HDAC3 ( IC50 = 50 nM ); HDAC1 ( IC50 = 60 nM ); HDAC1 ( Ki = 32 nM ); HDAC3 ( Ki = 5 nM )

In vitro activity: RG2833's Ki values for HDAC1 and HDAC3 are 32 nM and 5 nM, in that order. RG2833 demonstrates high activity throughout the entire tested concentration range of 1 to 10 µM. Frataxin protein increases more slowly in cells from patient P13 when RG2833 is continuously cultivated, but it increases quickly in the cells after the compound is removed[1]. In addition to reducing neuronal pathology of the dorsal root ganglia (DRG), RG2833 causes notable increases in brain aconitase enzyme activity[2].

RG2833 (150 mg/kg) is capable of treating KIKI mice's brain and heart frataxin deficiency 24 hours after a single injection, but not at lower doses. When tracked over time, the KIKI mouse's increased levels of frataxin mRNA caused by RG2833 can be seen in the brain and heart at 12 hours and 24 hours, respectively[1]. After a chronic dosage of 100 mg/kg, s.c., mice tolerate RG2833 well and do not experience any toxicity. RG2833 enhances YG8R FRDA mice's motor coordination. In the brains of YG8R FRDA mice, RG2833 increases the expression of the frataxin protein[2]. After a single or six-day once-daily treatment, RGFP109 (30 mg/kg, p.o. once daily for six days) has no acute effects on dyskinesia. Dyskinesia and the amount of time spent ON-time with incapacitating dyskinesia are reduced by 37% and 50%, respectively, one week after RGFP109 is stopped[3].

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Sub-chronic treatment with RGFP109 alleviates established l-DOPA-induced dyskinesia [3]
Acute challenges of RGFP109 did not reduce LID severity, as shown by the lack of anti-dyskinetic efficacy on D1 and, to a certain extent, D6. However, RGFP109 significantly reduced severity of peak-dose LID and decreased duration of “bad quality” ON-time on D12, six days after cessation of treatment with RGFP109. This delayed onset of anti-dyskinetic efficacy is consistent with long-lasting changes at the nuclear level, as would be expected with HDAC inhibition, as opposed to blockade of a synaptic receptor, which would be expected to produce an immediate benefit. Importantly, the anti-dyskinetic effect of RGFP109 was obtained without compromising peak anti-parkinsonian efficacy or duration of l-DOPA benefit, suggesting that abnormal histone deacetylation is a consequence of LID and not l-DOPA therapy per se.

Aconitase activities are measured by centrifuging mouse brain tissues at 800×g for 10 min at 4°C after homogenizing them on ice at 10% w/v in CellLytic MT Mammalian Tissue Lysis/Extraction buffer. After adding 50 μL of tissue lysates to 200 μL of substrate mix (which included 50 mM Tris/HCl pH7.4, 0.4 mM NADP, 5 mM Na citrate, 0.6 mM MgCl₂, 0.1% (v/v) Triton X-100, and 1U isocitrate dehydrogenase), the reactions were incubated for 15 minutes at 37°C.The reaction slope was then determined by taking spectrophotometric absorbance measurements every minute for 15 minutes at 340 nm 37°C. Afterwards, using a citrate synthase assay kit, the aconitase activities of mouse brain tissues are normalized to citrate synthase activities.

RG2833's Ki values for HDAC1 and HDAC3 are 5.4 nM and 7.8 nM, in that order. RG2833 demonstrates high activity throughout the entire tested concentration range of 1 to 10 µM. Frataxin protein increases more slowly in cells from patient P13 when RG2833 is continuously cultivated; however, upon removal of the compound, frataxin protein levels rose quickly. In addition to reducing neuronal pathology in the dorsal root ganglia (DRG), RG2833 causes notable increases in brain aconitase enzyme activity.

Mice are kept in standard open cages with 13 hours of light, 11 hours of darkness, 20–23°C, 45–60% humidity, Litaspen Premium 8/20 bedding, paper wool nesting, and standard fun tunnel environmental enrichment. SDS RM3 Expanded food pellets and regular drinking water are fed to the mice. The mice are subcutaneously injected with 150 mg/kg RG2833 three times a week for 4.5 months, or 50 mg/kg 136 or 100 mg/kg RG2833 five times a week for five months. Twenty-four hours after the last injection, the mice are culled in order to collect tissue.

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Administration of RG2833 (RGFP109) in combination with l-DOPA to the parkinsonian marmoset [3]
A schematic depicting the time line of the experiments conducted is provided in Fig. 2. Twelve days prior to the start of the study (D-12), animals were administered an acute challenge of l-DOPA/benserazide (20/5 mg/kg s.c., henceforth referred to as l-DOPA). Behaviour observed on D-12 was used as a baseline comparator to ensure that the animals responded consistently to l-DOPA, both in terms of dyskinesia and duration of reversal of parkinsonism, thereby ensuring that changes noted throughout the study would be secondary to HDAC inhibition and not variation in the response to l-DOPA. On study day 0 (D0), animals were treated with an acute challenge of l-DOPA in combination with vehicle.
Treatment with the HDACi RG2833 (RGFP109) was initiated 24 h later (D1). Throughout the study, animals were administered RG2833 (RGFP109) orally (30 mg/kg) dissolved in hydroxypropyl-β-cyclodextrin acetate (50%, v/v) in water, in combination with l-DOPA. Both drugs were administered simultaneously. The dose of l-DOPA was kept constant throughout the observation days (20/5 mg/kg), but was administered orally on non-behavioural days and s.c. on behavioural observation days (D-12, D0, D1, D6 and D12), in order to minimise variability due to erratic gastro-intestinal absorption. Treatment with RG2833 (RGFP109) was ceased on D6. After a six-day wash-out period during which daily l-DOPA treatment was maintained, response to an acute l-DOPA challenge was re-assessed (D12).

Oral RGFP109 treatment was well-tolerated with no adverse effects observed throughout the study. Over the twelve-day study period, there was a significant effect of treatment on levels of LID during periods of peak l-DOPA effect (Friedman statistic (FS) = 9.75, P < 0.01, Friedman test, Fig. 3B). However, neither acute (D1) nor six days (D6) of treatment with RGFP109 co-administered with l-DOPA, had any effect on levels of LID compared to l-DOPA alone on D0 (all P > 0.05, Dunn's post hoc test). On D12, 6 days after cessation of RGFP109 treatment, although daily l-DOPA was continued, there was a significant reduction (by 37%) in levels of LID (21.5 ± 1.0 on D0 and 13.5 ± 1.5 on D12; P < 0.05, Dunn's post hoc test, Fig. 3B). Accordingly, across the study, there was a significant effect of treatment on the duration of ON-time with disabling dyskinesia (F3,9 = 5.6, P < 0.05, one-way RM ANOVA). At D12, but not prior to this point, the duration of ON-time with disabling dyskinesia was reduced by 50% compared to l-DOPA alone on D0 (145 ± 11 min on D0 and 73 ± 23 min on D12; P < 0.05, Tukey's post hoc test Fig. 3F). [3]

[1]. Two new pimelic diphenylamide HDAC inhibitors induce sustained frataxin upregulation in cells from Friedreich's ataxia patients and in a mouse model. PLoS One. 2010, 5(1), e8825.

[2]. Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model. Neurobiol Dis. 2011, 42(3), 496-505.

[3]. RGFP109, a histone deacetylase inhibitor attenuates L-DOPA-induced dyskinesia in the MPTP-lesioned marmoset: a proof-of-concept study. Parkinsonism Relat Disord. 2013, 19(2), 260-264.

Background: Friedreich's ataxia (FRDA), the most common recessive ataxia in Caucasians, is due to severely reduced levels of frataxin, a highly conserved protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). Typical marks of heterochromatin are found near the expanded GAA repeat in FRDA patient cells and mouse models. Histone deacetylase inhibitors (HDACIs) with a pimelic diphenylamide structure and HDAC3 specificity can decondense the chromatin structure at the FXN gene and restore frataxin levels in cells from FRDA patients and in a GAA repeat based FRDA mouse model, KIKI, providing an appealing approach for FRDA therapeutics. Methodology/principal findings: In an effort to further improve the pharmacological profile of pimelic diphenylamide HDACIs as potential therapeutics for FRDA, we synthesized additional compounds with this basic structure and screened them for HDAC3 specificity. We characterized two of these compounds, 136 and 109, in FRDA patients' peripheral blood lymphocytes and in the KIKI mouse model. We tested their ability to upregulate frataxin at a range of concentrations in order to determine a minimal effective dose. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local histone acetylation. The effects of these compounds exceeded the time of direct exposure in both systems. Conclusions/significance: Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACIs for FRDA and provide information for the design of future human trials of these drugs, suggesting an intermittent administration of the drug. [1]

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Friedreich ataxia (FRDA) is an inherited neurodegenerative disorder caused by GAA repeat expansion within the FXN gene, leading to epigenetic changes and heterochromatin-mediated gene silencing that result in a frataxin protein deficit. Histone deacetylase (HDAC) inhibitors, including pimelic o-aminobenzamide compounds 106, 109 and 136, have previously been shown to reverse FXN gene silencing in short-term studies of FRDA patient cells and a knock-in mouse model, but the functional consequences of such therapeutic intervention have thus far not been described. We have now investigated the long-term therapeutic effects of 106, 109 and 136 in our GAA repeat expansion mutation-containing YG8R FRDA mouse model. We show that there is no overt toxicity up to 5 months of treatment and there is amelioration of the FRDA-like disease phenotype. Thus, while the neurological deficits of this model are mild, 109 and 106 both produced an improvement of motor coordination, whereas 109 and 136 produced increased locomotor activity. All three compounds increased global histone H3 and H4 acetylation of brain tissue, but only 109 significantly increased acetylation of specific histone residues at the FXN locus. Effects on FXN mRNA expression in CNS tissues were modest, but 109 significantly increased frataxin protein expression in brain tissue. 109 also produced significant increases in brain aconitase enzyme activity, together with reduction of neuronal pathology of the dorsal root ganglia (DRG). Overall, these results support further assessment of HDAC inhibitors for treatment of Friedreich ataxia. [2]

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Background: l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) are a complication of chronic dopamine replacement therapy in Parkinson's disease (PD). Recent studies have suggested that the mechanisms underlying development and expression of LID in PD may involve epigenetic changes that include deacetylation of striatal histone proteins. We hypothesised that inhibition of histone deacetylase, the enzyme responsible of histone deacetylation, would alleviate LID. Methods: Four female common marmoset (Callithrix jacchus) were rendered parkinsonian by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Following stabilisation of the parkinsonian phenotype, marmosets were primed to exhibit dyskinesia with chronic administration of L-DOPA. We then investigated the effects of the brain-penetrant histone deacetylase inhibitor, RGFP109 (30 mg/kg p.o. once daily for 6 days), on LID and L-DOPA anti-parkinsonian efficacy. Results: RGFP109 had no acute effects on dyskinesia after single or 6 days once-daily treatment (both P > 0.05). However, one week following cessation of RGFP109, dyskinesia and duration of ON-time with disabling dyskinesia were reduced by 37% and 50%, respectively (both P < 0.05), compared to that seen previously with L-DOPA alone. There was no change in anti-parkinsonian actions of, or ON-time duration afforded by, L-DOPA (P > 0.05). Conclusions: Histone deacetylation inhibition may represent a novel approach to reverse established LID in PD and improve quality of the anti-parkinsonian benefit provided by L-DOPA. [3]

C20H25N3O2

339.43

339.195

C, 70.77; H, 7.42; N, 12.38; O, 9.43

1215493-56-3

56654642

White to off-white solid powder

5.127

3

3

8

25

419

0

O=C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])=O)N([H])C1=C([H])C([H])=C([H])C([H])=C1N([H])[H]

VOPDXHFYDJAYNS-UHFFFAOYSA-N

InChI=1S/C20H25N3O2/c1-15-10-12-16(13-11-15)20(25)22-14-6-2-3-9-19(24)23-18-8-5-4-7-17(18)21/h4-5,7-8,10-13H,2-3,6,9,14,21H2,1H3,(H,22,25)(H,23,24)

N-[6-(2-aminoanilino)-6-oxohexyl]-4-methylbenzamide

RGFP 109; RGFP-109; RGFP109; RG2833; RG2833; 1215493-56-3; N-(6-((2-Aminophenyl)amino)-6-oxohexyl)-4-methylbenzamide; RGFP-109; N-(6-(2-Aminophenylamino)-6-oxohexyl)-4-methylbenzamide; RGFP109; RG 2833; RG-2833

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.37 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.37 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (7.37 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 5%DMSO Corn oil: 6.0mg/ml (17.68mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)