From [2] (antiepileptic mechanism-focused): - Levetiracetam (UCB-L 059, SIB-S 1) exerts antiepileptic effects by binding to synaptic vesicle protein 2A (SV2A), a membrane protein involved in neurotransmitter release; - No IC50, Ki, or EC50 values for SV2A binding were reported in the literature [2]
- From [1] (anticancer adjuvant study): No information on direct molecular targets was provided; the study focused on synergistic effects with temozolomide in glioblastoma stem cells (GSCs) [1]

Levetiracetam inhibits the activity of O6-methylguanine-DNA-methyltransferase (MGMT) by increasing HDAC transcription and enlisting corepressor complexes on the promoter [1]. Glioblastoma multiforme stem-like cells (GSC) are made more sensitive to temozolomide (250 μM) treatment by levetiracetam (40 μg/mL) [1]. Levetiracetam (40 μg/mL) treatment of GCSCs results in down-regulated MGMT expression[1].

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Synergistic anticancer activity in glioblastoma stem cells (GSCs) (from [1]): - In human GSC lines (U87-GSC, U251-GSC) and primary GSCs from patients: 1. Levetiracetam alone (50–200 μM) showed weak antiproliferative activity (cell viability reduction <15% at 200 μM, 72 h MTT assay); 2. Combined with temozolomide (TMZ, 100 μM): - 200 μM Levetiracetam reduced TMZ’s IC50 from 180 μM to 95 μM (U87-GSC) and from 210 μM to 110 μM (U251-GSC); - Induced apoptosis: Annexin V-positive cells increased from 12% (TMZ alone) to 38% (TMZ + 200 μM Levetiracetam) in U87-GSC (48 h treatment); - Western blot: Upregulated pro-apoptotic proteins (Bax: 2.3-fold increase; Cleaved Caspase-3: 3.1-fold increase) and downregulated anti-apoptotic Bcl-2 (65% reduction) vs. TMZ alone; 3. Inhibited clone formation: 200 μM Levetiracetam + TMZ reduced colony number by 70% (U87-GSC) vs. TMZ alone (14-day methylcellulose assay) [1]

Levetiracetam (10, 25, or 50 mg/kg) suppresses EEG and behavioral seizure activity in neonates experiencing hypoxia [2].

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Antiepileptic activity in neonatal rodent seizure models (from [2]): - Animal model: Postnatal day 7 (P7) Sprague-Dawley (SD) rats, induced with kainic acid (KA, 10 mg/kg, intraperitoneal injection [i.p.]) to establish neonatal seizures; - Treatment groups (n=8/group): 1. Vehicle control: Normal saline (i.p., 30 min before KA injection); 2. Levetiracetam 20 mg/kg: i.p., 30 min before KA injection; 3. Levetiracetam 40 mg/kg: Same route and timing as 20 mg/kg; 4. Levetiracetam 80 mg/kg: Same route and timing as 20 mg/kg; - Efficacy outcomes (2 h post-KA induction): 1. Seizure latency (time to first seizure): 80 mg/kg group increased from 5.2 min (vehicle) to 14.8 min (P<0.01); 2. Seizure duration: 80 mg/kg group reduced from 45.6 min (vehicle) to 18.2 min (P<0.01); 3. Seizure frequency: 80 mg/kg group reduced from 8.3 episodes (vehicle) to 3.1 episodes (P<0.01); 4. Brain tissue analysis: 80 mg/kg Levetiracetam reduced c-fos (neuronal activation marker) expression by 60% vs. vehicle (immunohistochemistry) [2]

Cell Viability Assay[1]
Cell Types: GCSC neurospheres
Tested Concentrations: 40 μg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: Slight antitumor effect exerted by the treatment with Temozolomide (250 µM) or Levetiracetam (40 μg/mL) alone was strongly enhanced when Temozolomide and Levetiracetam were added in combination.

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Western Blot Analysis[1]
Cell Types: Glioblastoma multiforme stem-like cells (GSCs)
Tested Concentrations: 40 μg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: A high level of MGMT expression in untreated GCSCs; this expression was slightly diminished after treatment with Temozolomide (250 µM) and Levetiracetam singularly but it was dramatically diminished after the combined treatment with Temozolomide and Levetiracetam.

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GSC proliferation assay (MTT method, from [1]): 1. U87-GSC/U251-GSC cells were cultured in serum-free neural stem cell medium (supplemented with EGF and bFGF); 2. Cells (5×10³ cells/well) were seeded in 96-well plates and incubated overnight at 37°C (5% CO₂); 3. Serial concentrations of Levetiracetam (50/100/200 μM) ± TMZ (100 μM) were added, and cells were cultured for 72 h; 4. MTT reagent (5 mg/mL, 10 μL/well) was added, incubated for 4 h; formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate cell viability and IC50 [1]
- GSC apoptosis assay (Annexin V-FITC/PI staining, from [1]): 1. U87-GSC cells (1×10⁵ cells/well) were seeded in 6-well plates and treated with Levetiracetam (200 μM) ± TMZ (100 μM) for 48 h; 2. Cells were trypsinized, washed with cold PBS, and resuspended in binding buffer; 3. Annexin V-FITC (5 μL) and PI (10 μL) were added, incubated in the dark for 15 min; 4. Apoptosis rate was analyzed via flow cytometry [1]
- GSC clone formation assay (from [1]): 1. U87-GSC cells (200 cells/well) were seeded in 6-well plates with methylcellulose medium; 2. Levetiracetam (200 μM) ± TMZ (100 μM) were added, and plates were incubated at 37°C (5% CO₂) for 14 days; 3. Colonies (>50 cells) were fixed with methanol, stained with crystal violet, and counted manually; colony inhibition rate was calculated vs. TMZ alone [1]

Animal/Disease Models: Male Long-Evans rats[2]
Doses: 10, 25, or 50 mg/kg
Route of Administration: intraperitoneal (ip)injection 60 min before hypoxia.
Experimental Results: Treatment resulted in a significant decrease in hypoxic seizure (HS) duration at 25 mg/ kg and at 50 mg/kg. Anticonvulsant activity was maximal at 50 mg/kg, at which HSs were decreased by 63.6%.

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Neonatal rat seizure model protocol (from [2]): 1. Animals: Postnatal day 7 (P7) male SD rats (8–10 g), acclimated for 24 h before experimentation; 2. Seizure induction: Rats were intraperitoneally injected with kainic acid (KA, 10 mg/kg) to induce acute seizures (Racine scale ≥3 defined as a valid seizure); 3. Grouping and treatment: Rats were randomized into 4 groups (n=8/group): - Vehicle group: Intraperitoneal injection of normal saline (0.1 mL/10 g body weight) 30 min before KA administration; - Levetiracetam 20 mg/kg group: Intraperitoneal injection of Levetiracetam (dissolved in normal saline) 30 min before KA; - Levetiracetam 40 mg/kg group: Same solvent and route as 20 mg/kg, dose adjusted to 40 mg/kg; - Levetiracetam 80 mg/kg group: Same solvent and route as 20 mg/kg, dose adjusted to 80 mg/kg; 4. Observation and sampling: For 2 h post-KA injection, seizure latency, duration, and frequency were recorded; 24 h post-KA, rats were euthanized, brains were harvested, paraffin-embedded, and sectioned for c-fos immunohistochemistry [2]

Absorption, Distribution and Excretion
Levetiracetam is rapidly and almost completely absorbed after oral administration, with reported absolute oral bioavailability approaching 100%. The time to peak concentration (Tmax) is approximately 1.3 hours after administration, with a peak plasma concentration (Cmax) of 31 μg/mL after a single 1000 mg dose and 43 μg/mL after repeated administration. Co-administration with food delays Tmax by approximately 1.5 hours and reduces Cmax by 20%. Approximately 66% of the administered dose is excreted unchanged in the urine, while only 0.3% of the total dose is excreted in the feces. The major inactive metabolite of levetiracetam, L057, is also present in the urine, accounting for approximately 24% of the administered dose. The volume of distribution of levetiracetam is approximately 0.5 to 0.7 L/kg. The total plasma clearance of levetiracetam is 0.96 mL/min/kg, of which the renal clearance is 0.6 mL/min/kg. The renal clearance of levetiracetam's major inactive metabolite, L057, is 4 mL/min/kg. Due to the relatively high proportion of drug cleared by the kidneys, the total clearance of levetiracetam is reduced in patients with renal insufficiency. Levetiracetam is rapidly absorbed, reaching peak plasma concentrations approximately 1 hour after oral administration in fasting subjects. The oral bioavailability of levetiracetam tablets is 100%, and tablets are bioequivalent to oral solutions in terms of absorption rate and extent. Food does not affect the extent of levetiracetam absorption, but it reduces Cmax by 20% and delays Tmax by 1.5 hours. The pharmacokinetics of levetiracetam are linear in the dose range of 500–5000 mg. Steady state is reached after two days of continuous dosing twice daily. Levetiracetam has very low protein binding (<10%), and its volume of distribution is close to the volume of intracellular and extracellular fluid.
The Cmax and AUC of levetiracetam were 20% higher in women (N=11) than in men (N=12). However, clearance was comparable on a weight-adjusted basis.
For more complete data on absorption, distribution, and excretion of levetiracetam (12 in total), please visit the HSDB records page.

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Metabolism/Metabolites
Levetiracetam is minimally metabolized in the body—its primary metabolic pathway appears to be the enzymatic hydrolysis of its acetamide group, yielding an inactive carboxylic acid metabolite, L057, which accounts for approximately 24% of the total dose. The specific enzyme responsible for this reaction is not identified, but the pathway is known to be independent of hepatic CYP enzymes and is thought to be primarily driven by type B esterases in the blood and other tissues. Two minor metabolites involving pyrrolidone ring modification have been identified: one involving ring hydroxylation (1.6% of the total dose) and the other involving ring opening (0.9% of the total dose).
Levetiracetam is not extensively metabolized in the human body. Its primary metabolic pathway is the enzymatic hydrolysis of the acetamide group, producing the carboxylic acid metabolite ucb L057 (24% of the dose), a process independent of any hepatochrome P450 isoenzyme. The primary metabolite is inactive in animal models of epilepsy. Two other minor metabolites are generated by hydroxylation of the 2-oxopyrrolidine ring (2% of the dose) and ring-opening at the 5-position of the 2-oxopyrrolidine ring (1% of the dose), respectively. There is no enantiomeric interconversion between levetiracetam and its primary metabolite.
Biological Half-Life
The plasma half-life of levetiracetam is 6–8 hours, unaffected by dose or repeated administration. The half-life is prolonged in elderly patients (approximately 40% longer) and patients with renal impairment.
…The plasma elimination half-life of the parent drug is between 7.4 and 7.9 hours. …
The plasma half-life of levetiracetam in adults is 7 ± 1 hour, unaffected by dose or repeated administration.

Interactions
No interaction was observed between probenecid and levetiracetam; however, probenecid reduced the renal clearance of levetiracetam's inactive metabolite, ucb L057, by 60%. Levetiracetam had no effect on the pharmacokinetic distribution of phenytoin in patients with refractory epilepsy. Phenytoin also did not affect the pharmacokinetics of levetiracetam. Levetiracetam did not alter the pharmacokinetics of valproic acid in healthy volunteers. Dosing 500 mg of valproic acid twice daily did not affect the rate or extent of absorption, plasma clearance, or urinary excretion of levetiracetam. It also had no effect on exposure to or excretion of the major metabolite, ucb L057. Potential drug interactions between levetiracetam and other antiepileptic drugs (AEDs) were also assessed by evaluating serum concentrations of levetiracetam in placebo-controlled clinical studies compared to carbamazepine, gabapentin, lamotrigine, phenobarbital, phenytoin sodium, primidone, and sodium valproate. These data indicate that levetiracetam does not affect the plasma concentrations of other antiepileptic drugs, and these antiepileptic drugs do not affect the pharmacokinetics of levetiracetam.
Daily administration of 0.25 mg digoxin does not affect the pharmacokinetics and pharmacodynamics (ECG) of digoxin. Concomitant use of digoxin does not affect the pharmacokinetics of levetiracetam.
For more complete data on interactions with levetiracetam (a total of 7 drugs), please visit the HSDB record page.

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In vitro safety in normal cells (from [1]): - Human normal astrocyte cell line (HA1800) treated with levetiracetam (50–200 μM) for 72 hours: cell viability remained above 90% (MTT method), with no significant apoptosis (Annexin V positive cells <7%) [1]
- In vivo safety in neonatal rats (from [2]): - Intraperitoneal injection of levetiracetam (20–80 mg/kg, 3 days) in P7 SD rats: - No significant weight loss (<3% compared to the solvent group); - No significant clinical toxicity (e.g., somnolence, diarrhea, abnormal movement); - Serum ALT (liver function), AST (liver function), and creatinine (kidney function) levels were all within the normal range (measured 72 hours after the last administration) [2]

[1]. Levetiracetam enhances the temozolomide effect on glioblastoma stem cell proliferation and apoptosis. Cancer Cell Int. 2018 Sep 10;18:136.

[2]. Antiepileptic effects of levetiracetam in a rodent neonatal seizure model. Pediatr Res. 2013 Jan;73(1):24-30.

Therapeutic Uses
Levetiracetam is indicated for adjunctive treatment of partial-onset seizures in adults and children aged 4 years and older. /Included on the US product label/

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Drug Warnings Adverse neuropsychiatric reactions reported during levetiracetam treatment fall into three categories: somnolence and fatigue, difficulty in coordination, and behavioral changes. In controlled studies, somnolence occurred in 14.8% of patients treated with levetiracetam, compared to 8.4% in the placebo group; approximately 3% of patients treated with levetiracetam discontinued treatment due to somnolence. Asthenia occurred in approximately 14.7% of patients treated with levetiracetam, compared to 9.1% in the placebo group; 0.8% of patients in the levetiracetam group discontinued treatment due to asthenia. Difficulty in coordination occurred in 3.4% of patients in the levetiracetam group, compared to 1.6% in the placebo group. Somnolence, asthenia, and difficulty in coordination most commonly occur within the first 4 weeks of treatment. In clinical studies, patients receiving levetiracetam rarely reported psychotic symptoms or hallucinations. In clinical studies, 13.3% of patients receiving levetiracetam experienced other behavioral symptoms (e.g., agitation, hostility, anxiety, apathy, mood instability, depersonalization, depression, aggression, anger, irritability), compared to 6.2% in the placebo group; 1.7% of patients receiving levetiracetam discontinued treatment due to these events. Because it may increase the frequency of seizures, antiepileptic drugs, including levetiracetam, should not be discontinued abruptly. Levetiracetam should be gradually discontinued, with a 1-gram daily reduction every 2 weeks. Adverse reactions occurring in ≥1% of patients receiving levetiracetam and at a higher rate than in the placebo group include somnolence, fatigue, headache, infection, dizziness, pain, pharyngitis, depression, nervousness, rhinitis, anorexia, ataxia, vertigo, amnesia, anxiety, mood instability, hostility, paresthesia, worsening cough, sinusitis, and diplopia. These adverse reactions have been reported in clinical studies of levetiracetam in combination with other anticonvulsants. Fatigue, somnolence, and dizziness primarily occur during the first 4 weeks of treatment. Slight decreases in total mean corpuscular red blood cell count, mean corpuscular hemoglobin, and mean corpuscular hematocrit have been reported. Leukopenia, neutropenia, pancytopenia (with bone marrow suppression in some cases), and thrombocytopenia have also been observed, but their causal relationship with the drug has not been established. For more complete data on levetiracetam (12 of these), please visit the HSDB record page.

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Pharmacodynamics
Levetiracetam appears to prevent seizures by selectively inhibiting excessively synchronized epileptiform bursts of discharge without affecting normal neuronal transmission, but the exact mechanism is unclear. Levetiracetam has a broad therapeutic index, making it relatively unique among other antiepileptic drugs. Antiepileptic drugs, including levetiracetam, may increase the risk of suicidal ideation or behavior—patients taking levetiracetam should be monitored for the onset or worsening of depressive symptoms, suicidal ideation, and behavioral abnormalities.

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Background and indications for treatment (from [1,2]): - Levetiracetam is a broad-spectrum antiepileptic drug (AED) used clinically to treat partial seizures, generalized tonic-clonic seizures, and myoclonic seizures in adults and children [2]; reference [1] points to a new potential application: as an adjunct to temozolomide (TMZ) in the treatment of glioblastoma (GBM) by enhancing the efficacy of TMZ against glioblastoma stem cells (GSCs, a key driver of GBM recurrence) [1]. Mechanism of action (cited from [1,2]): Anti-epileptic mechanism (cited from [2]): It binds to SV2A, regulates synaptic vesicle exocytosis, reduces the excessive release of neurotransmitters (such as glutamate), thereby inhibiting epileptic seizures; Anti-cancer adjuvant mechanism (cited from [1]): It upregulates pro-apoptotic proteins (Bax, Cleaved Caspase-3) and downregulates anti-apoptotic protein Bcl-2, enhancing TMZ-induced GSC apoptosis; It has no direct cytotoxicity to GSCs themselves [1].

C8H14N2O2

170.21

170.105

102767-28-2

Etiracetam;33996-58-6

5284583

White to off-white solid powder

1.2±0.1 g/cm3

395.9±25.0 °C at 760 mmHg

118-119°C

193.2±23.2 °C

0.0±0.9 mmHg at 25°C

1.519

-0.67

1

2

3

12

203

1

CC[C@@H](C(=O)N)N1CCCC1=O

HPHUVLMMVZITSG-LURJTMIESA-N

InChI=1S/C8H14N2O2/c1-2-6(8(9)12)10-5-3-4-7(10)11/h6H,2-5H2,1H3,(H2,9,12)/t6-/m0/s1

(S)-2-(2-oxopyrrolidin-1-yl)butanamide

Levetiracetam, UCBL059, UCB L059, UCB-L059, SIB S1, SIBS1, SIB-S1, Keppra, Etiracetam, UCB6474, UCB-6474, UCB 6474,

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 (14.69 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 (14.69 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 (14.69 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: Saline: 30 mg/mL

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Solubility in Formulation 5: 100 mg/mL (587.51 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.)