Kadowaki Atsushi, Sada Nagisa, Juge Narinobu, Wakasa Ayaka, Moriyama Yoshinori, Inoue Tsuyoshi
Department of Biophysical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Advanced Science Research Center, Okayama University, Okayama, Japan.
Epilepsia. 2017 May;58(5):845-857. doi: 10.1111/epi.13718. Epub 2017 Mar 11.
The ketogenic diet is clinically used to treat drug-resistant epilepsy. The diet treatment markedly increases ketone bodies (acetoacetate and β-hydroxybutyrate), which work as energy metabolites in the brain. Here, we investigated effects of acetoacetate on voltage-dependent Ca channels (VDCCs) in pyramidal cells of the hippocampus. We further explored an acetoacetate analog that inhibited VDCCs in pyramidal cells, reduced excitatory postsynaptic currents (EPSCs), and suppressed seizures in vivo.
The effects of acetoacetate and its analogs on VDCCs and EPSCs were evaluated using patch-clamp recordings from CA1 pyramidal cells of mouse hippocampal slices. The in vivo effects of these reagents were also evaluated using a chronic seizure model induced by intrahippocampal injection of kainate.
Acetoacetate inhibited VDCCs in pyramidal cells of hippocampal slices, and reduced EPSCs in slices exhibiting epileptiform activity. More potent EPSC inhibitors were then explored by modifying the chemical structure of acetoacetate, and 2-phenylbutyrate was identified as an acetoacetate analog that inhibited VDCCs and EPSCs more potently. Although acetoacetate is known to inhibit vesicular glutamate transporters (VGLUTs), 2-phenylbutyrate did not inhibit VGLUTs, showing that 2-phenylbutyrate is an acetoacetate analog that preferably inhibits VDCCs. In addition, 2-phenylbutyrate markedly reduced EPSCs in slices exhibiting epileptiform activity, and suppressed hippocampal seizures in vivo in a mouse model of epilepsy. The in vivo antiseizure effects of 2-phenylbutyrate were more potent than those of acetoacetate. Finally, intraperitoneal 2-phenylbutyrate was delivered to the brain, and its brain concentration reached the level enough to reduce EPSCs.
These results demonstrate that 2-phenylbutyrate is an acetoacetate analog that inhibits VDCCs and EPSCs in pyramidal cells, suppresses hippocampal seizures in vivo, and has brain penetration ability. Thus 2-phenylbutyrate provides a useful chemical structure as a lead compound to develop new antiseizure drugs originating from ketone bodies.
生酮饮食在临床上用于治疗耐药性癫痫。饮食治疗可显著增加酮体（乙酰乙酸和β-羟基丁酸），它们在大脑中作为能量代谢物发挥作用。在此，我们研究了乙酰乙酸对海马锥体细胞中电压依赖性钙通道（VDCCs）的影响。我们进一步探索了一种乙酰乙酸类似物，它能抑制锥体细胞中的VDCCs，减少兴奋性突触后电流（EPSCs），并在体内抑制癫痫发作。
使用来自小鼠海马切片CA1锥体细胞的膜片钳记录来评估乙酰乙酸及其类似物对VDCCs和EPSCs的影响。还使用海马内注射海藻酸诱导的慢性癫痫模型评估了这些试剂的体内作用。
乙酰乙酸抑制海马切片锥体细胞中的VDCCs，并降低表现出癫痫样活动的切片中的EPSCs。然后通过修饰乙酰乙酸的化学结构探索了更有效的EPSC抑制剂，2-苯丁酸被鉴定为一种能更有效地抑制VDCCs和EPSCs的乙酰乙酸类似物。尽管已知乙酰乙酸可抑制囊泡谷氨酸转运体（VGLUTs），但2-苯丁酸并不抑制VGLUTs，表明2-苯丁酸是一种优先抑制VDCCs的乙酰乙酸类似物。此外，2-苯丁酸显著降低表现出癫痫样活动的切片中的EPSCs，并在癫痫小鼠模型中体内抑制海马癫痫发作。2-苯丁酸的体内抗癫痫作用比乙酰乙酸更强。最后，将腹腔注射的2-苯丁酸输送到大脑，其脑内浓度达到足以降低EPSCs的水平。
这些结果表明，2-苯丁酸是一种乙酰乙酸类似物，它能抑制锥体细胞中的VDCCs和EPSCs，在体内抑制海马癫痫发作，并具有脑渗透能力。因此，2-苯丁酸作为一种先导化合物，提供了一种有用的化学结构，可用于开发源自酮体的新型抗癫痫药物。
