Admissible daily intake for glutamate.

PURPOSE OF REVIEW: Total glutamate (Glu) intake is 5-20 g/day in adults and about 40 mg/kg in breast-fed infant. Glu intake is constituted by Glu from protein and free Glu from certain foods and flavor-enhancing additive. The admissible intake of free Glu additive is addressed. RECENT FINDING: In the gut, Glu is actively metabolized by enterocytes and because of this metabolism, the systemic availability of ingested Glu remains relatively low. Human studies are preferred to assess the transfer in blood of dietary free Glu salts and their possible risks. When human data are not available, experimental animal models provide the basis to assess the risks to humans but toxicity studies in rodents remain for a part controversial. A No Observable Adverse Effect Level (NOAEL) in rodent of 3200 mg/kg/day and an uncertainty factor of 100 lead to an acceptable daily intake (ADI) of 30 mg/kg/day for free Glu salts used as additives, whereas a NOAEL higher than 6000 mg/kg/day and an uncertainty factor of 25 leads to an ADI of 240 mg/kg/day for free Glu salts. SUMMARY: Current discussions indicate an ADI from 30 to 240 mg/kg/day depending on the chosen NOAEL in animal model and compound-specific uncertainty factor (from 25 to 100).

Expression and Functional Characterization of Drug Transporters in Brain Microvascular Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

Brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs) have been proposed as a new blood-brain barrier model, but their transport function has not been fully clarified. Therefore, in this study, we investigated the gene expression and function of transporters in hiPS-BMECs by means of quantitative reverse transcription-PCR, in vitro transcellular transport studies, and uptake experiments. mRNAs encoding ABC and SLC transporters, such as BCRP, MCT1, CAT1, and GLAST, were highly expressed in hiPS-BMECs. Transcellular transport studies showed that prazosin, [14C]l-lactate, [3H]l-arginine, and [3H]l-glutamate (substrates of BCRP, MCT1, CAT1, and GLAST, respectively) were transported asymmetrically across the hiPS-BMEC monolayer. Substrates of LAT1, OCTN2, CAT1, GLAST, MCT1, and proton-coupled organic cation (H+/OC) antiporter were taken up by hiPS-BMECs in a time-, temperature-, and concentration-dependent manner, and the uptakes were markedly decreased by inhibitors of the corresponding transporter. These results indicate that hiPS-BMECs express multiple nutrient and drug transporters.

Metabolism of Dietary Glutamate in Adults.

BACKGROUND: Glutamate is a non-essential amino acid at the crossroads of nitrogen and energy metabolism. Glutamate metabolism is characterized by reactions that may be anabolic or catabolic in nature depending on the tissue (i.e., glutamate dehydrogenase, transaminases), and it can also be either the precursor or the metabolite of glutamine. Unlike glutamine, which is the form of interorgan ammonia transport, glutamate metabolism is mostly compartmentalized within the cells, its interorgan exchanges being limited to a flux from liver to muscle. SUMMARY: Glutamate catabolism is extremely intense in the splanchnic area, such that after a meal (rich in proteins) almost no glutamate appears in the systemic circulation. However, this process is saturable as after glutamate loading at a high dose level, glutamate appears dose-dependently in the circulation. This systemic glutamate -appearance is blunted if glutamate is co-ingested with a carbohydrate source. Key Messages: The underlying reason for this highly specific metabolism is that glutamate plays a key role in nitrogen homeostasis, and the organism does all it can to limit the bioavailability of glutamate, which can be neurotoxic in excess. As glutamate is never eaten alone, its bioavailability will be limited if not negligible, and no adverse effects are to be expected in adult humans.

Specific rescue by ortho-hydroxy atorvastatin of cortical GABAergic neurons from previous oxygen/glucose deprivation: role of pCREB.

The statin atorvastatin (ATV) given as a post-treatment has been reported beneficial in stroke, although the mechanisms involved are not well understood so far. Here, we investigated in vitro the effect of post-treatment with ATV and its main bioactive metabolite ortho-hydroxy ATV (o-ATV) on neuroprotection after oxygen and glucose deprivation (OGD), and the role of the pro-survival cAMP response element-binding protein (CREB). Post-OGD treatment of primary cultures of rat cortical neurons with o-ATV, but not ATV, provided neuroprotection to a specific subset of cortical neurons that were large and positive for glutamic acid decarboxylase (large-GAD(+) neurons, GABAergic). Significantly, only these GABAergic neurons showed an increase in phosphorylated CREB (pCREB) early after neuronal cultures were treated post-OGD with o-ATV. We found that o-ATV, but not ATV, increased the neuronal uptake of glutamate from the medium; this provides a rationale for the specific effect of o-ATV on pCREB in large-GABAergic neurons, which have a higher ratio of synaptic (pCREB-promoting) vs extrasynaptic (pCREB-reducing) N-methyl-D-aspartate (NMDA) receptors (NMDAR) than that of small-non-GABAergic neurons. When we pharmacologically increased pCREB levels post-OGD in non-GABAergic neurons, through the selective activation of synaptic NMDAR, we observed as well long-lasting neuronal survival. We propose that the statin metabolite o-ATV given post-OGD boosts the intrinsic pro-survival factor pCREB in large-GABAergic cortical neurons in vitro, this contributing to protect them from OGD.

Impact of oral supplementation of Glutamate and GABA on memory performance and neurochemical profile in hippocampus of rats.

Glutamate (GLU) and gamma-amino butyric acid (GABA) are essential amino acids (AA) for brain function serving as excitatory and inhibitory neurotransmitter respectively. Their tablets are available in market for improving gut function and muscle performance. Despite of having a major role during memory formation and processing, effects of these tablets on brain functioning like learning and memory have not been investigated. Therefore, present study is aimed to investigate the effects of orally supplemented GLU and GABA on learning and memory performance and further to monitor related effects of these orally supplemented GLU and GABA on brain levels of these AA. Three groups of rats were supplemented orally with drinking water (control group) or suspension of tablets of GABA and Glutamate, respectively for four weeks. Cognitive performance was determined using behavioral tests (Novel object recognition test, Morris water maze, Passive avoidance test) measuring recognition, spatial reference and aversive memory. Levels of GLU, GABA and acetylcholine (ACh) were estimated in rat hippocampus. Results showed that chronic oral administration of GLU and GABA tablets has a significant impact on brain function and can alter GLU and GABA content in rat hippocampus. Compared to GABA, GLU supplementation specifically enhances memory performance via increasing ACh. Thus, GLU can be suggested as a useful supplement for improving learning and memory performance and neurochemical status of brain and in future could be effective in the treatment of neurological disorders affecting learning and memory performance.

Positioning the Intracellular Salt Potassium Glutamate in the Hofmeister Series by Chemical Unfolding Studies of NTL9.

In vitro, replacing KCl with potassium glutamate (KGlu), the Escherichia coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu- in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to the effects of other stabilizers (KCl, KF, and K2SO4) and destabilizers (GuHCl and GuHSCN). GuHSCN titrations at 20 ° C, performed as a function of the concentration of KGlu or another salt and monitored by NTL9 fluorescence, are analyzed to obtain R-values quantifying the Hofmeister salt concentration (m3) dependence of the unfolding equilibrium constant K(obs) [r-value = −d ln K(obs)/dm3 = (1/RT) dΔG(obs) ° /dm3 = m-value/RT]. r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 ° C, we determine a KGlu r-value of 3.22 m(−1) and K2SO4, KF, KCl, GuHCl, and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38, and −3.00 m(−1), respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 ° C.

Novel bicyclo[3.1.0]hexane analogs as antagonists of metabotropic glutamate 2/3 receptors for the treatment of depression.

Negative modulators of metabotropic glutamate 2 & 3 receptors demonstrate antidepressant-like activity in animal models and hold promise as novel therapeutic agents for the treatment of major depressive disorder. Herein we describe our efforts to prepare and optimize a series of conformationally constrained 3,4-disubstituted bicyclo[3.1.0]hexane glutamic acid analogs as orthosteric (glutamate site) mGlu2/3 receptor antagonists. This work led to the discovery of a highly potent and efficacious tool compound 18 (hmGlu2 IC50 46±14.2nM, hmGlu3 IC50=46.1±36.2nM). Compound 18 showed activity in the mouse forced swim test with a minimal effective dose (MED) of 1mg/kg ip. While in rat EEG studies it exhibited wake promoting effects at 3 and 10mg/kg ip without any significant effects on locomotor activity. Compound 18 thus represents a novel tool molecule for studying the impact of blocking mGlu2/3 receptors both in vitro and in vivo.

Resolving confounding enrichment kinetics due to overlapping resonance signals from 13C-enriched long chain fatty acid oxidation and uptake within intact hearts.

PURPOSE: Long chain fatty acid (LCFA) oxidation measurements in the intact heart from 13C-NMR rely on detection of 13C-enriched glutamate. However, progressive increases in overlapping resonance signal from LCFA can confound detection of the glutamate 4-carbon (GLU-C4) signal. We evaluated alternative 13C labeling for exogenous LCFA and developed a simple scheme to distinguish kinetics of LCFA uptake and storage from oxidation. METHODS: Sequential 13C-NMR spectra were acquired from isolated rat hearts perfused with 13C LCFA and glucose. Spectra were evaluated from hearts supplied: U 13C LCFA, [2,4,6,8,10,12,14,16-(13) C8 ] palmitate, [2,4,6,8,10,12,14,16,18-(13) C9 ] oleate, [4,6,8,10,12,14,16-(13) C7 ] palmitate, or [4,6,8,10,12,14,16,18-(13) C8 ] oleate. RESULTS: 13C signal reflected the progressive enrichment at 34.6 ppm from GLU-C4, confounded by additional signal with distinct kinetics attributed to 13C-enriched LCFA 2-carbon (34.0 ppm). Excluding 13C at the 2-carbon of both palmitate and oleate eliminated signal overlap and enabled detection of the exponential enrichment of GLU-C4 for assessing LCFA oxidation. CONCLUSION: Eliminating enrichment at the 2-carbon of 13C LCFA resolved confounding kinetics between GLU-C4 and LCFA 2-carbon signals. With this enrichment scheme, oxidation of LCFA, the primary fuel for cardiac ATP synthesis, can now be more consistently examined in whole organs with dynamic mode, proton-decoupled (13C-NMR

Pharmacokinetic Properties of a New Glutamic Acid Derivative Glutaron.

Inhomogenous distribution of glutaron in organs and tissues was found after intravenous and peroral administration: the agent demonstrated high affinity to organs with high degree of vascularization (lungs and heart) and elimination (kidney). Glutaron easily penetrates through the blood-brain barrier, which is consistent with its concentration in the adipose tissue.

[ABSOLUTE AND RELATIVE BIOAVAILABILITY OF GLUTARON--A NEW DERIVATIVE OF GLUTAMIC ACID].

The pharmacokinetics of studies of 3-phenylglutamic acid hydrochloride (glutaron) has been studied in rats. The main pharmacokinetic parameters show low values of the half-life (T1/2 = 3.75 h), mean retention time in the body (MRT = 5.77 h). The medium rate of drug concentration decrease in the blood plasma leads to a low value of the area under pharmacokinetic curve (AUC = 41.18 mg · h/mL). The general volume of distribution (Vd = 3.42 L/kg) is 3.5 times greater than the volume of extracellular fluid in the rat body. These data indicate a high ability of the glutaron to be distributed and accumulated in animal tissues. The value of absolute bioavailability is 84%, and the relative bioavailabity is 100%.

Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake.

Human erythrocytes have a low basal permeability to L-glutamate and are not known to have a functional glutamate transporter. Here, treatment of human erythrocytes with arsenite was shown to induce the uptake of L-glutamate and D-aspartate, but not that of D-glutamate or L-alanine. The majority of the arsenite-induced L-glutamate influx was via a high-affinity, Na(+)-dependent system showing characteristics of members of the "excitatory amino acid transporter" (EAAT) family. Western blots and immunofluorescence assays revealed the presence of a member of this family, EAAT3, on the erythrocyte membrane. Erythrocytes infected with the malaria parasite Plasmodium falciparum take up glutamate from the extracellular environment. Although the majority of uptake is via a low-affinity Na(+)-independent pathway there is, in addition, a high-affinity uptake component, raising the possibility that the parasite activates the host cell glutamate transporter.

Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1.

Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure-function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)3 (CuPh) and cadmium ion (Cd(2+)) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + L-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.

Nitrogen in dietary glutamate is utilized exclusively for the synthesis of amino acids in the rat intestine.

Although previous studies have shown that virtually the entire carbon skeleton of dietary glutamate (glutamate-C) is metabolized in the gut for energy production and amino acid synthesis, little is known regarding the fate of dietary glutamate nitrogen (glutamate-N). In this study, we hypothesized that dietary glutamate-N is an effective nitrogen source for amino acid synthesis and investigated the fate of dietary glutamate-N using [(15)N]glutamate. Fischer male rats were given hourly meals containing [U-(13)C]- or [(15)N]glutamate. The concentration and isotopic enrichment of several amino acids were measured after 0-9 h of feeding, and the net release of each amino acid into the portal vein was calculated. Most of the dietary glutamate-C was metabolized into CO(2), lactate, or alanine (56, 13, and 12% of the dietary input, respectively) in the portal drained viscera (PDV). Most of the glutamate-N was utilized for the synthesis of other amino acids such as alanine and citrulline (75 and 3% of dietary input, respectively) in the PDV, and only minor amounts were released into the portal vein in the form of ammonia and glutamate (2 and 3% of the dietary input, respectively). Substantial incorporation of (15)N into systemic amino acids such as alanine, glutamine, and proline, amino acids of the urea cycle, and branched-chain amino acids was also evident. These results provide quantitative evidence that dietary glutamate-N distributes extensively to amino acids synthesized in the PDV and, consequently, to circulating amino acids.

Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions.

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 µM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti-inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A1 receptor, phosphatidylinositol-3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin-sensitive G-protein-coupled signaling, blockade of adenosine A2A receptors (A2A R), but not via A1 receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.

Relationship between thresholds to convulsions induced by a benzodiazepine inverse agonist and [³H]-L-glutamate binding in the membranes of brain regions.

Although some studies have investigated the influence of kindling model of epilepsy on the glutamatergic neurotransmission, the relation between glutamatergic receptors and seizure susceptibility remains unclear. The present study sought to determine if rats with high (HTR) and low (LTR) thresholds to clonic convulsions induced by the benzodiazepine inverse agonist DMCM differed in the [(3)H]-L-glutamate binding to membranes from discrete brain regions. Compared to the HTR subgroup, the LTR subgroup presented a lower binding of [(3)H]-L-glutamate in the hippocampus, frontal cortex and amygdala plus limbic cortex, suggesting that glutamatergic receptors in these brain regions may underlie the susceptibility to DMCM-induced convulsions.

Not all desensitizations are created equal: physiological evidence that AMPA receptor desensitization differs for kainate and glutamate.

AMPA receptor-mediated responses to the agonist kainate differ from those of glutamate in two important respects. Glutamate is a full agonist that elicits strongly desensitizing responses, whereas kainate is a partial agonist with responses that are often described as weakly desensitizing or non-desensitizing. The efficacy of kainate relative to glutamate has previously been shown to be increased by mutations in the AMPA receptor ligand-binding cleft (Mano et al., 1996) and by coexpression with the auxiliary subunit stargazin (Tomita et al., 2005; Turetsky et al., 2005), but much less is known about factors that affect kainate desensitization. We therefore designed experiments to compare kainate and glutamate desensitization and efficacy in wild-type and mutant AMPA receptors expressed with and without stargazin in HEK293 cells. Desensitization to the two agonists was differentially affected by mutations in the helices participating in bonds between two subunits in the active state of the receptor (Sun et al., 2002), indicating that the protein interactions maintaining the stability of the dimer interface differ depending on which agonist is bound. Kainate efficacy was affected by factors distinct from ligand-binding cleft closure, including mutations in the dimer interface and channel vestibule as well as receptor composition. The increase in kainate responses for AMPA receptors coexpressed with stargazin was the result of both reduced kainate desensitization and increased kainate efficacy. These results provide critical new insights into the agonist dependence of both AMPA receptor activation and desensitization and the mechanism of the effects of stargazin on responses of partial agonists.

mPEG-b-P(Glu-co-Phe) nanoparticles increase gastric retention time and gastric ulcer treatment efficacy of 20(S)-ginsenoside Rg3.

BACKGROUND: Gastric ulcer (GU) belongs to gastric mucosal irritation and damage. 20(S)-ginsenoside Rg3 (Rg3) has shown anti-oxidant, antiinflammation, and tissue repair effects which are essential for GU treatment. However, the solubility of Rg3 is poor and low gastrointestinal absorption may limit its anti-ulcer effects. As a result, we aim to increase the gastric retention time and gastric absorption of Rg3 to achieve better GU treatment efficacy. METHODS: The mPEG-b-P(Glu-co-Phe) nanoparticles loaded with Rg3 (Rg3-NPs) were developed. The characteristics of Rg3-NPs, including the morphology, diameter, and stability were analyzed. The Rg3 release profiles, gastric retention of Rg3, in vitro cytotoxicity, and pharmacokinetics of Rg3 were assessed. An alcohol-induced rats GU model was performed, and the rats were randomly separated into five treatment groups. Biochemical analysis, gross evaluation, histopathology, and immunohistochemical analysis were applied to further analyze the anti-ulcer effects of Rg3-NPs. RESULTS: Rg3-NPs were successfully prepared and the Rg3 release was pH sensitive. The gastric retention time of Rg3 is longer in Rg3-NPs group than that in Rg3 group. By slightly increasing nitric oxide (NO), obviously increasing epidermal growth factor (EGF), EGF receptor (EGFR), and superoxide dismutase (SOD), and decreasing endothelin-1 (ET-1) and nitric oxide synthase (NOS2), Rg3-NPs possess better GU treatment efficacy than Rg3. CONCLUSIONS: Rg3-NPs can increase gastric retention time and gastric absorption of Rg3 and promote its GU treatment efficacy.

Structural determinants for transport across the intestinal bile acid transporter using C-24 bile acid conjugates.

The human apical sodium dependent bile acid transporter (hASBT) reabsorbs gram quantities of bile acid daily and is a potential prodrug target to increase oral drug absorption. In the absence of a high resolution hASBT crystal structure, 3D-QSAR modeling may prove beneficial in designing prodrug targets to hASBT. The objective was to derive a conformationally sampled pharmacophore 3D-QSAR (CSP-SAR) model for the uptake of bile acid conjugates by hASBT. A series of bile acid conjugates of glutamyl chenodeoxycholate were evaluated in terms of K(m) and normalized V(max) (normV(max)) using hASBT-MDCK cells. All monoanionic conjugates were potent substrates. Dianions, cations and zwitterions, which bound with a high affinity, were not substrates. CSP-SAR models were derived using structural and physicochemical descriptors, and evaluated via cross validation. The best CSP-SAR model for K(m) included two structural and two physiochemical descriptors, where substrate hydrophobicity enhanced affinity. A best CSP-SAR model for K(m)/normV(max) employed one structural and three physicochemical descriptors, also indicating hydrophobicity enhanced efficiency. Overall, the bile acid C-24 region accommodated a range of substituted anilines, provided a single negative charge was present near C-24. In comparing uptake findings to prior inhibition results, increased hydrophobicity enhanced activity, with dianions and zwitterions hindering activity.

Involvement of glutamate in retinal protection against ischemia/reperfusion damage induced by post-conditioning.

Retinal ischemia could provoke blindness and there is no effective treatment against retinal ischemic damage. Brief intermittent ischemia applied during the onset of reperfusion (i.e., post-conditioning) protects the retina from ischemia/reperfusion injury. Multiple evidences support that glutamate is implicated in retinal ischemic damage. We investigated the involvement of glutamate clearance in post-conditioning-induced protection. For this purpose, ischemia was induced by increasing intra-ocular pressure for 40 min, and 5 min after reperfusion, animals underwent seven cycles of 1 min/1 min ischemia/reperfusion. One, three, or seven days after ischemia, animals were subjected to electroretinography and histological analysis. The functional and histological protection induced by post-conditioning was evident at 7 (but not 1 or 3) days post-ischemia. An increase in Müller cell glial fibrillary acidic protein (GFAP) levels was observed at 1, 3, and 7 days after ischemia, whereas post-conditioning reduced GFAP levels of Müller cells at 3 and 7 days post-ischemia. Three days after ischemia, a significant decrease in glutamate uptake and glutamine synthetase activity was observed, whereas post-conditioning reversed the effect of ischemia. The intravitreal injection of supraphysiological levels of glutamate mimicked electroretinographic and histological alterations provoked by ischemia, which were abrogated by post-conditioning. These results support the involvement of glutamate in retinal protection against ischemia/reperfusion damage induced by post-conditioning.

Double-blind, randomized, placebo-controlled study of trofinetide in pediatric Rett syndrome.

OBJECTIVE: To determine safety, tolerability, and pharmacokinetics of trofinetide and evaluate its efficacy in female children/adolescents with Rett syndrome (RTT), a debilitating neurodevelopmental condition for which no pharmacotherapies directed at core features are available. METHODS: This was a phase 2, multicenter, double-blind, placebo-controlled, parallel-group study, in which safety/tolerability, pharmacokinetics, and clinical response to trofinetide were characterized in 82 children/adolescents with RTT, aged 5 to 15 years. Sixty-two participants were randomized 1:1:1:1 to receive placebo twice a day (bid) for 14 days, followed by placebo, 50, 100, or 200 mg/kg bid of trofinetide for 42 days. Following blinded safety data review, 20 additional participants were randomized 1:1 to the 200 mg/kg or placebo bid groups. Safety assessments included adverse events, clinical laboratory tests, physical examinations, and concomitant medications. Clinician- and caregiver-based efficacy measurements assessed clinically relevant, phenotypic dimensions of impairment of RTT. RESULTS: All dose levels were well tolerated and generally safe. Trofinetide at 200 mg/kg bid showed statistically significant and clinically relevant improvements relative to placebo on the Rett Syndrome Behaviour Questionnaire, RTT-Clinician Domain Specific Concerns-Visual Analog Scale, and Clinical Global Impression Scale-Improvement. Exploratory analyses suggested that observed changes correlated with trofinetide exposure. CONCLUSION: These results, together with those from a previous adolescent/adult trial, indicate trofinetide's potential for treating core RTT symptoms and support further trials. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for children/adolescents with RTT, trofinetide was safe, well-tolerated, and demonstrated improvement over placebo at 200 mg/kg bid in functionally important dimensions of RTT.