Acetylcholinesterase (AChE)

The muscarinic antagonist activity of donepezil (E2020 free base) is demonstrated by its concentration-dependent inhibition of the carbachol-stimulated increase in intracellular Ca2+ concentration in human SHSY5Y neuroblastoma cells. Once rats received donepezil intraperitoneally, there was a dose-dependent rise in tremor and salivation, indicating overt cholinergic behavior, with an ED50 of 6 μmol/kg. With an ED50 of 50 μmol/kg, donepezil was found to be marginally less effective when taken orally [2]. According to a recent study, donepezil shields human umbilical vein endothelial cells (HUVEC) from cellular damage brought on by H2O2. This could potentially be used as a treatment for oxidative stress in diseases related to the heart and brain [3].

The in vitro and in vivo effects of the novel acetylcholinesterase inhibitors donepezil and NXX-066 have been compared to tacrine. Using purified acetylcholinesterase from electric eel both tacrine and donepezil were shown to be reversible mixed type inhibitors, binding to a similar site on the enzyme. In contrast, NXX-066 was an irreversible non-competitive inhibitor. All three compounds were potent inhibitors of rat brain acetylcholinesterase (IC50 [nM]; tacrine: 125 +/- 23; NXX-066: 148 +/- 15; donepezil: 33 +/- 12). Tacrine was also a potent butyrylcholinesterase inhibitor. Donepezil and tacrine displaced [3H]pirenzepine binding in rat brain homogenates (IC50 values [microM]; tacrine: 0.7; donepezil: 0.5) but NXX-066 was around 80 times less potent at this M1-muscarinic site. Studies of carbachol stimulated increases in [Ca2+]i in neuroblastoma cells demonstrated that both donepezil and tacrine were M1 antagonists. Ligand binding suggested little activity of likely pharmacological significance with any of the drugs at other neurotransmitter sites. Intraperitoneal administration of the compounds to rats produced dose dependent increases in salivation and tremor (ED50 [micromol/kg]; tacrine: 15, NXX-066: 35, donepezil: 6) with NXX-066 having the most sustained effect on tremor. Following oral administration, NXX-066 had the slowest onset but the greatest duration of action. The relative potency also changed, tacrine having low potency (ED50 [micromol/kg]; tacrine: 200, NXX-066: 30, donepezil: 50). Salivation was severe only in tacrine treated animals. Using in vivo microdialysis in cerebral cortex, both NXX-066 and tacrine were found to produce a marked (at least 30-fold) increase in extracellular acetylcholine which remained elevated for more than 2 h after tacrine and 4 h after NXX-066[2].

This study was designed to compare the in vitro inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) of donepezil and some other cholinesterase (ChE) inhibitors which have been developed for the treatment of Alzheimer's disease. The carbamate derivatives physostigmine and rivastigmine needed preincubation to exhibit appropriate anti-ChE activity. The maximum ChE inhibition by physostigmine developed within 30-60 min, while the inhibitory effect of rivastigmine on AChE and BuChE activities reached its peak after 48 and 6 h, respectively. The order of inhibitory potency (IC50) towards AChE activity under optimal assay conditions for each ChE inhibitor was: physostigmine (0.67 nM) > rivastigmine (4.3 nM) > donepezil (6.7 nM) > TAK-147 (12 nM) > tacrine (77 nM) > ipidacrine (270 nM). The benzylpiperidine derivatives donepezil and TAK-147 showed high selectivity for AChE over BuChE. The carbamate derivatives showed moderate selectivity, while the 4-aminopyridine derivatives tacrine and ipidacrine showed no selectivity. The inhibitory potency of these ChE inhibitors towards AChE activity may illustrate their potential in vivo activity[1].

The effect of cholinesterase inhibitors on calcium flux in SHSY5Y human neuroblastoma cells[2]
SHSY5Y cells were maintained in Eagles Minimum Essential Medium and Hams F-12 medium (1:1) supplemented by 10% foetal calf serum, 2% l-glutamine, 1% non-essential amino acids and 20 mM Hepes pH 7.4. To harvest the cells the medium was removed and the monolayer rinsed with 10 ml Hank’s balanced salt solution, and scraped from the base of the flask using a cell scraper. The resulting suspension was centrifuged at 250×g for 5 min at 4°C and the resulting pellet was resuspended in a loading buffer consisting of 6 ml phosphate buffered saline containing 2 mM EDTA, 10 μM Fluo-3AM and 0.02% pluronic F-127 pH 7.4. The cells were loaded with the acetoymethyl ester derivative of Fluo3 (Fluo-3AM) for 15 min at 37°C. Following centrifugation at 250×g for 5 min at 4°C the resulting pellet was resuspended in oxygenated Hepes-Ringer buffer pH 7.4 containing CaCl2 (0.5 mM) and glucose (10 mM). The cell suspension was then incubated at room temperature for a further 20 min to allow for hydrolysis of the Fluo-3AM, centrifuged at 250×g for 5 min and resuspended in oxygenated Hepes–Ringer buffer as described earlier. Aliquots (2 ml) of cell suspension were placed in quartz cuvettes nd equilibrated for 1 min with stirring at room temperature. A basal fluorescence was recorded after which test agents were added. Twenty microlitre additions were made with a Hamilton syringe, permitting continuous measurement of the fluorescence signal. The background fluorescence was unaffected by this procedure. Fluorescence was measured at excitation 505 nm: emission 530 nm. Maximal fluorescence was measured by addition of 10 μM calcium ionophore 4-bromo calcimycin. The fluorescence signal was then quenched using MnCl2 (1 mM).

Behavioural observations[2]
Tremor and salivation were assessed using the methods described by Hunter et al. (1989). Briefly, groups of animals were injected with various doses of cholinesterase inhibitor and observed. Tremor (score 0–3) and salivation (weight in 10 mg units) were noted. 2.9. Measurement of extracellular acetylcholine in rat brain using in vivo microdialysis.
Individually prepared concentric probes, essentially as described by Hutson et al. (1985), were used except that they were implanted without the use of a guide assembly and the internal glass capillary tubes were replaced by fused silica tubes (VS-150-075-1D). The dialysis membrane was 4mm long and had an approximate diameter of 0.2 mm. The in vitro efficiency of ACh recovery when the microdialysis probes were placed in an ACh (60 μM) solution at room temperature and perfused at 1.0 μl/min, was 17.9±2.4%.
Probes were implanted transversely into the cortex of rats anaesthetised with halothane (2%) in O2/N2O mixture (1: 2) and secured in Kopf stereotaxic frame with the tooth bar at −3.3 mm below interaural zero. Probes were implanted horizontally into the right cortex: +7.7 mm anterior and +4.2 mm lateral from interaural zero and −1.3 mm from the skull surface and secured to the skull with two screws and dental cement. Following surgery, the animals were housed in perspex boxes until the beginning of microdialysis procedures the next day. Placement of the probe was verified by visual inspection of the probe track at the end of each experiment by injecting Luxol Fast blue.
Dialysis probes were perfused at a rate of 1 μl/min with artificial CSF (composition mM: NaCl 125, KCl 2.5, MgCl2, 1.18 and CaCl2 1.26), or high K+CSF (composition mM: NaCl 27.5, KCl 100, MgCl2 1.18 and CaCl2 1.26) using a model 22 Harvard Microlitre syringe pump. The artificial CSF did not contain an acetylcholinesterase inhibitor. Thirty minute fractions were collected which were then stored on ice. Dialysate from the first 60 min was discarded and the next three collections of 30 min were baseline samples prior to the i.p administration of tacrine (21 μmol/kg) or NXX-066 (106 μmol/kg).
Acetylcholine was measured by a hplc method similar to that originally described by Potter et al. (1983). Following its separation on an analytical ion exchange column ACh was converted to hydrogen peroxide inside a 4.1×30 mm analytical column, filled with polymeric matrix to which AChE and choline oxidase enzymes has been covalently linked. The hydrogen peroxide formed was detected electrochemically by oxidation on a platinum electrode at +500 mV versus a Ag/AgCl reference. The mobile phase composition was 3.4 mM H3PO4 (85%) and 5 mM Kathon CG (1%); the pH was adjusted to 8.5 by addition of NaOH. The flow rate of 0.6 ml/min ensured quantitative conversion of ACh to H2O2 within an enzyme reactor. Peaks were recorded on a Kontron integrator. ACh in brain dialysates was quantified by the standard curve method. A lower level of 0.6 pmol of ACh on the column could be reliably detected.
Donepezil were dissolved in 0.9% w/v NaCl (saline) before injection.

Absorption, Distribution and Excretion
Donepezil is slowly absorbed via the gastrointestinal tract after oral administration. Tmax is 3 to 4 hours with a bioavailability of 100% and steady-state concentrations are attained within 15 to 21 days of administration. The Tmax in one pharmacokinetic study determined a Tmax of 4.1 ± 1.5 hours. The Cmax of 5 mg donepezil tablets is estimated to be 8.34 ng/mL, according to the Canadian monograph. The AUC of 5 mg donepezil tablets has been determined to be 221.90-225.36 ng.hr/mL.
In a study of radiolabeled administration donepezil in healthy adults, 57% of measured radioactivity was identified in the urine, and 5% was identified in the feces.
The volume of distribution of donepezil is 11.8 ± 1.7 L/kg for a 5-mg dose and 11.6 ± 1.91 L/kg for a 10-mg dose. It is largely distributed in the extravascular compartments. Donepezil crosses the blood-brain barrier and cerebrospinal fluid concentrations at the above doses have been measured at 15.7%. The volume of distribution at steady-state according to the FDA label for donepezil ranges from 12 - 16 L/kg.
According to the FDA label, the average apparent plasma clearance of this drug is 0.13 – 0.19 L/hr/kg. A 5 mg dose of donepezil in healthy patients was shown to have a plasma clearance of 0.110±0.02 L/h/kg. In 10 patients diagnosed with alcoholic cirrhosis, showed a mean decrease in clearance by 20% when compared to the clearance in 10 healthy subjects. In 4 patients with severe renal impairment compared to 4 healthy subjects, no significant change in clearance was noted.
Donepezil is well absorbed with a relative oral bioavailability of 100% and reaches peak plasma concentrations in 3 to 4 hours. Pharmacokinetics are linear over a dose range of 1 to 10 mg given once daily. Neither food nor time of administration (morning vs. evening dose) influences the rate or extent of absorption of donepezil hydrochloride tablets.
... The mean apparent plasma clearance (Cl/F) is 0.13 L/hr/kg. Following multiple dose administration, donepezil accumulates in plasma by 4 to 7 fold and steady state is reached within 15 days. The steady state volume of distribution is 12 L/kg.
In a study of 10 patients with stable alcoholic cirrhosis, the clearance of donepezil hydrochloride was decreased by 20% relative to 10 healthy age and sex matched subjects.
In a study of 11 patients with moderate to severe renal impairment (ClCr < 18 mL/min/1.73 sq m) the clearance of donepezil hydrochloride did not differ from 11 age and sex matched healthy subjects.
For more Absorption, Distribution and Excretion (Complete) data for Donepezil (12 total), please visit the HSDB record page.

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Metabolism / Metabolites
Donepezil is metabolized by first pass metabolism in the liver, primarily by CYP3A4, in addition to CYP2D6. After this, O-dealkylation, hydroxylation, N-oxidation, hydrolysis, and O-glucuronidation occur, producing various metabolites with similar half-lives to the unchanged parent drug. A study of the pharmacokinetics of radiolabeled donepezil demonstrated that about 53% of plasma radioactivity appeared as donepezil in the unchanged form, and 11% was identified as the metabolite 6-O-desmethyl donepezil, which exerts similar potency inhibition of the acetylcholinesterase enzyme. This drug is heavily metabolized to four primary metabolites, two of which are considered pharmacologically active, as well as to multiple inactive and unidentified metabolites.
Donepezil is both excreted in the urine intact and extensively metabolized to four major metabolites, two of which are known to be active, and a number of minor metabolites, not all of which have been identified. Donepezil is metabolized by CYP 450 isoenzymes 2D6 and 3A4 and undergoes glucuronidation. Following administration of 14C-labeled donepezil, plasma radioactivity, expressed as a percent of the administered dose, was present primarily as intact donepezil (53%) and as 6-O-desmethyl donepezil (11%), which has been reported to inhibit AChE to the same extent as donepezil in vitro and was found in plasma at concentrations equal to about 20% of donepezil.
The aim of this study was to investigate the metabolism and elimination of donepezil HCl in humans, following the administration of a single 5 mg (liquid) oral dose containing a mixture of unlabelled and 14C-labelled donepezil. ... Unchanged donepezil accounted for the largest component of the recovered dose in each matrix. Three metabolic pathways were identified: (i) O-dealkylation and hydroxylation to metabolites M1 and M2, with subsequent glucuronidation to metabolites M11 and M12; (ii) hydrolysis to metabolite M4; and (iii) N-oxidation to metabolite M6. In plasma, the parent compound accounted for about 25% of the dose recovered during each sampling period, as well as of the cumulative dose recovered. The recovered residue showed higher levels of the hydroxylated metabolites M1 and M2 than of their glucuronide conjugates M11 and M12, respectively. In urine, the parent compound accounted for 17%, on average, of the dose recovered from each pooled sample, as well as of the total recovered dose. The major metabolite was the hydrolysis product M4, followed by the glucuronidated conjugates M11 and M12. In feces, the parent compound also predominated, although it accounted for only 1%, of the recovered dose. A large percentage of the radioactivity in feces consisted of unidentified very polar metabolites, which were retained at the TLC origin. Of the extracted metabolites, the hydroxylation products M1 and M2 were the most abundant, followed by the hydrolysis product M4 and the N-oxidation product M6. Donepezil is hepatically metabolized and the predominant route for the elimination of both parent drug and its metabolites is renal, as 79% of the recovered dose was found in the urine with the remaining 21% found in the feces. Moreover, the parent compound, donepezil, is the predominant elimination product in urine. The major metabolites of donepezil include M1 and M2 (via O-dealkylation and hydroxylation), M11 and M12 (via glucuronidation of M1 and M2, respectively), M4 (via hydrolysis) and M6 (via N-oxidation).
Donepezil has known human metabolites that include 6-O-Desmethyl Donepezil, 5,6-dimethoxy-2-(piperidin-4-ylmethyl)-2,3-dihydroinden-1-one, and 5-O-Desmethyl Donepezil.
Donepezil is metabolized by CYP 450 isoenzymes 2D6 and 3A4 in the liver and also undergoes glucuronidation. The main metabolite, 6-O-desmethyl donepezil, has been reported to inhibit AChE to the same extent as donepezil in vitro.
Route of Elimination: Donepezil is both excreted in the urine intact and extensively metabolized to four major metabolites, two of which are known to be active, and a number of minor metabolites, not all of which have been identified.
Half Life: 70 hours

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Biological Half-Life
The average elimination half-life of donepezil is about 70 hours according to the results of various studies and the FDA label for donepezil.. One pharmacokinetic study determined the average terminal half-life to be 81.5±22.0 h
The elimination half life of donepezil is about 70 hours.
A 79-year-old woman with Alzheimer's disease was admitted due to acute cholinergic symptoms induced by overdose (45 mg) of donepezil (DPZ) ... The plasma concentration of DPZ was 54.6 ng/mL on admission and gradually decreased to the normal limits in about 90 hr. The calculative half-life of DPZ was about 55 hr ...

Toxicity Summary
Donepezil is a cholinesterase or acetylcholinesterase (AChE) inhibitor. A cholinesterase inhibitor (or 'anticholinesterase') suppresses the action of acetylcholinesterase. Because of its essential function, chemicals that interfere with the action of acetylcholinesterase are potent neurotoxins, causing excessive salivation and eye-watering in low doses, followed by muscle spasms and ultimately death. Nerve gases and many substances used in insecticides have been shown to act by binding a serine in the active site of acetylcholine esterase, inhibiting the enzyme completely. Acetylcholine esterase breaks down the neurotransmitter acetylcholine, which is released at nerve and muscle junctions, in order to allow the muscle or organ to relax. The result of acetylcholine esterase inhibition is that acetylcholine builds up and continues to act so that any nerve impulses are continually transmitted and muscle contractions do not stop. Among the most common acetylcholinesterase inhibitors are phosphorus-based compounds, which are designed to bind to the active site of the enzyme. The structural requirements are a phosphorus atom bearing two lipophilic groups, a leaving group (such as a halide or thiocyanate), and a terminal oxygen.

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Hepatotoxicity
In several large clinical trials, donepezil therapy was not associated with an increased rate of serum enzyme elevations compared to placebo treatment. Furthermore, escalation of the dose from 10 to 23 mg daily was not followed by an increased rate of ALT elevations compared to patients maintained on the lower dose. Nevertheless, since its introduction into clinical use, donepezil has been implicated in several isolated case reports of clinically apparent hepatotoxicity. The time to onset was short (1 to 6 weeks) and the pattern of serum enzyme elevations was cholestatic or mixed. The course of illness can be severe with prolonged jaundice and itching (Case 1), but fatal instances have not been published. Immunoallergic and autoimmune features are not common.
Likelihood score: D (possible, rare cause of clinically apparent liver injury).

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Protein Binding
Donepezil is 96% protein-bound, with approximately 75% binding to albumin and approximately 21% binding to alpha-1-glycoprotein.

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Interactions
Ketoconazole and quinidine, inhibitors of CYP450, 3A4 and 2D6, respectively, inhibit donepezil metabolism in vitro. Whether there is a clinical effect of quinidine is not known. In a 7 day crossover study in 18 healthy volunteers, ketoconazole increased mean donepezil concentrations (AUC0-24 and Cmax) by 36%. The clinical relevance of this increase in concentration is unknown.
A synergistic effect may be expected when cholinesterase inhibitors are given concurrently with succinylcholine, similar neuromuscular blocking agents or cholinergic agonists such as bethanechol.
Inducers of CYP 2D6 and CYP 3A4 (eg, phenytoin, carbamazepine, dexamethasone, rifampin, and phenobarbital) could increase the rate of elimination of donepezil hydrochloride.
A 75-year-old man with Alzheimer's disease, treated with the cholinesterase inhibitor donepezil for 14 months, was scheduled for left colectomy under general anesthesia. During the procedure, succinylcholine-induced relaxation was prolonged and the effect of atracurium besylate was inadequate even at higher doses than those indicated for the patient's weight. Cholinesterase blood tests performed 10 months, 1 month and 10 days before surgery had demonstrated a gradual decrease in the duration of activity of the enzyme. Such an effect, which has been described for cholinesterase inhibitors like neostigmine and donepezil, would explain the prolonged effect of succinylcholine. After ruling out other causes for resistance to atracurium, we conclude that donepezil or its metabolites acted on muscle plaque, blocking acetylcholine hydrolysis and antagonizing atracurium.
For more Interactions (Complete) data for Donepezil (7 total), please visit the HSDB record page.

[1]. Comparison of inhibitory activities of donepezil and other cholinesterase inhibitors on acetylcholinesterase and butyrylcholinesterase in vitro. Methods Find Exp Clin Pharmacol, 2000. 22(8): p. 609-13.

[2]. A comparative study in rats of the in vitro and in vivo pharmacology of the acetylcholinesterase inhibitors tacrine, donepezil and NXX-066. Neuropharmacology, 1999. 38(1): p. 181-93.

[3]. Donepezil protects endothelial cells against hydrogen peroxide-induced cell injury. CNS Neurosci Ther, 2012. 18(2): p. 185-7.

Therapeutic Uses
Cholinesterase Inhibitors; Nootropic Agents
Donepezil hydrochloride tablets are indicated for the treatment of dementia of the Alzheimer's type. Efficacy has been demonstrated in patients with mild to moderate Alzheimer's Disease. /Included in US product label/
/EXPTL Ther:/ ... Officially approved for mild-to-moderate and severe /Alzheimer's Disease/ (AD), donepezil has also been shown to be effective in early-stage AD, vascular dementia, Parkinson's disease dementia/Lewy body disease and cognitive symptoms associated with multiple sclerosis. In addition, one study suggested that donepezil may delay the onset of AD in subjects with mild cognitive impairment, a prodrome to AD.

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Drug Warnings
Donepezil hydrochloride tablets are contraindicated in patients with known hypersensitivity to donepezil hydrochloride or to piperidine derivatives.
Because of their pharmacological action, cholinesterase inhibitors may have vagotonic effects on the sinoatrial and atrioventricular nodes. This effect may manifest as bradycardia or heart block in patients both with and without known underlying cardiac conduction abnormalities. Syncopal episodes have been reported in association with the use of donepezil hydrochloride. Syncopal episodes have been reported in association with the use of donepezil hydrochloride.
Through their primary action, cholinesterase inhibitors may be expected to increase gastric acid secretion due to increased cholinergic activity. Therefore, patients should be monitored closely for symptoms of active or occult gastrointestinal bleeding, especially those at increased risk for developing ulcers, eg, those with a history of ulcer disease or those receiving concurrent non-steroidal anti-inflammatory drugs (NSAIDS).
Donepezil hydrochloride, as a predictable consequence of its pharmacological properties, has been shown to produce diarrhea, nausea and vomiting. These effects, when they occur, appear more frequently with the 10 mg/day dose than with the 5 mg/day dose. In most cases, these effects have been mild and transient, sometimes lasting one to three weeks, and have resolved during continued use of donepezil hydrochloride.
For more Drug Warnings (Complete) data for Donepezil (12 total), please visit the HSDB record page.

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Pharmacodynamics
By inhibiting the acetylcholinesterase enzyme, donepezil improves the cognitive and behavioral signs and symptoms of Alzheimer's Disease, which may include apathy, aggression, confusion, and psychosis.

C24H29NO3

379.492

379.214

C, 75.96; H, 7.70; N, 3.69; O, 12.65

120014-06-4

(R)-Donepezil;142698-19-9;(S)-Donepezil;142057-80-5;Donepezil-d7 hydrochloride;1261394-20-0;Donepezil-d5;1128086-25-8;Donepezil Hydrochloride;120011-70-3

3152

White to off-white solid powder

1.1±0.1 g/cm3

527.9±50.0 °C at 760 mmHg

207ºC

273.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.578

4.71

0

4

6

28

510

0

ADEBPBSSDYVVLD-UHFFFAOYSA-N

InChI=1S/C24H29NO3/c1-27-22-14-19-13-20(24(26)21(19)15-23(22)28-2)12-17-8-10-25(11-9-17)16-18-6-4-3-5-7-18/h3-7,14-15,17,20H,8-13,16H2,1-2H3

2-((1-benzylpiperidin-4-yl)methyl)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

HSDB 7743; HSDB-7743; HSDB7743

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~33.33 mg/mL (~87.83 mM)
H2O : ~2 mg/mL (~5.27 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.59 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 (6.59 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 (6.59 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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 (Please use freshly prepared in vivo formulations for optimal results.)