in inflammatory models such as CFA, but the topical drug usually has to be in a cream or lotion form, not a solution. For acute pain, the cream or lotion form should work in the tail flick test, but this has not been currently tested at Charles River.
In the dynamic weight bearing study format, how many animals were used?
8–10 animals per data set are recommended.
ION CHANNELS ASSOCIATED WITH PAIN
How many positive and negative controls should be used per plate to achieve good Z values?
We recommend using 16 wells with DMSO controls and 16 wells as a positive control for full inhibition. These are recommended controls for FLIPR®, IonWorks® and Qube™ systems.
What are some differences between fluorescence and radiometric outputs?
With the radiometric technique, a well-validated ligand needs to be used for displacement to be measured as the technique is quite sensitive. Fluorescent techniques are easier to develop, but there is a risk of false positives with compounds that interact with the fluorescent response. It is recommended to include an automated electrophysiology assay after the fluorescence-based screen to quickly identify false positives.
Do you have plans to create a Nav1.9 stable cell line?
Yes, that cell line development is in progress, but it is worthwhile to note that this channel is quite difficult to express stably.
Are post-translational modifications always required for ion channels to be functional in your cell lines?
Yes, ion channels need to be appropriately post-translationally modified to be functional so that current flow can be recorded.
Have you worked with iPSC-derived neurons?
Yes, we have a stem cell group that creates iPS-derived neurons.
What are the advantages and disadvantages of automated electrophysiology compared to fluorescent techniques?
Fluorescent techniques are easier to configure and faster than electrophysiology, and typically you can screen a hundred 384-well plates a day. Automated electrophysiology is comparatively slower—a typical assay takes 30–45 minutes to screen one plate, so 10–20 plates can be screened in a day. The advantage of automated electrophysiology is the generation of data to support a deeper understanding of how a compound interacts with a target. Depending on the target, we may recommend first performing a fluorescent screen followed by automated electrophysiology.
TRPC6 has been found to be involved in a genetic form of FSGS kidney disease where mutations increase influx of calcium. Have you done any research on TRPC6 or plan to do so?
We have a TRPC6 cell line and have done some work for specific clients using this cell line.
What's your electrophysiological data analysis tool for screening?
For certain automated electrophysiology platforms, we use the software that is supplied with the instrument (Sophion Qube™ or QPatch). For fluorescent assays, the output is analyzed using GeneData.
What are the typical levels of ion channel expression in your cell lines, in copies per cell?
The typical number of copies per cell is 10,000–30,000 copies.
Is there a preference for a specific method for specific models? For example, is von Frey better for nerve constriction or chemo-induced pain?
Some models show robust allodynia for mechanical stimulation, but may lack thermal allodynia. The actual model may have different features that determine allodynia response, so response to specific methods can be model specific. However, most models do not recapitulate all aspects of thermal and mechanical allodynia.
How long does pain from oxaliplatin persist after drug delivery is discontinued? Do any of the symptoms remain measurable for weeks/months after the treatment has ended?
When we evaluate the efficacy of a compound, there are periods of active dosing and testing to determine if thermal allodynia has been alleviated. The time period depends on the therapeutic molecule that is being tested. It can take a couple of hours to up to one week to return to normal. Oxaliplatin is mainly applied to symptomatic treatments, but in chronic conditions, the treatment can provide long-term alleviation of the allodynia.
Regarding diabetic models, the STZ-induced type 1 model has shown neuropathic manifestations. Do type 2 models, such as high-fat diet and genetic models, show similar manifestations?
The elevated blood and tissue glucose levels in poorly managed diabetes for both type 1 and type 2 diabetes cause peripheral nerve damage, so we can expect the neuropathic manifestations to be present in both type 1 and type 2 diabetes when glucose levels are high.
Can neuropathic pain models be used to test compounds intranasally?
Intranasal delivery is used at Charles River for various models. Molecules that have good penetrance through the nasal epithelia and show good exposure to the CNS can be tested intranasally. The nature and size of the molecule will help determine if a molecule can be tested intranasally.
How many available neuropathic pain drugs have been tested using kinematic analysis?
We have tested pregabalin, gabapentin and amitriptyline in our labs and are also working on validating the oxaliplatin mouse model using kinematic analysis.
Are there any restrictions on testing compounds for neuropathic pain using kinematic analysis?
Some molecules that cause high sedation or coordination issues can compromise sensitive kinematics data, so it may be necessary to use a less sensitive method to test compounds that cause large changes in motor coordination.
Can kinematic analysis be performed on rats?
Yes, kinematics analysis can be performed on rats and mice, so there is no species dependence. We have predominantly tested mice.