5-HT_1A Receptor ( Ki = 4.2 nM ); 5-HT_2A Receptor; 5-HT_2B Receptor; 5-HT_2C Receptor; D₂ Receptor; D₃ Receptor; D₄ Receptor

Aripiprazole reduces the levels of extracellular 5-HIAA in the striatum and medial prefrontal cortex of drug-naive rats, but not in rats that have been continuously treated with ripiprazole. [3] Rat hippocampal dopamine release is markedly increased by apiprimeze (0.1 mg/kg and 0.3 mg/kg). The release of dopamine in the medial prefrontal cortex is slightly but significantly increased by apipicazole (0.3 mg/kg), but not in the nucleus accumbens. Dopamine release in the nucleus accumbens is markedly reduced by apipicazole at doses of 3.0 mg/kg and 10 mg/kg, but not in the medical prefrontal cortex. In the medial prefrontal cortex, apipicazole (0.3 mg/kg) momentarily increases the release of dopamine induced by haloperidol (0.1 mg/kg), while inhibiting the release of dopamine in the nucleus accumbens.[4]

Radioligand Binding Assays [2]
A large number of transiently and stably transfected cloned human cDNAs, obtained via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP), were used for radioligand binding and functional assays as previously detailed (Rothman et al, 2000; Tsai et al, 2000). Conditions for radioligand binding assays, along with KD values for standard compounds, are listed in Table 1. In initial screening assays, Aripiprazole was tested at a concentration of 10 μM in quadruplicate at a large number of GPCRs, ion channels, and transporters. For molecular targets at which >50% inhibition was measured, Ki determinations were obtained using at least six concentrations of Aripiprazole; Ki values were calculated in quadruplicate using GraphPad Prism. [125I]DOI competition assays were performed as previously described (Choudhary et al, 1992) with the following changes: 12 dilutions of aAripiprazole spanning a range of 0.01–3000 nM were incubated with [125I]DOI (0.3 nM) in total volumes of 0.25 ml at 25°C for 1 h with 5–20 μg of membrane protein in binding buffer (50 mM Tris buffer, pH 7.4, 0.5 mM EDTA, 10 mM MgCl2). Membranes were harvested with a Brandel cell harvester by three ice-cold washes onto polyethyleneimine-pretreated (0.3%) Whatman GF/C filters. Radioactivity bound to filters was quantified by liquid scintillation counting.

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Aripiprazole is the first next-generation atypical antipsychotic with a mechanism of action that differs from currently marketed typical and atypical antipsychotics. Aripiprazole displays properties of an agonist and antagonist in animal models of dopaminergic hypoactivity and hyperactivity, respectively. This study examined the interactions of aripiprazole with a single population of human D2 receptors to clarify further its pharmacologic properties. In membranes prepared from Chinese hamster ovary cells that express recombinant D2L receptors, aripiprazole bound with high affinity to both the G protein-coupled and uncoupled states of receptors. Aripiprazole potently activated D2 receptor-mediated inhibition of cAMP accumulation. Partial receptor inactivation using the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) significantly reduced the maximum effect of aripiprazole on inhibition of cAMP accumulation. This effect was seen with concentrations of EEDQ that did not alter the maximal inhibitory effect of dopamine. Consistent with the expected effects of a partial agonist, increasing concentrations of aripiprazole blocked the action of dopamine with maximal blockade equal to the agonist effect of aripiprazole alone. The efficacy of aripiprazole relative to that of dopamine varied from 25% in cells that lacked spare receptors for dopamine to 90% in cells with receptor reserve. These results, together with previous studies demonstrating partial agonist activity at serotonin 5-hydroxytryptamine (5-HT)1A receptors and antagonist activity at 5-HT2A receptors, support the identification of aripiprazole as a dopamine-serotonin system stabilizer. The receptor activity profile may underlie the unique activity of aripiprazole in animals and its antipsychotic activity in humans. [2]

Effects of Aripiprazole on cAMP Production [2]
Inhibition of forskolin-stimulated cAMP production[2]
Inhibition of forskolin-stimulated 3′,5′-cyclic adenosine monophosphate (cAMP) production in stable D4 and 5-HT1A receptor expressing cell lines was measured as previously reported (Lawler et al, 1999; Zhang et al, 1994). In brief, cells were grown in 24-well plates and growth media were replaced with fresh F12 medium containing 100 μM IBMX and 100 μM forskolin (all on ice) just prior to experimentation. Serial dilutions (10-fold) of Aripiprazole ranging from 0.1 to 10.000 nM were added to the cells, which were then incubated 20 min at 37°C and 5% CO2. The reaction was terminated by aspiration and the addition of 0.5 ml of ice-cold 3% trichloroacetic acid. Plates were chilled for 1 h at 4°C and spun at 1000 g for 15 min. cAMP was quantified using a competitive binding assay adapted with minor modifications (Nordstedt and Fredholm, 1990). For measurement of cAMP content, trichloroacetic acid extracts (40 μl) were added to reaction tubes containing cAMP assay buffer (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA). [3H]cAMP (1 nM final concentration) was added to each tube, followed by cAMP-binding proteins (approximately 100 μg of crude extract from bovine adrenal cortex in 500 μl of cAMP buffer). The reaction tubes were incubated on ice for 2 h, then harvested with a Brandel cell harvester onto Whatman GF/C filters soaked in water. Filters were allowed to dry, and bound radioactivity was quantified by liquid scintillation counting. The concentration of cAMP in each sample was estimated from a standard curve ranging from 0.1 to 100 pmol of cAMP/assay.

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Stimulation of cAMP production[2]
Studies of the effects of serotonin and Aripiprazole at 5-HT6 and 5-HT7 receptors were carried out in stable transfectants using methods previously described (Max et al, 1995; Monsma et al, 1993; Shen et al, 1993).

Three to five days after cannulation, a dialysis probe was implanted into the medial prefrontal cortex, hippocampus or nucleus accumbens under slight anesthesia with isoflurane. Rats were then housed individually overnight in a dialysis cage. After the overnight perfusion at 0.4 μl/min of the probe, the flow was increased to 1.5 μl/min. One hour later, the dialysate samples were collected every 30 min. The perfusion medium was Dulbecco's phosphate-buffered saline solution including Ca2+ (138 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl, 1.2 mM CaCl2, pH 7.4). After stable baseline values in the dialysates were obtained, each rat received two injections, vehicle/Aripiprazole, WAY100635/aripiprazole or Aripiprazole/haloperidol. The locations of the dialysis probes were verified at the end of each experiment by brain dissection. [4]
Aripiprazole was dissolved in 45% 2-hydroxypropyl-β-cyclodextrin (HBC) [4]

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0.1 mg/kg and 0.3 mg/kg

Rats

Absorption, Distribution and Excretion
Tablet: Aripiprazole is well absorbed after administration of the tablet, with peak plasma concentrations occurring within 3 hours to 5 hours; the absolute oral bioavailability of the tablet formulation is 87%. ABILIFY can be administered with or without food. Administration of a 15 mg ABILIFY tablet with a standard high-fat meal did not significantly affect the Cmax or AUC of aripiprazole or its active metabolite, dehydro-aripiprazole, but delayed Tmax by 3 hours for aripiprazole and 12 hours for dehydro-aripiprazole. Oral Solution: Aripiprazole is well absorbed when administered orally as the solution. At equivalent doses, the plasma concentrations of aripiprazole from the solution were higher than that from the tablet formulation. In a relative bioavailability study comparing the pharmacokinetics of 30 mg aripiprazole as the oral solution to 30 mg aripiprazole tablets in healthy subjects, the solution-to-tablet ratios of geometric mean Cmax and AUC values were 122% and 114%, respectively. The single-dose pharmacokinetics of aripiprazole were linear and dose-proportional between the doses of 5 mg to 30 mg. Extended-release injectable suspension, bimonthly injection: Aripiprazole absorption into the systemic circulation is prolonged following gluteal intramuscular injection due to the low solubility of aripiprazole particles. The release profile of aripiprazole from ABILIFY ASIMTUFII results in sustained plasma concentrations over 2 months following gluteal injection(s). Following multiple doses, the median peak:trough ratio for aripiprazole following an ABILIFY ASIMTUFII dose is 1.3, resulting in a flat plasma concentration profile with Tmax ranging between 1 to 49 days following multiple gluteal administrations of 960 mg.
Following a single oral dose of [14C]-labeled aripiprazole, approximately 25% and 55% of the administered radioactivity was recovered in the urine and feces, respectively. Less than 1% of unchanged aripiprazole was excreted in the urine and approximately 18% of the oral dose was recovered unchanged in the feces.
The steady-state volume of distribution of aripiprazole following intravenous administration is high (404 L or 4.9 L/kg), indicating extensive extravascular distribution.
The clearance of aripiprazole was estimated to be 0.8mL/min/kg. Other studies have also reported a clearance rate of 3297±1042mL/hr.
Oral availability 87%. Aripiprazole is well absorbed and can be administered with or without food. Administration with a high fat meal did not affect the Cmax or AUC, but delayed Tmax by 3 hours for aripiprazole, and 12 hours for dehydro-aripiprazole.
Time to peak concentration: Peak plasma concentrations: within 3 to 5 hours.
The steady-state volume of distribution of aripiprazole following intravenous administration is high (404 L or 4.9 L/kg), indicating extensive extravascular distribution. At therapeutic concentrations, aripiprazole and its major metabolite are greater than 99% bound to serum proteins, primarily to albumin.
There was dose-dependent D2-receptor occupancy indicating brain penetration of aripiprazole in healthy human volunteers administered 0.5 to 30 mg per day.
For more Absorption, Distribution and Excretion (Complete) data for ARIPIPRAZOLE (8 total), please visit the HSDB record page.

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Metabolism / Metabolites
Aripiprazole is metabolized primarily by three biotransformation pathways: dehydrogenation, hydroxylation, and N-dealkylation. Based on in vitro studies, CYP3A4 and CYP2D6 enzymes are responsible for the dehydrogenation and hydroxylation of aripiprazole, and N-dealkylation is catalyzed by CYP3A4. Aripiprazole is the predominant drug moiety in systemic circulation. At steady-state, dehydro-aripiprazole, the active metabolite, represents about 40% of aripiprazole AUC in plasma.
Aripiprazole is extensively metabolized in the liver principally via dehydrogenation, hydroxylations, and N-dealkylation by the cytochrome P-450 (CYP) 2D6 and 3A4 isoenzymes. The major active metabolite of aripiprazole, dehydro-aripiprazole, exhibits affinity for D2 receptors similar to that of the parent compound and represents approximately 40% of the aripiprazole area under the concentration-time curve (AUC) in plasma. Steady-state plasma concentrations of both aripiprazole and dehydro-aripiprazole are achieved within 14 days.
ABILIFY activity is presumably primarily due to the parent drug, aripiprazole, and to a lesser extent, to its major metabolite, dehydro-aripiprazole, which has been shown to have affinities for D2 receptors similar to the parent drug and represents 40% of the parent drug exposure in plasma.
Aripiprazole has known human metabolites that include dehydro-aripiprazole, 4-[(2-oxo-3,4-dihydro-1H-quinolin-7-yl)oxy]butanal, 4-Hydroxyaripiprazole, and 2,3-dichlorophenylpiperazine.
Aripiprazole is metabolized primarily by three biotransformation pathways: dehydrogenation, hydroxylation,and N-dealkylation.Based on in vitro studies,CYP3A4 and CYP2D6 enzymes are responsible for dehydrogenation and hydroxylation of aripiprazole, and N-dealkylation is catalyzed by CYP3A4.Aripiprazole is the predominant drug moiety in the systemic circulation. At steady-state, dehydro-aripiprazole, the active metabolite, represents about 40% of aripiprazole AUC in plasma (RxList, A308).
Route of Elimination: Less than 1% of unchanged aripiprazole was excreted in the urine and approximately 18% of the oral dose was recovered unchanged in the feces.
Half Life: 75-146 hours

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Biological Half-Life
The mean elimination half-lives are about 75 hours and 94 hours for aripiprazole and dehydro-aripiprazole, respectively. For populations that are poor CYP2D6 metabolizers, the half-life of aripiprazole is 146 hours and these patients should be treated with half the normal dose. Other studies have reported a half-life of 61.03±19.59 hours for aripiprazole and 279±299 hours for the active metabolite.
The mean elimination half-lives are about 75 hours and 94 hours for aripiprazole and dehydro-aripiprazole, respectively.

Toxicity Summary
IDENTIFICATION AND USE: Aripiprazole is used IM for the acute management of agitation associated with schizophrenia or bipolar disorder, mixed or manic, in adults for whom treatment with aripiprazole is appropriate and who require an IM antipsychotic agent for rapid control of behaviors that interfere with diagnosis and care (e.g., threatening behaviors, escalating or urgently distressing behavior, self-exhausting behavior). Aripiprazole is used orally for the acute treatment of irritability associated with autistic disorder. Aripiprazole is used orally as an adjunct to antidepressants for the acute treatment of major depressive disorder in adults. Aripiprazole is used orally as monotherapy or as an adjunct to either lithium or valproate for the acute treatment of manic or mixed episodes associated with bipolar I disorder with or without psychotic features in adults and pediatric patients 10-17 years of age. The drug also is used orally as monotherapy or as adjunctive therapy with lithium or valproate for the maintenance treatment of bipolar I disorder in adults and pediatric patients 10-17 years of age. Aripiprazole is used orally for the acute and maintenance treatment of schizophrenia in adults and adolescents 13-17 years of age. HUMAN EXPOSURE AND TOXICITY: A potentially fatal symptom complex sometimes referred to as Neuroleptic Malignant Syndrome (NMS) has been reported in association with administration of antipsychotic drugs, including aripiprazole. Two possible cases of NMS occurred during aripiprazole treatment in the premarketing worldwide clinical database. Clinical manifestations of NMS are hyperpyrexia, muscle rigidity, altered mental status, and evidence of autonomic instability (irregular pulse or blood pressure, tachycardia, diaphoresis, and cardiac dysrhythmia). Additional signs may include elevated creatine phosphokinase, myoglobinuria (rhabdomyolysis), and acute renal failure. Elderly patients with dementia-related psychosis treated with antipsychotic drugs are at an increased risk of death. Antidepressants increased the risk compared to placebo of suicidal thinking and behavior (suicidality) in children, adolescents, and young adults in short-term studies of major depressive disorder (MDD) and other psychiatric disorders. ANIMAL STUDIES: In female mice, the incidences of pituitary gland adenomas and mammary gland adenocarcinomas and adenoacanthomas were increased at dietary dose of 3 to 30 mg/kg/day. Female rats were treated with oral doses of 2, 6, and 20 mg/kg/day of aripiprazole from 2 weeks prior to mating through day 7 of gestation. Estrus cycle irregularities and increased corpora lutea were seen at all doses, but no impairment of fertility was seen. Increased pre-implantation loss was seen at 6 and 20 mg/kg, and decreased fetal weight was seen at 20 mg/kg. Pregnant rabbits were treated with oral doses of 10, 30, and 100 mg/kg/day of aripiprazole during the period of organogenesis. Decreased maternal food consumption and increased abortions were seen at 100 mg/kg. Treatment caused increased fetal mortality (100 mg/kg), decreased fetal weight (30 and 100 mg/kg), increased incidence of a skeletal abnormality (fused sternebrae at 30 and 100 mg/kg) and minor skeletal variations (100 mg/kg). Aripiprazole and a metabolite (2,3-DCPP) were clastogenic in the in vitro chromosomal aberration assay in CHL cells with and without metabolic activation. The metabolite, 2,3-DCPP, produced increase in numerical aberrations in the in vitro assay in CHL cells in the absence of metabolic activation. A positive response was obtained in the in vivo micronucleus assay in mice, however,the response was shown to be due to a mechanism not considered relevant to humans.
Aripiprazole's antipsychotic activity is likely due to a combination of antagonism at D2 receptors in the mesolimbic pathway and 5HT2A receptors in the frontal cortex. Antagonism at D2 receptors relieves positive symptoms while antagonism at 5HT2A receptors relieves negative symptoms of schizophrenia. Aripiprazole exhibits high affinity for dopamine D2 and D3, serotonin 5-HT1A and 5- HT2A receptors, moderate affinity for dopamine D4, serotonin 5-HT2C and 5-HT7, alpha1-adrenergic and histamine H1 receptors and moderate affinity for the serotonin reuptake pump. Aripiprazole has no appreciable affinity for cholinergic muscarinic receptors. Aripiprazole functions as a partial agonist at the dopamine D2 and the serotonin 5-HT1A receptors, and as an antagonist at serotonin 5-HT2A receptor.

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Interactions
Substrates of Hepatic Microsomal Enzymes: Substrates of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4; pharmacokinetic interaction unlikely.
Hypotensive Agents: Potential pharmacologic interaction (additive hypotensive effects)
Famotidine: Coadministration of aripiprazole (given in a single dose of 15 mg) with a 40-mg single dose of the H2 antagonist famotidine, a potent gastric acid blocker, decreased the solubility of aripiprazole and, hence, its rate of absorption, reducing by 37% and 21% the Cmax of aripiprazole and dehydro-aripiprazole, respectively, and by 13% and 15%, respectively, the extent of absorption (AUC). No dosage adjustment of aripiprazole is required when administered concomitantly with famotidine.
Valproate: When valproate (500-1500 mg/day) and aripiprazole (30 mg/day) were coadministered at steady state, the C max and AUC of aripiprazole were decreased by 25%. No dosage adjustment of aripiprazole is required when administered concomitantly with valproate.
For more Interactions (Complete) data for ARIPIPRAZOLE (12 total), please visit the HSDB record page.

[1]. J Pharmacol Exp Ther. 2002 Jul;302(1):381-9.

[2]. Neuropsychopharmacology. 2003 Aug;28(8):1400-11.

[3]. Biochem Biophys Res Commun. 1999 Dec 20;266(2):560-3.

[4]. Eur J Pharmacol. 2004 Jun 16;493(1-3):75-83.

Therapeutic Uses
Antipsychotic Agents
Aripiprazole is used IM for the acute management of agitation associated with schizophrenia or bipolar disorder, mixed or manic, in adults for whom treatment with aripiprazole is appropriate and who require an IM antipsychotic agent for rapid control of behaviors that interfere with diagnosis and care (e.g., threatening behaviors, escalating or urgently distressing behavior, self-exhausting behavior).
Aripiprazole is used orally for the acute treatment of irritability associated with autistic disorder.
Aripiprazole is used orally as an adjunct to antidepressants for the acute treatment of major depressive disorder in adults.
For more Therapeutic Uses (Complete) data for ARIPIPRAZOLE (6 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ WARNING: INCREASED MORTALITY IN ELDERLY PATIENTS WITH DEMENTIA-RELATED PSYCHOSIS. Elderly patients with dementia-related psychosis treated with antipsychotic drugs are at an increased risk of death. Analyses of seventeen placebo-controlled trials (modal duration of 10 weeks), largely in patients taking atypical antipsychotic drugs, revealed a risk of death in drug-treated patients of between 1.6 to 1.7 times the risk of death in placebo-treated patients. Over the course of a typical 10-week controlled trial, the rate of death in drug-treated patients was about 4.5%, compared to a rate of about 2.6% in the placebo group. Although the causes of death were varied, most of the deaths appeared to be either cardiovascular (eg, heart failure, sudden death) or infectious (eg, pneumonia) in nature. Observational studies suggest that, similar to atypical antipsychotic drugs, treatment with conventional antipsychotic drugs may increase mortality. The extent to which the findings of increased mortality in observational studies may be attributed to the antipsychotic drug as opposed to some characteristic(s) of the patients is not clear. ABILIFY (aripiprazole) is not approved for the treatment of patients with dementia-related psychosis. /Included in label/
/BOXED WARNING/ WARNING: INCREASED SUICIDALTHOUGHTS AND BEHAVIORS. Antidepressants increased the risk compared to placebo of suicidal thinking and behavior (suicidality) in children, adolescents, and young adults in short-term studies of major depressive disorder (MDD) and other psychiatric disorders. Anyone considering the use of adjunctive ABILIFY or any other antidepressant in a child, adolescent, or young adult must balance this risk with the clinical need. Short-term studies did not show an increase in the risk of suicidality with antidepressants compared to placebo in adults beyond age 24; there was a reduction in risk with antidepressants compared to placebo in adults aged 65 and older. Depression and certain other psychiatric disorders are themselves associated with increases in the risk of suicide. Patients of all ages who are started on antidepressant therapy should be monitored appropriately and observed closely for clinical worsening, suicidality, or unusual changes in behavior. Families and caregivers should be advised of the need for close observation and communication with the prescriber. ABILIFY is not approved for use in pediatric patients with depression. /Included in label/
Contraindications: Known hypersensitivity reaction to aripiprazole or any ingredient in the formulation; such reactions have ranged from pruritus/urticaria to anaphylaxis.
Safety and effectiveness in pediatric patients with bipolar mania were established in a 4-week, placebo-controlled clinical trial in 197 pediatric patients aged 10 to 17 years. The incidence of discontinuation due to adverse reactions between aripiprazole-treated and placebo-treated pediatric patients (10 to 17 years) was 7% and 2%, respectively. Commonly observed adverse reactions associated with the use of aripiprazole in pediatric patients with bipolar mania (incidence of 5% or greater and aripiprazole incidence at least twice that for placebo) somnolence, extrapyramidal disorder, fatigue, nausea, akathisia, blurred vision, salivary hypersecretion, and dizziness. Although maintenance efficacy in pediatric patients has not been systematically evaluated, maintenance efficacy can be extrapolated from adult data along with comparisons of aripiprazole pharmacokinetic parameters in adult and pediatric patients.
For more Drug Warnings (Complete) data for ARIPIPRAZOLE (27 total), please visit the HSDB record page.

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Pharmacodynamics
Aripiprazole exhibits high affinity for dopamine D₂ and D₃, serotonin 5-HT_1a and 5-HT_2a receptors (Ki values of 0.34 nM, 0.8 nM, 1.7 nM, and 3.4 nM, respectively), moderate affinity for dopamine D₄, serotonin 5-HT_2c and 5-HT₇, alpha₁-adrenergic and histamine H₁ receptors (Ki values of 44 nM, 15 nM, 39 nM, 57 nM, and 61 nM, respectively), and moderate affinity for the serotonin reuptake site (Ki=98 nM). Aripiprazole has no appreciable affinity for cholinergic muscarinic receptors (IC₅₀>1000 nM).

C23H27CL2N3O2

448.39

447.148

C, 61.61; H, 6.07; Cl, 15.81; N, 9.37; O, 7.14

129722-12-9

Aripiprazole-d8; 1089115-06-9; Aripiprazole (1,1,2,2,3,3,4,4-d8); 1089115-04-7; Aripiprazole monohydrate; 851220-85-4; 1259305-26-4 (cavoxil)

60795

White to off-white solid powder

1.3±0.1 g/cm3

646.2±55.0 °C at 760 mmHg

139°C

344.6±31.5 °C

0.0±1.9 mmHg at 25°C

1.593

5.59

1

4

7

30

559

0

O=C1NC2=C(C=CC(OCCCCN3CCN(C4=CC=CC(Cl)=C4Cl)CC3)=C2)CC1

CEUORZQYGODEFX-UHFFFAOYSA-N

InChI=1S/C23H27Cl2N3O2/c24-19-4-3-5-21(23(19)25)28-13-11-27(12-14-28)10-1-2-15-30-18-8-6-17-7-9-22(29)26-20(17)16-18/h3-6,8,16H,1-2,7,9-15H2,(H,26,29)

7-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy]-3,4-dihydro-1H-quinolin-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (5.58 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 (5.58 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: 2.5 mg/mL (5.58 mM) in 10% DMF 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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: 2.5 mg/mL (5.58 mM) in 10% DMF 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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