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 oral Gaboxadol absorption in Sprague–Dawley rats [2]
The gaboxadol plasma concentration profiles after intravenous and oral administration to Sprague–Dawley rats are shown in Fig. 1. The time to maximum plasma concentration, Tmax, was 0.2 and 0.3 h for the dose groups of 0.5 and 5.0 mg/kg gaboxadol, respectively (Table 2). The oral absorption of 0.5 mg/kg gaboxadol appeared to be complete as the mean bioavailability, Fa, was 110%. The Fa of 5 mg/kg was 83 ± 5%, which is significant lower than the Fa determined for 0.5 mg/kg (p < 0.01). The clearance, CL/Fa, of gaboxadol increased from 1.93 to 2.56 l/h/kg for the doses of 0.5 and 5 mg/kg gaboxadol (p < 0.05), respectively. The peak plasma concentration, Cmax, increased 7 times when the dose increased 10-fold from 0.5 to 5 mg/kg and did not indicate dose proportionality at the investigated dosage range. Furthermore, the AUC only increased 7.5 times after a 10-fold increase in dose. The nonlinear pharmacokinetics suggested that a transport process in the absorption or the elimination of gaboxadol was carrier mediated. The carrier could be a saturable absorptive carrier in the intestine or a carrier involved in reabsorption of gaboxadol in the kidney. Administration of oral Gaboxadol and 5-HTP to Sprague–Dawley rats [2]
5-HTP administration significantly changed the gaboxadol plasma concentration profiles after both intravenous and oral dosing in rats (Fig. 1A–C) The absorption and elimination phases were altered, furthermore the AUC was significantly increased as a consequence of pre-dosing with 5-HTP in rats. During the initial absorption phase of gaboxadol (≤Tmax) the gaboxadol plasma concentrations with 5-HTP dosing were significantly lower as compared to the plasma concentrations in the animals not receiving 5-HTP (p < 0.05, Fig. 1B and C). 5-HTP did not significantly change the maximal plasma concentration, Cmax, of gaboxadol. Furthermore, 5-HTP caused a significant prolongation of the Tmax (p < 0.01 and p < 0.001) for 0.5 and 5.0 mg/kg, respectively, indicating that 5-HTP alters the intestinal absorption of gaboxadol in rats. The gaboxadol elimination phases shown in the semilogarithmic plots in Fig. 1A and B were approximately straight lines for rats dosed with 5-HTP and biphasic curves for gaboxadol administered alone. The presence of 5-HTP increased the mean AUC of 0.5 and 5.0 mg/kg gaboxadol by a factor of 5.5 (p < 0.01) and 3.6, respectively. The mean clearance, CL/Fa, of 0.5 and 5.0 mg/kg gaboxadol was decreased by 77% (p < 0.01) and 23% (not significant), respectively. The clearance, CL, of 2.5 mg/kg (IV) gaboxadol was decreased by and 66% (n = 2) in the presence of 5-HTP (Table 2). The oral absorption of paracetamol, used in the present study as a marker for gastric emptying, was not significantly changed after pre-dosing with 5-HTP or mannitol based on an evaluation of Cmax, Tmax, AUC, Fa and, CL/Fa (Fig. 2 and Table 3). Therefore, it is concluded that 5-HTP did not significantly prolong the gastric emptying. However, the elimination rate constant for paracetamol, ke (0.95 h−1), was significant lower in the presence of 5-HTP (0.53 h−1, p < 0.05). Gaboxadol is a substrate of rOat1, but 5-HTP is not [2]
Since 5-HTP had a significant effect on gaboxadol clearance in rats, the transport of gaboxadol by the renal organic anion transporter, rOat1 was investigated in the absence or presence of 5-HTP (Fig. 3, Fig. 4). The uptake of gaboxadol in both rOat1 cRNA and water injected oocytes was linear over the 90 min investigated (Fig. 3A). The gaboxadol uptake was significantly higher in rOat1 cRNA than in water injected oocytes (p ≤ 0.001). The concentration dependent uptake rate of gaboxadol in rOat1 cRNA injected oocytes was saturable (Fig. 3B). The data points could be described by Michaelis–Menten kinetics with a Km of 151.2 ± 58.19 μM and a Vmax of 0.78 ± 0.09 pmol oocyte−1 min−1. Gaboxadol is thus a substrate for rOat1 and we therefore investigated the rOat1-mediated uptake of gaboxadol in the presence of 5 mM 5-HTP, 5 mM l-proline, 1 mM PAH and 1 mM probenecid (Fig. 4). The uptake of gaboxadol was not changed by the presence of 5-HTP or l-proline, whereas the uptake of gaboxadol in the presence of PAH and probenecid (substrate and inhibitor of Oat1, respectively) was significantly reduced (p < 0.001). 5-HTP decreases Gaboxadol absorption in vitro and in vivo [2]
Transport of gaboxadol across Caco-2 cell monolayers was polarized in the A–B direction. The A–B gaboxadol transport was dependent on donor concentration and pH in the donor medium. This is consistent with gaboxadol transport across the luminal membrane of enterocytes mediated by hPAT1, a transport step which appears to be important for the resulting transepithelial absorption. Similar observations have been made in Caco-2 cell monolayers regarding the pH dependent transport of other PAT1 substrates such as l-proline (Thwaites et al., 1993) and GABA (Thwaites et al., 2000). Previously, hPAT1 was cloned from Caco-2 cells by Chen et al. and immunofluorescence showed that PAT1 is expressed at the apical membrane of the Caco-2 cells and of the small intestinal enterocytes in rats (Chen et al., 2003, Anderson et al., 2004). In Caco-2 cell monolayers 80–90% of gaboxadol transport was most likely mediated by hPAT1 and 5-HTP inhibited this transport. The Ki-values of gaboxadol and 5-HTP for inhibition of l-proline uptake in Caco-2 cells via hPAT1 were 6.6 mM (Larsen et al., 2009) and 2.3 mM, respectively (Larsen et al., 2008). The question is thus if the interaction between gaboxadol and 5-HTP is observed at relevant concentrations. Assuming an oral gaboxadol dose of 15 mg and a volume of 250 ml the resulting luminal concentration is 0.34 mM. For 5-HTP a normal dose is 50–100 mg resulting in a luminal concentration of 9–18 mM. In our experiment the lowest concentration of gaboxadol is 0.34 mM and that of 5-HTP is 18.8 mM. The concentrations are thus possible to obtain in vivo and the interaction between the two compounds likely to be relevant. In vivo the peak plasma concentration of gaboxadol was reached within 0.22–0.33 h after oral administration to Sprague–Dawley rats. During the initial absorption phase of gaboxadol, significantly lower plasma gaboxadol concentrations were observed in the rats pre-dosed with 5-HTP compared to the rats dosed only with gaboxadol. Furthermore, the presence of 5-HTP significantly increased Tmax of gaboxadol. A parallel study of paracetamol absorption, an often used marker for gastric emptying, showed that 5-HTP did not significantly delay the gastric emptying. This indicated that 5-HTP decreased the absorption rate of gaboxadol in the rat small intestine. Taken together with the in vitro findings of hPAT1-mediated transepithelial transport of gaboxadol, it is suggested that 5-HTP decreased the absorption rate of gaboxadol by its interaction with PAT1 in the rat small intestine. 4.2. Renal excretion of gaboxadol is likely to be dependent on transporters and the presence of 5-HTP

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In addition to decreasing the initial absorption of Gaboxadol, 5-HTP also increased AUC, mainly due to the changed the plasma profile of gaboxadol after Tmax. Previous studies have shown that gaboxadol has a low degree of plasma protein binding (Lund et al., 2006) furthermore, it is not a substrate of cytochrome P-450's (Schultz et al., 1981, Lund et al., 2006). Consequently, a decrease in gaboxadol clearance was likely to account for the increased gaboxadol AUC. The significantly prolonged Tmax is consistent with a decreased gaboxadol clearance however; Cmax was not significantly changed by 5-HTP pre-dosing. Perhaps, the simultaneous inhibition of gaboxadol absorption rate via rPat1 and a reduced gaboxadol clearance can explain the steady Cmax level observed. Gaboxadol is excreted predominantly via the kidneys, and variable amounts of metabolites can be found in urine i.g. in human urine, 34% of gaboxadol was found as an O-glucuronide (Lund et al., 2006). In rat urine, however, only 2–7% O-glucuronide has been found, and accordingly changes in metabolism do not seem to affect the results obtained the present study (Schultz et al., 1981).

Subsequent to identifying rPat1 as the most likely responsible intestinal transporter of Gaboxadol, we looked for transporters that could participate in renal handling of gaboxadol. The proton-coupled amino acid transporters rPat1 and rPat2 could be involved in reabsorption of gaboxadol from the urine, however the effect of 5-HTP would then likely be decreasing the reabsorption leading to an increased renal excretion and clearance. After 5-HTP pre-dosing the opposite was observed, i.e. a decrease in CL and CL/Fa, which points to a possible inhibition of an efflux transporter in the apical membrane or an influx transporter in the basolateral membrane of rat renal epithelial cells. A previous study on the pharmacokinetics and elimination of acyclovir in humans found similar effects of an increased AUC and a decreased drug excretion rate when the patients were also administered with probenecid (Laskin et al., 1982). As probenecid is an inhibitor of efflux transporters such as OAT1, and acyclovir is a substrate of rOat1, an interaction at hOAT1 most likely resulted in a decreased tubular secretion of the drug (Wada et al., 2000). Furthermore, it was recently suggested that the transport of gaboxadol into the human kidney happens via hOAT1 (Chu et al., 2009). Therefore, the rat basolateral renal organic anion transporter 1, rOat1, was investigated and it was demonstrated that gaboxadol is a substrate, whereas 5-HTP is not. Organic anion transporters are characterized by a broad substrate specificity and have been shown to transport especially small amphiphilic molecules across boundary epithelia (Rizwan and Burckhardt, 2007). The gaboxadol uptake rate in rOat1 cRNA injected oocytes was characterized by a mean Km value of 151 μM. This value is quite similar to the Km value of gaboxadol uptake (115 ± 27 μM) obtained in hOAT1 transfected CHO-K1 cells (Chu et al., 2009). In comparison, most antibiotics have lower affinities for hOAT1, whereas NSAIDs have higher affinities classifying gaboxadol as a medium affinity substrate for rOat1 (Apiwattanakul et al., 1999, Rizwan and Burckhardt, 2007). The murine and human orthologues of rOat1, mOat1 and hOAT1, respectively, have been observed to facilitate the transport of neuroactive tryptophan metabolites (Alebouyeh et al., 2003, Bahn et al., 2005). However, the uptake of gaboxadol in oocytes via rOat1 was not decreased by the presence of 5-HTP indicating that 5-HTP does not directly inhibit gaboxadol excretion via rOat1. Nevertheless, in rats, 5-HTP can be metabolised by the aromatic l-amino acid decarboxylase (LAAD) to 5-hydroxytryptamine (serotonin or 5-HT) and further by monoamine oxidase (MAO) to 5-hydroxyindoleacetic acid (5-HIAA) (Stier et al., 1984, Wang et al., 2001). 1 mM 5-HIAA has been reported to inhibit the uptake of 0.25 μM [3H]-PAH in mOat1 transfected COS-7 cells with approximately 90% (Bahn et al., 2005). Thus, depending on the formation rate of 5-HIAA in rats and affinity values of the compounds, interaction of 5-HIAA with gaboxadol at rOat1 might be possible.

The maximal Gaboxadol plasma concentration observed following doses of 5.0 mg/kg in the present in vivo study was approximately 2000 ng/ml, which corresponds to 14.3 μM. Thus, the clearance of gaboxadol was probably not limited by the capacity of rOat1. The effects on the clearance of gaboxadol observed with 5-HTP cannot be accounted for in terms of the transporters identified here. A possible explanation might be found in the effects of 5-HT on renal blood flow and glomerular filtration rate. In isolated perfused rat kidneys as well as in rat kidneys in vivo, 5-HT has been shown to mediate selective constriction of the afferent glomerular arterioles and significantly alter the renal function (Stier et al., 1984, Ding et al., 1989, Wang et al., 2001). In anesthetized rats, the glomerular filtration rate, effective plasma flow and urine flow were decreased by 26–41% during a 20 min infusion of 15 and 75 μg/min 5-HTP (Stier and Itskovitz, 1985). This would also account for the decreased paracetamol elimination rate constant observed in the present study, and generally for compounds with a certain extent of renal excretion, although interactions at Oat1 cannot be ruled out (Khamdang et al., 2002). [2]

Conclusion
The present study demonstrated that Gaboxadol transport across Caco-2 cell monolayers is most likely mediated via PAT1 and that 5-HTP inhibits this transport. The oral absorption of gaboxadol in rats might also be rPat1-mediated as the initial gaboxadol absorption was decreased by the presence of 5-HTP. The anion transporter rOat1 is suggested to be involved in the renal handling of gaboxadol in rats. The present study illustrates a possible drug–dietary supplement interaction as 5-HTP significantly changed the overall pharmacokinetic profile of gaboxadol. Although it is difficult to speculate about the consequences in humans taking gaboxadol for the treatment of insomnia or another indication along with 5-HTP supplements it could cause severe side effects. The identification of transporters involved in determining the pharmacokinetic profile of gaboxadol provides the basis for a further understanding of potential drug–drug interactions.

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Pharmacokinetic analysis of oral Gaboxadol absorption in dog [6]
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 [6]
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.

mouse LD50 intraperitoneal 98 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); SKIN AND 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)pyridin-3-ol is an oxazole.
Gaboxadol also known as 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP) is an experimental sleep aid drug developed by Lundbeck and Merck, who reported increased deep sleep without the reinforcing effects of benzodiazepines. Development of Gaboxadol was stopped in March 2007 after concerns regarding safety and efficacy. It acts on the GABA system, but in a seemingly different way from benzodiazepines and other sedatives.

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Drug Indication
Investigated for use/treatment in sleep disorders and insomnia.

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We have studied the actions of the δ-subunit preferring hypnotic drug THIP on neuronal GABAA receptors in the mouse neocortex. We found that Gaboxadol/THIP, at clinically relevant concentrations, induced a significant GABAA-mediated tonic current in neocortical neurons, without affecting intrasynaptic GABAA receptors. The magnitude of the tonic current correlated qualitatively with the δ-subunit expression in neocortical layers. Surprisingly, THIP caused a pronounced decrease in the frequency of activity driven sIPSCs in layer 2/3 neurons, while the frequency of sEPSCs increased. These results indicate that THIP primarily acts at extrasynaptic GABAA receptors in the neocortex, and may particularly depress interneuron activity to diminish inhibitory synaptic input. This may explain why THIP alters the cortical oscillatory activity (Vyazovskiy et al., 2005), since the spike timing, in part, depends on the networks of extensively connected interneurons (Tamas et al., 2000; Buhl et al., 1998).[1]

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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. [4]

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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.[5]

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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.[6]

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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.[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.)