ALK (IC50 = 0.2 nM); IGF-1R (IC50 = 8 nM); InsR (IC50 = 7 nM); STK22D (IC50 = 23 nM)

Ceritinib (LDK378) exhibits negligible levels of glutathione (GSH) adducts (<1%), which is intended to lessen the likelihood of producing reactive metabolites in liver microsomes. Ceritinib (LDK378) exhibits moderate inhibition of CYP3A4 (Midazolam substrate) and hERG, indicating comparatively good metabolic stability. When compared to liver blood flow, ceritinib (LDK378) shows poor plasma clearance in animals (mouse, rat, dog, and monkey), with oral bioavailability of greater than 55% in these species. In the Karpas299 and H2228 rat xenograft models, crizitinib (LDK378) causes a dose-dependent growth inhibition and tumor regression without causing any loss of body weight. After a chronic dosage of up to 100 mg/kg, ceretinib (LDK378) has no effect on insulin levels or plasma glucose utilization in mice[1].

All kinases are expressed using the baculovirus expression technology as either GST- or histidine-tagged fusion proteins, with the exception of untagged ERK2, which is made in E. coli. Using the LabChip mobility shift assay, the kinase activity is determined. For sixty minutes, the assay is run at 30°C. It is routinely possible to determine the effect of LDK378 on the enzymatic activity from a single reading (end point measurement) by analyzing the linear progress curves in both the presence and absence of LDK378.

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Enzymatic Kinase Profiling Description[1]
All kinases were expressed as either histidine- or GST-tagged fusion proteins using the baculovirus expression technology except for the untagged ERK2 which was produced in E. coli. AURORA-A, JAK2, MK2, SYK, ERK2 and PKA were purchased commercially, and all other kinases were supplied in-house. The kinase activity was measured in the LabChip mobility-shift assay. The assay was performed at 30 °C for 60 min. The effect of compound on the enzymatic activity was obtained from the linear progress curves in the absence and presence of compound and routinely determined from one reading (end point measurement).

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GSH-Trapping Assay[1]
For the characterization of the metabolic activation, 10 mmol/L DMSO stock solutions of the compounds were incubated at 37 °C for up to 60 min with human liver microsomes (50 μL), containing 1 mg protein/mL with phosphate buffer. Four microliters of 0.5 mM of 20 μL of DMSO stock solution of compound in 180 μL of a mixture of acetonitrile/water (ratio 1:1) was added to 50 μL of 1 mg/mL liver microsomes in phosphate buffer and preincubated for 3 min at 37 °C. After preincubation, the reaction was started by addition of 50 μL of the NADPH (1 mmol/L), UDPGA (1 mmol/L), MgCl (2 mmol/L), and ethyl ester GSH reduced (2 mmol/L). After 60 min, the reaction was stopped with 200 μL of ice-cold acetonitrile. The reaction mixture was stored at −20 °C. The mixture was centrifuged (10000g, 5 min), and 250 μL of 300 μL supernatant was transferred. From this solution, 20 μL aliquots were used for analysis. The liquid chromatographic separation was performed using an Agilent HP1100 pump and a Phenyl HexylRP column, 150 mm × 2.0 mm, particle size 4.6 μm. Gradient mobile phase programming was used with a flow rate of 350 μL/min. Eluent A was Milli-Q water with 0.1% formic acid. Eluent B was acetonitrile with 0.1% formic acid. The mobile phase was a linear gradient from 5% B to 95% B over 6 min and held for 2 min at 95% B for a total run time of 10 min. The column effluent was introduced directly into the ion source of a triple quadrupole MS instrument or ion trap MS instrument. The ionization technique employed was positive electrospray (ES). The TS-Quantum was used in a product ion scan mode, utilizing collision induced dissociation in Q2 (collision chamber). Collision gas was argon. The collision energy was set at 30 eV.

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Solubility Assays[1]
One-hundred microliter aliquots of 1 mM DMSO solutions were added to each of three glass vials and evaporated to dryness prior to addition of 500 μL of pH 6.8 buffer. Following 24 h of shaking, the solutions were vacuum filtered through MultiScreen Solubility 96-well plates with 0.4 μm modified PCTE membrane, and an aliquot of each filtrate is transferred to a UV plate for quantification as described in Uvarova et al.

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Metabolic Clearance Assays[1]
The metabolic clearance assays were conducted using the method described in Richmond et al.

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CYP Inhibition Assays[1]
The samples were prepared as 10 mM solutions in DMSO, then assayed and analyzed using the general LC–MS/MS method described by Bell et al.

LDK378 or DMSO are serially diluted and incubated with luciferase-expressing cells for two to three days. Using the Bright-Glo Luciferase Assay System, luciferase expression is measured as a proxy for cell proliferation and survival. The XLFit program is used to generate IC50 values.

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GI50 determination[3]
In order to calculate the half maximal growth inhibitory concentration (GI50) of individual compounds [Ceritinib (LDK-378)], neuroblastoma tumor cells were seeded into 96‐well plates in a total volume of 100 μL and allowed to attach overnight. Compound (e.g. Ceritinib (LDK-378) dissolved in DMSO) was added to wells in six replicates of 100 μL, across a concentration gradient including a DMSO‐only control, the next day. The cells were exposed to drug for 72 h. Thereafter, the cell number in treated versus control wells was estimated after cell fixation with 10% trichloroacetic acid and staining with sulforhodamine B in 1% acetic acid. The GI50 was calculated as the drug concentration that inhibits cell growth by 50% compared with control growth, according to nonlinear regression analysis, using graphpad prism.

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Preparation of protein lysates[3]
Cell lines were harvesting by scraping, spun at 500 g for 5 min, and washed once in phosphate‐buffered saline, and the cell pellets were resuspended in CHAPS lysis buffer [50 mm Tris/HCl pH 8.0, 1 mm EDTA, 150 mm NaCl, 1% CHAPS, 0.2 mm PMSF, 1 : 50 Phosphatase Inhibitor Cocktail 2 and 3, 1 : 100 Protease Inhibitor Cocktail. Frozen tissue samples were homogenized in CHAPS lysis buffer prepared as for cell lysates. After incubation for 30 min on ice, lysates were spun at 16 000 g for 15 min and the supernatant was collected. Protein concentrations were determined using BCA protein assay by comparison with bovine serine albumin standard.

RNU nude rats bearing the Karpas299/H2228 tumors
~50 mg/kg
o.g.

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Studies on in vivo PK are carried out on dogs, rats, mice, and cynomolgus monkeys. Male Balb/c mice are given cetinib (LDK378) (HCl salt) orally via gavage at a dose of 20 mg/kg (n=3) and intravenously via tail vein at a dose of 5 mg/kg (n= 3). Sprague-Dawley rats are dosed with Ceritinib (LDK378) (HCl salt) intravenously via the tail vein at 3 mg/kg (n=3) and orally via gavage at 10 mg/kg (n=3) using the same formulation. Serial blood samples are taken over the course of 24 hours following dosage at prearranged times. Ceritinib (phosphate salt) is given as a single intravenous (n = 2) or oral (n = 3) dose to male beagle dogs. The intravenous solution has a dosage of 5 mg/kg, while the oral suspension has a dosage of 20 mg/kg. A single intravenous (n = 2) or oral (n = 3) dose of Ceritinib (free base) is given to male Cynomologus monkeys. The intravenous solution has a dose of 5 mg/kg, while the oral suspension has a dose of 60 mg/kg. For plasma, blood is drawn at prearranged intervals over a period of 144 hours following dosage.

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PK Studies[1]
In vivo PK studies were conducted in mice, rats, dogs, and cynomolgus monkeys. 15b (HCl salt) was formulated as a solution in 75% PEG300/25% D5W and administered to male Balb/c mice intravenously via tail vein at 5 mg/kg (n = 3) and orally via gavage at 20 mg/kg (n = 3). By use of the same formulation, 15b (HCl salt) was dosed to Sprague–Dawley rats intravenously via the tail vein at 3 mg/kg (n = 3) and orally via gavage at 10 mg/kg (n = 3). Blood samples were collected serially at scheduled times over 24 h after dosing.Male beagle dogs received a single intravenous (n = 2) or oral (n = 3) dose of 15b (phosphate salt) as an intravenous solution in 30% propylene glycol/5% Solutol buffer at 5 mg/kg and an oral suspension in suspension in 0.5% (w/v) aqueous methylcellulose/0.5% Tween 80 at 20 mg/kg, respectively.Male cynomologus monkeys received single intravenous (n = 2) or oral (n = 3) dose of 15b (free base) as an intravenous solution in 30% propylene glycol/5% solutol at 5 mg/kg and an oral suspension in 0.5% (w/v) methylcellulose at 60 mg/kg, respectively. Blood samples for plasma were collected at prescheduled times over 144 h after dosing.

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In Vivo Experiments[1]
RNU nude rats bearing the Karpas299 tumors were randomized into five groups (n = 6 rats per group) with an average tumor size of 326 ± 128 mm3. 15b (phosphate salt) was formulated in 0.5% MC/0.5% Tween 80 and administered by oral gavage at a dosing volume of 10 μL/g of an animal body weight. Animals in each group received vehicle or 6.25, 12.5, 25, 50 mg/kg 15b every day for 14 consecutive days.RNU nude rats bearing the H2228 tumors were randomized into four groups (n = 4 rats per group) with an average tumor volume of 371 ± 139 mm3. 15b (phosphate salt) was formulated in 0.5% MC/0.5% Tween 80 and administered by oral gavage at a dosing volume of 10 μL/g of an animal body weight. Animals in each group received vehicle or 5, 10, 25 mg/kg 15b (phosphate salt) every day for 14 consecutive days.RNU nude rats bearing the Karpas299 tumors were dosed with 15b (phosphate salt) at 50 mg/kg. Tumor and plasma samples were collected 7, 24, 48, and 72 h after dosing. Two tumor pieces were collected from each animal, one piece for protein extraction and the other for PK analysis. Proteins were extracted from tumor samples and then subjected to SDS–PAGE followed by Western blot with phospho-STAT3 antibody (pSTAT3, Tyr705).

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HOMA-IR[1]
Homeostatic model assessment (HOMA) of insulin resistance (IR) is a technical method for assessing IR from basal (fasting) glucose and insulin or C-peptide concentrations. The model is widely used to estimate insulin resistance. Groups of wild-type mice (n = 8 mice per group) were randomized into treatment groups based on their initial body weight. Mice were housed four per cage and dosed with vehicle orally, or ALK inhibitor (15b or Ceritinib (LDK-378)), orally once per day for 7 days. On day 7, compound was administered 180 min prior to a 3 g/kg glucose bolus. OGTT evaluations were performed in conscious mice that were 11 weeks of age. The mice were fasted by removing food at 6 p.m. the day before. A baseline blood sample was taken at t = −180 min, and the mice were then dosed orally with the compounds. A baseline blood sample was taken at t = 0 min, and the animals were then administered an oral glucose bolus (3 g/kg) immediately. Blood was obtained (via tail bleeding) to measure blood glucose (using glucometer). A single drop of blood from the tail was measured for glucose using a glucometer at t = −180, 0, 20, 40, 60, 120 min. Approximately 40 μL samples of blood were collected separately for insulin analysis 3 h prior to dosing (on day 0 and day 7) into chilled collection tubes containing EDTA. Plasma was isolated and stored at −70 °C until further analysis. The homeostatic model assessment-insulin resistance index (HOMA-IR) was used as a measure of insulin resistance and was calculated using the formula HOMA-IR = (FPG × FPI)/22.5 where FPG (mM) is the fasting plasma glucose concentration and FPI (μU/mL) is the fasting plasma insulin concentration. Insulin levels were determined using a detection assay kit from Mesoscale Discovery (MSD): catalog no. K112BZC-2. Higher values indicate insulin resistance.

Absorption, Distribution and Excretion
Peak plasma concentrations of ceritinib are reached approximately 4 to 6 hours after oral administration. Following a single oral dose of 750 mg of radiolabeled ceritinib, 92.3% of the administered dose is excreted in feces (68% of which is unmetabolized parent compound), and 1.3% is excreted in urine. The apparent volume of distribution (Vd/F) after a single 750 mg dose is 4230 L. The steady-state geometric mean apparent clearance (CL/F) after daily administration of 750 mg ceritinib (33.2 L/h) is lower than that after a single 750 mg dose (88.5 L/h). Metabolism/Metabolites In vitro studies have shown that CYP3A is the major enzyme involved in metabolic clearance. Ceritinib. Following a single oral dose of 750 mg of radiolabeled ceritinib, ceritinib, as the parent compound, is the major circulating component in human plasma (82%).

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Biological half-life
The terminal half-life is 41 hours.

Hepatotoxicity
Elevated serum transaminase levels are common during ceritinib treatment, occurring in 20% to 50% of patients, but only 1% to 2% of patients have transaminase levels exceeding five times the upper limit of normal. Liver failure has been reported in 0.2% of patients, leading to several deaths. Hepatotoxicity appears to be a class effect of ALK inhibitors, although liver injury caused by crizotinib appears to be more common and severe than that caused by ceritinib or alectinib. Specific details regarding ceritinib-related liver injury, such as latency, serum enzyme profiles, clinical features, and course, have not been published. Other ALK inhibitors typically cause liver injury within days or weeks of starting treatment, manifesting as a sudden increase in hepatocyte enzymes, with a moderate to severe course. Immune hypersensitivity and autoimmune features are uncommon. Patients with a history of cirrhosis or liver dysfunction due to hepatic tumor burden have an increased risk of clinically significant liver injury and liver failure. Relapses following re-administration have been reported. Probability Score: D (likely to cause clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the clinical use of ceritinib during lactation. Because ceritinib binds to plasma proteins at a rate as high as 97%, its concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during ceritinib treatment and for two weeks after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Ceritinib binds to human plasma proteins at a rate of 97%, regardless of drug concentration.

[1]. Synthesis, structure-activity relationships, and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials. J Med Chem. 2013 Jul 25;56(14):5675-90.

[2]. Ceritinib-a second-generation ALK inhibitor overcoming resistance in ALK-rearranged non-small celllung cancer. Transl Lung Cancer Res. 2014 Dec;3(6):379-81.

Ceritinib belongs to the aminopyrimidine class of drugs, with the chemical name 2,6-diamino-5-chloropyrimidine, where the amino groups at positions 2 and 6 are respectively substituented with 2-methoxy-4-(piperidin-4-yl)-5-methylphenyl and 2-(isopropylsulfonyl)phenyl. It is used to treat ALK-positive metastatic non-small cell lung cancer. Ceritinib is an antitumor drug and also an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor. It is an aminopyrimidine, aromatic ether, organochlorine compound, secondary amino compound, piperidine compound, and sulfone compound. Ceritinib is used to treat adult patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have failed prior crizotinib treatment (secondary to resistance or intolerance). Approximately 4% of NSCLC patients have chromosomal rearrangements, leading to the formation of an EML4 (echinoderm microtubule-associated protein-like 4) and ALK (anaplastic lymphoma kinase) fusion gene, resulting in constitutive kinase activity that promotes tumorigenesis and drives malignant phenotypes. Ceritinib exerts its therapeutic effect by inhibiting ALK autophosphorylation, ALK-mediated phosphorylation of the downstream signaling protein STAT3, and the proliferation of ALK-dependent cancer cells. Following treatment with crizotinib (a first-generation ALK inhibitor), most tumors develop resistance due to mutations in key "gatekeeper" residues of the enzyme. This situation prompted the development of novel second-generation ALK inhibitors, such as ceritinib, to overcome crizotinib resistance. Due to the unexpectedly high efficacy (56%) of ceritinib in crizotinib-resistant tumors, the FDA approved it for marketing in April 2014 and granted it orphan drug designation. Ceritinib is a kinase inhibitor. Its mechanism of action is as a tyrosine kinase inhibitor, cytochrome P450 3A inhibitor, and cytochrome P450 2C9 inhibitor. Ceritinib is a small-molecule tyrosine kinase receptor inhibitor and anti-tumor drug used to treat certain types of advanced non-small cell lung cancer (NSCLC). Elevated serum transaminases during ceritinib treatment occur at a moderate rate, and clinically observable cases of acute liver injury are rare. Ceritinib is an oral anaplastic lymphoma kinase (ALK) receptor tyrosine kinase activity inhibitor with anti-tumor activity. After administration, ceritinib binds to and inhibits the activity of wild-type ALK kinase, ALK fusion protein, and ALK point mutants. ALK inhibition leads to disruption of ALK-mediated signaling and inhibits the growth of ALK-overexpressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK dysregulation and gene rearrangement are associated with various tumor cell types. Drug Indication Ceritinib is a kinase inhibitor indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have not responded to or are intolerant of crizotinib. This indication received accelerated approval based on tumor response rate and duration of response. Ceritinib has not been shown to improve survival or disease-related symptoms. Continued approval for this indication may be contingent upon validation and description of clinical benefit in confirmatory trials. FDA Label Zykadia is indicated for the treatment of adult patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC) who have previously received crizotinib. Mechanism of Action Ceritinib inhibits anaplastic lymphoma kinase (ALK), also known as the ALK tyrosine kinase receptor or CD246 (differentiation cluster 246), an enzyme encoded by the ALK gene. Approximately 4-5% of NSCLCs exhibit chromosomal rearrangements leading to the fusion of EML4 (echinoderm microtubule-associated protein-like 4) and ALK (anaplastic lymphoma kinase), resulting in constitutive kinase activity that promotes carcinogenesis and drives malignant phenotypes. Ceritinib exerts its therapeutic effect by inhibiting ALK autophosphorylation, phosphorylation of the ALK-mediated downstream signaling protein STAT3, and the proliferation of ALK-dependent cancer cells. In vitro experiments have shown that ceritinib can inhibit the proliferation of cell lines expressing the EML4-ALK and NPM-ALK fusion proteins, and also exhibits dose-dependent inhibitory effects on the growth of EML4-ALK-positive NSCLC xenografts in mice and rats.

C28H38CL3N5O3S

631.0570

629.176

C, 53.29; H, 6.07; Cl, 16.85; N, 11.10; O, 7.61; S, 5.08

1380575-43-8

Ceritinib;1032900-25-6;Ceritinib-d7;1632484-77-5

67973512

Light yellow to yellow solid powder

8.87

5

8

9

40

835

0

ClC1=C([H])N=C(N=C1N([H])C1=C([H])C([H])=C([H])C([H])=C1S(C([H])(C([H])([H])[H])C([H])([H])[H])(=O)=O)N([H])C1C([H])=C(C([H])([H])[H])C(=C([H])C=1OC([H])(C([H])([H])[H])C([H])([H])[H])C1([H])C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H].Cl[H].Cl[H]

WNCJOPLFICTLPT-UHFFFAOYSA-N

InChI=1S/C28H36ClN5O3S.2ClH/c1-17(2)37-25-15-21(20-10-12-30-13-11-20)19(5)14-24(25)33-28-31-16-22(29)27(34-28)32-23-8-6-7-9-26(23)38(35,36)18(3)4;;/h6-9,14-18,20,30H,10-13H2,1-5H3,(H2,31,32,33,34);2*1H

5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine;dihydrochloride

Ceritinib dihydrochloride; LDK-378; Ceritinib HCl; 1380575-43-8; Ceritinib dihydrochloride; LDK378 dihydrochloride; LDK378 (dihydrochloride); Ceritinib 2hcl; LDK378 2HCl (Ceritinib); LDK-378 hydrochloride; 2,4-Pyrimidinediamine, 5-chloro-N4-[2-[(1-methylethyl)sulfonyl]phenyl]-N2-[5-methyl-2-(1-methylethoxy)-4-(4-piperidinyl)phenyl]-, hydrochloride (1:2); LDK 378; LDK378

2934.99.03.00

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, avoid exposure to moisture.

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

DMSO: 13~100 mg/mL (20.60~158.5 mM)
Ethanol: ~10 mg/mL(~15.9 mM)

Solubility in Formulation 1: 2.5 mg/mL (3.96 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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 (3.96 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 (3.96 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: 33.33 mg/mL (52.82 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.)