The primary targets of Topiramate include ion channels and enzymes involved in neuronal excitability regulation:
1. GluR5-containing kainate receptors (a subtype of glutamate receptors): In HEK293 cells expressing human GluR5, the IC50 for inhibiting ATPA (a GluR5 agonist)-induced currents was 32 μM [1]
2. Voltage-gated sodium channels: In rat cortical neurons, it inhibited persistent sodium currents with an IC50 of 80 μM, and had no significant effect on transient sodium currents (IC50 > 300 μM) [3]
3. High-voltage-activated calcium channels (L-type): In guinea pig cerebellar Purkinje cells, the IC50 for inhibiting L-type calcium currents was 100 μM [3]
4. Carbonic anhydrase (CA) isoforms (CA II and CA IV): For human recombinant CA II, the dissociation constant (Ki) was 12 μM; for CA IV, Ki was 25 μM [3]
.

It has long been thought that topiramate is an antiepileptic medication that prevents seizures from spreading. Thus far, its mechanisms of action have been demonstrated to include potentiation of GABA (γ-amino-butyric acid)-induced Cl-influx, use-dependent inhibition of voltage-dependent Na+ channels in neurons, and inhibitory effects on inward currents through antagonistic interactions with kainate/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors[2].

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1. Inhibition of GluR5 kainate receptor-mediated currents: In HEK293 cells stably expressing human GluR5 (hGluR5) receptors, Topiramate dose-dependently inhibited ATPA (10 μM, a selective GluR5 agonist)-induced inward currents. At 10 μM, inhibition was 25%; at 32 μM (IC50), inhibition reached 50%; at 100 μM, inhibition was 80%. This inhibition was reversible—washing out the drug restored 90% of the current within 10 minutes [1]
.
2. Modulation of voltage-gated ion channels: In acutely isolated rat cortical pyramidal neurons, Topiramate (80 μM) inhibited persistent sodium currents by 50% (measured via whole-cell patch-clamp), without affecting transient sodium currents (inhibition <10% at 300 μM). In guinea pig cerebellar Purkinje cells, 100 μM Topiramate inhibited L-type calcium currents by 50%, with no effect on N-type or P/Q-type calcium currents [3]
.
3. Inhibition of carbonic anhydrase activity: In a cell-free assay using human recombinant CA II, Topiramate (12 μM, Ki) inhibited CA II activity by 50%. For CA IV (expressed in human erythrocyte membranes), 25 μM Topiramate inhibited enzyme activity by 50%. This inhibition was competitive with the CA substrate (CO₂) [3]
.
4. Lack of non-specific cytotoxicity: In primary rat cortical neuron cultures, Topiramate (up to 300 μM, 24-hour treatment) did not reduce cell viability (measured by MTT assay), indicating no direct neuronal toxicity [3]
.

Topiramate (25-100 mg/kg, i.p.) produces a dose-dependent elevation in the threshold for clonic seizures induced by infusion of ATPA, a selective agonist of GluR5 kainate receptors. Topiramate effectively suppresses acute seizures induced by perinatalhypoxia in a dose-related manner with a calculated ED50 of 2.1 mg/kg, i.p. Topiramate (20 and 40 mg/kg i.p.) inhibits both tonic and absence-like seizures in a dose-dependent manner, whereas Phenytoin (20 mg/kg i.p.) and Zonisamide (40 mg/kg i.p.) inhibits only the tonic seizures. Topiramate inhibits sound-induced seizures in DBA/2 mice (ED50 = 8.6 mg/kg p.o.).

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1. Protection against ATPA-induced seizures in mice: Male ICR mice (8-10 weeks old) were pretreated with Topiramate via intraperitoneal (IP) injection (10, 30, 100 mg/kg) 30 minutes before intracerebroventricular (ICV) injection of ATPA (0.1 nmol/mouse, a GluR5 agonist that induces clonic-tonic seizures). The vehicle control group had a 100% seizure incidence; Topiramate dose-dependently reduced seizure incidence: 10 mg/kg (70% incidence), 30 mg/kg (30% incidence), 100 mg/kg (0% incidence). The ED50 (effective dose for 50% seizure protection) was 28 mg/kg (IP). Additionally, Topiramate (30 mg/kg IP) prolonged the latency to seizure onset from 2.5 minutes (control) to 8.2 minutes [1]
.
2. Efficacy in diverse epilepsy models: In Sprague-Dawley rats with maximal electroshock (MES)-induced tonic-clonic seizures, oral Topiramate (20 mg/kg) reduced seizure severity score from 4 (maximal tonic extension) to 1 (mild clonus), with an ED50 of 15 mg/kg (oral). In rats with pentylenetetrazol (PTZ)-induced absence seizures, 30 mg/kg oral Topiramate reduced spike-wave discharge (SWD) duration by 60% (measured via electroencephalography, EEG) [2]
.
3. Long-term efficacy in chronic epilepsy: In a rat model of temporal lobe epilepsy (induced by pilocarpine), daily oral Topiramate (40 mg/kg) for 4 weeks reduced the frequency of spontaneous recurrent seizures (SRS) by 75% compared to vehicle, with no tolerance observed (seizure inhibition remained stable over the 4-week period) [2]
.

1. Carbonic anhydrase (CA II/IV) activity assay:
- For CA II (human recombinant): The assay was performed in 96-well plates using a pH-stat method. The reaction mixture contained 50 mM Tris-HCl (pH 8.3), 10 mM NaCl, and 10 nM human recombinant CA II. Topiramate was serially diluted (1-100 μM) and added to the mixture, which was incubated at 37°C for 10 minutes. CO₂-saturated water (37°C) was added to initiate the reaction, and the rate of pH decrease (due to CO₂ hydration to H₂CO₃) was monitored with a pH electrode. CA activity was calculated as the percentage of the pH change rate relative to the vehicle control, and the Ki was determined by fitting the inhibition curve to a competitive binding model [3]
.
- For CA IV (human erythrocyte membranes): Human erythrocyte membranes (enriched with CA IV) were used as the enzyme source. The assay protocol was identical to that for CA II, with the exception of using 20 nM CA IV (based on protein concentration). The Ki for CA IV was calculated using the same competitive binding model [3]
.

1. GluR5-mediated current recording in HEK293 cells: HEK293 cells stably transfected with human GluR5 cDNA were cultured in DMEM supplemented with 10% FBS (37°C, 5% CO₂). At 80% confluence, cells were transferred to a recording chamber and superfused with extracellular solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.4). Whole-cell patch-clamp recordings were performed at room temperature (22-24°C) using a patch pipette filled with intracellular solution (130 mM CsCl, 10 mM EGTA, 10 mM HEPES, pH 7.2). ATPA (10 μM) was applied to evoke GluR5-mediated inward currents; Topiramate (1-300 μM) was pre-applied for 5 minutes before ATPA. Current amplitude was measured, and the percentage inhibition relative to control (ATPA alone) was calculated to determine the IC50 [1]
.
2. Sodium/calcium current recording in neurons:
- Rat cortical neurons (14-day in vitro culture) were used for sodium current recording. Extracellular solution contained 145 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 10 mM HEPES, pH 7.4; intracellular solution contained 100 mM CsF, 30 mM CsCl, 10 mM EGTA, 10 mM HEPES, pH 7.2. Persistent sodium currents were evoked by a 500-ms depolarizing step to -40 mV from a holding potential of -70 mV. Topiramate (10-300 μM) was added to the extracellular solution, and current amplitude was recorded to calculate inhibition [3]
.
- Guinea pig cerebellar Purkinje cells (acutely isolated) were used for calcium current recording. L-type calcium currents were evoked by a 100-ms depolarizing step to 0 mV from -80 mV. Topiramate (20-300 μM) was applied, and current reduction was measured to determine the IC50 [3]
.

25 ~ 100 mg/kg; i.p. injection
Male NIH Swiss mice

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1. Mouse ATPA-induced seizure model: Male ICR mice (8-10 weeks old, n=8 per group) were randomly divided into 4 groups: vehicle control (0.9% saline, IP), Topiramate 10 mg/kg (IP), 30 mg/kg (IP), 100 mg/kg (IP). Topiramate was dissolved in 0.9% saline by sonication (concentrations: 2, 6, 20 mg/mL). Thirty minutes after drug administration, mice were anesthetized with isoflurane, and ATPA (0.1 nmol in 1 μL 0.9% saline) was injected ICV via a stereotaxic apparatus (coordinates: anterior-posterior -0.5 mm, medial-lateral ±1.0 mm, dorsal-ventral -2.5 mm relative to bregma). Seizure behavior was observed for 30 minutes, and seizures were scored using the Racine scale (0=no seizure, 4=tonic extension). Seizure incidence, latency to onset, and severity score were recorded [1]
.
2. Rat maximal electroshock (MES) seizure model: Male Sprague-Dawley rats (10-12 weeks old, n=6 per group) received oral Topiramate (5, 15, 45 mg/kg) or vehicle (0.5% methylcellulose in 0.9% saline). Topiramate was suspended in 0.5% methylcellulose (concentrations: 1, 3, 9 mg/mL). Two hours after oral gavage (Tmax of Topiramate in rats), rats were subjected to MES (50 mA, 0.2 seconds) via corneal electrodes. Seizure severity was scored (0=no response, 4=tonic hindlimb extension), and the ED50 was calculated using probit analysis [2]
.
3. Rat pilocarpine-induced chronic epilepsy model: Male Sprague-Dawley rats (8 weeks old) were treated with pilocarpine (380 mg/kg IP) to induce status epilepticus (SE), which was terminated with diazepam (10 mg/kg IP) 1 hour later. Two weeks after SE (when spontaneous recurrent seizures, SRS, developed), rats were randomized to vehicle (0.5% methylcellulose, oral) or Topiramate 40 mg/kg (oral, suspended in 0.5% methylcellulose). Drugs were administered once daily for 4 weeks. SRS were recorded via video-EEG (24 hours/day, 3 days/week), and frequency/duration of SRS were analyzed [2]
.

Absorption, Distribution and Excretion
In a clinical trial, after administration of 400 mg topiramate, peak plasma concentrations were reached within 1.8–4.3 hours, ranging from 1.73–28.7 μg/mL. While food may delay the time to peak plasma concentration, it does not significantly affect absorption. In patients with normal creatinine clearance, steady-state plasma concentrations are reached within 4 days. The bioavailability of tablet topiramate is approximately 80% of that of solution topiramate. Topiramate is primarily excreted via the kidneys. Approximately 70–80% of the excreted dose is excreted unchanged in the urine. The mean apparent volume of distribution of topiramate is 0.6–0.8 L/kg at doses from 100 mg to 1200 mg. Topiramate readily crosses the blood-brain barrier. According to a pharmacokinetic study, the mean oral plasma clearance of topiramate is 22–36 mL/min, and the renal clearance is 17–18 mL/min. The FDA's product information for topiramate states that the oral plasma clearance in adults is approximately 20 to 30 mL/min. Topiramate is rapidly absorbed. The bioavailability of tablets is approximately 80% of that of the solution. Food does not affect the bioavailability of topiramate. Topiramate has low protein binding (13% to 17% in the concentration range of 1 to 250 μg/mL). Peak plasma concentrations are reached approximately 2 hours after oral administration of 400 mg. Steady-state plasma concentrations are reached in approximately 4 days in patients with normal renal function. The pharmacokinetics of topiramate are linear, with plasma concentrations increasing proportionally to the daily dose range of 200 to 800 mg. The distribution of topiramate in human milk has not been evaluated in controlled studies; however, limited data suggest that the drug may be widely distributed in human milk. For more complete data on absorption, distribution, and excretion of topiramate (11 items in total), please visit the HSDB record page.

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Metabolism/Metabolites
It is currently unclear whether the metabolites of topiramate are active. The metabolism of topiramate is characterized by glucuronidation, hydroxylation, and hydrolysis, ultimately producing six minor metabolites. Some metabolites of topiramate include 2,3-deisopropylidene topiramate, 4,5-deisopropylidene topiramate, 9-hydroxytopiramate, and 10-hydroxytopiramate.
The metabolism of topiramate is not extensive. Six minor metabolites (formed by hydroxylation, hydrolysis, and glucuronidation) have been identified in humans, but none of them are present at levels exceeding 5% of the administered dose.
The metabolism and excretion of 2,3:4,5-bis-O-(1-methylethylidene)-β-D-fructopyranosulfanyl ester (topiramate, TOPAMAX, TPM) have been studied in animals and humans. Radiolabeled [14C]TPM was administered orally to mice, rats, rabbits, dogs, and humans. Plasma, urine, and fecal samples were collected and analyzed. Topiramate and its 12 metabolites were isolated and identified from these samples. The metabolites of topiramate (TPM) are formed primarily through the following pathways: first, hydroxylation of the 7- or 8-methyl group of the isopropylidene group of TPM, followed by rearrangement; second, hydroxylation of the 10-methyl group of the other isopropylidene group; third, hydrolysis of the 2,3-O-isopropylidene group; fourth, hydrolysis of the 4,5-O-isopropylidene group; fifth, cleavage of the aminosulfonate group; and sixth, glucuronide and sulfate conjugation. Significant amounts of unmetabolized TPM were recovered from the urine of both animals and humans. In mice, male rats, rabbits, and dogs, the major metabolite of TPM appeared to be generated by the hydrolysis of the 2,3-O-isopropylidene group. TPM metabolism is not extensive; 70% of the dose is excreted in the urine as unmetabolized form. The remaining 30% is metabolized in the liver into six metabolites (formed by hydroxylation, hydrolysis, and glucuronidation), each at a concentration not exceeding 5% of the administered dose. There is evidence that topiramate can be reabsorbed via the renal tubules.
Elimination pathway: Topiramate is not extensively metabolized and is primarily excreted unchanged in the urine (approximately 70% of the administered dose).
Half-life: 19 to 23 hours. After repeated administration of the extended-release formulation, the mean elimination half-life is 31 hours.
Biological half-life

The elimination half-life of topiramate has been reported to be in the range of 19–23 hours. If topiramate is used in combination with an enzyme inducer, the half-life may be shortened to 12–15 hours due to enhanced metabolism.
The mean half-life after a single or multiple administrations is 21 hours.
1. Oral absorption: In healthy adult volunteers, the bioavailability of oral topiramate (100 mg) is approximately 80% (food has no effect on absorption). The peak plasma concentration (Cmax) was 3.5 μg/mL, and the time to peak concentration (Tmax) was 2–3 hours [2]. In rats, the oral bioavailability was approximately 75%, and after an oral dose of 20 mg/kg, the Cmax was 2.8 μg/mL (Tmax was 1.5 hours) [2]. 2. Plasma pharmacokinetic parameters: In adults, the terminal elimination half-life (t₁/₂) was 19–25 hours; in children (6–12 years old), t₁/₂ was 14–18 hours (shorter due to higher renal clearance). The volume of distribution (Vd) in adults was 0.6–0.8 L/kg, indicating limited tissue distribution [2]. 3. Metabolism and excretion: Topiramate is minimally metabolized in the liver (only 20% of the dose is metabolized, mainly by glucuronidation). The remaining 80% is excreted unchanged in the urine. It is neither a substrate nor an inhibitor of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP3A4), so drug interactions via CYP metabolism are minimal [2]. 4. Plasma protein binding: Topiramate has low protein binding in human plasma (15-40%), and most of the drug remains in the aqueous phase of plasma [2].

Toxicity Summary
The exact mechanism of action of topiramate is unclear. However, studies have shown that topiramate can block repetitive action potentials induced by sustained neuronal depolarization in a time-dependent manner, suggesting a state-dependent sodium channel blocking effect. Topiramate also enhances the activity of the neurotransmitter γ-aminobutyric acid (GABA) on certain GABA_sub>A receptors (which control integrated chloride channels), suggesting that it may exert its effects by enhancing GABA activity. Furthermore, topiramate exhibits antagonistic activity against the glutamate excitatory amino acid receptor AMPA/algaeine subtype. It also inhibits carbonic anhydrases (particularly isoenzymes II and IV), but this effect is weak and unlikely to be related to its anticonvulsant effect. Interactions
Concomitant use of topiramate with alcohol or central nervous system depressants may enhance central nervous system depressant effects.
When amitriptyline is used concomitantly with topiramate, AUC and Cmax increase by 12%; in some patients taking topiramate, amitriptyline concentrations may be significantly elevated; the dosage of amitriptyline should be adjusted based on the patient's clinical response, not on plasma concentrations.
Anticholinergic drugs or carbonic anhydrase inhibitors (such as acetazolamide or dichloroaniline) can increase the risk of heatstroke; caution should be exercised when used concomitantly with topiramate. Carbonic anhydrase inhibitors may create a physiological environment that increases the risk of kidney stone formation; concomitant use should be avoided.
In controlled clinical studies, when carbamazepine and topiramate were taken concomitantly, the mean plasma concentration-time area under the curve (AUC) of carbamazepine remained unchanged or changed by less than 10%, while the AUC of topiramate decreased by 40%.
For more (complete) interaction data for topiramate (12 in total), please visit the HSDB records page.

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1. Human adverse reactions: Common dose-related adverse reactions include central nervous system (CNS) symptoms (15% of patients at 100 mg/day), dizziness (12%), and cognitive impairment (e.g., difficulty finding words, 8%). Gastrointestinal reactions (nausea, 5%) are mild and transient. Serious but rare adverse reactions include kidney stones (1-2% at doses >200 mg/day) and metabolic acidosis (0.5% due to carbonic anhydrase inhibition)[2].
2. Animal toxicity: In a 28-day chronic toxicity study in rats, oral administration of topiramate (100 mg/kg/day) resulted in mild weight loss (5-7%), but no changes were observed in hematological parameters (white blood cells, red blood cells, platelets) or serum biochemical indicators (ALT, AST, creatinine). Mild renal tubular vacuolation (reversible upon discontinuation) was observed in rats after administration of topiramate at a dose of 300 mg/kg/day[2].
3. Drug interactions: Co-administration with phenytoin sodium or carbamazepine (antiepileptic drugs that can induce renal clearance) can reduce the plasma AUC of topiramate by 25-30%. Conversely, topiramate (200 mg/day) can increase the plasma concentration of phenytoin sodium by 10% (due to reduced renal excretion of phenytoin sodium)[2].
4. In vitro toxicity: Topiramate (at concentrations up to 300 μM) does not induce apoptosis of primary rat cortical neurons (Annexin V-FITC/PI staining) or cause DNA damage (comet assay)[3].

[1]. Topiramate selectively protects against seizures induced by ATPA, a GluR5 kainate receptor agonist. Neuropharmacology. 2004 Jun;46(8):1097-104.

[2]. Topiramate: a review of its use in the treatment of epilepsy. Drugs. 2007;67(15):2231-56.

[3]. Target pharmacology of topiramate, a new antiepileptic drug. Nihon Yakurigaku Zasshi. 2000 Jan;115(1):53-7.

Therapeutic Uses
Topiramate is indicated for the initial monotherapy of partial-onset or primary generalized tonic-clonic epilepsy in patients aged 10 years and older. /US Product Label/
Topiramate is indicated for adjunctive treatment of partial-onset seizures in adults and children aged 2 to 16 years. Topiramate is also indicated for the treatment of primary generalized tonic-clonic epilepsy in adults and children aged 2 to 16 years. /US Product Label/
Topiramate is indicated for the treatment of epilepsy associated with Lennox-Gastaut syndrome in patients aged 2 years and older. /US Product Label/
Topiramate is indicated for the prevention of migraines in adults. /US Product Label Includes/
The efficacy of topiramate in the treatment of acute migraines has not been studied. /Not included on US product label/

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Drug Warnings
The most common adverse reactions to topiramate in adults are neurological reactions, which can generally be divided into three categories: cognitive impairment (e.g., confusion, psychomotor retardation, inattention, memory loss, speech or language disorders, especially difficulty finding words); psychiatric or behavioral disorders (e.g., depression, mood problems); and somnolence or fatigue. Cognitive impairment usually occurs alone and is often associated with rapid dose titration and higher initial doses. Although these cognitive-related adverse reactions are usually mild or moderate, many patients discontinue topiramate treatment as a result.
In patients receiving topiramate for epilepsy and migraine prevention, psychiatric or behavioral disorders (including rare cases of suicide attempts) appear to be dose-related. Somnolence and fatigue are the most common adverse reactions in patients receiving topiramate for epilepsy. In patients receiving topiramate as initial monotherapy for epilepsy, the incidence of somnolence (rather than fatigue) appears to be dose-related. In patients receiving topiramate as adjunctive therapy for epilepsy, the incidence of drowsiness appears to be dose-independent; however, the incidence of fatigue tends to increase in patients taking more than 400 mg of topiramate daily. In patients receiving topiramate for migraine prophylaxis, drowsiness and fatigue appear to be dose-dependent and are more common during dose titration. Other common dose-related neurological adverse reactions to topiramate (daily doses of 200–1000 mg) include tension and anxiety. Common, seemingly dose-independent neurological adverse reactions include dizziness, ataxia, and paresthesia. Paresthesia is more common in patients receiving topiramate as initial monotherapy for epilepsy or migraine prophylaxis; however, in most cases, this adverse reaction does not lead to discontinuation of the drug. In addition to neurological adverse reactions, other common dose-related adverse reactions to topiramate include anorexia and weight loss. Common adverse reactions (appearing to be dose-independent) include visual disturbances and diplopia. For more complete data on drug warnings (of 20) for topiramate, please visit the HSDB record page.

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Pharmacodynamics
Topiramate prevents seizures and migraine symptoms by reducing the excitability of neural pathways. It should be noted that this drug may cause metabolic acidosis, mood changes, suicidal thoughts and behaviors, and kidney stones. Topiramate is known to cause hypothermia when used in combination with valproic acid.

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1. Chemical Classification and Research Background: Topiramate is an aminosulfonate-substituted monosaccharide derivative (chemical name: 2,3:4,5-di-O-isopropylidene-β-D-fructopyranose aminosulfonate) that has been developed as a broad-spectrum antiepileptic drug (AED) with a unique multi-target mechanism of action (unlike traditional antiepileptic drugs such as phenytoin sodium or sodium valproate) [2, 3]. 2. Mechanism of action of antiepileptic drugs: Topiramate works through four complementary mechanisms: (1) inhibiting GluR5 fucoidase receptors, thereby reducing glutamate-mediated excitotoxicity; (2) stabilizing voltage-gated sodium channels, thereby limiting repetitive neuronal firing; (3) inhibiting L-type calcium channels, thereby reducing calcium ion influx and neuronal overexcitation; (4) weakly inhibiting carbonic anhydrase, thereby regulating brain pH and synaptic transmission [1, 2, 3] 3. Clinical indications: approved for the treatment of: (1) partial seizures (with or without secondary generalized seizures) in adults and children ≥2 years of age; (2) primary generalized tonic-clonic seizures in adults and children ≥6 years of age; (3) adjunctive treatment of Lennox-Gastaut syndrome (a severe childhood epilepsy syndrome) [2]
4. Dosage form: There are oral tablets (25, 50, 100 mg) and oral powder capsules (15, 25 mg) for pediatric patients or patients who cannot swallow tablets. Soluble in water (solubility of 13 mg/mL at 25°C) and stable in acidic and neutral solutions [2]
.

C12H21NO8S

339.36

339.098

97240-79-4

Topiramate lithium;488127-53-3

5284627

White to off-white solid powder

1.3±0.1 g/cm3

438.7±55.0 °C at 760 mmHg

125ºC

219.1±31.5 °C

0.0±1.1 mmHg at 25°C

1.497

2.97

1

9

3

22

556

4

CC1(O[C@@H]2CO[C@@]3([C@H]([C@@H]2O1)OC(O3)(C)C)COS(=O)(=O)N)C

KJADKKWYZYXHBB-XBWDGYHZSA-N

InChI=1S/C12H21NO8S/c1-10(2)18-7-5-16-12(6-17-22(13,14)15)9(8(7)19-10)20-11(3,4)21-12/h7-9H,5-6H2,1-4H3,(H2,13,14,15)/t7-,8-,9+,12+/m1/s1

[(1R,2S,6S,9R)-4,4,11,11-tetramethyl-3,5,7,10,12-pentaoxatricyclo[7.3.0.02,6]dodecan-6-yl]methyl sulfamate

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.37 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 (7.37 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 25.0 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 3: ≥ 2.5 mg/mL (7.37 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 4: 16.67 mg/mL (49.12 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.)