Repurposing Vorinostat for the Treatment of Disorders Affecting Brain
Athira K. V.1 · Prashant Sadanandan2 · Sumana Chakravarty3

Received: 10 October 2020 / Accepted: 9 April 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Based on the findings in recent years, we summarize the therapeutic potential of vorinostat (VOR), the first approved histone deacetylase (HDAC) inhibitor, in disorders of brain, and strategies to improve drug efficacy and reduce side effects. Scien- tific evidences provide a strong case for the therapeutic utility of VOR in various disorders affecting brain, including stroke, Alzheimer’s disease, frontotemporal dementia, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, spinal muscular atrophy, X‐linked adrenoleukodystrophy, epilepsy, Niemann-Pick type C disease, and neuropsychiatric disorders. Further elucidation of the neuroprotective and neurorestorative properties of VOR using proper clinical study designs could provide momentum towards its clinical application. To improve the therapeutic prospect, concerns on sys- temic toxicity and off-target actions need to be addressed along with the improvement in formulation and delivery aspects, especially with respect to solubility, permeability, and pharmacokinetic properties. Newer approaches in this regard include poly(ethylene glycol)-b-poly(DL-lactic acid) micelles, VOR-pluronic F127 micelles, encapsulation of iron complexes of VOR into PEGylated liposomes, human serum albumin bound VOR nanomedicine, magnetically guided layer-by-layer assembled nanocarriers, as well as convection-enhanced delivery. Even though targeting specific class or isoform of HDAC is projected as advantageous over pan-HDAC inhibitor like VOR, in terms of adverse effects and efficacy, till clinical validation, the idea is debated. As the VOR treatment-related adverse changes are mostly found reversible, further optimization of the therapeutic strategies with respect to dose, dosage regimen, and formulations of VOR could propel its clinical prospects.
Keywords Brain disorders · Blood–brain barrier · Histone deacetylase inhibitor · Neuroprotection · Suberoylanilide hydroxamic acid · Drug delivery

ABCD1 ATP‐binding cassette subfamily D member 1 BBB Blood–brain barrier
BDNF Brain-derived neurotrophic factor cAMP Cyclic adenosine monophosphate

 Athira K. V.
[email protected]; [email protected]
 Sumana Chakravarty
[email protected]; [email protected]
1 Department of Pharmacology, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Amrita Institute of Medical Sciences Health Sciences Campus, Kochi 682 041, Kerala, India
2 Department of Pharmaceutical Chemistry & Analysis, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Amrita Institute of Medical Sciences Health Sciences Campus, Kochi 682 041, Kerala, India
3 Applied Biology Division, CSIR- Indian Institute of Chemical Technology, Tarnaka, Uppal Road, Hyderabad 500007, Telangana, India
CREB CAMP response element binding protein CRTC1 CREB-regulated transcription coactivator 1 CBP CREB binding protein
CNS Central nervous system CTCL Cutaneous T-cell lymphoma DMSO Dimethyl sulfoxide
GBM Glioblastoma multiforme
GDNF Glial cell-derived neurotrophic factor HATs Histone acetyltransferases
HDACs Histone deacetylases Hsp Heat shock protein
iPSC Induced pluripotent stem cells NPC Niemann-Pick Type C
NR2B N-Methyl-D-aspartate receptor 2 subunit B PTCL Peripheral T-cell lymphoma
SAHA Suberoylanilide hydroxamic acid SMA Spinal muscular atrophy
SMN Survival motor neuron VLCFA Very long‐chain fatty acids VOR Vorinostat

Recent findings implicate the therapeutic potential of epi- genetic modulators in various disorders affecting brain (Athira et al., 2020; Gangisetty & Murugan, 2016; Jako- vcevski & Akbarian, 2012; Jhelum, 2017). These disorders are characterized by structural, cellular, and molecular alterations of the central or peripheral nervous systems which eventually lead to some functional impairment (Chakravarty et al., 2014b). Epigenetic modifications of chromatin play a key role in altering neural circuitries by cell-specific gene expression (Chakravarty et al., 2014b; Ziemka-Nalecz et al., 2018). The fundamental subunit of chromatin is nucleosome, which is DNA wrapped around histone proteins. It is the chemical modification of histone protein complexes by different enzymes that regulate the state of chromatin structure, in turn activating or repress- ing the gene transcription. An important modification includes the acetylation and deacetylation of lysine resi- dues within the amino terminal tails of histone proteins by the enzymes, histone acetyltransferases (HATs), and histone deacetylases (HDACs), respectively. In general, histone acetylation loosens up the condensed chromatin to activate transcription while histone deacetylation pro- motes chromatin condensation, repressing gene transcrip- tion. Normally, the balanced levels and activities of HATs and HDACs maintain homeostasis, ensuring coordinated regulation of gene expression and neuro-physiological
outputs. However, disruption of the acetylation homeosta- sis has been evident in multiple disorders affecting brain (Mai et al., 2009). HDAC inhibitors have, therefore, gained increasing attention owing to their promising role in miti- gating various brain disorders (Ziemka-Nalecz et al., 2018). Based on the findings in recent years, here, we summarize the therapeutic potential of vorinostat (VOR), the first approved HDAC inhibitor, in disorders of brain, and strategies to improve drug efficacy and reduce side effects (Fig. 1).

Histone Deacetylases
Gene activation is associated with acetylation of histone lysine residues which imparts a negative charge, thus facilitating the relaxation of chromatin locally. Although the acetylation reaction can occur on all four core his- tones, such as H2A, H2B, H3 and H4, the well-known sites include those on H3 and H4 (Grayson et al., 2010). Acetylation promotes transcription not only via an open conformation of chromatin that is favorable for the bind- ing of transcriptional machinery to the DNA, but also by offering recognition sites for transcription factors (Shahbazian & Grunstein, 2007). HDACs are known to possess a catalytic site having a narrow tubular pocket made up of hydrophobic walls and a Zn2+ cation located at the bottom. HDACs (except class III) mediate deacety- lation through Zn2+ catalyzed hydrolysis of acetamido

Fig. 1 Therapeutic potential of
vorinostat in disorders of brain Stroke
Traumatic brain injury

Alzheimer’s disease

Rubinstein-Taybi syndrome

Multiple sclerosis

Neuropsychiatric disorders

Niemann-Pick type C disease
(Suberoylanilide hydroxamic acid (SAHA)

Molecular Weight: 264.32 g/mol
Solubility: very slightly soluble in water,
slightly soluble in ethanol, isopropanol and acetone, freely soluble in dimethyl sulfoxide and insoluble in methylene chloride
Approval: ZOLINZA (vorinostat capsules, for oral use) in cutaneous T-cell lymphoma

Parkinson’s disease

Huntington’s disease

Amyotrophic lateral sclerosis

Spinal muscular atrophy




group of ε-carbon of lysine residues that fit into its cata- lytic pocket, thus decreasing the approachability of tran- scription factors to DNA and inhibiting gene expression (Grayson et al., 2010). HDACs are incapable of directly binding to chromatin; their sequence specificity is mostly determined by other proteins that associate with them. Although HDACs via deacetylation is generally thought to repress transcription by reinstating the positive charge on lysine and thus decreasing the affinity of acetyl-lysine binding transcription factors and co-activators, recent evi- dence suggests it could also activate some genes (Haber- land et al., 2009).
Based upon the structure, function, and expression pat- terns, HDACs are distributed in four classes. The class I HDACs (HDAC 1, 2, 3, and 8) are constitutively nuclear proteins, but HDAC3 is exceptional, being found at the plasma membrane, cytoplasm, and nucleus, whereas the class II HDACs (HDAC 4, 5, 6, 7, 9, and 10; can be fur- ther subdivided into class IIa—HDAC 4, 5,7, 9 or class IIb—HDAC 6, 10) can move between the nucleus and cytoplasm (Morris & Monteggia, 2013; Ruijter et al., 2003). The class III HDACs, called sirtuins, are func- tionally unrelated to remaining HDACs and require nico- tinamide adenine dinucleotide (NAD+) as opposed to zinc (Zn2+) as a cofactor, in spite of possessing deacetylase activity (Ruijter et al., 2003). Class IV contains only one member, HDAC11, whose function is much less illus- trated (Gao et al., 2002). All the 11 HDACs are expressed to some extend in the adult rodent brain (Broide et al., 2007). HDACs are important regulators of diverse biolog-
Histone Deacetylase Inhibitors
A number of HDAC inhibitors have been developed belong- ing to various classes (Table 1) (Didonna & Opal, 2015; Grayson et al., 2010; Souza, 2015; Wash et al., 2008). While most of these are pan-HDAC inhibitors, newer HDAC inhib- itors are selective and include rocilinostat (ACY1215)— inhibits class II HDAC, CHR-3996—inhibits class I HDAC, and ACY-738 and ACY-775—both inhibit HDAC6 (Eck- schlager et al., 2017; Jochems et al., 2014). While vorinostat (VOR) and romidepsin are approved for cutaneous T-cell lymphoma (CTCL), other approvals include belinostat for peripheral T-cell lymphoma (PTCL) and panobinostat for multiple myeloma (Mann et al., 2007; VanderMolen et al., 2011). Although mostly investigated for the anticancer activ- ity, an increasing numbers of studies are evaluating HDAC inhibitors for central nervous system (CNS) diseases, such as neurodegenerative disorders and psychiatric disorders (Abel & Zukin, 2008; Chakravarty et al., 2014a; Golden et al., 2013; Kazantsev & Thompson, 2008; Kilgore et al., 2010; Price & Dyke, 2007). The mechanism of HDAC inhibitors is not uniform as it can vary depending upon several factors including the nature of the illness, type of HDAC inhibi- tor, and dose (Kretsovali et al., 2012). The advantage with HDAC inhibitors is that they not only modify downstream translational endophenotypes, but also modulate anomalous cell transcription (Seo et al., 2014).

Table 1 Classification of HDAC inhibitors

ical functions that vary from tissue specific developmen-

tal programming, apoptosis and cancer to synaptogenesis, cognition, and neurodegeneration (Morris & Monteggia, 2013). As majority of the existing HDAC inhibitors tar- get multiple isoforms of HDACs, determining the bio- logical role of individual HDACs has been cumbersome. However, strategies including conditional knockout and small-interfering RNA could unravel the unique function- ality of specific HDAC isoform, particularly, in a region- dependent manner (Morris & Monteggia, 2013).
Chemical class Examples

Hydroxamates Trichostatin A
Vorinostat (suberoylanilide hydroxamic acid (SAHA))
Belinostat (PXD-101) Panobinostat (LBH-589) Givinostat (ITF- 2357) Resminostat (4SC-201 or
Abexinostat (PCI-24781)

While the initial studies focused on the activity of HDACs on histones and chromatin, they are currently known to deacetylate nonhistone substrates, such as tran-
Short-chain fatty acids
Tubacin (BML-GR362)
Valproic acid Phenyl butyrate Butyrate

scription factors, metabolic enzymes, cytoskeletal pro- teins, and chaperones, thereby playing an extensive role in cell biology (Choudhary et al., 2009; Dokmanovic et al.,
2007). Some of the prominent nonhistone HDAC targets
Cyclic peptides Apicidine
Romidepsin (depsipeptide)
Benzamides Entinostat (MS-275) Mocetinostat (MGCD0103)

include α-tubulin, β-catenin, p53, E2F, heat shock protein 90 (Hsp90), retinoblastoma, NF-κB, and STAT proteins (Buchwald et al., 2009). Moreover, their effects can be transcription-dependent as well as transcription-inde- pendent mechanisms (McLaughlin & Thangue, 2004).

Mercaptoketone Sirtuin inhibitors
Tacedinaline (CI994) KD5170
Nicotinamide Sirtinol AGK‐2 AK‐7

VOR (SAHA) was developed in collaboration between the chemistry group of Ronald Breslow, Columbia University, New York, and the cancer biology group of Paul A Marks and Richard Rifkind, Memorial Sloan-Kettering Cancer Center, New York. The development was initiated based on the observation of the cytodifferentiation and growth arrest properties of a polar, small molecule solvent dime- thyl sulfoxide (DMSO), which led to the identification that simple amides were more potent than DMSO, which in turn led to the development of bis-, tri-, and tetra-aceta- mides, and finally a series of derivatives of N-hydroxy- amide, a species known as a hydroxamic acid (Richon et al., 1996; Tanaka et al., 1975). The structure activity relationship of these ligands was indicative of their bind- ing to a receptor site containing metal ion. This led to the eventual development of VOR which not only had a hydroxamic acid group capable of binding to a metal ion, as well as a hydrophobic group for hydrophobic binding (Marks & Breslow, 2007). On discovery, VOR was found to induce growth arrest and cell death in transformed cells, and later, its biological mechanism was found to be inhibi- tion of HDAC, an enzyme containing Zn2+ cation. HDAC inhibition resulted in accumulation of acetylated histones and certain transcription factor proteins regulating gene expression and thereby altering the transcription of genes (Marks & Dokmanovic, 2005; Peixoto & Lansiaux, 2006). While some increase or decrease in transcription of spe- cific genes was beneficial in the anticancer effects, it was thought that undesirable effects might stem through con- tinuous long-term modulation by the slow release of the drug from the receptor pocket, which could strongly bind to other nonspecific targets. However, the effective dose of VOR was found to be tolerated by patients (Marks & Breslow, 2007).
Further, it was revealed that the hydroxamic acid group of VOR gets doubly coordinated to the Zn2+ atom in HDACs, efficiently inactivating it (Finnin et al., 1999). Later, preclinical studies revealed multiple targets for VOR, such as histones and nonhistone proteins includ- ing proteins in transcription factor complexes, proteins regulating cell proliferation, tumor suppressors, apopto- sis, structural protein, chaperone protein, angiogenesis, and oxidative stress (Drummond et al., 2005; Johnstone & Licht, 2003; Marks & Breslow, 2007). VOR was found to inhibit class I and II HDACs (HDAC-1, 2, 3 and 6; IC50 < 86 nM). A remarkable observation was that, at concentrations in which VOR induce growth arrest in both normal and transformed cells in culture, it causes cell death only of transformed cells. “Cell context” of the transformed cell was reported to be an important
determining factor of the VOR-induced phenotype (Marks & Dokmanovic, 2005). Moreover, VOR was demonstrated to selectively alter the transcription of expressed genes. Microarray analysis of the gene expression of human multiple myeloma cells treated with VOR revealed that changes in gene expression occurred in only a few genes, such as those related to insulin-like growth factor (IGF)/ IGF-1 receptor (IGF-1R) and IL-6 receptor (IL-6R) signal- ing, caspase inhibitors, oncogenic kinases, DNA synthesis/ repair enzymes, and transcription factors (Mitsiades et al., 2004). While the proapoptotic genes, Apaf-1, Caspase-9, DRAK1, and DFF45, were upregulated, a constellation of antiapoptotic genes, including FLIP and Survivin, were downregulated (Mitsiades et al., 2004). It was suggested that the composition and structure of the transcription fac- tor complex might be important in determining the gene selectivity of VOR.
Further, in vivo studies demonstrated anticancer activ- ity alone and in synergy with other agents (Marks & Bres- low, 2007). As preclinical studies demonstrated favorable toxicology and pharmacokinetics, intravenous VOR was tested in phase I clinical trial for the treatment of refrac- tory hematologic and solid tumors, in which acetylated his- tones were found to accumulate in tumor as well as normal tissues (Kelly et al., 2003). VOR was well tolerated, with the exception of thrombocytopenia and neutropenia at high dose, exclusively in hematologic tumor patients. The oral preparation also demonstrated good availability and favora- ble pharmacokinetics (Kelly et al., 2005). Further, VOR demonstrated effectiveness against hematologic malignan- cies, CTCL, and solid tumors. Common toxicities such as fatigue, diarrhea, anorexia, and dehydration were revers- ible on termination of therapy for 4–7 days. Especially, the effects of VOR were remarkable against CTCL as it selec- tively induced apoptosis of malignant T-cell and modulated acetylated histones, p21waf1, Bax, Stat6, and caspase 3 (Olsen et al., 2006). Finally, VOR (ZOLINZA, Merck & Co., Inc.) was approved for the treatment of CTCL by the USFDA in 2006 (Mann et al., 2007; Marks & Breslow, 2007; Zagni et al., 2017).
In one of the earlier study analyzing the effects of VOR
against B cell lymphoma in mice, administration of VOR at the dose of 200 mg/kg, intraperitoneally (i.p.), for 7 days, followed by 150 mg/kg, i.p., for 14–21 days, was shown to mediate a therapeutic response via activation of the intrinsic apoptotic pathway (Lindemann et al., 2007). Further, VOR was demonstrated to inhibit brain metastatic colonization in a model of triple-negative breast cancer and induce DNA double-strand breaks (Palmieri et al., 2009). In this study, brain distribution of VOR was confirmed by the administra- tion of 150 mg/kg [14C]VOR intravenously. VOR showed a significant uptake into brain, reaching a brain/blood con- centration ratio at 30 min that exceeded the brain residual

blood volume by 5- to 7-fold. In addition, VOR was shown to potentiate the anticancer effects of radiation in neuroblas- toma, via downregulation of DNA repair enzyme, in Ku-86 mice treated with VOR, 150 mg/kg, i.p., every other day, three doses total, in combination with 1 Gy of radiation fol- lowing each dose of VOR (Mueller et al., 2011). Moreover, in a phase II trial of VOR in recurrent glioblastoma multi- forme (GBM), administration of a dose of 200 mg orally twice a day for 14 days, followed by a 7-day rest period, was found to be well tolerated in patients but with modest single- agent activity (Galanis et al., 2009). However, the ability of VOR to target pathways in GBM was demonstrated in histone acetylation analysis and RNA expression profiling. Further, a mouse model of Niemann-Pick Type C (NPC) disease demonstrated that treatment with the maximum tol- erated dose of VOR (150 mg/kg, i.p., 5 days/week) led to the reversal of liver dysfunction that typifies the NPC disease, albeit the absence of detectable alteration in NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity (Munkacsi et al., 2017).

Vorinostat in Disorders Affecting Brain
A number of animal studies have tested the activity of VOR in various diseases affecting brain. Even though VOR is reported to possess low blood–brain barrier (BBB) perme- ability (Hanson et al., 2013a; Lee et al., 2015), numerous studies have demonstrated it to be capable of crossing the BBB, as demonstrated directly by measuring the amount of VOR in the brain or indirectly by changes in histone acety- lation in brain (Eyüpoglu et al., 2005; Guan et al., 2009; Hockly et al., 2003; Palmieri et al., 2009; Ugur et al., 2007; Wei et al., 2012; Yin et al., 2007). Notably, these studies have utilized various drug carriers and delivery systems as well as solubility and permeability enhancing agents. Fur- ther, a main difference regarding the use of VOR in cancer and CNS diseases was found to be the dose level adminis- tered. While the range of doses used in cancer study was around 150–200 mg/kg, for the treatment of diseases affect- ing brain, it was about 12.5–100 mg/kg. So the optimization of doses, dosing regimen, and formulation of VOR are very much important for its effectiveness and tolerability in vari- ous brain disorders. As described below, increasing scientific evidence provides a strong case for the therapeutic utility of VOR in brain disorders.

With respect to neurodegeneration in stroke, a mice model of intracerebral hemorrhage demonstrated that the admin- istration of VOR (70 mg/kg, i.p., 1 h post-induction) has the potential to reduce neurodegeneration with concomitant
improvement in neurological outcomes via downregulation of glial activation and the expression of heme oxygenase-1 (Sukumari-Ramesh et al., 2016). Further, in an experimental model of ischemic brain injury, administration of two i.p. injections of VOR straightaway and 6 h post-ischemia, was shown to reduce ischemic neurodegeneration characterized by a bell-shaped dose–response curve (Faraco et al., 2006). The infarct volume was decreased by 25 mg/kg VOR (28.5% reduction), while it was unchanged at the dose of 12.5 mg/ kg. While a 50 mg/kg dose of VOR led to 29.8% reduction, 100 mg/kg dose caused loss of ischemic neuroprotection. Along with the reversal of the decline in histone acetylation in the ischemic brain tissues, VOR increased expression of neuroprotective proteins, Hsp70 and Bcl-2. A theoretical proposition for the bell-shaped dose–response curve of VOR was that, at high doses, transcription might be impaired by extreme histone acetylation leading to the activation of neurotoxic genes. The authors also added a possibility that high doses of VOR might result in excessive acetylation of nonhistone proteins, such as p53 involved in cellular death mechanisms, leading to loss of neuroprotection as the dose of VOR goes above a limit. Furthermore, Li et al., evaluated the effects of VOR (50 mg/kg, i.p., single dose) administered instantaneously or 12 h post-ischemia in a mice model of transient middle cerebral artery occlusion. The immediate treatment showed a comparatively better outcome, associ- ated with a reduction in activated microglia and pro-inflam- matory cytokines. Remarkably, the activated microglia was primed towards a protective phenotype, evident by the increase in the transcription of M2 cytokines (Arg-1 and IL-10) and improved CD206, a M2 marker; along with the suppression of M1 cytokine expression (IL-6, TNF-α, and iNOS) and reduction in CD86, a M1 marker (Li et al., 2019).
Alzheimer’s Disease

Chronic systemic administration of VOR (50 mg/kg, i.p., in 10% DMSO in saline) was found to rescue cognitive deficits and restore and stabilize contextual memory in APPswe/PS1dE9 mice, a model of Alzheimer’s disease (Kilgore et al., 2010). Further, Hanson et al., observed that in vivo VOR treatment (25 and 50 mg/kg, i.p., 35 days, in DMSO) to Tg2576 mice, an Alzheimer’s disease model, failed to improve memory deficits, in spite of in vitro syn- aptic enhancements. The pharmacokinetic analysis revealed low brain concentrations of free VOR, and administration of higher dose (150 mg/kg) prolonged VOR exposure but did not enhance the peak concentration achieved in the brain (Hanson et al., 2013b). This discrepancy could be accounted to the solubility of VOR; a proper vehicle or a drug deliv- ery medium is required to get sufficient concentration of VOR in the brain after systemic administration. Further, VOR and the flavonoid, curcumin demonstrated synergistic

neuroprotective effects against amyloid-β25–35-induced neu- ronal damage in PC12 cells. Based on the network analysis, Meng et al., suggested that the combination could restore the functionality of the impaired Akt and the CBP/p300 path- ways that are implicated in Alzheimer’s disease (Meng et al., 2014). Synergistic effects were also found for the combina- tion of VOR with tadalafil, a phosphodiesterase-5 inhibitor (VOR (12.5 mg/kg, i.p.) + tadalafil (1 mg/kg, p.o.), 4 weeks). The combination not only improved the compromised long- term potentiation in slices from APP/PS1 mice, but also reversed the cognitive deficits, amyloid, and tau pathology, as well as reduced hippocampal dendritic spine density, in aged Tg2576 mice. As only a low dose of VOR was required in the combination therapy, this strategy could offer optimal safety profiles for HDAC inhibitors in chronic treatments. With respect to obtaining an effective functional response such as H3K9 acetylation, it was suggested that HDAC inhi- bition in a potent manner is unnecessary and even molecules having a short half-life and residence time might produce optimal therapeutic effects (Cuadrado-Tejedor et al., 2015). Further, VOR administered orally (2 mg intake per day, in drinking water containing β-cyclodextrin) to APP/PS1-21 mice that showed age-associated decline in memory and amyloid deposition was able to partially reinstate the spa- tial memory function. VOR could cross the BBB, as evident from the concomitant restoration of neuronal H4K12ac in CA1 region of the hippocampus. This study by utilizing a systems biology approach, tried to address the concern that pan-HDAC inhibitors like VOR would cause excessive alterations in the expression of genes, in a nonspecific man- ner (Benito et al., 2015). Remarkably, VOR ameliorated only prevailing deficits and did not alter the homeostasis of cells maintaining normal histone acetylation status. Further, no evidences were found to confirm VOR administration lead- ing to massive and nonselective alterations in gene expres- sion of the hippocampus. VOR was able to reestablish the physiological levels of expression of the plasticity genes in the aging and amyloid model by modulating gene expression and RNA splicing. Further, in human astrocytes, VOR was reported to significantly upregulate the expression of clus- terin, a gene implicated in late-onset Alzheimer’s disease (Nuutinen et al., 2010). Furthermore, a three-drug combina- tion of curcumin, VOR, and silibinin has shown to reduce Aβ25–35-induced toxicity by decreasing oxidative stress and apoptosis, particularly by modulating AKT/MDM2/p53 pathway (Meng et al., 2019).
Frontotemporal Dementia

VOR was also projected as a promising approach to treat frontotemporal dementia in humans, as it was found to res- cue the expression of progranulin in haploinsufficient cells from human subjects (Cenik et al., 2011). VOR was also
shown to restore progranulin content and prevent the cyto- solic TDP-43 accumulation in lymphoblasts from patients having frontotemporal lobar degeneration with TDP-43 protein inclusions and carrying a null progranulin gene mutation (Alquezar et al., 2015). Moreover, VOR increased progranulin production in induced pluripotent stem cells (iPSC)-derived cortical neurons of frontotemporal demen- tia patients (Almeida et al., 2016). However, the authors proposed the necessity of further studies to identify the unwanted effects of VOR administration as it resulted in massive alterations in the overall transcriptome, apart from the reversal of gene expression changes caused by progran- ulin haploinsufficiency. Furthermore, VOR demonstrated marked induction of progranulin mRNA and protein pro- duction in human iPSC-derived neural progenitor cells and differentiated neurons (She et al., 2017).
Parkinson’s Disease

Treatment of VOR to drosophila with pan-neuronal expres- sion of α-synuclein, a neuronal protein involved geneti- cally in Parkinson’s disease, was found to rescue neurons in a dose-dependent manner. HDAC inhibition was found to reduce targeting of α-synuclein to the nucleus, thereby ameliorating the toxicity of α-synuclein in both cell cul- ture and transgenic flies (Kontopoulos et al., 2006). VOR could offer neuroprotection to dopaminergic neurons as it offered partial protection from MPP(+)-mediated apopto- sis in human-derived SK-N-SH and rat-derived MES 23.5 cells, with concomitant increase in histone acetylation (Kidd & Schneider, 2010). The neuroprotective role of VOR was further demonstrated against MPP(+)- and LPS-induced neurotoxicity. Astroglia, but not microglia or neurons, was involved in promoting the survival of dopaminergic neuron by enhancing the release of neurotrophic factors, such as glial cell-derived neurotrophic factor (GDNF) and brain- derived neurotrophic factor (BDNF), through inhibition of histone deacetylation (Chen et al., 2012).
Huntington’s Disease

In a R6/2 Huntington’s disease mouse model, VOR admin- istered orally in drinking water in the form of cyclodextrin complex showed improvement in the motor impairment. VOR was also shown to cross the BBB and increase brain histone acetylation (Hockly et al., 2003). VOR administered in a similar manner (0.67 mg/ml) was shown to decrease HDAC2 and HDAC4 in the cortex and brain stem, but not in the hippocampus, without affecting mRNA levels, of both wild-type and R6/2 mice. In addition, VOR treatment decreased Hdac11 expression in R6/2 but not wild type, while downregulating Hdac7 expression in both wild-type and R6/2 brains. The authors suggested that the discrepancy

might be related to the variation in cell-type composition, region-dependent exposure to VOR, or differences in the regional expression levels of HDACs. VOR also reduced disease progression as indicated by reduced load of SDS- insoluble aggregate in the cortex and brain stem, but not in the hippocampus of the R6/2 brains, along with the restora- tion of cortical Bdnf expression (Mielcarek et al., 2011).
Amyotrophic Lateral Sclerosis

VOR was found to exert neuroprotection in amyotrophic lat- eral sclerosis models by acting as HSP co-inducer in motor neurons in response to proteotoxic stress. Remarkably, com- bination of VOR with arimoclomol enhanced stress-induced Hsp70 expression. Further, VOR mitigated loss of nuclear FUS, a disease hallmark, and HDAC inhibition was found to rescue the DNA repair response in iPSC-derived motor neurons with FUSP525L mutation (Kuta et al., 2020).
Spinal Muscular Atrophy

VOR was found as an effective as well as a nontoxic can- didate to treat spinal muscular atrophy (SMA), an inherited disorder affecting lower motor neurons, caused by insuf- ficient survival motor neuron (SMN) protein levels. VOR upregulated survival motor neuron gene 2 (SMN2), the target gene for therapy of SMA, and enhanced SMN lev- els in multiple neuroectodermal tissues, including rat hip- pocampal brain slices, motoneurone-rich cell fractions, and novel human brain slice culture, while inhibiting HDACs at submicromolar doses (Hahnen et al., 2006). VOR was also shown to elevate SMN2 mRNA levels in SMA-patient- specific iPSC-derived neuronal cell line containing homog- enous Tuj1 + neurons (Lai et al., 2017). VOR was also able to bypass long transcript—survival motor neuron gene 2 (LT-SMN2) silencing by DNA methylation in spinal mus- cular atrophy fibroblasts. As HDAC isoenzyme-selective inhibitors displayed only moderate effects, pan-HDAC inhibitor like VOR was proposed as functionally superior to activate SMN2 (Hauke et al., 2009). The prospective of VOR as a suitable candidate for SMA therapy was further elucidated in two severe mouse models each carrying two SMN2 transgenes: US-SMA mice with one SMN2 per allele (Smn(−/−);SMN2(tg/tg)) and Taiwanese-SMA mice with two SMN2 per allele (Smn(−/−);SMN2(tg/wt)). The US- SMA mice which were embryonically lethal consisted of heterozygous males with markedly diminished fertility and VOR treatment (200 mg/kg /day, in drinking water with hydroxypropyl-β-cyclodextran as carrier) of pregnant moth- ers could reverse it. VOR (25 mg/kg twice daily, in DMSO, orally administered using feeding needle) ameliorated the progression of disease in SMA mice as evident by the increase in life span, increase in the size of neuromuscular
junctions and muscle fibers, and elevated SMN RNA and protein levels along with the decrease in motor neuron degeneration in spinal cord. This particular dosage regimen was optimized for newborn SMA mice to achieve maximum therapeutic efficacy, as the previous dose of 200 mg/kg/day was found to be toxic in newborn SMA mice as well as het- erozygous mice (Riessland et al., 2010).
X‐linked Adrenoleukodystrophy
VOR was also found promising against X‐linked adreno- leukodystrophy, a neurodegenerative disease character- ized by mutations in the ATP‐binding cassette subfamily D member 1 (ABCD1) gene, leading to accumulation of very long‐chain fatty acids (VLCFA) in tissues and body fluids (Berger & Gärtner, 2006). If untreated, it causes progressive inflammatory destruction of brain white matter that can even lead to death (Musolino et al., 2015). VOR was found to induce ABCD2 expression, peroxisomal ß‐oxidation, and ameliorate VLCFA accumulation in differentiated human macrophages to compensate for ABCD1 deficiency. These effects were mediated via modulating IL12B expression and decreasing monocyte differentiation. Further, VOR was able to normalize the blood-CSF/BBB integrity in a cerebral X‐ linked adrenoleukodystrophy patient. However, it could not offer significant clinical benefits in advanced disease state. Better therapeutic prospect may require the commencement of treatment at earlier disease stages. Further, safety con- cerns such as dose-limiting thrombocytopenia need to be addressed (Zierfuss et al., 2020).

VOR also holds potential utility in epilepsies caused by the α1A295D, N40S, R43Q, P44S, and R138G mutations, by enhancing proteostasis, increasing functional surface expression of GABAA receptor subunits, and thereby rec- tifying inhibitory synaptic deficits (Di et al., 2013; Durisic et al., 2018). Currently, a phase II clinical trial is testing the safety, tolerability, and efficacy of VOR (once daily at a dose of 230 mg/m2/day for a total of 6 weeks) in addition to standard of care antiepileptic drugs in pediatric patients with drug-resistant epilepsy (ClinicalTrials.gov Identifier: NCT03894826).
Niemann‑Pick Type C (NPC) Disease

VOR also demonstrated beneficial role in the treatment of NPC disease, a rare progressive genetic disorder, character- ized by abnormal accumulation of lipids in various tissues of the body, including brain. The mutations carried by NPC patients could be corrected by treatment with HDAC inhibi- tors. VOR as well as panobinostat was found to increase

expression of the mutant NPC1 protein in NPC-patient- derived NPC1I1061T fibroblasts, leading to correction of the cholesterol storage (Pipalia et al., 2017). HDAC inhibitors might be altering the proteostatic environment via increasing the expression of protein chaperonins or directly modulat- ing the activity of chaperones by modifying their acetyla- tion status (Pipalia et al., 2017; Rauniyar et al., 2015; Yang et al., 2013). Further, administration of VOR (150 mg/kg, i.p., 5 days/week) to Npc1nmf164 mice, a murine model of NPC disease which expresses a missense mutation in the Npc1 gene, demonstrated significant improvement in liver pathology and function. As VOR concentrations in plasma were > 200 μM, but were almost 100-fold lower in brain tis- sue, the beneficial effects of VOR were found limited to visceral NPC disease. Hence, to relieve the neurological symptoms of NPC disease, strategies to improve BBB pen- etration would be required (Munkacsi et al., 2017).
Neuropsychiatric Disorder

In case of neuropsychiatric disorders, majority of reports validate the pursuit of VOR as an antidepressant (Athira et al., 2020). A continuous infusion of VOR (osmotic deliv- ery of 100 μm in 5% hydroxypropyl β-cyclodextrin vehi- cle) into the nucleus accumbens in social defeat stress mice demonstrated antidepressant effects (Covington et al., 2009). Subchronic VOR administration (25 mg/kg, i.p.) to mice exposed to chronic ultra-mild stress also showed reversal of depressive-like behavior (Uchida et al., 2011). Further, chronic systemic treatment with VOR (25 mg/kg, i.p., in 10% DMSO in saline, 28 days) partly rescued the depres- sive-like behavior of mice deficient in cyclic adenosine monophosphate (cAMP) response element binding protein (CREB)-regulated transcription coactivator 1 (CRTC1) (Meylan et al., 2016). However, chronic systemic treatment with VOR (25 mg/kg, i.p., in vehicle (10% DMSO, 45% PEG-400, 45% saline), 10 days) did not improve behavior in acute amphetamine challenge and forced swim test in wild- type mice (Schroeder et al., 2013). Thus, the duration of treatment with VOR could be considered critical to modu- late behavioral and molecular changes (Berton et al., 2006; Tsankova et al., 2006). Further, administration of VOR (25 mg/kg, i.p., in vehicle (10% DMSO, 45% propylene gly- col and 45% normal saline), 2 weeks)) in a chronic corticos- terone-induced stress model demonstrated antidepressant- like activity via modulation of multiple targets related to inflammation and oxidative stress, as well as HDAC2 (Athira & Madhana, 2018), whereas the same dosage regimen of VOR in a chronic social defeat stress model demonstrated antidepressant-like responses via modulation of cytoplasmic HDAC6 level (Athira et al., 2020). Furthermore, VOR facili- tated fear extinction in rats by increasing histone acetylation and enhancing hippocampal N-methyl-D-aspartate receptor
2 subunit B (NR2B) expression in a contextual fear condi- tioning paradigm (Fujita et al., 2012). Notably, depression is reported in approximately 15% of glioma patients (Rooney et al., 2011). The mood-enhancing property of VOR along with its anticancer activity might be of interesting applica- tion in clinical depression in patients with glioblastomas. For instance, changes in the levels of metabolites such as myo- inositol and N-acetyl aspartate were explored as biomarkers to evaluate anticancer and antidepressant response in glio- blastoma patients treated with vorinostat and temozolomide after progression on standard-of-care radiation therapy and temozolomide or new experimental antiangiogenic therapy (Shim et al., 2014). Further studies in this line are required, to come up with conclusive evidence.
Other Brain Disorders

VOR has emerged as a promising candidate for the treat- ment of neurodegenerative, neurodevelopmental as well as neuropsychiatric illnesses, apart from the anticancer activity. Further elucidation of the neuroprotective and neurorestora- tive properties of VOR could provide momentum towards its clinical application. A number of related studies have shed more light towards the utility of VOR in disorders affecting brain. For instance, VOR might treat multiple sclerosis as it mitigated experimental autoimmune encephalomyelitis in mice by suppressing dendritic cell-mediated Th1 and Th17 cell functions, demyelination, and CNS inflammation (Ge et al., 2013). Further, VOR ameliorated the synaptic plastic- ity deficit observed in CREB binding protein (CBP) mutant, a mouse model of the haploinsufficiency form of Rubin- stein-Taybi syndrome, a condition characterized by skeletal abnormalities as well as mental retardation (Alarcón et al., 2004). VOR could be useful to reverse the negative regula- tory effects on synaptic plasticity in HIV-infected nicotine abusers as it inhibited HDAC2 upregulation resulting in HIV latency breakdown (Atluri et al., 2014). Corroborating the procognitive role of VOR, a recent study (90 mg/kg, i.p.) demonstrated its ability to reverse the impaired learning and memory in offspring of maternal rats exposed to sevoflu- rane during the late gestation. This was accompanied by downregulation of the expression of HDAC2 along with the upregulation of the expression of the CREB and the NR2B mRNA and protein in the hippocampus of offspring in a temporal manner (Yu et al., 2019). Further, chronic adminis- tration of VOR was shown to improve learning and memory via modulation of HDAC2 (Guan et al., 2009). In this study, wild-type mice were injected intraperitoneally with 25 or
16.6 mg/kg of VOR for 10 days and a better response was
found for 25 mg/kg dose in the contextual fear conditioning test. VOR (25 mg/kg, i.p.) was also found to reverse cogni- tive dysfunction via modulation of ER stress markers and HDAC2 (Kv et al., 2020). Neuroprotective role of VOR was

also elucidated in a mice model of traumatic brain injury, in which VOR (100 mg/kg, i.p.) demonstrated the potential to modulate altered level of oxidative stress and inflammatory response markers (Xu et al., 2018). Clinically, neurorestora- tive potential of VOR was projected in myeloablative con- ditioning allogeneic hematopoietic cell transplantation; however, confirmatory large randomized controlled trial is required (Hoodin et al., 2019). Hence, future studies need to focus more on clinically validating the preclinical findings using proper clinical study designs. The clinical translation of VOR into disorders of brain seems promising with ongo- ing clinical trial to determine the tolerable doses of VOR in patients with mild Alzheimer disease (Clinicaltrials.gov identifier: NCT03056495). The mechanistic role of VOR in disorders affecting brain is summarized in Fig. 2.

Conclusions and Future Perspectives
Improving the Pharmaceutical Aspects of VOR

Current efforts on developing HDAC inhibitors are mostly focused on isoform selectivity, identification of Zn2+ bind- ing groups with better pharmacokinetic properties and high potency, as well as concomitantly targeting another biologi- cal target along HDACs (Faria Freitas et al., 2018). Further,
to improve the therapeutic prospect of HDAC inhibitors, concerns on systemic toxicity and off-target actions need to be addressed along with the improvement in formulation and delivery aspects, especially with respect to solubility and permeability of VOR, as well as pharmacokinetic prop- erties (Souza & Lindstrom, 2020; Suraweera et al., 2018). For example, poly(ethylene glycol)-b-poly(DL-lactic acid) micelles of VOR provided sustained exposure and improved the oral and intravenous pharmacokinetics and bioavail- ability (Mohamed et al., 2012). Further, encapsulation of iron complexes of VOR into PEGylated liposomes demon- strated enhanced drug aqueous solubility, in vitro release, and in vivo pharmacokinetic properties (Wang et al., 2014). A relevant approach to achieve localized delivery and tissue specific distribution is to load HDAC inhibitors into bio- compatible nanocarriers (Gryder et al., 2012). Nanocarrier- based drug delivery vehicles such as liposomes, dendrim- ers, hydrogels, solid lipid nanoparticles, synthetic polymeric nanoparticles, polymeric micelles, lipid-polymer hybrid nanoparticles, carbon nanotubes, silica nanoparticles, metal nanoparticles, and organic–inorganic hybrid nanoparticles can be used to improve the solubility of hydrophobic drugs, thereby prolonging their half-life and increasing the drug concentration in targeted tissues (Souza & Lindstrom, 2020). For instance, a nanomedicine comprising of VOR encap- sulated within human serum albumin matrix demonstrated

Fig. 2 The mechanistic role of VOR in disorders affecting brain based on the preclini- cal findings. VOR modulated
specific targets pertinent to each disorder, where ABCD2 = ATP‐ binding cassette subfamily
D member 2, BDNF = brain- derived neurotrophic factor, GABAAR = γ-aminobutyric acid type A receptor,
HDAC = histone deacetylase, Hsp70 = heat shock protein 70, NPC1 = Niemann-Pick disease, type C1, NR2B = N-methyl-D- aspartate receptor 2 subunit B, SMN = survival motor neuron protein, VLCFA = very long‐ chain fatty acids

Table 2 Protective role of vorinostat in disorders affecting brain
CNS disorder Model system Dosing of VOR Effects Reference

Stroke Mice model of intracerebral hemorrhage 70 mg/kg, i.p., dissolved in DMSO and reconstituted in PBS, 1 h post- induction
↓ glial activation & the expression of heme oxygenase-1
Sukumari-Ramesh et al. (2016)

Stroke Permanent distal middle cerebral artery occlusion model of ischemic brain injury in mice

Stroke Mice model of transient middle cerebral artery occlusion
12.5, 25, 50 & 100 mg/kg, in a solution
containing 25% DMSO & 75% PBS, two i.p. injections straightaway and 6 h post-ischemia
50 mg/kg, i.p., in a solution containing 25% DMSO & 75% PBS, single dose, instantaneously or 12 h post-ischemia
25 & 55 mg/kg: ↑ histone acetylation, Hsp70 & Bcl-2

↓ activated microglia & pro-inflamma- tory cytokines,
↑ Arg-1, IL-10 & CD206,
↓ IL-6, TNF-α, iNOS & CD86
Faraco et al. (2006)

Li et al. (2019)

Alzheimer’s disease APPswe/PS1dE9 double-transgenic
50 mg/kg, i.p., in 10% DMSO in saline, 14 days
↑ contextual memory Kilgore et al. (2010)

Alzheimer’s disease Tg2576 mice 25 and 50 mg/kg, i.p., in DMSO, 35 days
Failed to improve memory deficits Hanson et al. (2013b)

Alzheimer’s disease 20-month-old mice and 10-month-old
APP/PS1-21 mice
2 mg intake per day orally from 0.67 g dissolved in 1 L of drinking water containing 18 g of β-cyclodextrin
Partial reinstatement of spatial memory function,
↑ H4K12ac in CA1 region of the hip- pocampus
Benito et al. (2015)

Alzheimer’s disease Human astrocytes 1 & 10 µM, in DMSO ↑ clusterin Nuutinen et al. (2010)

Frontotemporal dementia Neuro-2a and HEK293 cells, haploinsuf-
ficient cells from human subjects
0.1–3 µM, in DMSO ↑ progranulin Cenik et al. (2011)

Frontotemporal dementia Stable GRN knockdown neuroblastoma
SH-SY5Ycells, lymphoblasts from patients having frontotemporal lobar degeneration with TDP-43 protein inclusions and carrying a null pro- granulin gene mutation
0.25–2 µM, in DMSO ↑ progranulin,
↓ TDP-43 accumulation
Alquezar et al. (2015)

Frontotemporal dementia iPSC-derived cortical neurons of fronto-
temporal dementia patients
Frontotemporal dementia Human iPSC-derived neural progenitor
cells & differentiated neurons
1 µM, in DMSO ↑ progranulin Almeida et al. (2016)

10 µM, in DMSO ↑ progranulin She et al. (2017)

Cognitive dysfunction Contextual fear conditioning test in
wild-type mice
Cognitive dysfunction Chronic corticosterone-injected mice

Parkinson’s disease Drosophila with pan-neuronal expres-
sion of α-synuclein, transfected SH- SY5Y cells
25 mg/kg or 16.6 mg/kg, i.p., in 10% DMSO/90% saline, 10 days
25 mg/kg, i.p., in vehicle (10% DMSO, 45% propylene glycol and 45% normal
saline), 14 days
10 & 20 µ M SAHA, in DMSO, 20 days
10 µM, in DMSO
↑ learning and memory via modulation of HDAC2
↑ learning and memory via modulation of ER stress markers & HDAC2

↓ toxicity to dorsomedial dopamine neurons,
↓ targeting of α-synuclein to the nucleus
Guan et al. (2009) Kv et al. (2020)
Kontopoulos et al. (2006)

Parkinson’s disease MPP(+)-mediated apoptosis in human-
derived SK-N-SH and rat-derived
5 µM Partial neuroprotection,
↑ histone acetylation
Kidd and Schneider, (2010)

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MES 23.5 cells

Table 2 (continued)
CNS disorder Model system Dosing of VOR Effects Reference

Parkinson’s disease MPP(+)- and LPS-induced neurotoxic-
ity in rat mesencephalic neuron–glia cultures
0.3–10 µM, 7 days ↑ histone acetylation,
Chen et al. (2012)

Huntington’s disease R6/2 mice model 0.67 g/L, orally in drinking water in the form of cyclodextrin complex (0.67 g in a solution of 18 g of 2-hydroxypro- pyl -β-cyclodextrin in 1L of water)
Huntington’s disease R6/2 mice model 0.67 mg/ml, orally in drinking water in the form of cyclodextrin complex
↑ motor function,
↑ histone acetylation

↓ load of SDS-insoluble aggregate in the cortex and brain stem, but not in the hippocampus,
↑ cortical Bdnf expression,
↓ HDAC2 and HDAC4 in the cortex and brain stem, but not in the hippocam- pus,
↓ Hdac11 & Hdac7 expression
Hockly et al. (2003)

Mielcarek et al. (2011)

Amyotrophic lateral sclerosis Spinal cord–dorsal root ganglion cul-
iPSC-derived motor neurons with FUSP525L mutation
2 µM, in DMSO ↑ nuclear FUS & DNA repair response, induced HSP in motor neurons
Kuta et al. (2020)

Spinal muscular atrophy F98 rat glioma cells,
primary SMN1‐deleted fibroblast lines ML5 and ML16, rat hippocampal brain slice, human brain slice, motoneurone‐ enriched co‐cultures
Spinal muscular atrophy Patient specific iPSC-derived neuronal
cell line containing homogenous Tuj1 + neurons
8 µM, in DMSO ↑ SMN2 Hahnen et al. (2006)

0.1 & 1 µM, in DMSO ↑ SMN2 Lai et al. (2017)

Spinal muscular atrophy US-SMA mice with one SMN2 per
allele (Smn(−/−);SMN2(tg/tg)) and Taiwanese-SMA mice with two SMN2 per allele (Smn(−/−);SMN2(tg/wt))
pregnant mothers: 200 mg/kg /day, in drinking water with hydroxypropyl-β- cyclodextran as carrier;
new born SMA mice: 25 mg/kg twice daily, in DMSO, orally administered using feeding needle
↑ life span, size of neuromuscular junc- tions and muscle fibers, SMN RNA and protein levels,
↓ motor neuron degeneration in spinal cord
Riessland et al. (2010)

X‐linked adrenoleukodystrophy ABCD1 deficient differentiated human
Epilepsy HEK293 cells stably expressing α1(A322D)β2γ2 GABAA receptors
2.5 µM, in DMSO ↑ ABCD2 expression & peroxisomal ß‐ oxidation, ↓ VLCFA accumulation
2.5 µΜ in DMSO, 24 h ↑ proteostasis & functional surface expression of GABAA receptor subunits
Zierfuss et al. (2020) Di et al. (2013)

Niemann-Pick type C disease Human NPC1 fibroblasts GM05659,
GM18453 (homozygous NPC1 mutant I1061T), & GM03123 (heterozygous
10 µM from a 5 mM stock in DMSO ↑ mutant NPC1 protein Pipalia et al. (2017)

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NPC1 mutations P237S and I1061T)

Table 2 (continued)
CNS disorder Model system Dosing of VOR Effects Reference

Niemann-Pick type C disease Npc1nmf164 mice 150 mg/kg, i.p., 5 days/week, in 45% PEG-400 + 10% DMSO
Improvement in liver pathology & function
Munkacsi et al. (2017)

Depression Chronic social defeat stress mice model osmotic delivery of 100 μm in 5% hydroxypropyl β-cyclodextrin vehicle) into the nucleus accumbens
Antidepressant effects Covington et al. (2009)

Depression CRTC1-deficient mice 25 mg/kg, i.p., in 10% DMSO in saline,
28 days
Partially rescued the depressive-like behavior
Schroeder et al. (2013)

Depression Acute amphetamine challenge and forced swim test in wild-type mice
25 mg/kg, i.p., in vehicle (10% DMSO, 45% PEG-400, 45% saline), 10 days
Failed to improve behavior Schroeder et al. (2013)

Depression Chronic corticosterone-induced depres- sion
25 mg/kg, i.p., in vehicle (10% DMSO, 45% propylene glycol and 45% normal
saline), 2 weeks
Antidepressant-like activity via modula- tion of multiple targets related to inflammation, oxidative stress & HDAC2
Athira and Madhana (2018)

Depression Chronic social defeat stress model 25 mg/kg, i.p., in vehicle (10% DMSO,
45% propylene glycol and 45% normal
saline), 2 weeks
Antidepressant-like activity via modula- tion of HDAC6
Athira et al. (2020)

Multiple sclerosis Experimental autoimmune encephalo- myelitis in mice,
human CD14 + monocyte-derived den- dritic cells,
DC2.4 culture
100 mg/kg daily from 3 days before MOG35–55 immunization, intragastric,
1 or 2.5 μM
↓ dendritic cell-mediated Th1 and Th17 cell functions, demyelination and CNS inflammation
Ge et al. (2013)

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improvement in solubility, sustained drug release, as well as absence of adverse effects on normal cells, albeit being potent antileukemic (Chandran et al., 2014). Recently, novel methods are emerging that can directly delivery drugs to the brain facilitating bypass of the BBB. For instance, con- vection-enhanced delivery via a chronic implantable drug delivery system is reported to be beneficial for the treat- ment of CNS disease, including glial malignancies and nonmalignant inflammatory and neurodegenerative dis- ease of the brain. The brilliant aspect is the water-soluble nature of formulation which comprises a water-insoluble HDAC inhibitor encapsulated in a micelle, thereby ensur- ing enhanced efficacy and decreased damage to brain paren- chyma (Gill, 2019). Studies are ongoing to understand the safety and tolerability aspects, such as a phase I/II trial of the panobinostat nanoparticle formulation MTX110 by convection-enhanced delivery infusion in newly diagnosed diffuse intrinsic pontine glioma (ClinicalTrials.gov Identi- fier: NCT03566199). With respect to VOR, a stable and safe nanoparticulate delivery system comprising of 1:50 VOR- pluronic F127 micelles, demonstrated enhanced effective- ness in inhibiting tumor growth with prolonged release and reduced hepatic and renal toxicities (Mohamed et al., 2017). Another approach was to use magnetically guided layer-by- layer assembled nanocarriers for the coencapsulation of ten- ofovir, an anti-HIV drug and VOR, a latency-breaking agent, for the treatment of neuroAIDS. This arrangement not only provided sustained release, but also demonstrated good BBB transmigration ability and cell viability (Jayant et al., 2015). Hence, the improved efficacy and safety features provided by the nanoformulations of VOR may be beneficial to improve the patient’s adherence and compliance.
Addressing the Safety and Tolerability Issues of VOR

Adverse effect is indeed an important parameter that needs to be taken care for optimal clinical application of VOR. As the targets of VOR action are important components of the physiology of various cell types and organs, appropri- ate strategies need to be devised to reduce nonspecificity and adverse effects on long-term use. Even though targeting specific class or isoform of HDAC is projected as advan- tageous over pan-HDAC inhibitor like VOR in terms of adverse effects and efficacy, till clinical validation, the idea is debated (Kazantsev & Thompson, 2008; Marks & Bres- low, 2007). We suppose a characterization of the changes in different HDACs is important to identify proper HDAC treatment for a specific patient. Furthermore, an intermittent dosing of VOR rather than a continuous one might be com- paratively well tolerated. For example, daily administration of VOR at doses of 50–150 mg/kg, i.p. (average treatment length of 20 days) for treatment of metastatic neuroblastoma was reported to be associated with excessive toxicities, while
a less frequent dosing was well tolerated even with a high dose (Mueller et al., 2011). As the VOR treatment-related adverse changes are mostly found reversible, further opti- mization of the therapeutic strategies with respect to dose, dosage regimen, and formulations of VOR could propel its clinical prospects. Novel strategies are emerging in order to improve the efficacy and reduce toxicity of HDAC inhibi- tors, such as strategic crafting of the key pharmacophoric elements of VOR and tubastatin-A into architecting a single molecule. Such carbazole-based HDAC inhibitors are prom- ising to treat psychiatric disorders by virtue of enhancing neurite outgrowth (Reddy et al., 2019).
In conclusion, current evidences point towards the promising role of VOR in various disorders affecting brain (Table 2). However, various pharmaceutical concerns per- taining to solubility, blood–brain barrier penetrability, and metabolic stability need to be addressed. Novel strategies to improve the efficacy and limit the toxicity can improve the therapeutic prospects of VOR.

Author Contributions AKV and PS involved in conceptualization, data curation, formal analysis, funding acquisition, and writing–reviewing and editing. SC participated in formal analysis, funding acquisition, supervision, and writing—review and editing.

Funding The work was jointly supported by CSIR-Indian Institute of Chemical Technology, Hyderabad, and Amrita Vishwa Vidyapeetham, Kochi. IICT communication number generated by KIM division, CSIR- IICT [IICT/Pubs./2020/244], for this manuscript is duly acknowledged.

Conflict of interest The authors declare no conflict of interests.

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