SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer’s disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology

SAHA enhances synaptic function and plasticity in vitro but has limited brain availability in vivo and does not impact cognition.

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer's disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology

Excitatory synaptic function is selectively enhanced in CA1 of hippocampal brain slices following <i>in vitro</i> SAHA treatment.

<p>(A) The median amplitude of mEPSCs was significantly increased in SAHA-treated slices (p<0.05, n = 14 vehicle, 14 SAHA), while there was no significant change to mEPSC frequency as measured by the median interval between events (p>0.05). Example mEPSC traces from vehicle and SAHA treated slices are shown inset (scale bar represents 10 pA and 250 ms). (B) SAHA treatment did not significantly alter the amplitude or frequency of mIPSCs (p>0.05, n = 14, 11). Example mIPSC traces from vehicle and SAHA treated slices are shown inset (scale bar represents 10 pA and 500 ms). All data points are plotted as mean ±SEM.</p

SAHA IC₅₀ values for each HDAC isozyme are shown as measured using <i>in vitro</i> enzymatic assays.

<p>Values are in µM and confidence intervals are in parentheses.</p

Acute or chronic SAHA treatment does not produce significant drug class activity signatures as assessed by the SmartCube®.

<p>A. Groups of mice were treated acutely with a single injection of 50 mg/kg or 150 mg/kg SAHA or vehicle. In addition, a group was treated with valproate (225 mg/kg). Both does of SAHA were behaviorally inactive without a clear therapeutic signal. In contrast valproate was behaviorally active (p<0.001, discrimination index = 100%) with a strong anxiolytic signature and a mild psychostimulant signature. B. Groups of mice were treated daily for 14 days with SAHA or Valproate. While the lower dose of SAHA appeared behaviorally active (p<0.001, discrimination index = 88%), the activity was not consistent with any known therapeutic signal and the higher dose was not behaviorally active. In contrast valproate showed a strong behavioral activity (p<0.001, discrimination index = 98%) with a predominantly anxiolytic signature. C. The legend shows the 15 classes of behavioral activity that were assessed.</p

Intrinsic membrane properties are unaltered by SAHA treatment.

<p>(A) Representative traces from vehicle (black) and SAHA (red) treated slices during a series of hyperpolarizing and depolarizing current injection steps (scale bar represents 20 mV and 100 ms). There was no difference between vehicle and SAHA treated slices in the number of action potentials elicited by 500 ms current injection pulses at any of the current injection levels (p>0.05, n = 7,7). (B) Action potential threshold, input resistance, and membrane sag reflecting the hyperpolarization-induced inward current, were all unaltered following SAHA treatment (p>0.05). Data are plotted as mean ±SEM.</p

Pharmacokinetic analysis of SAHA following i.p. injection.

<p>A) Bioanalysis of the time course of total (top) and unbound (bottom) plasma, CSF, and brain levels of SAHA following a single 50 mg/kg ip injection (n = 3 mice/time point). The dotted red lines represent the SAHA concentration imposed on the <i>in vitro</i> slice cultures for the electrophysiological studies. B) Total (top) and unbound (bottom) SAHA levels are shown following a 150 mg/kg ip injection (n = 3/time point). All data is shown as mean ± SD.</p

Fear memory deficits in Tg2576 mice are not rescued by SAHA treatment.

<p>A) Compared to non-transgenic littermates (n = 15), Tg2576 (n = 14) mice showed significantly less freezing than wt mice when returned to the context in which conditioning occurred (context, p<0.001), when placed in an altered context (altered, p<0.01), or in response to the cue used for conditioning (cue, p<0.05). B) Tg2576 mice were treated daily for 33 days prior to, as well as during fear conditioning with either vehicle (n = 14), 25 mg/kg SAHA (n = 13), or 50 mg/kg SAHA (n = 13). There was no effect of treatment on the percentage of time Tg2576 mice spent freezing in response to the context, altered context, or cue (p>0.05). C) There was no effect of treatment on the percentage of time spent freezing during conditioning (p>0.05). D) There was no effect of treatment on the distance traveled in the open field test (total distance = 38.0±7.7 m for vehicle, 39.6±6.1 m for 25 mg/kg SAHA, and 34.2±5.2 m for 50 mg/kg SAHA, p>0.05). All data are plotted as mean ±SEM.</p

SAHA treated slices exhibit enhanced induction of LTP and impaired LTD.

<p>(A) An induction protocol that was subthreshold in vehicle treated slices readily evoked LTP in SAHA treated slices (p<0.05, n = 5,5). Example traces before and after LTP induction are shown in red for SAHA and black for vehicle treated slices (scale bars represent 20 pA and 20 ms). (B) An induction protocol that readily induced LTD in vehicle treated slices could not produce LTD in SAHA treated slices (p<0.05, n = 9 vehicle, 8 SAHA). Example traces before and after LTD induction are shown in red for SAHA and black for vehicle treated slices (scale bars represent 25 pA and 20 ms). Data are plotted as mean ±SEM.</p