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closeReply to Beconi et al.
Posted by joelg on 19 Oct 2012 at 18:39 GMT
Reply to Beconi et al.
Joel M. Gottesfeld and Elizabeth A. Thomas
Department of Molecular Biology
The Scripps Research Institute
La Jolla, CA 92037
Email: joelg@scripps.edu
We are writing to point out several inconsistencies and misstatements made by Beconi et al. [1] with regard to our previous publication [2] and omissions by Beconi et al. in citation of other relevant papers from our laboratories [3,4]. First, we agree with Beconi et al. that there are pharmacology liabilities for drug development in the first generation of pimelic 2-aminobenzamide histone deacetylase inhibitors, exemplified by compound 4b. We have previously noted that these compounds have two problems for use as orally available CNS acting drugs; namely, less than optimal brain penetration and metabolic instability (formation of an inactive benzimidazole; Xu et al. [3]). Although the Xu et al. [3] study was published prior to submission of the final revised version of the Beconi et al., it was not discussed or cited by Beconi et al. In Xu et al., we reported extensive chemical optimization studies focusing on modifications of the 4b scaffold to improve on pharmacological properties. We presented a novel chemical strategy based on click chemistry to derive a new generation of compounds that retain potency as HDAC inhibitors but have improvements in both brain penetration and stability. Brain penetration was improved by removal of the “left” amide linkage, and benzimidazole formation was virtually eliminated by placing a double bond adjacent to the “right” amide linkage. A heterocycle in the linker region, afforded by the click reaction, was tolerated without loss of class I HDAC inhibition. Pharmacological properties of the new compounds were also reported by Xu et al. [3].
Further, we stand by the data presented in Thomas et al [2] and the thesis that class I HDAC inhibitors have beneficial therapeutic effects in HD mouse models. Other findings from our group and collaborating laboratories have indeed demonstrated beneficial effects of 4b after subcutaneous administration in HD mice [5], increased histone acetylation in the mouse brain by subcutaneous injection [4,6,7], and changes in gene expression by this route of administration have also been reproduced [4]. Beconi et al. compare the activity of SAHA by subcutaneous injection, as previously reported by Hockly et al. [8], to that of oral administration of 4b. This comparison is not valid and conclusions drawn from this comparison are not warranted. Indeed, Hockly et al. only found increased histone acetylation in the mouse brain after subcutaneous injection of SAHA and not via drinking water. We have also shown that compounds that target class I HDACs, including HDACs 1 and 3, are of benefit in HD mouse models [4,5] and in mouse models for the related neurodegenerative disease Friedriech’s ataxia [6,7,9], contrary to the claims put forward by Beconi et al. Lastly, the conclusions that the benzamide chemotype of HDAC inhibitors are unsuitable for use in CNS diseases, and that HDAC3 inhibition should not be considered a target for the treatment of Huntington’s disease are not warranted by the data presented by Beconi et al. These authors failed to present any HD mouse model studies, and as noted above, newer generations of compounds do hold promise for treatment of HD and other CNS indications [3,4,6,9,10].
1. Beconi M, Aziz O, Matthews K, Moumne L, O'Connell C, et al. (2012) Oral Administration of the Pimelic Diphenylamide HDAC Inhibitor HDACi 4b Is Unsuitable for Chronic Inhibition of HDAC Activity in the CNS In Vivo. PLoS One 7: e44498.
2. Thomas EA, Coppola G, Desplats PA, Tang B, Soragni E, et al. (2008) The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in Huntington's disease transgenic mice. Proc Natl Acad Sci U S A 105: 15564-15569.
3. Xu C, Soragni E, Jacques V, Rusche JR, Gottesfeld JM (2011) Improved Histone Deacetylase Inhibitors as Therapeutics for the Neurodegenerative Disease Friedreich’s Ataxia: A New Synthetic Route. Pharmaceuticals 4: 1578-1590.
4. Jia H, Pallos J, Jacques V, Lau A, Tang B, et al. (2012) Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease. Neurobiol Dis 46: 351-361.
5. Jia H, Kast RJ, Steffan JS, Thomas EA (2012) Selective histone deacetylase (HDAC) inhibition imparts beneficial effects in Huntington's disease mice: implications for the ubiquitin-proteasomal and autophagy systems. Hum Mol Genet. In press.
6. Rai M, Soragni E, Chou CJ, Barnes G, Jones S, et al. (2010) Two new pimelic diphenylamide HDAC inhibitors induce sustained frataxin upregulation in cells from Friedreich's ataxia patients and in a mouse model. PLoS One 5: e8825.
7. Rai M, Soragni E, Jenssen K, Burnett R, Herman D, et al. (2008) HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model. PLoS ONE 3: e1958 doi:1910.1371/journal.pone.0001958.
8. Hockly E, Richon VM, Woodman B, Smith DL, Zhou X, et al. (2003) Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease. Proc Natl Acad Sci U S A 100: 2041-2046.
9. Sandi C, Pinto RM, Al-Mahdawi S, Ezzatizadeh V, Barnes G, et al. (2011) Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model. Neurobiol Dis 42: 496-505.
10. McQuown SC, Barrett RM, Matheos DP, Post RJ, Rogge GA, et al. (2011) HDAC3 is a critical negative regulator of long-term memory formation. J Neurosci 31: 764-774.
Reply to Dr Gottesfeld and Thomas re their commentary "Reply to Beconi et al."
vahri replied to joelg on 11 Mar 2013 at 17:30 GMT
In response to the comments by Drs. Joel M. Gottesfeld and Elizabeth A. Thomas regarding our publication [1], we would like to thank them for bringing the Xu et al. [2] reference to our attention; we were not aware of this publication since it was not available in PubMed at the time our paper was published (and still is not, as of March 11th 2013). However, it should be noted that the work by Xu et al. relates to a new chemical series that was not the focus of our paper and has no bearing on the thesis of our publication.
With regard to the route of administration used in the comparison of 4b activity that Drs Gottesfeld and Thomas mention, our studies show that systemic exposures to 4b following 5 mg/kg subcutaneous (sc) and 50 mg/kg per oral (po) are comparable. 4b is unstable in mouse plasma and so likely undergoes rapid systemic hydrolysis regardless of route of administration. Further, the brain-to-plasma (B:P) ratios experimentally determined are negligible, likely because most 4b is rapidly metabolized in plasma and therefore will not reach the brain; if some 4b does reach the brain, it is likely to be exported via Pgp. As the study in [3] used very high sc doses in the HD models (and other FRDA models mentioned both by the commentators and comprehensively referenced in [1]) for pharmacodynamic studies, far in excess to those we tested subcutaneously, this could have resulted in adequate brain exposure to centrally inhibit Class I HDACs, although this is conjecture on our part since we did not test formally. We raise this point in our discussion. This does not refute our major findings and the conclusion of our work: that oral administration of 4b delivered at the same ‘estimated daily dose’ in drinking water as acute sc administration cannot reach the same levels in the brain and is unlikely to engage central Class 1 HDACs. In other words, a mismatch exists between the R6/2 efficacy study and the pharmacodynamic studies performed in [3] such that brain concentrations of 4b in those two experiments are likely different and not comparable. These authors concluded that the mechanism of action of 4b in improving R6/2 phenotype per chronic oral drinking water study was due to central HDAC inhibition and correlated to the reversal of transcriptional dysregulation [3]. We stand by our statement that we consider this is highly unlikely.
The point raised by us above is also relevant to the comments by Drs Gottesfeld and Thomas on the inclusion of SAHA in our studies. As stated in our Results, SAHA was used only as a positive control to demonstrate the validity of the technique used to show differences in ex vivo histone acetylation. In no way was this meant to be correlated to a mechanism of action for the SAHA efficacy described in Hockly et al, 2003 [4]. Indeed, for the reasons stated above, we currently have no reason to think that a modest enhancement of histone acetylation could not be obtained with a very high acute sc dose of undegraded 4b, sufficient to provide adequate brain exposure, despite the poor B:P ratio.
We would also like to clarify that formation of the inactive benzimidazole is not an in vivo phenomenon, and is not due to “metabolic instability” as described in the comments from Drs Gottesfeld and Thomas and in [2]. Rather it is due to chemical instability, as described in detail [1]. Indeed the metabolic stabilities of either 4b or first-generation analogs, or the second-generation analogs described in [2], have to our knowledge never been published. The published descriptions of 4b pharmacokinetics (restricted to assertions of B:P ratio) are very difficult to reconcile with our findings; Jia et al 2012 [5] reported a B:P ratio for 4b of 0.45, for which they cited reference [6], although there is no description of the methodology employed, species used, dose level or route of administration in support of these findings in either publication. This B:P ratio substantially contradicts our findings. Indeed the premise of the second generation series reported in [2] was to improve suboptimal brain penetration, benchmarked in that paper with a “series 1” compound (109) to be 0.15, with the resultant Click-1 compound described as affording improved B:P ratio of 0.3 (5 mg/kg rat IV determination), but not the Click-2 compound described (B:P ratio of 0.03) [2]. We would welcome further clarification of this from the authors in future publications.
To our knowledge, our paper [1] provides the first detailed investigation of the metabolic stability and pharmacokinetic properties of 4b in the mouse, the preclinical species of choice for both the FRDA and HD models. We suggest that this is now the published benchmark for preclinical mouse work against which improvements in future series should be demonstrated.
Regarding the suggestion by Drs Gottesfeld and Thomas that our conclusions are “not warranted” because we did not proceed to in vivo efficacy evaluation, we direct interested readers to a very recent publication from Professor Mike Levine’s group [7]. It reports an attempt to replicate the findings of the published study [3] on the effects of treating the R6/2 HD mouse model with 4b. The authors were unable to replicate the significant behavioral effects of oral 4b treatment in the R6/2 mice previously reported in [3], but were able to replicate the protection from striatal atrophy in the R6/2 mice. In addition, Chen et al [7] did not observe any beneficial effects of 4b treatment in the N171-82Q mice. Although the behavioral procedures were replicated and an automated activity assessment was added, the authors of this study reported complications regarding solubility and stability of the drug, which corroborates our findings. CAG-repeat length and sex differences in the HD models used and subsequent progression of the phenotype could also have affected outcomes. Chen et al [7] conclude that further studies are required using different delivery methods and assessing effects in more slowly progressing HD models to better evaluate the effects of this HDAC inhibitor. Our conclusions remain the same from our initial evaluation; that appropriate ADME, PK/PD relationships of compound activity remain a critical determinant of target validation and the subsequent interpretation of preclinical in vivo efficacy studies (whether the outcomes are positive or negative) and, given the ADME and PK/PD profile we have determined for 4b, we consider that it is not a suitable molecule to cleanly validate CNS HDAC target engagement in mice.
We thank PLOS ONE for the opportunity to respond in this public forum to the comments by Drs. Gottesfeld and Thomas on our paper.
1. Beconi M, Aziz O, Matthews K, Moumne L, O'Connell C, et al. (2012) Oral administration of the pimelic diphenylamide HDAC inhibitor HDACi 4b is unsuitable for chronic inhibition of HDAC activity in the CNS in vivo. PLoS One 7: e44498.
2. Xu C SE, Jacques V, Rusche JR, Gottesfeld JM (2011) Improved Histone Deacetylase Inhibitors as Therapeutics for the Neurodegenerative Disease Friedreich’s Ataxia: A New Synthetic Route. Pharmaceuticals 4, : 1578-1590.
3. Thomas EA, Coppola G, Desplats PA, Tang B, Soragni E, et al. (2008) The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in Huntington's disease transgenic mice. Proc Natl Acad Sci U S A 105: 15564-15569.
4. Hockly E, Richon VM, Woodman B, Smith DL, Zhou X, et al. (2003) Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease. Proc Natl Acad Sci U S A 100: 2041-2046.
5. Jia H, Kast RJ, Steffan JS, Thomas EA (2012) Selective histone deacetylase (HDAC) inhibition imparts beneficial effects in Huntington's disease mice: implications for the ubiquitin-proteasomal and autophagy systems. Hum Mol Genet 21: 5280-5293.
6. Jia H, Pallos J, Jacques V, Lau A, Tang B, et al. (2012) Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease. Neurobiol Dis 46: 351-361.
7. Chen J, Wang E, Galvan L, Huynh M, Joshi P, et al. (2013) Effects of the Pimelic Diphenylamide Histone Deacetylase Inhibitor HDACi 4b on the R6/2 and N171-82Q Mouse Models of Huntington’s Disease. PLOS Currents Huntington Disease Feb 5 [last modified: 2013 Feb 2015]. Edition 2011. doi: 2010.1371/currents.hd.ec3547da2011c2012a2520ba2959ee2017bf2018bdd2202.