GABA receptor

盐酸加波沙朵/Gaboxadol（0.34、3.5和7.0 μM）以剂量依赖性方式降低Caco-2单层细胞的通透性，平均Papp值为8.1 × 10-6 cm·s-1、6.1 × 10 -1 cm·s-1（0.34、3.5 和 7 μM 加波沙朵），5.6 × 10-6 cm·s-1（0.34、3.5 和 7 μM 加波沙朵）[3]。

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Gaboxadol在体外通过hPAT1转运的特性 [3]
在Gaboxadol/加博沙多浓度增加的情况下，通过测量hPAT1底物脯氨酸进入cco -2细胞单层的顶端摄取，研究了加博沙多和hPAT1之间的相互作用（图1）。加博沙多降低cco -2细胞单层的顶端脯氨酸摄取，估计抑制剂亲和力（Ki值）为6.6 mmol·L−1。同样，已知的PAT1抑制剂色氨酸也降低了脯氨酸的顶端吸收，Ki值为7.7 mmol·L−1。研究了三种浓度（0.34、3.5和7.0 mmol·L−1）下加博沙多在caco2细胞单层上的经上皮转运（A-B）通量。对于0.34、3.5和7 mmol·L−1的加博沙多，加博沙多转运的平均Papp值分别为8.1 × 10−6 cm·s−1、6.1 × 10−6 cm·s−1和5.6 × 10−6 cm·s−1（图2）。因此，随着加博沙多浓度的增加，加博沙多通过Caco-2细胞单层的通透性降低（P < 0.05）。在35 mmol·L−1色氨酸存在的情况下，研究了加博沙多在caco2细胞单层中使用3.5 mmol·L−1加博沙多的顶端浓度，有或没有pH梯度，双向运输（图2）。加博沙多在A-B方向的转运量约为B-A方向的5倍（P < 0.005）。色氨酸的存在使加博沙多的通透性降低了53% （Papp为2.9 × 10−6 cm·s−1,P < 0.005）。在没有质子梯度的情况下，加博沙多的渗透率降低了82%，为1.1 × 10−6 cm·s−1 （P < 0.005）。在色氨酸存在、质子梯度不存在的情况下，加博沙多在B-A方向的渗透性与[3H]-甘露醇的渗透性相似（Papp为1.6±0.36 × 10−6 cm·s−1）。此外，在转运实验中，加博沙多和色氨酸的存在并没有改变美托洛尔或甘露醇在Caco-2细胞单层间转运的通透性。甘露醇和美托洛尔的渗透率分别为1.6±0.36 × 10−6 cm·s−1和6.9±0.99 × 10−6 cm·s−1。综上所述，加博沙多通过Caco-2细胞单层的上皮转运是ph依赖的，可以被色氨酸抑制，并在A-B方向极化。综上所述，这些观察结果表明，hPAT1介导了加博沙多在人肠上皮细胞腔膜上的转运，这一转运步骤在很大程度上决定了加博沙多的上皮转运。

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加博沙多/Gaboxadol转运通过hPAT1体外的表征[3]
在加博沙多浓度增加的情况下，通过测量hPAT1底物脯氨酸进入cco -2细胞单层的顶端摄取，研究了加博沙多和hPAT1之间的相互作用（图1）。加博沙多降低cco -2细胞单层的顶端脯氨酸摄取，估计抑制剂亲和力（Ki值）为6.6 mmol·L−1。同样，已知的PAT1抑制剂色氨酸也降低了脯氨酸的顶端吸收，Ki值为7.7 mmol·L−1。研究了三种浓度（0.34、3.5和7.0 mmol·L−1）下加博沙多在caco2细胞单层上的经上皮转运（A-B）通量。对于0.34、3.5和7 mmol·L−1的加博沙多，加博沙多转运的平均Papp值分别为8.1 × 10−6 cm·s−1、6.1 × 10−6 cm·s−1和5.6 × 10−6 cm·s−1（图2）。因此，随着加博沙多浓度的增加，加博沙多通过Caco-2细胞单层的通透性降低（P < 0.05）。在35 mmol·L−1色氨酸存在的情况下，研究了加博沙多在caco2细胞单层中使用3.5 mmol·L−1加博沙多的顶端浓度，有或没有pH梯度，双向运输（图2）。加博沙多在A-B方向的转运量约为B-A方向的5倍（P < 0.005）。色氨酸的存在使加博沙多的通透性降低了53% （Papp为2.9 × 10−6 cm·s−1,P < 0.005）。在没有质子梯度的情况下，加博沙多的渗透率降低了82%，为1.1 × 10−6 cm·s−1 （P < 0.005）。在色氨酸存在、质子梯度不存在的情况下，加博沙多在B-A方向的渗透性与[3H]-甘露醇的渗透性相似（Papp为1.6±0.36 × 10−6 cm·s−1）。此外，在转运实验中，加博沙多和色氨酸的存在并没有改变美托洛尔或甘露醇在Caco-2细胞单层间转运的通透性。甘露醇和美托洛尔的渗透率分别为1.6±0.36 × 10−6 cm·s−1和6.9±0.99 × 10−6 cm·s−1。综上所述，加博沙多通过Caco-2细胞单层的上皮转运是ph依赖的，可以被色氨酸抑制，并在A-B方向极化。综上所述，这些观察结果表明，hPAT1介导了加博沙多在人肠上皮细胞腔膜上的转运，这一转运步骤在很大程度上决定了加博沙多的上皮转运。

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Gaboxadol是Caco-2细胞单层中质子偶联氨基酸转运体hPAT1的底物[3]
加博沙多抑制cco -2细胞对hPAT1底物脯氨酸的顶端吸收，Ki值为6.6 mmol·L−1。这种亲和力与最近观察到的其他hPAT1底物如GABA （3.1 mmol·L−1）和GABA类似物muscimol （1.7 mmol·L−1）和THPO （11.3 mmol·L−1）的亲和力相当（Larsen et al., 2008）。色氨酸的亲和力与加博沙多相当，为7.7 mmol·L−1。Metzner等人（2005）先前将色氨酸描述为PAT1的抑制剂，并报道了cco -2细胞中通过hPAT1摄取脯氨酸的Ki值为4.7 mmol·L−1。考虑到两个实验室对hPAT1脯氨酸亲和力的微小差异；Metzner等人报告的Kt值为1.4 mmol·L−1，而Larsen等人报告的Km值分别为3.6 mmol·L−1，两项研究中色氨酸对hPAT1的亲和力相当(Metzner等人，2005；Larsen et al., 2008)。根据其他小组发表的结果(Thwaites et al., 1993；Metzner et al., 2005)，我们发现ccao -2细胞的大部分顶端脯氨酸运输是由hPAT1介导的，没有观察到脯氨酸的其他钠依赖性或钠依赖性转运体的证据（Larsen et al., 2008）。其他氨基酸转运蛋白，如顶钠依赖性氨基酸转运蛋白B0 (B0AT1), B0,+ (ATB0,+)和系统（ASC） （ASCT2）不太可能参与加博沙多的转运。它们都存在于Caco-2细胞中，但它们的底物移位不是质子偶联的。此外，与PAT1相比，这些转运蛋白对底物的亲和力值更高，例如ASC (ASCT2)，约为100 μ mol·L−1 (Uchiyama et al., 2005)；B0 (B0AT1), 500-700µmol·L−1 （Broer et al., 2004）和B0，+ (ATB0,+)，约150µmol·L−1 （Hatanaka et al., 2002）。

Gaboxadol在Caco-2细胞单层间的上皮运输呈尖向基底侧极化。色氨酸可以抑制加博沙多的转运，并依赖于顶端供体溶液的pH。此外，随着加博沙多浓度的增加，加博沙多在根尖-基底侧方向的Papp降低。这与加博沙多通过hPAT1转运一致，该途径约占总上皮转运的80%。在小鼠小肠和Caco-2细胞中发现了氨基酸转运系统b0，+，分别占丙氨酸和精氨酸总转运量的15-85% (Wenzel et al., 2001；Dave et al., 2004)。然而，色氨酸与系统b0 +的结合尚未明确显示(Su et al., 1992；Tate et al., 1992)，此外，阳离子氨基酸、两性离子氨基酸和胱氨酸对体系b0，+具有µmol·L−1的亲和性（Palacin, 1994）。因此，如果加博沙多通过系统b0，+运输到任何显著程度，它在Caco-2细胞中应该是明显的。在体外，经上皮的加博沙多转运还具有ph依赖性，而PAT1是目前已知的肠道中唯一的质子偶联氨基酸转运蛋白。加巴喷丁（也是两性离子γ-氨基类似物）在大鼠小肠中的渗透性被证明是质子独立的（Nguyen et al., 2007），因此加巴喷丁和加博沙多存在不同的根尖转运机制。结果表明，加博沙多是Caco-2细胞单层中hPAT1的底物，hPAT1介导加博沙多通过肠道肠细胞的管腔膜运输，这似乎对由此产生的上皮运输很重要。加博沙多跨基底外膜外排的机制尚不清楚。

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加博沙多/Gaboxadol的体内吸收 在狗[3]
口服给药后，加博沙多在犬体内吸收迅速，Tmax约为0.46 h，生物利用度高达85%。这些观察结果与先前关于人体口服吸收的研究一致，表明加博沙多的Tmax约为0.5 h，生物利用度约为90% (Schultz等人，1981；Lund et al., 2006)。一旦被吸收，加博沙多主要以加博沙多的形式从尿液中排出，而一小部分以葡萄糖醛酸偶联物的形式排出，在大鼠和小鼠中占2-7%，在两个人类受试者中占30-35% (Schultz et al., 1981；Lund et al., 2006；Shadle et al., 2006)。总的来说，这表明在狗体内，加博沙多被迅速和完全吸收，可能在小肠近端，吸收后代谢最小。

盐酸加波沙朵/Gaboxadol（腹膜内注射；0.5、1、1.5、2、3、4 或 5 mg/kg；每天三次；间隔三天）使 Fmr1 KO2 小鼠的步行距离正常化至 0.5 mg/kg WT 活动水平，此外，该化学物质对 Fmr1 KO2 小鼠的运动活性没有影响 [2]。

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在这里，我们试图评估Gaboxadol（也称为OV101和THIP），一种选择性和有效的含有δ亚基的突触外GABAA受体（dSEGA）的激动剂，通过评估其在相对未表征的FXS小鼠模型（Fmr1 KO2小鼠）中的异常行为正常化能力，作为FXS的治疗剂。四个行为领域（多动、焦虑、攻击和重复行为）通过一系列行为分析进行了探讨。结果显示，与野生型（WT）相比，Fmr1 KO2小鼠过度活跃，具有异常焦虑样行为，更易怒和更具攻击性，重复行为频率增加，这些都是FXS个体的行为缺陷。加博沙多/Gaboxadol治疗使Fmr1 KO2小鼠中观察到的所有异常行为恢复到WT水平，为其治疗FXS的潜在益处提供了证据。我们发现，仅加博沙多就能增强突触外GABA受体，足以使FXS模型中的许多行为缺陷正常化，这些缺陷的终点可直接转化为FXS的临床表现。综上所述，这些数据支持未来对FXS患者加博沙多的评估，特别是关于多动症、焦虑、易怒、攻击和重复行为的症状。[2]

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加博沙多使Fmr1 KO2小鼠的过度活动正常化[2]
多动是人类FXS的一个显著特征(Bailey et al., 2008；Wheeler et al., 2014；Hagerman et al., 2017)，并已在先前表征的荷兰-比利时Fmr1 KO小鼠中可靠地复制(Olmos-Serrano et al., 2010；Kazdoba et al., 2014)。为了测试Fmr1 KO2小鼠是否表现出运动亢进，以及加博沙多是否能使这种异常行为正常化，我们给Fmr1 KO2小鼠注射了载药或加博沙多（0.5-5 mg/kg, i.p），并在OFT测试前30分钟给WT窝仔注射载药。记录30 min在OFT内行走的总距离（cm）。结果显示，Fmr1 KO2小鼠的行走距离较WT窝中对照组显著增加（图1，F(8,81) = 21.27, p < 0.0001），与其他FXS模型的结果一致。加博沙多（0.5 mg/kg）使Fmr1 KO2小鼠的行走距离正常化到WT活性水平（图1）。高剂量的加博沙多（1 - 5 mg/kg, ig）对Fmr1 KO2小鼠的运动活性没有影响（图1）。这些结果不能归因于加博沙多的镇静作用，因为在WT C57Bl/6或BALB/c小鼠中，加博沙多高达2.0 mg/kg的剂量在60分钟的OFT中对运动活动没有影响（数据未显示），这与先前的研究一致，表明加博沙多对WT小鼠（Olmos-Serrano等人，2011）或大鼠（Silverman等人，2016）的运动没有影响。

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Fmr1 KO2小鼠焦虑样行为经加博沙多归一化 [2]
为了评估加博沙多对Fmr1 KO2小鼠焦虑样行为的影响，采用了三种不同的行为测试：OFT、LDT和SAT的中心移动距离。中心移动距离的增加被解释为焦虑的减少，并利用了小鼠在进入新环境时保持在周边的固有偏好。Fmr1 KO2小鼠注射加博沙多（0.5-5 mg/kg, i.p）， WT窝仔在放置于OFT中30分钟前注射载药。与WT对照组相比，Fmr1 KO2小鼠在中心行走的总距离显著增加（图2A， F(8,81) = 21.32, p < 0.0001）。加博沙多治疗（0.5 mg/kg, i.p.p）使Fmr1 KO2对中心距离的影响正常化，达到与WT对照组相当的水平（图2A）。在本实验中，高剂量的加博沙多（1-5 mg/kg）对Fmr1 KO2小鼠没有影响（图2A）。

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加博沙多使Fmr1 KO2小鼠的易怒和攻击行为正常化 [2]
与其他形式的综合征自闭症一样，很大一部分FXS患者表现出易怒、社交焦虑和攻击性。这些异常行为可以在啮齿类动物中建模，通过表征测试小鼠和新笼伴侣之间的si。为了验证Fmr1 KO2突变体易怒和攻击性增加的假设，我们量化了摇尾、咬人行为、攀爬行为和攻击延迟的实例。小鼠入笼前30 min分别注射载药或加博沙多（0.5 ~ 5mg /kg, i.p）。 尾巴嘎嘎作响，或尾巴的快速振动，反映了攻击性和战斗倾向。与WT对照组相比，Fmr1 KO2小鼠的摇尾频率显著增加（图3A， F(8,81) = 16.03, p < 0.0001）。加博沙多（0.5、1.5和5.0 mg/kg）使Fmr1 KO2小鼠的效果正常化，达到与WT对照组相当的水平（图3A）。

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Gaboxadol使Fmr1 KO2小鼠的重复行为正常化[2]
坚持和重复行为在FXS患者中很常见，并且具有高度破坏性(Arron et al., 2011；Leekam et al., 2011；Hall et al., 2016)。为了验证这些特征可能在Fmr1 KO2动物中观察到的假设，我们量化了WT和Fmr1 KO2突变小鼠的打圈、自我梳理和刻板印象。小鼠分别注射载药或加博沙多（0.5 ~ 5mg /kg, i.p）后，在实验室内测量逆时针转数（CCW）。与WT对照组相比，Fmr1 KO2小鼠在5分钟测试期间CCW转数显著增加（图4A， F(8,81) = 25.46, p < 0.0001）。向Fmr1 KO2小鼠注射加博沙多（0.5、1.0 mg/kg）后，CCW转数恢复到WT水平（图4A）。基因型对顺时针旋转无影响（p = 0.386，数据未显示）。

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加博沙多的体内吸收 在狗[3]
口服给药后，加博沙多在犬体内吸收迅速，Tmax约为0.46 h，生物利用度高达85%。这些观察结果与先前关于人体口服吸收的研究一致，表明加博沙多的Tmax约为0.5 h，生物利用度约为90% (Schultz等人，1981；Lund et al., 2006)。一旦被吸收，加博沙多主要以加博沙多的形式从尿液中排出，而一小部分以葡萄糖醛酸偶联物的形式排出，在大鼠和小鼠中占2-7%，在两个人类受试者中占30-35% (Schultz et al., 1981；Lund et al., 2006；Shadle et al., 2006)。总的来说，这表明在狗体内，加博沙多被迅速和完全吸收，可能在小肠近端，吸收后代谢最小。

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联合给药色氨酸后，Gaboxadol/加博沙多的体内吸收[3]
同时给药hPAT1抑制剂色氨酸对加博沙多的吸收谱有剂量依赖性，导致Cmax降低和Tmax增加。加博沙多吸收率的降低可由胃排空率的改变引起。在人类中，胃排空的速度随着一餐所摄入的卡路里数量的增加而降低(Calbet和MacLean, 1997；Sunesen et al., 2005)，在狗的实验中，膳食成分也被证明可以延长胃排空（Mizuta et al., 1990）。为了排除观察到的对加博沙多吸收的影响是胃排空改变的结果，研究了常被用作胃排空标志的扑热息痛的累积吸收曲线(Calbet and MacLean, 1997；Sunesen et al., 2005)，在色氨酸存在的情况下进行了研究。高剂量色氨酸对扑热息痛的胃排空无显著影响。然而，色氨酸对加博沙多的ka有显著影响。由于共给色氨酸改变了加博沙多Tmax， Cmax和ka，而Fa， ke和AUC不变，色氨酸的作用可能是色氨酸和加博沙多在吸收部位相互作用的结果，而不是由于胃排空的改变。其他研究表明，加博沙多与血浆蛋白结合程度较低，不被细胞色素p -450代谢（Lund et al., 2006）。因此，根据体外实验结果表明，hPAT1介导了Caco-2单层中大部分加博沙多的腔内转运，似乎体内观察也可以解释为pat1介导的狗对加博沙多的吸收，而这种吸收被色氨酸的共同给药所减少。从色氨酸对加博沙多Cmax的影响或对肠道吸收速率常数ka的影响来估计色氨酸抑制加博沙多肠道运输的体内亲和值。IC50值分别为10.1和12.6 mmol·L−1。如前所述，在Caco-2细胞中，hPAT1对色氨酸的体外亲和力（通过hPAT1抑制脯氨酸运输）为7.7 mmol·L−1。考虑到色氨酸的抑制作用是针对两种不同的化合物（脯氨酸和加博沙多）测量的，并且体内转运不仅包括腔内转运，还包括体循环中的外观，其中遇到了加博沙多跨几个膜的转移，IC50值彼此惊人地接近。hPAT1底物的特点是在毫摩尔范围内具有亲和力，并且在整个肠道中表达的转运蛋白具有高容量（Chen et al., 2003）。加博沙多和色氨酸的摩尔给药比高达1:41，由于它们对hPAT1的亲和力相当，Cmax和ka的降低可能是由于加博沙多和色氨酸在吸收部位，即小肠肠细胞管腔膜上的PAT1蛋白处的竞争性相互作用。因此，最大血浆浓度随着Tmax的延长而出现，但由于容量过大和肠道中PAT1的表达，随着吸收沿着肠道的长度进行，吸收分数保持不变。因此，加博沙多的峰值血浆浓度可以通过修改吸收过程来降低，如这里所示，或者通过更经典的缓释制剂方法，如前面所建议的（Kjaer和Nielsen， 1983）。

细胞培养和体外实验方案如前所述（Larsen等人，2008年）。将第20 ~ 29代Caco-2细胞接种到Transwell™插入物（1.12 cm2, 0.4µm孔径）上，于接种后第25 ~ 28天进行实验。在汉克斯平衡盐溶液（HBSS）缓冲液中，测定了Gaboxadol/加博沙多在Caco-2细胞单分子层从根尖向基底侧方向（A-B）和基底侧向根尖方向（B-A）的顶端摄取和上皮转运。在所有实验中，基底外侧的缓冲液pH为7.4。除非另有说明，在加入Gaboxadol/加博沙多盐酸或35 mmol·L−1色氨酸后，将应用于顶室的缓冲液调至pH 6.0。研究了0.34、3.5或7.0 mmol·L−1Gaboxadol的转运情况。这些浓度的选择是基于人单次睡前口服15mg加博沙多，提供约0.34 mmol·L−1的通径浓度，以及所获得的加博沙多对hPAT1的亲和力。在根尖室中加入含有12.5 nmol·L−1(0.5µCi) L-(3H)脯氨酸和0-30 mmol·L−1加博沙多或0-35 mmol·L−1色氨酸的新鲜根尖HBSS培养基，进行根尖摄取实验。5 min后终止根尖摄取实验，对样品进行闪烁计数分析。[3]

Animal/Disease Models: Fmr1 KO2 mice (the Fmr1 promoter and first exon are deleted, resulting in mice with missing mRNA and protein) [2]
Doses: 0.5, 1, 1.5, 2, 3, 4 or 5 mg/kg given Medication: intraperitoneal (ip) injection
Experimental Results: Normalized hyperactivity was observed in Fmr1 KO2 mice.

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Mice in the same cage were injected with the same dose of Gaboxadol or vehicle, and mutants and controls were housed separately. All mice were group-housed in plastic cages (35 × 30 × 12 cm), five per cage, and habituated to the animal facility for at least a week before testing. The room temperature (21 ± 2°C), relative humidity (55 ± 5%), a 12 h light-dark cycle (lights on 7 am–7 pm) and air exchange (16 times per hour) were automatically controlled. All mice had ad libitum access to food and water. All testing was conducted in the light-phase by an investigator blind to genotype and drug treatment. [2]

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Gaboxadol Treatment and Experimental Timeline: Fmr1 KO2 mice were injected with vehicle (0.9% sterile saline) or Gaboxadol (0.5, 1, 1.5, 2, 3, 4, or 5 mg/kg, i.p.) 30 min prior to behavioral testing on each testing day, with a three-day interval between each test to avoid any cumulative effect of the drug administration. Wild-type mice injected with vehicle at the same time point were also included in all experiments. Behavioral screening of the mice (n = 10 per group) was conducted in the following order with 2–3 days between each test: Open Field Test (OFT; day 1), successive alleys (day 4), light/dark box (day 7), social tests and aggression (day 10), and self-grooming and stereotypy (day 12). [2]

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Absorption of Gaboxadol in dogs [3]
All animal care and experimental studies were approved by the Animal Welfare Committee, appointed by the Danish Ministry of Justice, and were carried out in compliance with EC Directive 86/609/EEC, the Danish law regulating experiments on animals and NIH Guidelines for the Care and Use of Laboratory Animals. Six full-grown male beagle dogs (body weight 15.9–21.7 kg) were selected and allocated into a Roman quadrant design and assigned to receive all the six formulations of Gaboxadol hydrochloride randomly during 6 weeks. The dogs were fasted for 20–24 h before the initiation of the experiment and fed again 10 h after the administration. The gaboxadol dose was given either as an intravenous injection (1.0 mL·kg−1) or as an oral solution given by gavage (5.0 mL·kg−1) directly into the stomach using a soft tube. All dogs received 2.5 mg·kg−1 gaboxadol. In addition to gaboxadol, the oral formulations contained 0, 2.5, 10, 50 or 150 mg·kg−1 of tryptophan to ensure simultaneous co-administration of the two compounds. All solutions were adjusted to a pH of 5.2, and osmolarity was checked with a Vapro vapor pressure osmometer (model 552O, Wescor Inc., Logan, UT, USA), the intravenous solutions were adjusted to iso-osmolarity with glucose. Blood samples (2 mL) were taken from the cephalic vein by individual venepuncture and collected into Eppendorf tubes containing 200 IE heparin as an anticoagulant. Samples were collected before administration of gaboxadol and after 5, 15, 30, 60, 90 min, and 2, 3, 4, 6, 8 and 10 h after gaboxadol administration. The plasma was harvested immediately by centrifugation for 15 min at 2200 g and 4–8°C and stored at −80°C until further analysis. The animals had a 6-day washout period between treatments.

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Investigation of gastric emptying in dog [3]
A protocol similar to the one described earlier using paracetamol as a marker was used to evaluate the influence of tryptophan on the gastric emptying rate in dogs. Six dogs (body weight 16.1–21.5 kg) were selected and randomly allocated to receive three formulations of paracetamol in a crossover study. The dogs received 50 mg·kg−1 paracetamol as an intravenous injection (1 mL·kg−1) or as an oral solution (5 mL·kg−1) containing 2.5 mg·kg−1 Gaboxadol and 0 or 150 mg·kg−1 tryptophan. Fasting of the dogs, drug administration, blood sampling and washout were done as described earlier.

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Analytical methods [3]
Quantification of Gaboxadol in plasma and buffer: Gaboxadol was extracted from plasma and buffer samples by liquid extraction. 100 µL HBSS or plasma samples were mixed with 25 µL internal standard (d4-gaboxadol) and 25 µL purified water. Protein precipitation was carried out by addition of 400 µL cold acetonitrile. After centrifugation at 10 000 g for 15 min, 425 µL of supernatant was transferred to glass tubes and evaporated to dryness under nitrogen at 45°C. The samples were redissolved in 80 µL of methanol/acetonitrile (30:70), whirl-mixed for 10 min and centrifuged for 3 min at 3300× g. Gaboxadol was subsequently quantified by hydrophilic interaction chromatography followed by tandem mass spectrometry (MS/MS) detection using a protocol modified from Kall et al. (2007). The liquid chromatography (LC) system comprised by an Agilent 1100 series pump and degasser. An Asahipak amino column, (NH2P-50, 150 × 2 mm) from Phenomenex (Torrance, CA, USA) was used with a mobile phase of 20.0 mmol·L−1 ammonium acetate (pH 4): acetonitrile (30:70) and a flow rate of 0.2 mL·min−1. Twenty-microlitre samples were injected onto the column, which was kept at room temperature. The total run time was 10 min with the first 5 min of elution let to waste. The elution time of gaboxadol on the column was approximately 8 min. The MS/MS system used consisted of a Sciex API 4000 MS/MS detector with a Turbo Ion Spray and Turbo V source (Applied Biosystems, Foster City, CA, USA). The signals were linear between 0.5 and 2500 ng·mL−1, and the limit of quantification by this procedure was 0.5 ng·mL−1. The software was from Analyst™ (Applied Biosystems, version 4.0).

Pharmacokinetic analysis of oral Gaboxadol absorption in dog [3]
Gaboxadol plasma concentration profiles following oral or intravenous administration of 2.5 mg·kg−1 gaboxadol in beagle dogs were monitored over 10 h (Figure 3). The bioavailability, Fa, of gaboxadol following oral administration in dog was high (over 80%) (Table 1). Oral co-administration of 2.5–150 mg·kg−1 tryptophan did not change the AUC of gaboxadol significantly, and the mean relative bioavailability of the formulations varied between 75 (10 mg·kg−1 tryptophan) and 86.1% (2.5 mg·kg−1 tryptophan). Also, the elimination rate constant (ke) and the clearance (CL) of gaboxadol did not change with co-administration of tryptophan. However, co-administration of 150 mg·kg−1 tryptophan decreased the maximal gaboxadol plasma concentration, Cmax, from 2502 to 1419 ng·mL−1, that is, 57%. Furthermore, the time required to reach the maximal plasma concentration, Tmax, was increased from 0.46 h to 1.5 h (P < 0.01). The Cmax values of the five dose groups were subsequently fitted to a dose-response curve (Figure 4), which indicated a direct interaction between gaboxadol absorption and tryptophan concentration. The in vivo IC50 value of tryptophan on gaboxadol Cmax was estimated to be 12.6 mg·kg−1, which is equivalent to a concentration of 12.3 mmol·L−1 tryptophan (not corrected for dilution in gastric and intestinal fluids).

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Absorption rate constants of Gaboxadol and paracetamol [3]
Co-administration of increasing tryptophan doses gradually changed the mean cumulative fraction of absorbed gaboxadol as seen in the deconvolution profiles in Figure 5A. The absorption of gaboxadol in the presence of 150 mg·kg−1 tryptophan was significantly decreased at time points 0.5–1.25 h compared with the absorption of gaboxadol alone. An oral dose of 91.5 ± 3.3% of paracetamol was absorbed after 60 min (Figure 5B) indicating that gastric emptying happens mainly within the first hour after administration. Co-administration of 150.0 mg·kg−1 of tryptophan did not significantly change the gastric emptying rate, as the fraction of absorbed paracetamol in the absence or presence of tryptophan was not significantly different at the time points tested. The pharmacokinetic parameters Tmax, AUC and CL of plasma paracetamol concentrations were not significantly different from parameters obtained after co-administration of paracetamol and tryptophan (results not shown). Based on the profiles shown in Figure 5A, the absorption rate constant, ka, of gaboxadol were calculated and these are depicted as a function of the logarithmic tryptophan dose in Figure 6A. The ka of gaboxadol was decreased by co-administration of tryptophan with an in vivo IC50 value on gaboxadol absorption of 10.3 mg·kg−1, which corresponds to an oral solution with a concentration of 10.1 mmol·L−1 tryptophan. Figure 6B shows that 150 mg·kg−1 of the PAT1 inhibitor tryptophan significantly decreased the absorption rate constant of gaboxadol (P < 0.01), whereas it had no significant effect on the absorption rate constant of paracetamol.

[1]. Neurochemicals for the investigation of GABA(C) receptors. Neurochem Res. 2010 Dec;35(12):1970-7.

[2]. Gaboxadol Normalizes Behavioral Abnormalities in a Mouse Model of Fragile X Syndrome.Front Behav Neurosci. 2019 Jun 25;13:141.

[3]. Intestinal gaboxadol absorption via PAT1 (SLC36A1): modified absorption in vivo following co-administration of L-tryptophan.Br J Pharmacol. 2009 Aug;157(8):1380-9.

GABA(C) receptors are being investigated for their role in many aspects of nervous system function including memory, myopia, pain and sleep. There is evidence for functional GABA(C) receptors in many tissues such as retina, hippocampus, spinal cord, superior colliculus, pituitary and the gut. This review describes a variety of neurochemicals that have been shown to be useful in distinguishing GABA(C) receptors from other receptors for the major inhibitory neurotransmitter GABA. Some selective agonists (including (+)-CAMP and 5-methyl-IAA), competitive antagonists (such as TPMPA, (±)-cis-3-ACPBPA and aza-THIP), positive (allopregnanolone) and negative modulators (epipregnanolone, loreclezole) are described. Neurochemicals that may assist in distinguishing between homomeric ρ1 and ρ2 GABA(C) receptors (2-methyl-TACA and cyclothiazide) are also covered. Given their less widespread distribution, lower abundance and relative structural simplicity compared to GABA(A) and GABA(B) receptors, GABA(C) receptors are attractive drug targets. [1]

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Gaboxadol normalized all of the tested behavioral deficits of Fmr1 KO2 mice at a dose of 0.5 mg/kg. While higher doses also normalized irritability and aggressive behaviors, this was not observed for other behavioral domains evaluated. One explanation for the somewhat narrow efficacy window observed here may come from previous work showing compromised information processing by either insufficient or excess tonic inhibition, the physiological process that Gaboxadol potentiates. Under this model, the behavioral benefit of drug at high doses would be offset by pharmacologically introduced FXS-independent deficits (Duguid et al., 2012). Our results provide robust evidence of the potential benefit of Gaboxadol in reversing ASD related behaviors, aggression and sociability. Taken together, these results support the hypothesis that potentiation of extrasynaptic GABAA receptors by gaboxadol may be of benefit in individuals with FXS. In conclusion, these data support the future evaluation of gaboxadol in individuals with FXS, particularly with regard to symptoms of hyperactivity, anxiety, ASD related stereotypy, sociability, irritability, aggression, and cognition.[2]

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Background and purpose: Gaboxadol has been in development for treatment of chronic pain and insomnia. The clinical use of Gaboxadol has revealed that adverse effects seem related to peak serum concentrations. The aim of this study was to investigate the mechanism of intestinal absorption of gaboxadol in vitro and in vivo.
Experimental approach: In vitro transport investigations were performed in Caco-2 cell monolayers. In vivo pharmacokinetic investigations were conducted in beagle dogs. Gaboxadol doses of 2.5 mg.kg(-1) were given either as an intravenous injection (1.0 mL.kg(-1)) or as an oral solution (5.0 mL.kg(-1)).
Key results: Gaboxadol may be a substrate of the human proton-coupled amino acid transporter, hPAT1 and it inhibited the hPAT1-mediated L-[(3)H]proline uptake in Caco-2 cell monolayers with an inhibition constant K(i) of 6.6 mmol.L(-1). The transepithelial transport of gaboxadol was polarized in the apical to basolateral direction, and was dependent on gaboxadol concentration and pH of the apical buffer solution. In beagle dogs, the absorption of gaboxadol was almost complete (absolute bioavailability, F(a), of 85.3%) and T(max) was 0.46 h. Oral co-administration with 2.5-150 mg.kg(-1) of the PAT1 inhibitor, L-tryptophan, significantly decreased the absorption rate constant, k(a), and C(max), and increased T(max) of gaboxadol, whereas the area under the curve and clearance of gaboxadol were constant.
Conclusions and implications: The absorption of Gaboxadol across the luminal membrane of the small intestinal enterocytes is probably mediated by PAT1. This knowledge is useful for reducing gaboxadol absorption rates in order to decrease peak plasma concentrations.[3]

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In conclusion, the present study shows for the first time that the high permeability of Gaboxadol across Caco-2 cell monolayers is most likely due to PAT1-mediated transport across the luminal membrane resulting in a high transepithelial transport. The in vitro gaboxadol transport kinetics and the pharmacokinetics observed in dogs support the conclusion that PAT1 mediates transport of gaboxadol across the mucosal membrane both in vitro as well as in vivo. In addition, the present study indicates that it is possible to exploit transporter activity in order to modify or control the intestinal absorption of drug substances. The formulation design provides a simple basis for decreasing peak plasma concentration of gaboxadol, while maintaining a high bioavailability. This may aid in reducing side effects related to high plasma peak concentrations.[3]

C6H9CLN2O2

176.6009

176.035

C, 40.81; H, 5.14; Cl, 20.07; N, 15.86; O, 18.12

85118-33-8

THIP;64603-91-4

5702253

White to off-white solid powder

295.7ºC at 760 mmHg

236 °C

132.6ºC

0.744

58.03

3

3

0

11

210

0

O=C1NOC2=C1CCNC2.[H]Cl

ZDZDSZQYRBZPNN-UHFFFAOYSA-N

InChI=1S/C6H8N2O2.ClH/c9-6-4-1-2-7-3-5(4)10-8-6/h7H,1-3H2,(H,8,9)1H

4,5,6,7-Tetrahydroisoxazolo(5,4-c)pyridin-3(2H)-one monohydrochloride

OV-101; OV101; Lu-02-030; MK-0928; GABOXADOL HYDROCHLORIDE; 85118-33-8; 478RVH3TVD; EINECS 285-687-7; 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride; DTXSID90234251; GABOXADOL HYDROCHLORIDE [MI]; ISOXAZOLO(5,4-C)PYRIDIN-3(2H)-ONE, 4,5,6,7-TETRAHYDRO-, HYDROCHLORIDE (1:1); MK 0928; Lu-02030; OV 101; Lu02-030; MK0928; Lu02030; THIP

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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

H2O : ≥ 100 mg/mL (~566.25 mM)
DMSO : ~75 mg/mL (~424.69 mM)

配方 1 中的溶解度: ≥ 1.47 mg/mL (8.32 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 14.7 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.47 mg/mL (8.32 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 14.7mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1.47 mg/mL (8.32 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 14.7 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 100 mg/mL (566.25 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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