GABA receptor

In layer 2/3 neurons, THIP (1 µM; 5 s) generates strong GABAA-mediated currents [1]. In layer 2/3 neurons, THIP (1 µM; 1 s) had no influence on micro-IPSCs [1].

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THIP/Gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a selective GABA(A) receptor agonist with a preference for delta-subunit containing GABA(A) receptors. THIP is currently being tested in human trials for its hypnotic effects, displaying advantageous tolerance and addiction properties. Since its cellular actions in the neocortex are uncertain, we studied the effects of THIP on neurons in slices of frontoparietal neocortex of 13- to 19-day-old (P13-19) mice. Using whole-cell patch-clamp recordings, we found that the clinically relevant THIP concentration of 1 muM induced a robust tonic GABA(A)-mediated current in layer 2/3 neurons. In comparison, only a minute tonic current was induced by mimicking in vivo endogenous GABA levels. Miniature IPSCs were not affected by 1 muM THIP suggesting an extrasynaptic site of action. The EC(50) for THIP was 44 muM. In accordance with the stronger expression of delta-containing receptors in superficial neocortical layers, THIP induced a 44% larger tonic current in layer 2/3 than in layer 5 neurons. Finally, monitoring spontaneously active neocortical neurons, THIP caused an overall depression of inhibitory activity, while enhancing excitatory activity prominently. Our studies suggest that THIP activates an extrasynaptic GABA(A) receptor-mediated conductance in the neocortex, which may alter the cortical network activity. [1]

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Characterization of Gaboxadol transport across Caco-2 cell monolayers [2]
The transepithelial transport of gaboxadol across Caco-2 cell monolayers was investigated at different gaboxadol concentrations, apical pH values and in the absence or presence of 5-HTP (Table 1). In the presence of a proton gradient across the monolayers (pH values of the apical and basolateral chamber were 6.0 and 7.4, respectively) the mean A–B flux of gaboxadol increased whereas the permeability of gaboxadol decreased from 8.0 × 10−6 to 6.1 × 10−6 and 5.6 × 10−6 cm s−1 (p < 0.05) for 0.34, 3.5 and 7.0 mM gaboxadol, respectively (Table 1). Hence, the permeability of gaboxadol across Caco-2 cells was concentration dependent, which is consistent with a partial saturation of hPAT1. In the absence of a proton gradient across the monolayers (pH values of the apical and basolateral chamber were 7.4 and 7.4, respectively) the permeability of 3.5 and 7 mM gaboxadol was reduced approximately a 5- and 7-fold (p < 0.01 and p < 0.001), respectively. This is consistent with gaboxadol transport via hPAT1, since the transporter is proton-coupled. In the presence of 5-HTP the gaboxadol permeability was decreased approximately 5-fold (p < 0.001). Gaboxadol transport in the B–A direction was 21% of the transport in the A–B direction, hence the transport of gaboxadol was polarized in the A–B direction (p < 0.01). The permeability of gaboxadol in the absence of a proton gradient or in the presence of 5-HTP was quite similar to the permeability of gaboxadol in the B–A direction and the permeability of [14C]-mannitol. Together, these observations suggest that the proton-dependent amino acid transporter, hPAT1, mediates the transport of gaboxadol across the brush-border membrane of enterocytes. Furthermore, the results indicate that hPAT1 is responsible for 80–90% of the total transepithelial gaboxadol transport in Caco-2 cell monolayers. The permeability of Caco-2 cell monolayers was lowered in a dose-dependent manner by gaboxadol hydrochloride (0.34, 3.5, and 7.0 μM), with mean Papp values of 8.1 × 10-6 cm·s-1, 6.1 × 10 -1 cm·s-1 for 0.34, 3.5, and 7 μM gaboxadol, and 5.6 × 10-6 cm·s-1 for 0.34, 3.5, and 7 μM gaboxadol, respectively[6].

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Characterization of Gaboxadol transport via hPAT1 in vitro [6]
The interaction between gaboxadol and hPAT1 was investigated by measuring the apical uptake of the hPAT1 substrate proline into Caco-2 cell monolayers in the presence of increasing gaboxadol concentrations (Figure 1). Gaboxadol decreased apical proline uptake in Caco-2 cell monolayers with an estimated inhibitor affinity (Ki value) of 6.6 mmol·L−1. Similarly, the known PAT1 inhibitor, tryptophan, also decreased the apical uptake of proline with a Ki value of 7.7 mmol·L−1. The transepithelial (A-B) flux of gaboxadol transport across Caco-2 cell monolayers was investigated at three apical concentrations (0.34, 3.5 and 7.0 mmol·L−1). The mean Papp values of gaboxadol transport were 8.1 × 10−6 cm·s−1, 6.1 × 10−6 cm·s−1 and 5.6 × 10−6 cm·s−1 for 0.34, 3.5 and 7 mmol·L−1 gaboxadol, respectively (Figure 2). Thus, the gaboxadol permeability across Caco-2 cell monolayers decreased with increasing gaboxadol concentrations (P < 0.05). The gaboxadol transport across Caco-2 cell monolayers using an apical concentration of 3.5 mmol·L−1 gaboxadol was investigated in the presence of 35 mmol·L−1 tryptophan, with or without a pH gradient, and bidirectional (Figure 2). Transport of gaboxadol in the A–B direction was approximately five times higher than in the B–A direction (P < 0.005). The presence of tryptophan reduced the permeability of gaboxadol by 53% (to a Papp of 2.9 × 10−6 cm·s−1, P < 0.005). In the absence of a proton gradient across the monolayer, the permeability of gaboxadol was reduced by 82% to 1.1 × 10−6 cm·s−1 (P < 0.005). Gaboxadol permeability in the presence of tryptophan, in absence of a proton gradient, and in the B–A direction was similar to the permeability of [3H]-mannitol (Papp of 1.6 ± 0.36 × 10−6 cm·s−1). Furthermore, the presence of gaboxadol and tryptophan in the transport experiments did not change the permeability of metoprolol or mannitol transport across Caco-2 cell monolayers. The permeabilities of mannitol and metoprolol were 1.6 ± 0.36 × 10−6 cm·s−1 and 6.9 ± 0.99 × 10−6 cm·s−1, respectively. In summary, the transepithelial gaboxadol transport across Caco-2 cell monolayers was pH-dependent, could be inhibited by tryptophan and was polarized in the A-B direction. Together these observations suggest that hPAT1 mediates gaboxadol transport across the luminal membrane of human intestinal epithelial cells, and that this transport step to a large degree determines the resulting transepithelial transport of gaboxadol.

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Gaboxadol is a substrate for the proton-coupled amino acid transporter, hPAT1, in Caco-2 cell monolayers [6]
Gaboxadol inhibited the apical uptake of the hPAT1 substrate proline in Caco-2 cell monolayers with a Ki value of 6.6 mmol·L−1. This affinity is comparable with those recently observed for other hPAT1 substrates such as GABA (3.1 mmol·L−1) and the GABA analogues muscimol (1.7 mmol·L−1) and THPO (11.3 mmol·L−1) (Larsen et al., 2008). The affinity of tryptophan was found to be quite similar to gaboxadol, that is, 7.7 mmol·L−1. Metzner et al. (2005) previously characterized tryptophan as an inhibitor of PAT1, and reported a Ki value of 4.7 mmol·L−1 for proline uptake via hPAT1 in Caco-2 cells. Considering minor differences in the proline affinities for hPAT1 between the two laboratories; Metzner et al. reports a Kt of 1.4 mmol·L−1 whereas Larsen et al. reports a Km of 3.6 mmol·L−1, respectively, the affinities of tryptophan for hPAT1 are quite comparable between the two studies (Metzner et al., 2005; Larsen et al., 2008). In accordance with results published by other groups (Thwaites et al., 1993; Metzner et al., 2005), we found that the majority of apical proline transport in Caco-2 cells was mediated by hPAT1, and no evidence of other sodium-dependent or sodium-independent transporters of proline was observed (Larsen et al., 2008). Other amino acid transporters such as the apical sodium-dependent amino acid transporters B0 (B0AT1), B0,+ (ATB0,+) and system (ASC) (ASCT2) are not likely to be involved in the transport of gaboxadol. They are all present in Caco-2 cells but their translocation of substrate is not proton-coupled. Furthermore, these transporters are characterized by higher affinity values for their substrates than observed for PAT1, for example, ASC (ASCT2), approximately 100 µmol·L−1 (Uchiyama et al., 2005); B0 (B0AT1), 500–700 µmol·L−1 (Broer et al., 2004) and B0,+ (ATB0,+), approximately 150 µmol·L−1 (Hatanaka et al., 2002).

The transepithelial transport of Gaboxadol across Caco-2 cell monolayers was polarized in the apical to basolateral direction. Gaboxadol transport could be inhibited by tryptophan and was dependent on the pH of the apical donor solution. Furthermore, the Papp of gaboxadol in the apical–basolateral direction decreased with increasing gaboxadol concentration. This is consistent with transport of gaboxadol via hPAT1, and this pathway accounted for approximately 80% of the total transepithelial transport. The amino acid transport system b0,+ has been identified in the small intestine of mouse and in Caco-2 cells, where it accounts for 15–85% of the total transport of alanine and arginine respectively (Wenzel et al., 2001; Dave et al., 2004). However, tryptophan binding to system b0,+ has not been unequivocally shown (Su et al., 1992; Tate et al., 1992), and furthermore cationic amino acids, zwitterionic amino acids and cystine have µmol·L−1 affinities for system b0,+ (Palacin, 1994). Therefore, if gaboxadol is transported via system b0,+ to any significant degree, it should be evident in Caco-2 cells. In vitro the transepithelial gaboxadol transport was furthermore pH-dependent, and PAT1 is the only currently known proton-coupled amino acid transporter in the intestine. The permeability of gabapentin (also a zwitterionic γ-amino analogue) in rat small intestine was shown to be proton independent (Nguyen et al., 2007), hence different apical transport mechanisms exist for gaboxadol and gabapentin. The results indicate that gaboxadol is a substrate for hPAT1 in Caco-2 cell monolayers, and that hPAT1 mediates gaboxadol transport across the luminal membrane of the intestinal enterocytes, which appear to be important for the resulting transepithelial transport. The mechanism for gaboxadol efflux across the basolateral membrane is still unknown.

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In vivo absorption of Gaboxadol in dogs [6]
The in vivo absorption of gaboxadol in dogs occurred rapidly following oral administration with a Tmax of approximately 0.46 h and a high bioavailability of 85%. These observations are consistent with previous studies on the oral absorption in humans showing a gaboxadol Tmax of approximately 0.5 h and a bioavailability >90% (Schultz et al., 1981; Lund et al., 2006). Once absorbed, gaboxadol is mainly excreted in the urine in the form of gaboxadol, whereas a minor fraction is excreted in the form of a glucuronic acid conjugate comprising of 2–7% in rat and mouse and 30–35% in two human subjects (Schultz et al., 1981; Lund et al., 2006; Shadle et al., 2006). Collectively, this indicates that in dogs gaboxadol is quickly and completely absorbed, probably in the proximal small intestine, with a minimal post-absorptive metabolism.

Gaboxadol (THIP; 0.5, 5.0 mg/kg; oral; single dosage) exhibits good oral availability; at 0.5 and 5.0 mg/kg, Fa values are 83% and 110%, respectively [2].

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The in vivo pharmacokinetic profile of Gaboxadol after oral administration to rats was investigated in the absence and presence of a pre-dose of 5-HTP. In Caco-2 cell monolayers >80% of the absorptive gaboxadol transport was suggested to be hPAT1-mediated. In rats, the initial absorption rate of gaboxadol was decreased in the presence of 5-HTP. The AUC of gaboxadol was increased by a factor of 3.6-5.5 when rats were pre-dosed with 5-HTP. Gaboxadol was a substrate for the renal transporter rOat1 with a K(m)-value of 151 microM. 5-HTP did not interact with rOat1. In conclusion, gaboxadol acts as a substrate for hPAT1 and is a substrate of rOat1. In rats, 5-HTP decreased the initial absorption rate and increased AUC of gaboxadol. 5-HTP thus had a significant impact on the pharmacokinetic profile of gaboxadol.[2]

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Gaboxadol hydrochloride (intraperitoneal injection; 0.5, 1, 1.5, 2, 3, 4, or 5 mg/kg; thrice daily; three days apart) normalizes walking distance in Fmr1 KO2 mice to 0.5 mg/kg WT activity level, in addition, the chemical had no effect on locomotor activity in Fmr1 KO2 mice [5].

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Here, we sought to evaluate the potential of Gaboxadol (also called OV101 and THIP), a selective and potent agonist for delta-subunit-containing extrasynaptic GABAA receptors (dSEGA), as a therapeutic agent for FXS by assessing its ability to normalize aberrant behaviors in a relatively uncharacterized mouse model of FXS (Fmr1 KO2 mice). Four behavioral domains (hyperactivity, anxiety, aggression, and repetitive behaviors) were probed using a battery of behavioral assays. The results showed that Fmr1 KO2 mice were hyperactive, had abnormal anxiety-like behavior, were more irritable and aggressive, and had an increased frequency of repetitive behaviors compared to wild-type (WT) littermates, which are all behavioral deficits reminiscent of individuals with FXS. Treatment with gaboxadol normalized all of the aberrant behaviors observed in Fmr1 KO2 mice back to WT levels, providing evidence of its potential benefit for treating FXS. We show that the potentiation of extrasynaptic GABA receptors alone, by gaboxadol, is sufficient to normalize numerous behavioral deficits in the FXS model using endpoints that are directly translatable to the clinical presentation of FXS. Taken together, these data support the future evaluation of gaboxadol in individuals with FXS, particularly with regard to symptoms of hyperactivity, anxiety, irritability, aggression, and repetitive behaviors. [5]

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Gaboxadol Normalizes Hyperactivity Observed in Fmr1 KO2 Mice [5]
Hyperactivity is a salient feature of human FXS (Bailey et al., 2008; Wheeler et al., 2014; Hagerman et al., 2017) and has been reliably reproduced in the previously characterized Dutch-Belgian Fmr1 KO mouse (Olmos-Serrano et al., 2010; Kazdoba et al., 2014). To test whether the Fmr1 KO2 mice showed locomotor hyperactivity and whether gaboxadol could normalize this aberrant behavior, Fmr1 KO2 mice were injected with vehicle or gaboxadol (0.5–5 mg/kg, i.p.), and WT littermates were injected with vehicle 30 min before testing in the OFT. The total distance traveled (cm) in the OFT was recorded for 30 min. The results showed that the distance traveled by Fmr1 KO2 mice was significantly increased compared to WT littermate controls (Figure 1, F(8,81) = 21.27, p < 0.0001), consistent with results from other models of FXS. Treatment with gaboxadol (0.5 mg/kg) normalized the distance traveled by Fmr1 KO2 mice to WT activity levels (Figure 1). Higher doses of gaboxadol (1–5 mg/kg, i.p.) had no effect on locomotor activity in Fmr1 KO2 mice (Figure 1). These results were not attributable to sedative effects of gaboxadol because in WT C57Bl/6 or BALB/c mice, gaboxadol doses up to 2.0 mg/kg, i.p. have no effect on locomotor activity in a 60 min OFT (data not shown), consistent with previous work showing no effect of gaboxadol on locomotion in WT mice (Olmos-Serrano et al., 2011) or rats (Silverman et al., 2016).

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Anxiety-Like Behaviors in Fmr1 KO2 Mice Are Normalized by Gaboxadol [5]
To assess the effect of gaboxadol on anxiety-like behaviors in the Fmr1 KO2 mice, three different behavioral tests were employed: center distance traveled in the OFT, the LDT and the SAT. Increased distance traveled in the center is interpreted as decreased anxiety and takes advantage of the inherent preference of mice to remain in the perimeter when introduced to a novel environment. Fmr1 KO2 mice were injected with gaboxadol (0.5–5 mg/kg, i.p.), and WT littermates were injected with vehicle 30 min before being placed in the OFT for 30 min. The total distance traveled in the center was significantly increased in Fmr1 KO2 mice compared to WT controls (Figure 2A, F(8,81) = 21.32, p < 0.0001). Treatment with gaboxadol (0.5 mg/kg, i.p.) normalized the effect of Fmr1 KO2 on center distance traveled to levels comparable to WT controls (Figure 2A). Higher doses of gaboxadol (1–5 mg/kg) had no effect on Fmr1 KO2 mice in this test (Figure 2A).

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Irritability and Aggressive Behaviors in Fmr1 KO2 Mice Are Normalized by Gaboxadol [5]
As with other forms of syndromic autism, a large proportion of individuals with FXS show irritability, social anxiety and aggression. These aberrant behaviors can be modeled in rodents through characterization of SIs between a test mouse and a novel cage-mate. To test the hypothesis that irritability and aggression were increased in Fmr1 KO2 mutants, we quantified instances of tail rattling, biting behavior, mounting behavior, and latency to attack. Mice were injected with vehicle or gaboxadol (0.5–5 mg/kg, i.p.) 30 min before being placed into the test cage. Tail rattling, or rapid vibrations of the tail, reflects aggressiveness and fight tendency. Fmr1 KO2 mice showed significantly increased tail rattling frequency compared to WT controls (Figure 3A, F(8,81) = 16.03, p < 0.0001). Gaboxadol (0.5, 1.5, and 5.0 mg/kg) normalized the effect in Fmr1 KO2 mice to levels comparable to WT controls (Figure 3A).

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Gaboxadol Normalizes Repetitive Behaviors in Fmr1 KO2 Mice [5]
Perseveration and repetitive behaviors are common in individuals with FXS and are highly disruptive (Arron et al., 2011; Leekam et al., 2011; Hall et al., 2016). To test the hypothesis that such features might be observed in Fmr1 KO2 animals, we quantified circling, self-grooming, and stereotypy in WT and Fmr1 KO2 mutant mice. Counter-clockwise (CCW) revolutions were measured in the testing chamber after mice were injected with vehicle or gaboxadol (0.5–5 mg/kg, i.p.). Fmr1 KO2 mice showed significantly increased CCW revolutions during the 5 min test compared with WT controls (Figure 4A, F(8,81) = 25.46, p < 0.0001). Injection of gaboxadol (0.5, 1.0 mg/kg) into Fmr1 KO2 mice restored the number of CCW revolutions to WT levels (Figure 4A). There was no effect of genotype on clockwise circling (p = 0.386, data not shown).

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In vivo absorption of Gaboxadol in dogs [6]
The in vivo absorption of gaboxadol in dogs occurred rapidly following oral administration with a Tmax of approximately 0.46 h and a high bioavailability of 85%. These observations are consistent with previous studies on the oral absorption in humans showing a gaboxadol Tmax of approximately 0.5 h and a bioavailability >90% (Schultz et al., 1981; Lund et al., 2006). Once absorbed, gaboxadol is mainly excreted in the urine in the form of gaboxadol, whereas a minor fraction is excreted in the form of a glucuronic acid conjugate comprising of 2–7% in rat and mouse and 30–35% in two human subjects (Schultz et al., 1981; Lund et al., 2006; Shadle et al., 2006). Collectively, this indicates that in dogs gaboxadol is quickly and completely absorbed, probably in the proximal small intestine, with a minimal post-absorptive metabolism.

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In vivo absorption of Gaboxadol following co-administration of tryptophan [6]
Co-administration of the hPAT1 inhibitor tryptophan had a dose-dependent effect on the absorption profile of gaboxadol resulting in a decreased Cmax and an increased Tmax. A reduction in the gaboxadol absorption rate could be caused by an alteration of the gastric emptying rate. In humans, the rate of gastric emptying decreases as a function of the number of calories in an ingested meal (Calbet and MacLean, 1997; Sunesen et al., 2005), and also in dogs the composition of meals has been shown to prolong gastric emptying (Mizuta et al., 1990). To rule out that the observed effect on gaboxadol absorption was a result of altered gastric emptying, the cumulative absorption curve of paracetamol, which is often used as a marker of gastric emptying (Calbet and MacLean, 1997; Sunesen et al., 2005), was investigated in the presence of tryptophan. The gastric emptying of paracetamol was not significantly affected by high doses of tryptophan. However, a significant effect of tryptophan on the ka of gaboxadol was observed. As co-administration of tryptophan changed gaboxadol Tmax, Cmax and ka whereas the Fa, ke and the AUC were constant, the effect of tryptophan is likely to be a result of interaction between tryptophan and gaboxadol at the site of absorption, and not due to changes in gastric emptying. From other studies it is known that gaboxadol has a low degree of plasma protein binding and is not metabolized by cytochrome P-450s (Lund et al., 2006). Thus, on the basis of the findings in vitro suggesting that hPAT1 mediates the majority of the luminal gaboxadol transport in Caco-2 monolayers, it seems likely that also the in vivo observation can be explained by a PAT1-mediated gaboxadol absorption in dogs, which is reduced by the co-administration of tryptophan. The observed in vivo affinity values of tryptophan inhibition of gaboxadol intestinal transport were estimated from either the effect of tryptophan on gaboxadol Cmax or on the intestinal absorption rate constant, ka. This gave IC50 values of 10.1 and 12.6 mmol·L−1, respectively. As discussed earlier, the in vitro affinity of hPAT1 for tryptophan measured as inhibition of proline transport via hPAT1 was 7.7 mmol·L−1 in Caco-2 cells. The IC50 values are surprisingly close to each other considering that tryptophan inhibition is measured for two different compounds (proline and gaboxadol), and that the in vivo transport is composed of not only luminal transport but appearance in the systemic circulation, which encounters the transfer of gaboxadol across several membranes. hPAT1 substrates are characterized by having affinities in the millimolar range, and the transporter, which is expressed along the entire intestinal tract, has a high capacity (Chen et al., 2003). The molar dosing ratio between gaboxadol and tryptophan was up to 1:41, and as they both have comparable affinities for hPAT1, the reduced Cmax and ka may be due to the competitive interaction between gaboxadol and tryptophan at the site of absorption, that is, at the PAT1 protein in the luminal membrane of the small intestinal enterocytes. The maximal plasma concentration thus occurs with a prolonged Tmax, but due to the excessive capacity and the intestinal expression of PAT1, the absorption fraction remains unchanged as the absorption proceeds along the length of the intestine. The peak plasma concentration of gaboxadol may thus be reduced by modifying the absorption process as illustrated here, or through a more classical sustained release formulation approach as suggested earlier (Kjaer and Nielsen, 1983).

Cell Viability Assay[1]
Cell Types: Layer 2/3 neurons (from male C57BL/6 mice, postnatal days 13-19)
Tested Concentrations: 1 µM
Incubation Duration: 5 s
Experimental Results: Induced a tetanus current of 43.2 pA.

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Cell viability assay[1]
Cell Types: Layer 2/3 Neurons (from male C57BL/6 mice, postnatal days 13-19)
Tested Concentrations: 1 µM
Incubation Duration: 1 s
Experimental Results: Amplitude and frequency of mIPSCs only Minor changes occur.

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In vitro transepithelial transport in Caco-2 cells [2]
Cell cultures and in vitro transport experiments were performed as previously described (Larsen et al., 2008). In brief, cells of passages 24 through 29 were seeded in Transwell™ inserts and experiments were conducted on days 25–28 after seeding. The transepithelial transport of Gaboxadol across Caco-2 cell monolayers in the apical to basolateral (A–B) or basolateral to apical (B–A) direction was measured in HBSS buffers. In all experiments HBSS applied to the basolateral side was pH 7.4 while apical buffers were adjusted to pH 6.0 or 7.4 after the addition of test compounds. The volumes of the apical and basolateral chambers were 0.5 and 1.0 ml, respectively. The addition of fresh solutions of 0.34, 3.5 or 7.0 mM Gaboxadol to the donor chambers initiated the transport experiments. The transport of gaboxadol was measured in the absence and presence of 18.8 mM 5-HTP. The transport of mannitol in the A–B direction was measured with 18 μM [14C]-mannitol. 100 μl and 20 μl samples were taken from basolateral and apical receiver chambers, respectively, after 20, 40, 60, 80 and 120 min.

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Uptake of Gaboxadol into rOat-injected oocytes [2]
Uptake of gaboxadol via rOat1 was investigated in BD Gentest™ Transportocytes; Xenopus oocytes pre-injected with rOat1 cRNA. The transportocytes were handled according the manufactures instructions. As a negative control, the uptake of gaboxadol was also measured in water injected oocytes i.e. in oocytes not expressing rOat1. For each data point 5–10 oocytes were placed in a 5 ml test tube. The oocytes were washed 3 times with 1 ml of Na+ buffer before they were incubated in 100 μl of test compound solutions. The time dependent uptake of 100 μM gaboxadol was investigated by sampling after 15, 30, 60 or 90 min in rOat1 cRNA injected oocytes and after 30, 60 and 90 min in control oocytes. The uptake of 10, 50, 100, 300, 500 and 1000 μM gaboxadol was investigated for 60 min in rOat1 cRNA injected oocytes whereas the uptake of 100, 500 and 1000 μM gaboxadol was studied in the water injected oocytes as a control. The uptake of 100 μM gaboxadol in the presence of 5 mM 5-HTP, 5 mM l-proline, 1 mM PAH or 1 mM probenecid was measured in rOat1 cRNA injected oocytes as well as in water injected oocytes for 60 min. After incubation, the oocytes were washed 3 times in ice-cold Na+ buffer and each oocyte was placed in an Eppendorf tube with 30 μl MilliQ water and 25 μl internal standard. The oocytes were then kept at −80 °C until time of quantification where they were lysed by thawing and whirl mixing. Subsequent to centrifugation at 10,000 × g for 15 min, gaboxadol was extracted from the supernatant and prepared for analysis as described below except for following steps: 250 μl cold (4 °C) acetonitrile was used to precipitate proteins and the dry samples were re-dissolved in 60 μl (30:70) MeOH/MeCN.

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Quantification of Gaboxadol in plasma and buffers [2]
Gaboxadol was extracted from plasma and HBSS samples by liquid extraction. 100 μl HBSS (80 μl purified water were added to the 20 μl samples) or 100 μl 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 supernatant was transferred to glass tubes and evaporated to dryness under nitrogen at 45 °C. The samples were dissolved in 80 μl methanol/acetonitrile (30:70), whirl mixed for 10 min and centrifuged for 3 min at 3000 rpm. Standards were added to blind plasma and prepared similar to the plasma samples. Gaboxadol was quantified by hydrophilic interaction chromatography followed by MS/MS detection using a protocol modified from (Kall et al., 2007). The LC system comprised of an Agilent 1100 series pump and degasser. An Asahipak amino column (NH2P-50, 150 mm × 2 mm) from Phenomenex was used with a mobile phase of 20.0 mM ammonium acetate pH 4:acetonitrile (30:70) and a flow rate of 0.2 ml/min. 20 μl samples were injected onto the column, which was kept at room temperature. The total runtime was 10 min with the first 5 min of elution let to waste. The elution time of gaboxadol 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. The detection was performed in negative ionization mode where gaboxadol (precursor 139.1 Da, product 110.1 Da) and d4-gaboxadol (precursor 143.0, product 112.2 Da) were measured by multiple-reaction-monitoring (MRM). The signals were linear over the concentration range of 1.7–1000.0 ng/ml and the limit of quantification by this procedure was 1.7 ng/ml. The software was from Analyst™.

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Protocols for cell culturing and in vitro experiments were as previously described (Larsen et al., 2008). Caco-2 cells of passages 20 through 29 were seeded onto Transwell™ inserts (1.12 cm2, 0.4 µm pore size) and experiments were conducted on day 25–28 after seeding. The apical uptake and the transepithelial transport of Gaboxadol across Caco-2 cell monolayers in apical to basolateral direction (A–B) and basolateral to apical direction (B–A) were measured in Hanks' balanced salt solution (HBSS) buffers. In all experiments, buffer applied to the basolateral side was pH 7.4. Unless otherwise stated, buffer applied in the apical chamber was adjusted to pH 6.0 after the addition of Gaboxadol hydrochloride or 35 mmol·L−1 tryptophan. The transport of 0.34, 3.5 or 7.0 mmol·L−1 Gaboxadol was investigated. These concentrations were selected based on a single bedtime oral dose of 15 mg gaboxadol to humans, which provides an approximate luminal concentration of 0.34 mmol·L−1, and the obtained gaboxadol affinity for hPAT1. Apical uptake experiments were initiated by adding fresh apical HBSS medium with 12.5 nmol·L−1 (0.5 µCi) L-(3H)proline and 0–30 mmol·L−1 gaboxadol or 0–35 mmol·L−1 tryptophan to the apical chamber. The apical uptake experiments were terminated after 5 min. Samples were analysed by scintillation counting.[6]

Animal/Disease Models: SD (SD (Sprague-Dawley)) rat (255-276 g) [2].
Doses: 2.5 mg/kg (intravenous (iv) (iv)injection); 0.5 and 5.0 mg/kg (oral).
Route of Administration: po (oral gavage); intravenous (iv) (iv)injection; single.
Experimental Results: 1.19 pharmacokinetic/PK/PK parameters of THIP 4 in SD (SD (Sprague-Dawley)) rats [2]. IV (2.5 mg/kg) PO (0.5 mg/kg) PO (5.0 mg/kg) AUC (ng/mL·h) 1193 263 1988 Tmax (h) - 0.22 0.33 Cmax (ng/mL) 4350 291 2061 CL/ Fa (mL/h/kg) - 1939 2558 CL (mL/h/kg) 2043 - - Fa (%) 100 110 83

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In vivo absorption of Gaboxadol in Sprague–Dawley rats [2]
Male Sprague–Dawley rats were housed and acclimated for 7 days before the experiments. The animals weighed 255–276 g at the day of the experiment. The rats were maintained on standard food and water until 16–20 h prior to dosing where food was retrieved. Water was available to the animals until beginning of experiment and again 2 h after dosing. Each animal was randomly assigned to receive one of the intravenous or oral formulations, in total 6 parallel groups of 6 animals (n = 6) unless otherwise stated. The oral solutions contained 0.5 or 5.0 mg/kg of gaboxadol and the intravenous solutions contained 2.5 mg/kg gaboxadol. The animals were orally dosed with 10.0 ml/kg (i.e. concentrations of 0.35 and 3.5 mM gaboxadol) or intravenously with 5 ml/kg (3.5 mM). The gaboxadol doses were given 30 min after an oral pre-dose of isotonic saline or 100.0 mg/kg 5-HTP (for IV gaboxadol) or 200.0 mg/kg 5-HTP (for oral gaboxadol) by oral gavage (10.0 ml/kg). All solutions were adjusted to a pH of 5.2 before the osmolarity was checked on a Vapro vapour pressure osmometer and adjusted with mannitol to iso-osmolarity. Blood samples (0.2 ml) were taken from the tail vein by individual vein puncture and collected into Eppendorf tubes containing 20 IE heparin. Blood samples were collected at 5, 15, 30, 45, 60 min and 2, 3, 4, 6, 8 h after gaboxadol administration. The plasma was harvested immediately by centrifugation for 10 min at 3600 × g and stored at −80 °C until further analysis. After the experiment the animals were euthanized.

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Investigation of gastric emptying in rats [5]
A protocol similar to the one described above using paracetamol as a marker was used to investigate paracetamol absorption and evaluate the influence of 5-HTP on the gastric emptying rate in rats. 26 rats were selected and allocated randomly to receive one of 5 different formulations of paracetamol. The rats received 120.0 mg/kg paracetamol and 2.5 mg/kg Gaboxadol (10 ml/kg) as an intravenous injection or oral solution. The oral solutions were given 30 min after an oral pre-dose of isotonic solutions of saline, mannitol or 200 mg/kg 5-HTP. The intravenous injection of gaboxadol was given 30 min after an oral pre-dose of isotonic saline (n = 6) or 200.0 mg/kg 5-HTP (n = 2). Drug administration, blood sampling and handling of animals was done as described above.Animal/Disease Models: Fmr1 KO2 mice (the Fmr1 promoter and first exon are deleted, resulting in mice with missing mRNA and protein) [5]
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). [5]

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Absorption of Gaboxadol in dogs [6]
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 [6]
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 [6]
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 Gaboxadol in Sprague-Dawley rats after oral administration [2] Figure 1 shows the plasma concentration curves of Gaboxadol after intravenous and oral administration in Sprague-Dawley rats. The time to peak plasma concentration (Tmax) was 0.2 h and 0.3 h for the 0.5 mg/kg and 5.0 mg/kg dose groups, respectively (Table 2). Oral absorption appeared to be complete in the 0.5 mg/kg dose group, with a mean bioavailability (Fa) of 110%. The Fa in the 5 mg/kg dose group was 83 ± 5%, which was significantly lower than that in the 0.5 mg/kg dose group (p < 0.01). The clearance of Gaboxadol (CL/Fa) increased from 1.93 L/h/kg to 2.56 L/h/kg at the 0.5 mg/kg and 5 mg/kg doses, respectively (p < 0.05). When the dose was increased from 0.5 mg/kg to 5 mg/kg (a 10-fold increase), the peak plasma concentration (Cmax) increased 7-fold, indicating that the peak concentration was not proportional to the dose within the studied dose range. In addition, the AUC increased only 7.5-fold after a 10-fold increase in dose. Nonlinear pharmacokinetics suggests that the absorption or elimination of gabosaccharide may involve carrier-mediated transport. This carrier may be a saturated absorption carrier in the intestine or a carrier involved in the multiple absorption of gabosaccharide by the kidneys. In Sprague-Dawley rats, oral administration of gabosaccharide and 5-hydroxytryptophan [2] followed by intravenous and oral administration significantly altered the concentration profiles of gabosaccharide in rat plasma (Figure 1A-C). Both the absorption and elimination phases were altered, and pre-administration of 5-hydroxytryptophan significantly increased the AUC in rats. During the initial absorption phase (≤Tmax) of gabosadal, the plasma concentration of gabosadal was significantly lower in animals receiving 5-hydroxytryptophan compared to those not receiving 5-hydroxytryptophan (p < 0.05, Figures 1B and 1C). 5-HTP had no significant effect on the peak plasma concentration (Cmax) of gabosadal. Furthermore, 5-HTP significantly prolonged the time to peak concentration (Tmax) of gabosadal at doses of 0.5 mg/kg and 5.0 mg/kg (p < 0.01 and p < 0.001), indicating that 5-HTP alters the intestinal absorption of gabosadal in rats. The semi-logarithmic plots in Figures 1A and 1B show that the elimination curve of gabosadal in rats administered 5-HTP was approximately linear, while that in rats administered gabosadal alone was biphasic. The presence of 5-HTP increased the mean AUC of gabocadol at 0.5 mg/kg and 5.0 mg/kg doses by 5.5-fold (p < 0.01) and 3.6-fold, respectively. The mean clearance CL/Fa of gabocadol at 0.5 mg/kg and 5.0 mg/kg doses decreased by 77% (p < 0.01) and 23% (not statistically significant), respectively. In the presence of 5-HTP, the clearance CL of gabocadol at 2.5 mg/kg (IV) decreased by 66% (n = 2) (Table 2). In this study, oral absorption of acetaminophen (as a marker of gastric emptying) did not change significantly after pre-administration of 5-HTP or mannitol, as assessed by Cmax, Tmax, AUC, Fa, and CL/Fa (Figure 2 and Table 3). Therefore, it can be concluded that 5-HTP does not significantly prolong gastric emptying. However, the elimination rate constant ke (0.95 h−1) of acetaminophen was significantly reduced in the presence of 5-HTP (0.53 h−1, p < 0.05). Gabosad is a substrate of rOat1, but 5-HTP is not [2] Since 5-HTP has a significant effect on the clearance of gabosad in rats, the transport of gabosad by the renal organic anion transporter rOat1 was studied with or without 5-HTP (Fig. 3, Fig. 4). Within the 90 minutes studied, the uptake of gabosad by oocytes in both the rOat1 cRNA injection group and the water injection group was linear (Fig. 3A). The uptake of gabosad by oocytes in the rOat1 cRNA injection group was significantly higher than that in the water injection group (p ≤ 0.001). In oocytes injected with rOat1 cRNA, the concentration-dependent uptake rate of gaboxadol was saturated (Fig. 3B). The data points conformed to Michaelis-Menten kinetics, with Km values of 151.2 ± 58.19 μM and Vmax values of 0.78 ± 0.09 pmol oocyte−1 min−1. Therefore, gaboxadol is a substrate of rOat1. We then investigated rOat1-mediated gaboxadol uptake in the presence of 5 mM 5-HTP, 5 mM L-proline, 1 mM PAH, and 1 mM probenecid (Fig. 4). The presence of 5-HTP or L-proline did not affect gaboxadol uptake, while the presence of PAH and probenecid (substrate and inhibitor of Oat1, respectively) significantly reduced gaboxadol uptake (p < 0.001). 5-HTP can reduce the absorption of gaboxadol both in vitro and in vivo [2]. Gabosador transport in Caco-2 cell monolayers exhibits AB-directional polarization. This AB-directional transport is dependent on donor concentration and the pH of the donor medium. This is consistent with the hPAT1-mediated transluminal transport of gabosador across the intestinal cell membrane, a step crucial for eventual transepithelial uptake. Similar phenomena, namely pH-dependent transport of other PAT1 substrates (such as L-proline (Thwaites et al., 1993) and GABA (Thwaites et al., 2000)), have also been observed in Caco-2 cell monolayers. Previously, Chen et al. cloned hPAT1 from Caco-2 cells, and immunofluorescence showed that PAT1 is expressed on the apical membrane of Caco-2 cells and rat small intestinal epithelial cells (Chen et al., 2003; Anderson et al., 2004). In Caco-2 cell monolayers, 80-90% of gabosarcol transport is likely mediated by hPAT1, while 5-HTP inhibits this transport. The Ki values for gabosarcol and 5-hydroxytryptophan (5-HT) inhibiting L-proline uptake in Caco-2 cells via hPAT1 are 6.6 mM (Larsen et al., 2009) and 2.3 mM (Larsen et al., 2008), respectively. Therefore, the question is whether an interaction between gabosarcol and 5-HT can be observed at relevant concentrations. Assuming an oral dose of 15 mg gabosarcol in a 250 ml volume, the intraluminal concentration is 0.34 mM. The commonly used dose of 5-HT is 50-100 mg, with an intraluminal concentration of 9-18 mM. In this experiment, the lowest concentration of gabosarcol was 0.34 mM, and the lowest concentration of 5-HT was 18.8 mM. Therefore, these concentrations can be measured in vivo, and the interaction between the two compounds may be correlated. In vivo, after oral administration to Sprague-Dawley rats, peak plasma concentrations of gabosadal were reached within 0.22–0.33 hours. During the initial absorption phase of gabosadal, rats pre-administered with 5-HTP showed significantly lower plasma concentrations of gabosadal compared to rats administered gabosadal alone. Furthermore, the presence of 5-HTP significantly prolonged the time to peak concentration (Tmax) of gabosadal. A parallel study on the absorption of acetaminophen (a commonly used marker of gastric emptying) showed that 5-HTP did not significantly delay gastric emptying. This suggests that 5-HTP reduces the absorption rate of gabosadal in the rat small intestine. Combined with the results of in vitro studies on hPAT1-mediated transepithelial transport of gabosadal, this suggests that 5-HTP may reduce the absorption rate of gabosadal by interacting with PAT1 in the rat small intestine. 4.2. Renal excretion of gabosadole may depend on the presence of transport proteins and 5-HTP. In addition to reducing initial absorption of gabosadole, 5-HTP increased AUC, primarily due to its alteration of the plasma concentration profile of gabosadole after time to peak concentration (Tmax). Previous studies have shown that gabosadole has low plasma protein binding (Lund et al., 2006) and is not a substrate of cytochrome P-450 (Schultz et al., 1981; Lund et al., 2006). Therefore, the decreased clearance of gabosadole may be the cause of its increased AUC. The significant prolongation of Tmax is consistent with the decreased clearance of gabosadole; however, pre-dose with 5-HTP did not significantly alter Cmax. Perhaps the simultaneous action of rPat1-mediated inhibition of gabosadole absorption and decreased clearance can explain the observed stable Cmax levels. Gabosadole is primarily excreted via the kidneys, and varying amounts of metabolites are detectable in the urine. In human urine, 34% of gaboxadol exists as O-glucuronide (Lund et al., 2006). However, only 2–7% of O-glucuronide was detected in rat urine, so metabolic changes do not appear to affect the results of this study (Schultz et al., 1981). After identifying rPat1 as the most likely transporter responsible for intestinal transport of gaboxadol, we searched for other transporters that might be involved in renal processing of gaboxadol. The proton-coupled amino acid transporters rPat1 and rPat2 may be involved in the reabsorption of gaboxadol in urine, but the role of 5-HTP may be to reduce reabsorption, thereby increasing renal excretion and clearance. Pre-administration of 5-HTP resulted in the opposite effect, with both CL and CL/Fa decreasing, suggesting that either efflux transporters on the apical membrane of rat renal epithelial cells or influx transporters on the basolateral membrane may be inhibited. A previous study on the pharmacokinetics and elimination of acyclovir in humans found a similar increase in AUC and a decrease in drug excretion when patients were concurrently taking probenecid (Laskin et al., 1982). Since probenecid is an inhibitor of efflux transporters such as OAT1, and acyclovir is a substrate of rOAT1, their interaction at hOAT1 is likely to lead to reduced renal tubular secretion of the drug (Wada et al., 2000). Furthermore, recent studies have shown that the transport of gabosadol into the human kidney is mediated via hOAT1 (Chu et al., 2009). Therefore, we investigated the basolateral organic anion transporter 1 (rOat1) in the rat kidney, and the results showed that gabosadol is its substrate, while 5-hydroxytryptophan (5-HTP) is not. Organic anion transporters have broad substrate specificity and have been shown to transport, especially small amphiphilic molecules, across boundary epithelial cells (Rizwan and Burckhardt, 2007). In oocytes injected with rOat1 cRNA, the uptake rate of gabosadol was characterized by a mean Km value of 151 μM. This value is very close to the gabosadol uptake Km value (115 ± 27 μM) obtained in CHO-K1 cells transfected with hOAT1 (Chu et al., 2009). In contrast, most antibiotics have low affinity for hOAT1, while nonsteroidal anti-inflammatory drugs (NSAIDs) have high affinity, thus gabosadol is considered a medium-affinity substrate for rOat1 (Apiwattanakul et al., 1999; Rizwan and Burckhardt, 2007). The murine homologs of rOat1, mOat1 and hOAT1, have been observed to promote the transport of neuroactive tryptophan metabolites (Alebouyeh et al., 2003; Bahn et al., 2005). However, the presence of 5-hydroxytryptophan (5-HTP) did not reduce oocyte uptake of gabosadol via rOat1, indicating that 5-HTP does not directly inhibit gabosadol excretion via rOat1. However, in rats, 5-HTP can be metabolized to serotonin (serotonin or 5-HT) by aromatic L-amino acid decarboxylase (LAAD), and further metabolized to 5-hydroxyindoleacetic acid (5-HIAA) by monoamine oxidase (MAO) (Stier et al., 1984; Wang et al., 2001). It has been reported that 1 mM 5-HIAA inhibits approximately 90% of the uptake of 0.25 μM [3H]-PAH in mOat1-transfected COS-7 cells (Bahn et al., 2005). Therefore, based on the rate of 5-HIAA production and compound affinity in rats, 5-HIAA may interact with gabosadol on rOat1.
In in vivo studies, the highest plasma concentration of gabosadol observed after administration of a dose of 5.0 mg/kg was approximately 2000 ng/ml, equivalent to 14.3 μM. Therefore, the clearance of gabosadol may not be limited by the transport capacity of rOat1. The effect of 5-HTP on gabosadol clearance cannot be explained by the transporter identified in this study. One possible explanation is the effect of 5-HT on renal blood flow and glomerular filtration rate. In isolated perfused rat kidneys and in vivo rat kidneys, serotonin (5-HT) has been shown to mediate selective constriction of glomerular arterioles and significantly alter renal function (Stier et al., 1984; Ding et al., 1989; Wang et al., 2001). In anesthetized rats, continuous infusion of 5-hydroxytryptophan (5-HTP) at rates of 15 and 75 μg/min for 20 minutes resulted in a 26%–41% decrease in glomerular filtration rate, effective plasma flow, and urine flow, respectively (Stier and Itskovitz, 1985). This could also explain the decrease in the acetaminophen elimination rate constant observed in this study, and is generally applicable to compounds with some degree of renal excretion, although the interaction with the Oat1 receptor cannot be ruled out (Khamdang et al., 2002). [2] Conclusion This study suggests that the transport of Gaboxadol on the Caco-2 cell monolayer is likely mediated by PAT1, and that 5-HTP inhibits this transport. Oral absorption of Gaboxadol in rats may also be mediated by rPat1, as the presence of 5-HTP reduces the initial absorption of Gaboxadol. The anion transporter rOat1 is thought to be involved in the processing of Gaboxadol by the rat kidneys. This study reveals a possible drug-supplement interaction, as 5-HTP significantly alters the overall pharmacokinetic profile of Gaboxadol. While it is difficult to speculate what the consequences would be if humans were to take Gaboxadol for insomnia or other conditions while taking 5-HTP supplements, it could lead to serious side effects. Identifying the transporter involved in the pharmacokinetic profile of Gaboxadol lays the foundation for further understanding of potential drug interactions.

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Pharmacokinetic analysis of oral absorption of Gaboxadol in dogs [6]
In beagle dogs, plasma concentration curves were monitored over 10 hours after oral or intravenous administration of 2.5 mg kg−1 Gaboxadol (Figure 3). The bioavailability Fa of oral Gaboxadol in dogs was high (over 80%) (Table 1). Oral co-administration of 2.5–150 mg kg−1 tryptophan did not significantly alter the AUC of Gaboxadol, and the mean relative bioavailability of each formulation ranged from 75% (10 mg kg−1 tryptophan) to 86.1% (2.5 mg kg−1 tryptophan). Furthermore, the elimination rate constant (ke) and clearance rate (CL) of gabosadal were not altered by co-administration with tryptophan. However, after co-administration with tryptophan at 150 mg kg⁻¹, the peak plasma concentration (Cmax) of gabosadal decreased from 2502 ng mL⁻¹ to 1419 ng mL⁻¹, a reduction of 57%. Additionally, the time to reach peak plasma concentration (Tmax) was prolonged from 0.46 h to 1.5 h (P < 0.01). Subsequently, the Cmax values from the five dose groups were fitted to dose-response curves (Figure 4), indicating a direct interaction between gabosadal absorption and tryptophan concentration. The in vivo IC50 value of tryptophan on gabosadal Cmax was estimated to be 12.6 mg kg⁻¹, equivalent to a tryptophan concentration of 12.3 mmol L⁻¹ (uncorrected for gastrointestinal dilution).

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Absorption rate constants of gabosadol and acetaminophen[6]
As shown in the deconvolution curves in Figure 5A, the mean cumulative absorption fraction of gabosadol was gradually altered by the administration of escalating doses of tryptophan. Compared with gabosadol alone, the absorption of gabosadol was significantly reduced at time points from 0.5 to 1.25 hours in the presence of 150 mg kg−1 tryptophan. The absorption rate of acetaminophen 60 minutes after oral administration was 91.5 ± 3.3% (Figure 5B), indicating that gastric emptying mainly occurred within the first hour after administration. The administration of 150.0 mg kg−1 tryptophan did not significantly alter the gastric emptying rate, as there was no significant difference in the absorption fraction of acetaminophen at the time points tested, regardless of the presence of tryptophan. The pharmacokinetic parameters Tmax, AUC, and CL of acetaminophen in plasma were not significantly different from those obtained after co-administration of acetaminophen and tryptophan (results not shown). Based on the curve shown in Figure 5A, the absorption rate constant ka of gabosadal was calculated and plotted as a function of the logarithm of tryptophan dose in Figure 6A. Co-administration with tryptophan reduced the ka value of gabosadal, with an in vivo IC50 of 10.3 mg kg−1, equivalent to an oral solution of tryptophan at a concentration of 10.1 mmol L−1. Figure 6B shows that 150 mg kg−1 of the PAT1 inhibitor tryptophan significantly reduced the absorption rate constant of gabosadal (P < 0.01), while having no significant effect on the absorption rate constant of acetaminophen.

Intraperitoneal LD50 in mice: 98 mg/kg. Sensory organs and special senses: ptosis; Behavior: somnolence (overall activity inhibition); Skin and its appendages (skin); Hair; Other neuropharmacology, 21(803), 1982 [PMID:7121752]

[1]. THIP, a hypnotic and antinociceptive drug, enhances an extrasynaptic GABAA receptor-mediated conductance in mouse neocortex. Cereb Cortex. 2006 Aug;16(8):1134-41.

[2]. 5-Hydroxy-L-tryptophan alters gaboxadol pharmacokinetics in rats: involvement of PAT1 and rOat1 in gaboxadol absorption and elimination. Eur J Pharm Sci. 2010 Jan 31;39(1-3):68-75.

[3]. Probing α4βδ GABAA receptor heterogeneity: differential regional effects of a functionally selective α4β1δ/α4β3δ receptor agonist on tonic and phasic inhibition in rat brain. J Neurosci. 2014 Dec 3;34(49):16256-72.

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

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

[6]. 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.

4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridine-3-ol is an oxazole compound. Gaboxadol, also known as 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP), is an experimental sleep aid developed by Lundbeck and Merck. It was reported to increase deep sleep without the stimulating effects of benzodiazepines. Development of Gaboxadol was halted in March 2007 due to concerns about safety and efficacy. It acts on the GABA system, but its mechanism of action appears to differ from benzodiazepines and other sedatives.

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Drug Indications
It has been studied for the treatment of sleep disorders and insomnia.

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We investigated the effects of the δ-subunit-preferred hypnotic drug THIP on GABAA receptors in mouse neocortical neurons. We found that at clinically relevant concentrations, Gaboxadol/THIP induced significant GABAA receptor-mediated tetanic currents in neocortical neurons without affecting intrasynaptic GABAA receptors. The intensity of these tetanic currents was qualitatively correlated with the expression of the δ subunit in each layer of the neocortex. Unexpectedly, THIP significantly reduced the frequency of activity-driven spontaneous inhibitory postsynaptic currents (sIPSCs) in neurons of layers 2/3, while increasing the frequency of spontaneous excitatory postsynaptic currents (sEPSCs). These results suggest that THIP primarily acts on extrasynaptic GABAA receptors in the neocortex and may reduce inhibitory synaptic input by inhibiting interneuronal activity. This may explain why THIP alters cortical oscillatory activity (Vyazovskiy et al., 2005), since neuronal firing time is partly dependent on the extensively connected network of interneurons (Tamas et al., 2000; Buhl et al., 1998). [1] The role of GABA(C) receptors in many aspects of nervous system function, including memory, myopia, pain, and sleep, is being extensively studied. Evidence has been established that functional GABA(C) receptors exist in a variety of tissues, including the retina, hippocampus, spinal cord, superior colliculus, pituitary gland, and intestine. This article reviews a number of neurochemicals that have been shown to be useful in distinguishing GABA(C) receptors from those of the major inhibitory neurotransmitter GABA. This article describes some selective agonists (including (+)-cAMP and 5-methyl-IAA), competitive antagonists (e.g., TMPPA, (±)-cis-3-ACPBPA, and aza-THIP), positive modulators (allogrenolone), and negative modulators (epigrenolone, lorecrix). In addition, neurochemicals that may help distinguish homologous p1 and p2 GABA(C) receptors (2-methyl-TACA and cyclothiazide) are also introduced. Because GABA(C) receptors are less abundant and structurally simpler than GABA(A) and GABA(B) receptors, they are highly attractive drug targets. [4] Gaboxadol at a dose of 0.5 mg/kg restored all behavioral deficits in Fmr1 KO2 mice to normal. While higher doses also restored irritability and aggressive behavior, this was not observed in other behavioral domains assessed. The therapeutic window observed here is somewhat narrow, and one explanation may be that previous studies have shown that impaired information processing is due to persistent under- or over-inhibition, and Gaboxadol enhances this physiological process. According to this model, the behavioral benefits of high-dose drugs would be offset by drug-induced deficits unrelated to Fragile X syndrome (Duguid et al., 2012). Our results provide strong evidence for the potential benefits of Gaboxadol in reversing behaviors, aggression, and social competence associated with autism spectrum disorder. In summary, these results support the hypothesis that Gaboxadol's enhancement of extrasynaptic GABAA receptor activity may be beneficial for patients with Fragile X syndrome. In conclusion, these data support future evaluation of Gaboxadol in patients with Fragile X syndrome (FXS), particularly in terms of symptoms such as hyperactivity, anxiety, autism spectrum disorder (ASD)-related stereotyped behaviors, social skills, irritability, aggression, and cognitive function. [5]

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Background and Objectives: Gaboxadol has been in development for the treatment of chronic pain and insomnia. Clinical use has shown that adverse effects of Gaboxadol appear to be related to peak serum concentrations. This study aimed to investigate the intestinal absorption mechanism of Gaboxadol in vitro and in vivo.
Experimental Methods: In vitro transport studies were conducted in Caco-2 cell monolayers. In vivo pharmacokinetic studies were conducted in beagle dogs. Subjects were administered 2.5 mg/kg⁻¹ of Gaboxadol via intravenous injection (1.0 mL/kg⁻¹) or oral solution (5.0 mL/kg⁻¹). Main results: Gaboxadol is likely a substrate of the human proton-coupled amino acid transporter hPAT1, which inhibits hPAT1-mediated L-[³H]proline uptake in Caco-2 cell monolayers with an inhibition constant K₁ of 6.6 mmol/L⁻¹. Transepithelial transport of Gaboxadol exhibits apical-to-basal polarization, dependent on Gaboxadol concentration and the pH of the apical buffer. In beagle dogs, Gaboxadol absorption was nearly complete (absolute bioavailability F(a) 85.3%), with a time to peak concentration (T(max)) of 0.46 hours. Oral administration of 2.5–150 mg·kg⁻¹ of the PAT1 inhibitor L-tryptophan significantly reduced the absorption rate constants k(a) and C(max) of gabosadol and prolonged its T(max), while the area under the curve and clearance remained unchanged. Conclusion and significance: The absorption of gabosadol across the intestinal luminal membrane may be mediated by PAT1. This study contributes to reducing the absorption rate of gabosadol, thereby reducing the peak plasma concentration. [6] In summary, this study is the first to show that the high permeability of gabosadol on the Caco-2 cell monolayer is likely due to PAT1-mediated transluminal membrane transport, resulting in high transepithelial transport. In vitro gabosadol transport kinetics and pharmacokinetics observed in dogs support the conclusion that PAT1-mediated transmucosal transport of gabosadol is possible both in vitro and in vivo. Furthermore, this study shows that the intestinal absorption of drugs can be modulated or controlled by the activity of transport proteins. The formulation is designed to provide a simple method for reducing peak plasma concentrations of gabozador while maintaining high bioavailability. This may help reduce side effects associated with high peak plasma concentrations. [6]

C₆H₈N₂O₂

140.14

140.058

64603-91-4

Gaboxadol hydrochloride;85118-33-8

3448

White to yellow solid powder

1.3±0.1 g/cm3

340.5±42.0 °C at 760 mmHg

159.7±27.9 °C

0.0±0.8 mmHg at 25°C

1.551

-0.61

2

3

0

10

210

0

C1CNCC2=C1C(=O)NO2

ZXRVKCBLGJOCEE-UHFFFAOYSA-N

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

4,5,6,7-Tetrahydroisoxazolo(5,4-c)pyridin-3(2H)-one4,5,6,7-Tetrahydro-[1,2]oxazolo[5,4-c]pyridin-6-ium-3-one

Gaboxadol; OV-101; MK0928; Lu02030; OV101; Lu-02-030; gaboxadol; 64603-91-4; Gaboxadolum; Gaboxadolum [Latin]; Gaboxadol [USAN:INN]; 4,5,6,7-Tetrahydroisoxazolo(5,4-c)pyridin-3-ol; Lu 02-030; MK-0928; Lu-02030

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)

DMSO : ~50 mg/mL (~356.79 mM)
H2O : ~23.33 mg/mL (~166.48 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (17.84 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 (17.84 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 3: ≥ 2.08 mg/mL (14.84 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 20.8 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 4: 50 mg/mL (356.79 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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