1. Stem Cells and Regenerative Medicine

Comment on 'Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity'

  1. Maurizio Pacifici  Is a corresponding author
  2. Eileen M Shore  Is a corresponding author
  1. The Children's Hospital of Philadelphia, United States
  2. University of Pennsylvania, United States
https://doi.org/10.7554/eLife.43173
Share this article
Cite this article
  1. Maurizio Pacifici
  2. Eileen M Shore
(2019)
Comment on 'Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity'
eLife 8:e43173.
https://doi.org/10.7554/eLife.43173
  2. Download BibTeX
  3. Download .RIS

Abstract

We are writing to communicate our concerns regarding the recently published study by Lees-Shepard et al. (2018).

https://doi.org/10.7554/eLife.43173.001

Introduction

Lees-Shepard et al. reported that palovarotene (a retinoid agonist) had severe side effects on growth plate and limb joints in an Acvr1R206H knock-in mouse model of fibrodysplasia ossificans progressiva (FOP; Lees-Shepard et al., 2018). We have a number of concerns about this publication. Lees-Shepard et al. write that “it is important for FOP therapeutics to exhibit an acceptable safety profile in juvenile patients”: however, they used a retinoid drug delivery method that is never used in the treatment of patients, thus undermining their goal. Moreover, the mouse model used does not sufficiently mimic FOP patients.

Discussion

Route of palovarotene administration

Palovarotene was originally designed for oral administration (Hind and Stinchcombe, 2009), allowing for low and steady drug adsorption by the digestive system and an overall absorption efficiency of 50% to 65% of administered dose, typical of retinoids (Blaner and Olson, 1994). Instead, Lees-Shepard et al. injected the drug intraperitoneally (IP) every day for 4 weeks. This IP route of administration is known to invariably cause a sharp and sudden increase in circulating drug levels, resulting in a higher and faster Cmax compared to oral delivery (Eaton and Gilbert, 2013). Indeed, palovarotene Cmax levels were found to be about 6-fold higher after IP injection compared to oral administration in mice (Clementia Pharmaceuticals, internal data). Circulating retinoids are quickly taken up by tissues and cells (Cullum and Zile, 1985), and cells accumulate retinoids in large amounts intracellularly (Kurlandsky et al., 1995; Randolph and Simon, 1997) where their action on cell function and phenotype can last hours to days (Chen and Gudas, 1996). Thus, daily IP injections are expected to result in ever increasing and possibly confounding drug effects over time. Lees-Shepard et al. stated that they used “palovarotene doses….that correspond to approximate adult human equivalent doses of 3.6 mg and 7.2 mg” under testing in ongoing efficacy and safety study of palovarotene for the treatment of FOP being conducted by Clementia Pharmaceuticals (https://clinicaltrials.gov/ct2/show/NCT03312634. This trial has recently entered phase 3). However, the IP administration changed the effective dose and undermined the goal of testing a clinically relevant treatment.

FOP mouse model

Lees-Shepard et al. utilized a conditional mouse model in which the FOP causative gene was activated in Pdgfrα-expressing cells by mating floxed Acvr1R206H mice with Pdgfrα-Cre mice. Those cells were described as a subpopulation of progenitor cells particularly sensitive to effects of the mutation. This mouse model is at sharp variance with FOP patients in which the mutant ACVR1R206H gene characterizes the majority of, if not all, cells. Thus, data obtained with the Pdgfrα-Cre mouse model may be skewed by targeting a specific subpopulation of cells that could also be affected by wild-type cells, with the wild-type cells likely outnumbering the cells in the specific Acvr1R206H subpopulation and interacting with them. Thus, the model does not faithfully reproduce the overall genetic makeup and cell and tissue responses in FOP patients. While Lees-Shepard et al. observed that Pdgfrα+ cells contributed to heterotopic ossification (HO) in their model, it is not known whether this specific cell population is clinically relevant in FOP patients. Further, Lees-Shepard et al. presented no data showing that targeted Pdgfrα+ cells were present in the growing skeleton.

Effects on growth plates and joints

These latter issues are particularly relevant to the severe effects on growth plates and joints reported by Lees-Shepard et al. Given that the Pdgfrα gene is not known to be expressed in chondrocytes (Hamilton et al., 2003), it is nearly certain that growth plate and articular chondrocytes were not targeted by Pdgfrα-Cre and remained largely, if not totally, wild-type. Lees-Shepard et al. included no data to demonstrate that Pdgfrα-Cre had targeted growth plate and joint chondrocytes. Studies dating back decades have established that cartilage is sensitive to systemic retinoid levels (Wolbach and Hegsted, 1953). In our studies (Chakkalakal et al., 2016; Shimono et al., 2011), we did observe a slight growth retardation of about 10% in control wild type mice after oral palovarotene administration, although their growth plates remained open and there were no obvious effects on their joints. Notably, there were no obvious growth retardation and growth plate effects after oral palovarotene administration in our Prrx1-Cre; Acvr1R206H mutant mice in which the entire limb mesenchymal cell population was targeted, including skeletal cells. The higher tolerance of mutant growth plates was likely due to higher basal levels of canonical BMP signaling activity. As we pointed out previously (Chakkalakal et al., 2016), this provides support to the notion that palovarotene may be quite safe for – and well tolerated by – FOP patients, as supported by the announcement of Phase 2 Part B data from the ongoing efficacy and safety study being conducted by Clementia (Clementia Pharmaceuticals, 2018).

Conclusions

In sum, the severe side effects on growth plates and joints observed by Lees-Shepard et al. are likely the results of: (i) the route of drug administration; (ii) excessive palovarotene levels and effects likely caused by the daily IP administration; and (iii) reliance on a mouse model that does not mimic FOP patients, including apparent lack of mutation expression in cartilage. We strongly recommend that data and conclusions in that study should be considered and interpreted with caution with regard to drug effects and action in skeletal tissues and for relevance to ongoing clinical studies.

References

  1. Book
    1. Blaner WS
    2. Olson JA
    (1994)
    Retinol and retinoic acid metabolism
    In: Sporn MB, Roberts AB, Goodman DS, editors. The Retinoids: Biology, Chemistry, and Medicine (Second Edition). New York: Raven Press. pp. 229–255.
    1. Cullum ME
    2. Zile MH
    (1985)
    Metabolism of all-trans-retinoic acid and all-trans-retinyl acetate. Demonstration of common physiological metabolites in rat small intestinal mucosa and circulation
    The Journal of Biological Chemistry 260:10590–10596.
    1. Eaton DL
    2. Gilbert SG
    (2013)
    Casarett and Doull's Toxicology: The Basic Science of Poisons
    Principles of toxicology, Casarett and Doull's Toxicology: The Basic Science of Poisons, Eighth Edition, London, McGraw-Hill.
    1. Hind M
    2. Stinchcombe S
    (2009)
    Palovarotene, a novel retinoic acid receptor gamma agonist for the treatment of emphysema
    Current Opinion in Investigational Drugs 10:1243–1250.
    1. Randolph RK
    2. Simon M
    (1997)
    Metabolism of all-trans-retinoic acid by cultured human epidermal keratinocytes
    Journal of Lipid Research 38:1374–1383.
    1. Wolbach SB
    2. Hegsted DM
    (1953)
    Hypervitaminosis A in young ducks: The epiphyseal cartilage
    Archives of Pathology 55:47–54.

Article and author information

Author details

  1. Maurizio Pacifici

    Translational Research Program in Pediatric Orthopaedics, The Children's Hospital of Philadelphia, Philadelphia, United States
    Contribution
    Conceptualization, Formal analysis
    For correspondence
    pacificim@email.chop.edu
    Competing interests
    MP is a consultant for Clementia Pharmaceuticals
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6854-4942

  2. Eileen M Shore

    Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, United States
    Contribution
    Conceptualization, Formal analysis
    For correspondence
    shore@pennmedicine.upenn.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2609-6971

Copyright

© 2019, Pacifici and Shore

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,083
    views
  • 74
    downloads
  • 2
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Maurizio Pacifici
  2. Eileen M Shore
(2019)
Comment on 'Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity'
eLife 8:e43173.
https://doi.org/10.7554/eLife.43173

Share this article

https://doi.org/10.7554/eLife.43173

Categories and tags

Research organism

Further reading

    1. Stem Cells and Regenerative Medicine

    Response to comment on 'Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity'

    David J Goldhamer, John B Lees-Shepard
    Scientific Correspondence

    We respond to concerns expressed by Pacifici and Shore (2019) about a recent paper (Lees-Shepard and Goldhamer, 2018a) in which we reported that the drug palovarotene can have severe side effects in a mouse model of fibrodysplasia ossificans progressiva.

    1. Stem Cells and Regenerative Medicine

    Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity

    John B Lees-Shepard, Sarah-Anne E Nicholas ... David J Goldhamer
    Research Article

    Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by debilitating heterotopic ossification (HO). The retinoic acid receptor gamma agonist, palovarotene, and antibody-mediated activin A blockade have entered human clinical trials, but how these therapeutic modalities affect the behavior of pathogenic fibro/adipogenic progenitors (FAPs) is unclear. Using live-animal luminescence imaging, we show that transplanted pathogenic FAPs undergo rapid initial expansion, with peak number strongly correlating with HO severity. Palovarotene significantly reduced expansion of pathogenic FAPs, but was less effective than activin A inhibition, which restored wild-type population growth dynamics to FAPs. Palovarotene pretreatment did not reduce FAPs’ skeletogenic potential, indicating that efficacy requires chronic administration. Although palovarotene inhibited chondrogenic differentiation in vitro and reduced HO in juvenile FOP mice, daily dosing resulted in aggressive synovial joint overgrowth and long bone growth plate ablation. These results highlight the challenge of inhibiting pathological bone formation prior to skeletal maturation.

    1. Genetics and Genomics
    2. Stem Cells and Regenerative Medicine

    A multiplexed, single-cell sequencing screen identifies compounds that increase neurogenic reprogramming of murine Muller glia

    Amy Tresenrider, Marcus Hooper ... Thomas A Reh
    Research Article

    Retinal degeneration in mammals causes permanent loss of vision, due to an inability to regenerate naturally. Some non-mammalian vertebrates show robust regeneration, via Muller glia (MG). We have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. With the goal of improving this in mammals, we designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single-nucleus RNA-seq conditions into a single experiment. We used this technology to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease.