The Renovo Remyelination Advantage

 

Why Renovo?

Renovo is a designer CRO that brings state-or-the art technologies to investigate therapeutic enhancement of remyelinating therapies in rodent models of multiple sclerosis. We have over 5 years of experience in offering the rodent model of cuprizone induced demyelination to Big Pharma, Biotech and academic institutions. This experience provides a valuable data base of outcome measures which includes thousands of mice at each time point. We have an accurate indication of the baseline for demyelination and the rate of spontaneous remyelination. We have documented that endogenous remyelination can be enhanced by therapeutic approaches that are documented by robust primary outcomes. Several aspects of our protocol distinguish our model from others. During cuprizone-mediated demyelination, we inhibit spontaneous remyelination with daily injections of rapamycin. This provides a baseline of near total demyelination at the beginning of therapeutic treatment. Without rapamycin treatment it is impossible to detect a therapeutic effect. The following outline will provide additional insight to the unique nature of the Renovo Augmented Cuprizone Model and why it is superior to conventional cuprizone-only studies.

While cuprizone has been used in demyelination studies for some time, the traditional acute cuprizone study model suffers from multiple shortcomings which limit the value of this model in evaluating potential therapies for Multiple Sclerosis. Renovo Neural, Inc. has optimized an augmented cuprizone model to provide a robust and repeatable means to reliably evaluate the remyelination potential of a therapeutic compound. This method, developed from the pioneering work of Bruce Trapp and Wendy Macklin (see link), allows researchers to have greater confidence in the validity of their study results. Renovo Neural has conducted this specialized model for over 5 years and has accumulated data from thousands of study animals.
Our expertise in this field is unparalleled, and we have, and continue to conduct studies for clients in major multinational pharmaceutical companies as well as mid-size and small biotech and virtual firms.

 

The history of our technology…

Cuprizone is a widely used toxin for demyelination of neuronal tissue. This toxin strips the protective myelin sheath around the CNS neurons via destruction of oligodendrocytes. However, the conventional cuprizone study design suffers from significant shortcomings. Renovo has addressed these shortcomings via our unique design, providing the following benefits:

  • The Renovo Augmented Cuprizone Model provides a demyelination process WITHOUT the spontaneous remyelination shortcomings of conventional cuprizone,
  • The Renovo Augmented Cuprizone Model provides more uniform and more robust demyelination WITHOUT the rapid autonomous remyelination which follows cuprizone in conventional studies, allowing a more robust assessment of therapeutic effect,
  • Renovo’s Augmented Cuprizone Model provides reliable insight into remyelination of BOTH white matter and gray matter, versus other cuprizone models which focus only on white matter.

As a result, the Renovo model generates results that offer greater confidence regarding the ability of the Sponsor’s compound to promote remyelination.

 

Why rapamycin?

The Renovo Augmented Cuprizone model incorporates co-administration of rapamycin, a compound known to inhibit spontaneous remyelination, along with the cuprizone-laced chow. As a result, the Augmented Cuprizone model leads to near complete demyelination of axonal tissues in the regions of interest. This creates a cleaner and more reliable basis for measurement of remyelination when compared to conventional cuprizone models (See: Figure A and B).Consequently, results from these studies utilizing the Augmented Cuprizone model are more translatable, and can be more confidently attributed to the therapeutic effects of the test compound. This can lead to a significant savings in time, money, and overall resources.

In contrast, conventional cuprizone studies use cuprizone alone to promote demyelination. However, because cuprizone does not kill the oligodendrocyte precursor cells, a significant amount of spontaneous remyelin ation occurs, leading to confounding results. Rapamycin does not have an effect on the remyelinating phase of our protocol because it is not administered during the remyelinating phase. It is rapidly cleared from the mice and has no lingering effect on oligodendrogenesis and remyelination.

 

 

 

Why 12-weeks of demyelination?

The Renovo Augmented Cuprizone model uses a chronic demyelination period with a much slower innate remyelination when compared to conventional demyelination models. This allows a wider window to evaluate remyelination from the therapeutic effect of the test compound. (See: Figure C). Renovo has pioneered this technique and has found a 12 week demyelination period to be optimal.

By comparison, conventional cuprizone studies may incorporate a demyelination period as short as 6 weeks. Animals subjected to this short-term demyelination paradigm tend to display rapid ‘rebound’ remyelination regardless of treatment, leading to a very narrow window for therapeutic evaluation. The rate of spontaneous remyelination following 6 weeks of cuprizone is rapid and difficult, if not impossible, to enhance by therapeutics.

 

 

 

Why gray matter?

The Renovo Augmented Cuprizone model uses histological endpoints which have been optimized for this assay. We use a robust hybridoma-derived PLP stain for examination of myelin in and cortex and hippocampus (See: Figures D and E, respectively). Quantification of remyelination using these specific techniques leads to more predictable translation of these results into the clinic.

Because of the aforementioned limitations, remyelination effects in gray matter cannot be accurately measured in conventional cuprizone models. Because of the impact of MS on myelin in gray matter is becoming increasingly relevant, this is a serious shortcoming of traditional cuprizone studies. The uniform demyelination characteristic of our model is particularly quantifiable in the gray matter of both the hippocampus and cortex.

Conventional cuprizone studies focus only on white matter, not gray matter. Clearly, white matter is important, and Renovo uses superior staining and quantitation techniques for white matter as well. Toward this, Renovo uses a PPD (p-Phenylenediamine) stain to provide a specific, clear, definitive, and quantifiable staining of myelinated axons, as opposed to general stains such as Luxol Fast Blue. Because PPD staining in the corpus callosum is specific to myelin surrounding axons in white matter, it is the most directly applicable means to evaluate remyelination in corpus callosum. Only the Renovo Augmented Cuprizone Model provides consistent and reliable assessment of both white matter and gray matter.

 

Superior Histology in White Matter as well:

The most popular target is the corpus callosum. This a relatively large white matter structure in the mouse, but it is populated by small diameter axons. Therefore, it is impossible to distinguish individual myelinated and remyelinated fibers by routine histological stains (LFB, black gold). To overcome this obstacle, we quantify myelinated and remyelinated fibers in micron-thick Epon sections. We quantify the number of myelinated and remyelinated axons and do not rely on staining intensities that are inaccurate and nonlinear. By comparison, Luxol Fast Blue will indicate myelin content, but does not discriminate between myelinated axons or other myelin which may simply be debris resulting from demyelination.  Only Renovo provides such differentiation via superior white-matter histology (see more about PPD staining here).