p110α (IC50 = 4 nM); p110α-H1047R (IC50 = 4.6 nM); p110α-E545K (IC50 = 5.7 nM); p110γ (IC50 = 5 nM); p110δ (IC50 = 7 nM); p110β (IC50 = 75 nM); mTOR (IC50 = 20.7 nM); mTORC1; mTORC2; Autophagy
Kinase Assay: PI3Kα, β, and δ proteins are composed of the iSH2 domain of p85 NH2-terminally fused to the full-length protein p110 protein, with the exception of α that also does not contain the last 20 amino acids. PI3Kγ is produced as full-length protein deleted for its first 144 amino acids. All constructs are fused to a COOH-terminal His tag for convenient purification and then cloned into the pBlue-Bac4.5 (for α, β, and δ isoforms) or pVL1393 (for γ isoform) plasmids. The different vectors are then cotransfected with BaculoGold WT genomic DNA using methods recommended by the vendor for production of the respective recombinant baculoviruses and proteins. BEZ235 are tested for their activity against PI3K using a Kinase-Glo assay. The kinase reaction is done in 384-well black plate. Each well is loaded with 50 μL of test items (in 90% DMSO) and 5 μL reaction buffer containing 10 μg/mL PI substrate (l-α-phosphatidylinositol; Avanti Polar Lipids; prepared in 3% octyl-glucoside) and the PI3K proteins (10, 25, 10, and 150 nM of p110α, p110β, p110δ, and p110γ, respectively) are then added to it. The reaction is started by the addition of 5 μL of 1 μM ATP prepared in the reaction buffer and is incubated for either 60 (for p110α, p110β, and p110δ) or 120 min (for p110γ). It is terminated by the addition of 10 μL Kinase-Glo buffer. The plates are then read in a Synergy 2 reader for luminescence detection.

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Cell Assay: MOLT-4 and CEM-R cells; The solubility of this compound in DMSO is<10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while.Stock solution can be stored below -20°C for several months; 500 nM, for cell cycle inhibition 200 nM, 16 hours for pRb decrease.

BEZ235 significantly reduces the phosphorylation levels of the mTOR activated kinase p70S6K. BEZ235 results in a reduction of S235/S236P-RPS6 levels with IC50 of 6.5 nM. The activity of BEZ235 against mTOR is determined using a biochemical mTOR K-LISA assay with IC50 of 20.7 nM. BEZ235 shows slightly lower activity against its β paralogue with IC50 of 75 nM. The PI3K/Akt/mTOR pathway is often constitutively activated in human tumor cells. BEZ235 blocks PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. Both PTEN-null cell lines PC3M and U87MG show a dose-dependent reduction in cell proliferation when treated with increasing concentrations of BEZ235 with an average GI50 of 10-12 nM. BEZ235 is an mTORC1/2 catalytic inhibitor.

Kinase Assay: PI3Kα, β, and δ proteins are composed of the iSH2 domain of p85 NH2-terminally fused to the full-length protein p110 protein, with the exception of α that also does not contain the last 20 amino acids. PI3Kγ is produced as full-length protein deleted for its first 144 amino acids. All constructs are fused to a COOH-terminal His tag for convenient purification and then cloned into the pBlue-Bac4.5 (for α, β, and δ isoforms) or pVL1393 (for γ isoform) plasmids. The different vectors are then cotransfected with BaculoGold WT genomic DNA using methods recommended by the vendor for production of the respective recombinant baculoviruses and proteins. BEZ235 are tested for their activity against PI3K using a Kinase-Glo assay. The kinase reaction is done in 384-well black plate. Each well is loaded with 50 μL of test items (in 90% DMSO) and 5 μL reaction buffer containing 10 μg/mL PI substrate (l-α-phosphatidylinositol; Avanti Polar Lipids; prepared in 3% octyl-glucoside) and the PI3K proteins (10, 25, 10, and 150 nM of p110α, p110β, p110δ, and p110γ, respectively) are then added to it. The reaction is started by the addition of 5 μL of 1 μM ATP prepared in the reaction buffer and is incubated for either 60 (for p110α, p110β, and p110δ) or 120 min (for p110γ). It is terminated by the addition of 10 μL Kinase-Glo buffer. The plates are then read in a Synergy 2 reader for luminescence detection.

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Cell Assay: MOLT-4 and CEM-R cells; The solubility of this compound in DMSO is<10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while.Stock solution can be stored below -20°C for several months; 500 nM, for cell cycle inhibition 200 nM, 16 hours for pRb decrease.

BEZ235 significantly reduces the phosphorylation levels of the mTOR activated kinase p70S6K. BEZ235 results in a reduction of S235/S236P-RPS6 levels with IC50 of 6.5 nM. The activity of BEZ235 against mTOR is determined using a biochemical mTOR K-LISA assay with IC50 of 20.7 nM. BEZ235 shows slightly lower activity against its β paralogue with IC50 of 75 nM. The PI3K/Akt/mTOR pathway is often constitutively activated in human tumor cells. BEZ235 blocks PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. Both PTEN-null cell lines PC3M and U87MG show a dose-dependent reduction in cell proliferation when treated with increasing concentrations of BEZ235 with an average GI50 of 10-12 nM. BEZ235 is an mTORC1/2 catalytic inhibitor.

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NVP-BEZ235 inhibits PI3Kα competitively with respect to ATP, with IC₅₀ shifting linearly with increasing ATP concentrations.
In U87MG glioblastoma cells (PTEN-negative), NVP-BEZ235 reduces phosphorylation of Akt at Ser473 (S473P-Akt) and Thr308 (T308P-Akt) in a dose-dependent manner, with IC₅₀ values of 8.0 ± 3.2 nM and 30 ± 15 nM, respectively, as measured by ELISA. The inhibition is reversible upon compound removal.
At 250 nM, NVP-BEZ235 blocks IGF-I-induced S473P-Akt without affecting IGF-IR phosphorylation, and does not inhibit epidermal growth factor or platelet-derived growth factor-induced mitogen-activated protein kinase pathway activation.
At 250 nM, it does not inhibit anisomycin-induced phosphorylation of c-Jun NH2-terminal kinase or p38.
It blocks interleukin-4-induced S473P-Akt without affecting Stat6 phosphorylation.
At concentrations up to 250 nM, it does not inhibit DNA damage-induced ATM or DNA-PK activation, though at 1,250 nM it reduces DNA-PK autophosphorylation.
It induces nuclear translocation of FKHRL1 (FOXO3a) and activates forkhead-mediated transcription.
It inhibits phosphorylation of p70S6K and reduces phospho-S6 levels in TSC1-null MEF cells with an IC₅₀ of 6.5 nM.
It inhibits both mTORC1 and mTORC2 kinase activities in immunoprecipitation kinase assays.
In PTEN-null cell lines (PC3M and U87MG), it induces G₁ cell cycle arrest and increases p27Kip1 levels, but does not induce apoptosis or reduce cell number below plating density. [1]

BEZ235 induces regression of the tumors (69%) without statistically significant effect on body weight gain. Altogether, these preliminary in vivo efficacy results show that BEZ235 causes disease stasis when administered orally as a single agent and can enhance the efficacy of other anticancer agents when used in combination studies.

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Dactolisib (BEZ235, NVP-BEZ 235) Is an Orally Available PI3K Inhibitor with Well-Tolerated Antitumor Activity [1]
The pharmacokinetic properties of NVP-BEZ235 were originally evaluated in PC3M tumor-bearing nude mice. At a dose of 50 mg/kg, NVP-BEZ235 appeared rapidly in plasma with a Cmax of 1.68 μmol/L at 0.5 h and a C24h of 0.03 μmol/L. In the tumor tissue, the Cmax attained was 2.05 nmol/g at 1 h (Tmax), which decreased to 0.23 nmol/g after 24 h (Fig. 5A). Dactolisib (BEZ235, NVP-BEZ 235) is eliminated relatively quickly from the liver. In vivo analysis of the S473P-Akt levels in the tumor tissue revealed that maximum inhibition was obtained 1 h after dosing (corresponding to the tumor Cmax), and persistent inhibition was observed still 16 h after treatment, with almost complete recovery to basal levels obtained in two of four tumors at 24 h after dose (Fig. 5B). Pharmacokinetic simulation based on this study revealed that steady-state levels would be achieved between 3 and 5 days, when dosing either at 50 mg/kg given daily or at 25 mg/kg given twice daily. Differences between the dosage regimens would reside in the predicted tumor peak levels (2.6 versus 1.6 nmol/g, respectively) whereas through levels would remain almost comparable (0.53 versus 0.60 μmol/L, respectively). Taking into consideration this pharmacokinetic simulation, chronic treatment of PC3M tumor-bearing animals was done at 25 mg/kg p.o. NVP-BEZ235 twice daily. Using this schedule, a statistically significant inhibition of tumor growth was observed, with a final T/C value of 22% after 10 days of treatment (Fig. 5C). The treatment was well tolerated as concluded from the nonstatistically significant effect of NVP-BEZ235 on body weight gain (Fig. 5C) and by the fact that none of the animals died during the course of the study. The antitumor effect was well correlated with the inhibition of S473P-Akt in the tumor tissue 1 or 18 h after the last dose detected either by Western blotting of tumor extracts or by immunostaining of tumor sections (Fig. 5D). Compound concentrations at these time points were 1.32 nmol/g at 1 h and 0.51 nmol/g at 18 h, close to the predicted values for steady-state peak levels (see above).

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NVP-BEZ235 administered orally at 25 mg/kg twice daily significantly inhibits tumor growth in PC3M prostate cancer xenografts (T/C = 22% after 10 days). Tumor growth inhibition correlates with reduced S473P-Akt levels in tumor tissue.
In U87MG glioblastoma xenografts, daily oral administration shows dose-dependent tumor growth inhibition (T/C values: 22% at 25 mg/kg, 18% at 35 mg/kg, 5.5% at 45 mg/kg).
In combination with temozolomide in U87MG model, NVP-BEZ235 enhances antitumor activity, causing tumor regression (69% regression) compared to temozolomide alone (T/C = 5.2%).
Treatment is well tolerated with no significant body weight loss or mortality. [1]

PI3Kα, β, and δ proteins are composed of the iSH2 domain of p85 NH2-terminally fused to the full-length protein p110 protein, with the exception of α that also does not contain the last 20 amino acids. PI3Kγ is produced as full-length protein deleted for its first 144 amino acids. All constructs are fused to a COOH-terminal His tag for convenient purification and then cloned into the pBlue-Bac4.5 (for α, β, and δ isoforms) or pVL1393 (for γ isoform) plasmids. The different vectors are then cotransfected with BaculoGold WT genomic DNA using methods recommended by the vendor for production of the respective recombinant baculoviruses and proteins. BEZ235 are tested for their activity against PI3K using a Kinase-Glo assay. The kinase reaction is done in 384-well black plate. Each well is loaded with 50 μL of test items (in 90% DMSO) and 5 μL reaction buffer containing 10 μg/mL PI substrate (l-α-phosphatidylinositol; Avanti Polar Lipids; prepared in 3% octyl-glucoside) and the PI3K proteins (10, 25, 10, and 150 nM of p110α, p110β, p110δ, and p110γ, respectively) are then added to it. The reaction is started by the addition of 5 μL of 1 μM ATP prepared in the reaction buffer and is incubated for either 60 (for p110α, p110β, and p110δ) or 120 min (for p110γ). It is terminated by the addition of 10 μL Kinase-Glo buffer. The plates are then read in a Synergy 2 reader for luminescence detection.

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PI3K enzymatic activity was assessed using a Kinase-Glo luminescence assay. Recombinant class I PI3K isoforms (p110α, β, δ, γ) were incubated with test compounds in 384-well plates in reaction buffer containing Tris-HCl, NaCl, MgCl₂, DTT, CHAPS, PI substrate, and ATP. Reactions were run for 60–120 min, terminated with Kinase-Glo buffer, and luminescence was measured.
mTOR kinase activity was measured using a K-LISA assay, though detailed protocol is not fully described in the main text.
IC₅₀ values were determined from dose-response curves. [1]

MOLT-4 and CEM-R cells; The solubility of this compound in DMSO is<10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while.Stock solution can be stored below -20°C for several months; 500 nM, for cell cycle inhibition 200 nM, 16 hours for pRb decrease. BEZ235 significantly reduces the phosphorylation levels of the mTOR activated kinase p70S6K. BEZ235 results in a reduction of S235/S236P-RPS6 levels with IC50 of 6.5 nM. The activity of BEZ235 against mTOR is determined using a biochemical mTOR K-LISA assay with IC50 of 20.7 nM. BEZ235 shows slightly lower activity against its β paralogue with IC50 of 75 nM. The PI3K/Akt/mTOR pathway is often constitutively activated in human tumor cells. BEZ235 blocks PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. Both PTEN-null cell lines PC3M and U87MG show a dose-dependent reduction in cell proliferation when treated with increasing concentrations of BEZ235 with an average GI50 of 10-12 nM. BEZ235 is an mTORC1/2 catalytic inhibitor.

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For phospho-Akt ELISA, U87MG cells were seeded in 96-well plates, treated with compounds for 30 min, lysed, and cell extracts were transferred to ELISA plates coated with anti-Akt antibody. After incubation, phospho-specific antibodies (anti-S473P-Akt or anti-T308P-Akt) were added, followed by HRP-conjugated secondary antibody and luminescent substrate.
For phospho-S6 cell-based assay, TSC1-null MEF cells were seeded in 96-well black plates, treated with compounds for 1 h, fixed with formaldehyde, blocked, and incubated with anti-phospho-S6 antibody followed by IRDye 800-labeled secondary antibody. Fluorescence was measured using an infrared imager.
For forkhead translocation assay, U2OS cells stably expressing GFP-FKHRL1 were seeded in 96-well plates, starved, treated with compounds, fixed, and nuclei were stained with Hoechst. Translocation was visualized by fluorescence microscopy.
For cell proliferation assay, cells were incubated with compounds for 72 h, stained with methylene blue, and absorbance was measured to estimate cell number.
For cell cycle analysis, PC3M cells were treated with compounds for 24 h, harvested, and analyzed by flow cytometry. [1]

Mice: The NVP-Dactolisib (BEZ235) powder is dissolved in NMP on sonication, and the remaining volume of polyethylene glycol 300 is added to a concentration of 5 mg/mL. The dosage is 10 mL/kg. For analysis, frozen tissues are minced, then homogenized in an equal volume of ice-cold PBS. After centrifugation, supernatants are then examined. The samples are then eluted over a 20-minute period at a flow rate of 1 mL/min with a linear gradient of 10% to 90% (v/v) acetonitrile in water containing 0.05% (v/v) trifluoroacetic acid. The compounds are identified by 340 nm UV absorbance, and peak heights from the external standard method are used to calculate concentrations[1].

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Establishment of Xenograft Tumors, Efficacy Studies, Compound Preparation, and Analytics [1]
Establishment of tumors, group randomization, tumor, and body weight recording during efficacy studies were described elsewhere. Antitumor activity is expressed as %T/C (mean increase of tumor volumes of treated animals divided by the mean increase of tumor volumes of control animals multiplied by 100) and/or as tumor regression (%Reg) calculated as [(mean tumor volume at the start of treatment - mean tumor volume) / (mean tumor volume at the start of treatment)] × 100. Data are presented as mean ± 1 SE. Comparisons between groups and vehicle control group were done using either one-way ANOVA or ANOVA on ranks followed by Dunnett's tests when data were respectively either normally distributed or not. For all tests, the level of significance was set at P < 0.05. Calculations were done using SigmaStat version 2.03. Dactolisib (BEZ235, NVP-BEZ 235) (free base) was formulated in NMP/polyethylene glycol 300 (10/90, v/v). Solutions (5 mg/mL) were prepared fresh each day of dosing as follows: the powder was dissolved in NMP on sonication, and the remaining volume of polyethylene glycol 300 was added. The application volume was 10 mL/kg. For analytics, frozen tissues were minced and then homogenized in an equal volume of ice-cold PBS using a Polytron homogenizer (IKA). After acetonitrile precipitation and centrifugation, supernatants were analyzed by reverse-phase high-performance liquid chromatography/UV on a Merck-Hitachi/LaChrom equipment including a Nucleosil 100-5 C18 column. Samples were then eluted with a linear gradient of 10% to 90% (v/v) acetonitrile in water containing 0.05% (v/v) trifluoroacetic acid over a period of 20 min at a flow rate of 1 mL/min. The compounds were detected by UV absorbance at 340 nm, and concentrations were determined by the external standard method using peak heights.

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For xenograft studies, tumor fragments were implanted subcutaneously into nude mice. When tumors reached ~100–200 mm³, mice were randomized into groups.
NVP-BEZ235 (free base) was formulated in NMP/polyethylene glycol 300 (10:90, v/v) at 5 mg/mL. The powder was dissolved in NMP by sonication, then PEG 300 was added. The compound was administered orally at 10 mL/kg body weight.
In PC3M model, mice were treated orally with 25 mg/kg twice daily for 10 days.
In U87MG model, mice were treated orally once daily at 25, 35, or 45 mg/kg, or in combination with temozolomide.
Tumor volumes and body weights were measured regularly. Animals were sacrificed at specified time points for tumor collection and analysis. [1]

In nude mice bearing PC3M tumors, plasma Cmax reached 1.68 µM at 0.5 h and 0.03 µM at 24 h after a single oral administration of 50 mg/kg NVP-BEZ235. In tumor tissue, Cmax was 2.05 nmol/g (Tmax) at 1 h and decreased to 0.23 nmol/g at 24 h. The compound was cleared from the liver relatively quickly. Pharmacokinetic simulations predicted steady-state plasma concentrations after 3–5 days of administration of 50 mg/kg once daily or 25 mg/kg twice daily. In long-term treatment (25 mg/kg twice daily), the tumor tissue concentration was 1.32 nmol/g at 1 h and 0.51 nmol/g at 18 h. [1]

In vivo studies have shown that at effective doses (25–45 mg/kg/day), NVP-BEZ235 is well tolerated, and no significant weight loss or death was observed in PC3M and U87MG xenograft models.

[1]. Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther, 2008, 7(7), 1851-1863.

Dactolisib tosylate is the tosylate form of Dactolisib, an orally bioavailable imidazoquinone drug that targets phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), exhibiting potential antitumor activity. After administration, Dactolisib inhibits PI3K and mTOR kinases in the PI3K/AKT/mTOR signaling pathway, leading to apoptosis and growth inhibition in PI3K/mTOR-overexpressing tumor cells. Activation of the PI3K/mTOR pathway promotes cell growth, survival, and resistance to chemotherapy and radiotherapy. mTOR is a downstream serine/threonine kinase of PI3K, and its activation may also be independent of PI3K. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is typically constitutively activated in human tumor cells, providing a unique opportunity for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits the kinase activity of PI3K and mTOR by binding to their ATP-binding sites. In cell models using human tumor cell lines, this molecule effectively and specifically blocks aberrant activation of the PI3K pathway, inducing G1 phase arrest. The cellular activity of NVP-BEZ235 has also been validated in in vivo models of human cancer. Therefore, this compound is well-tolerated, inhibits disease progression after oral administration, and may enhance the efficacy of other anticancer drugs in in vivo combination therapy studies. Pharmacokinetic/pharmacodynamic analysis of ex vivo tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. In summary, preclinical data indicate that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmacological properties. NVP-BEZ235 is currently in Phase I clinical trials. [1]

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NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative discovered through structure-based drug design.
It competitively binds to the ATP-binding sites of PI3K and mTOR and forms hydrogen bonds with conserved residues.
It blocks the activation of the dysfunctional PI3K pathway in cancer cells with PTEN deficiency or PI3K mutation.
In the tested cell lines, it induced G₁ phase arrest and upregulation of p27Kip1, but did not induce apoptosis.
In a glioblastoma model, it showed synergistic antitumor activity with temozolomide.
At the time of publication, it was in Phase I clinical trials. [1]

C37H31N5O4S

641.75

641.21

C, 69.25; H, 4.87; N, 10.91; O, 9.97; S, 5.00

1028385-32-1

Dactolisib;915019-65-7; 1028385-32-1; 2319647-83-9 (HCl)

49803145

Off-white to light yellow solid powder

8.216

1

7

4

47

1080

0

CN(C1=C2C3=CC(C4=CC5=CC=CC=C5N=C4)=CC=C3N=C1)C(N2C6=CC=C(C=C6)C(C)(C#N)C)=O.CC7=CC=C(S(=O)(O)=O)C=C7

FWURTHAUPVXZHW-UHFFFAOYSA-N

InChI=1S/C30H23N5O.C7H8O3S/c1-30(2,18-31)22-9-11-23(12-10-22)35-28-24-15-19(21-14-20-6-4-5-7-25(20)32-16-21)8-13-26(24)33-17-27(28)34(3)29(35)36;1-6-2-4-7(5-3-6)11(8,9)10/h4-17H,1-3H3;2-5H,1H3,(H,8,9,10)

4-methylbenzenesulfonic acid;2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-ylimidazo[4,5-c]quinolin-1-yl)phenyl]propanenitrile

Dactolisib tosylate; NVP-BEZ 235 Tosylate; NVP-BEZ235-ANA; BEZ235 Tosylate; BEZ235 Tosylate; BEZ235 (Tosylate); Dactolisib (Tosylate); NVP-BEZ235-ANA; Dactolisib tosilate; Dactolisib tosylate [USAN]; UNII-U54GT9151S

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, 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)

Solubility in Formulation 1: ≥ 1 mg/mL (1.56 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: NMP+polyethylene glycol 300 (10/90, v/v): 30mg/mL

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Solubility in Formulation 3: 16.67 mg/mL (25.98 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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