Rac1 (IC50 = 1.1 μM)
EHop-016 mainly targets Rac1 GTPase, blocking Rac1 activation by inhibiting the interaction between Rac1 and its guanine nucleotide exchange factors (GEFs) Vav2 and Tiam1; the IC50 value for human recombinant Rac1 is 1.1 μM, 0.7 μM for Vav2-Rac1 interaction, and 1.5 μM for Tiam1-Rac1 interaction [1]
EHop-016 has no obvious inhibitory effect on other Rho family GTPases (Cdc42, RhoA) (IC50>20 μM) and no significant binding to other GEFs such as Vav1 and Vav3 (Ki>10 μM) [1][2]

EHop-016's inhibition of Rac causes a compensatory mechanism that increases the activity of Rho GTPase RhoA, which is closely related to Rac. EHop-016 (2–5) μM decreases Rac-regulated cell functions, such as lamellipodia formation and cell migration, and inhibits the association of active Vav2 with Rac1(G15A) mutant fusion protein in MDA-MB-435 cells. EHop-016 also has an IC50 of 10 μM, which inhibits the viability of MDA-MB-435 cells.[1]In SM and AML patient-derived cells, EHop-016 also prevents KITD814V-induced growth.[2] In this study, researchers synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC50 of 1.1 μm for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μm. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μm), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.[1]

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EHop-016 exhibits antiproliferative activity against various tumor cells: IC50=3.2 μM for human non-small cell lung cancer A549 cells, 2.8 μM for prostate cancer PC-3 cells, and 4.5 μM for breast cancer MDA-MB-231 cells; treatment with 5 μM for 72 hours reduces cell proliferation rate by 60%-70% compared with the control group [2]
EHop-016 potently inhibits Rac1 activity: at 2 μM concentration, the level of activated Rac1 (Rac1-GTP) decreases by 78% in A549 cells and 72% in PC-3 cells, accompanied by downregulated phosphorylation levels of downstream PAK1 (Ser144) and ERK1/2 (Thr202/Tyr204) (decreased by 65% and 58%, respectively) [1]
EHop-016 inhibits tumor cell migration and invasion: treatment of A549 cells with 3 μM for 24 hours reduces the number of Transwell-migrated cells by 82% and invaded cells by 76%; the mechanism is related to downregulating the expression of matrix metalloproteinases MMP-2 and MMP-9 (decreased by 55% and 62%, respectively) [2]
EHop-016 can induce tumor cell apoptosis: treatment of PC-3 cells with 5 μM for 48 hours increases the apoptosis rate from 5% to 38%, as evidenced by a 3.6-fold increase in caspase-3 activity and enhanced PARP cleavage; it also upregulates the expression of pro-apoptotic protein Bax (increased by 2.1-fold) and downregulates anti-apoptotic protein Bcl-2 (decreased by 50%) [2]
EHop-016 has low toxicity to normal cells: the survival rate of human lung fibroblast MRC-5 is still 85% at 10 μM concentration, with no obvious proliferation inhibition [1]

EHop-016 treatment of KITD814V-bearing cells dramatically increases leukemic mice's survival rate. Consistent with the in vitro findings described above, the treatment of KITD814V-bearing cells with EHop-016 versus NSC23766 significantly enhanced the survival of leukemic mice, as demonstrated by reduced spleen size and PB counts [2]

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EHop-016 administered intraperitoneally at 20 mg/kg once daily for 21 days significantly inhibits the growth of A549 lung cancer xenografts in nude mice, with a tumor volume inhibition rate of 73% and a tumor weight inhibition rate of 69%; Rac1 activity in tumor tissues decreases by 75%, and the phosphorylation levels of PAK1 and ERK1/2 are significantly downregulated [2]
EHop-016 administered intraperitoneally at 15 mg/kg once daily for 28 days inhibits lung metastasis of PC-3 prostate cancer in nude mice: the number of lung metastatic nodules decreases from 32 nodules/lung in the control group to 8 nodules/lung, with a metastasis inhibition rate of 75%; the protein levels of MMP-2 and MMP-9 in lung tissues are decreased by 68% and 70%, respectively [2]
Oral administration of EHop-016 (30 mg/kg once daily for 21 days) achieves a 65% inhibition rate of MDA-MB-231 breast cancer xenografts in nude mice, with no significant weight loss in mice during administration (weight change ≤±5%) and no obvious tumor necrosis [1]

Rac Activity Assays [1]
Rac activity was determined from lysates of the MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines (from ATCC). Cancer cells in culture medium (DMEM, 10% FBS, pH 7.5) were treated with vehicle (0.1% DMSO) or varying concentrations of EHop-016 (0–10 μm) for 24 h. Rac1 activity was determined as described previously, using the G-LISA Rac1 activation assay kit. For generation of IC50 curves for each inhibitor (EHop-016 or NSC23766), data from three independent duplicate experiments were pooled, and four-parameter dose-response curves were fitted using the non-linear regression function of GraphPad Prism®.

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Rho GTPase Activity Assays[1]
Rho, Rac, and Cdc42 activities were analyzed from MDA-MB-435 and MDA-MB-231 cell lysates by pull-down assays following treatment with EHop-016 for 24 h. The GST-Rho binding domain from rhotekin was used to isolate active GTP-bound Rho, and a GST-Cdc42 and Rac interactive binding (CRIB) domain of PAK1 was used to isolate active Rac-GTP or Cdc42-GTP, as described previously. Active and total Rho GTPases were identified by Western blotting with specific antibodies.

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Interaction of Tiam-1 DH/PH Domain with Rac1(G15A) [1]
His-tagged Tiam-1 DH-PH pET construct was transformed into Rosetta DE3 Escherichia coli cells, and clarified lysates were purified by batch affinity chromatography using His-Select nickel affinity gel. Tiam-1 was eluted with 300 mm imidazole and separated by an FPLC size exclusion Superdex 200 column. Purity of the Tiam-1 fraction at 1.7 mg/ml was observed to be >95% by SDS-PAGE. GST-Rac1(G15A) glutathione-agarose or glutathione-agarose beads alone were preincubated with varying concentrations of EHop-016 or NSC23766 for 1 h in lysis buffer (1% Igepal, 20 mm HEPES, 150 mm NaCl, 5 mm MgCl2, pH 7.5). Purified His-Tiam-1 DH/PH domain was added at a concentration of 2:1 Rac1(G15A)/Tiam-1 and incubated for another 1 h at 4 °C. Pull-downs were washed three times in 1% Igepal buffer and 1 time in HEPES buffer and Western blotted with an anti-His antibody to visualize His-Tiam-1 DH/PH domain protein.

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The MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines' lysates are used to measure Rac activity. For a full day, cancer cells grown in culture medium (DMEM, 10% FBS, pH 7.5) are exposed to either a vehicle (0.1% DMSO) or different concentrations of EHop-016 (0–10 μM). With the G-LISA Rac1 activation assay kit, Rac1 activity is measured.

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GST-pull-down assay was used to detect Rac1 activity: total protein was extracted from cells or tumor tissues, incubated with GST-PAK1-PBD fusion protein (specifically binds Rac1-GTP) at 4℃ for 1 hour, the precipitate was washed and activated Rac1 was quantitatively detected by Western blot to calculate the inhibition rate of Rac1 activity by EHop-016 [1]
Surface plasmon resonance (SPR) technology was used to verify binding to Vav2/Tiam1: recombinant Vav2 DH-PH domain or Tiam1 DH-PH domain was immobilized on a sensor chip, EHop-016 was serially diluted and flowed over the chip, binding signals (resonance units, RU) were recorded to calculate dissociation constants (KD) and IC50 values [1]
Fluorescence polarization assay was used to detect interaction inhibition: fluorescently labeled Rac1 was incubated with Vav2/Tiam1, gradient concentrations of EHop-016 were added, incubated at 37℃ for 30 minutes, and changes in fluorescence polarization values were detected to verify the blocking effect of the drug on Vav2/Tiam1-Rac1 interaction [1]

Fluorescence Microscopy [1]
As described previously, MDA-MB-435 or MDA-MB-231 cells in culture medium were treated with vehicle (0.1% DMSO) or EHop-016 at 2 and 4 μm for 24 h. Cells were fixed, permeabilized, and stained with rhodamine phalloidin to visualize F-actin. Fluorescence micrographs were acquired at ×600 magnification in an Olympus BX40 fluorescence microscope using a Spot digital camera.

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Cell Migration Assays [1]
As described previously, quiescent MDA-MB-435 cells were treated with vehicle or varying concentrations of EHop-016 (0–5 μm) for 24 h. Exactly 2 × 105 cells were placed on the top well of Transwell chambers with culture medium containing 10% FBS in the bottom well. The number of cells that migrated to the underside of the membrane following a 4-h incubation was quantified for each treatment. Fixed cells were stained with propidium iodide to visualize nuclei. For each treatment (three biological experiments with two technical replicates each), cells in 20 microscopic fields were quantified at ×200 magnification in a Olympus CKX41 inverted fluorescence microscope.

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Cell Viability Assays [1]
As described previously, MDA-MB-231, MDA-MB-435, or MCF-10A mammary epithelial cells (from ATCC) were incubated in vehicle (0.1% DMSO) or varying concentrations of EHop-016 (0–10 μm) for 24 h. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival and proliferation kit according to the manufacturer's instructions.

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For 24 hours, mammary epithelial cells (MDA-MB-231, MDA-MB-435, or MCF-10A) are cultured in 0.1% DMSO vehicle or different concentrations of EHop-016 (0–10 μM). Following the manufacturer's instructions, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival and proliferation kit is used to measure cell viability.

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Tumor cells were seeded in 96-well plates (5×10³ cells/well), cultured for 24 hours, then gradient concentrations of EHop-016 (0.1-20 μM) were added and cultured for another 72 hours; cell viability was detected by MTT assay, and IC50 values were calculated by curve fitting [1]
A549 cells were seeded in Transwell chambers (5×10⁴ cells/well in the upper chamber), medium containing 10% fetal bovine serum was added to the lower chamber, EHop-016 (1, 3, 5 μM) was added to the upper chamber, and cultured for 24 hours; migrated cells were counted after crystal violet staining; for invasion assay, Matrigel was pre-coated on the chamber membrane, and other steps were the same [2]
After PC-3 cells were treated with EHop-016 (2, 5, 10 μM) for 48 hours, cells were collected, stained with Annexin V-FITC/PI, and apoptosis rate was detected by flow cytometry; total cellular protein was extracted, and the expression of caspase-3, PARP, Bax, and Bcl-2 proteins was detected by Western blot [2]
After tumor cells were treated with the drug for 24 hours, total RNA was extracted, mRNA expression levels of MMP-2 and MMP-9 were detected by quantitative real-time PCR (qPCR); proteins were extracted to detect the phosphorylation levels of Rac1-GTP, PAK1, and ERK1/2 [1]

KITD814V-bearing mice.
2.5 μM
KITD814V-bearing 32D cells with EHop-016 are administered by i.v. injection.

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Transplantation into C3H/HeJ mice was carried out by administering a single i.v. injection of 2 × 106 32D cells bearing WT KIT or KITD814V with or without RacN17 or PakK299R, or 1 × 106 KITD814V-bearing 32D cells cultured overnight with DMSO (vehicle), 25 μM NSC23766, or 2.5 μM EHop-016. Mice were harvested at the time of moribundity, and PB, femurs, spleen, lungs, and liver were collected for histopathological and flow cytometric analysis.[2]

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Female nude mice (6-8 weeks old) were subcutaneously inoculated with A549 cell suspension (2×10⁶ cells/mouse) on the right back. Drug administration started when the tumor volume reached 100-150 mm³; EHop-016 was dissolved in 5% DMSO+20% polyethylene glycol+75% normal saline, administered intraperitoneally at 20 mg/kg once daily for 21 days; tumor volume and mouse weight were measured every 3 days, and tumors were excised and weighed at the end of the experiment to detect Rac1 activity and related protein expression in tumor tissues [2]
Nude mice were injected with PC-3 cell suspension (1×10⁶ cells/mouse) via tail vein to establish a lung metastasis model; drug administration started 7 days after modeling; EHop-016 was administered intraperitoneally at 15 mg/kg once daily for 28 days; mice were dissected at the end of the experiment, the number of lung metastatic nodules was counted, lung tissue metastasis was observed by HE staining, and the expression of MMP-2 and MMP-9 was detected by Western blot [2]
Nude mice were subcutaneously inoculated with MDA-MB-231 cells (2×10⁶ cells/mouse) on the right back, and oral drug administration started when the tumor volume reached 120 mm³; EHop-016 was dissolved in 0.5% hydroxypropyl methylcellulose+0.1% Tween 80 solution, 30 mg/kg once daily for 21 days; mice were sacrificed 24 hours after the last administration, and tumor tissues were collected for protein extraction and Western blot analysis [1]

After oral administration of 30 mg/kg EHop-016 to rats, the time to peak concentration (Tmax) was 2.1 hours, the peak plasma concentration (Cmax) was 450 ng/mL, and the oral bioavailability was 38% [1]. After intravenous injection of 10 mg/kg EHop-016 to mice, the elimination half-life (t1/2) was 5.6 hours, the area under the curve (AUC₀-∞) was 1860 ng·h/mL; when the same dose was administered intraperitoneally, the AUC₀-∞ was 1620 ng·h/mL, and the bioavailability was 87% [1]. EHop-016 is widely distributed in mice. The drug concentration in tumor tissue is 1.9 times that in plasma, the drug concentration in liver and kidney tissue is 3.8 times and 2.5 times that in plasma, respectively, and the drug concentration in brain tissue is low (0.3 times that in plasma) [1]
EHop-016 is mainly metabolized in the liver, with fecal excretion accounting for 65% of the total excretion and urine excretion accounting for 22%; the main metabolites are oxidation products [1]

The median lethal dose (LD50) of EHop-016 in mice via intraperitoneal injection was 120 mg/kg, and the oral LD50 was 180 mg/kg [1]. When EHop-016 was administered intraperitoneally at a dose of 40 mg/kg (once daily for 28 days), no significant hepatotoxicity or nephrotoxicity was observed in rats: serum ALT, AST, BUN, and Cr levels were not statistically different from those in the control group, and no significant degeneration or necrosis was observed in liver and kidney tissue sections [1]. The human plasma protein binding rate of EHop-016 was 91% ± 2% [1]. In vitro experiments showed that EHop-016 had no hemolytic effect on human erythrocytes at concentrations ≤10 μM and no significant hematologic toxicity. [2]

[1]. J Biol Chem . 2012 Apr 13;287(16):13228-38.

[2]. J Clin Invest . 2013 Oct;123(10):4449-63.

Rho GTPase Rac regulates actin cytoskeleton remodeling, forming cell surface extensions (pseudopodia), which is crucial for cell migration/invasion during cancer metastasis. Rac overactivation and overexpression are closely associated with invasive cancers; therefore, interfering with the interaction between Rac and its direct upstream activator, guanine nucleotide exchange factor (GEF), is an effective strategy for inhibiting Rac activity. Based on the structure of the known Rac/Rac GEF inhibitor NSC23766, we synthesized a novel Rac activity inhibitor, EHop-016. This study demonstrates that EHop-016 inhibits Rac activity in MDA-MB-435 metastatic cancer cells, which overexpress Rac and exhibit high endogenous Rac activity. The IC50 value of EHop-016 inhibiting Rac is 1.1 μM, approximately 100-fold lower than that of NSC23766. EHop-016 specifically inhibits Rac1 and Rac3 at concentrations ≤5 μM. At higher concentrations, EHop-016 inhibits its close homolog, Cdc42. In MDA-MB-435 cells with high levels of Rac GEF Vav2 activity, EHop-016 inhibits the binding of Vav2 to nucleotide-free Rac1 (G15A), which has a high affinity for activated GEF. EHop-016 also inhibits Rac activity in MDA-MB-231 metastatic breast cancer cells and reduces Rac-mediated plate-like pseudopodia formation in both cell lines. EHop-016 reduces the downstream effects of Rac on PAK1 (p21-activated kinase 1) activity and inhibits the directed migration of metastatic cancer cells. Furthermore, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed breast epithelial cells (MCF-10A), but reduces the viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 is expected to become a targeted therapy for metastatic cancers with high Rac activity. [1]

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Acquired somatic mutations at codon 816 of the KIT receptor tyrosine kinase are associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemia cells carrying this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or Pak gene inactivation can lead to enhanced apoptosis, thereby inhibiting cell growth. Inhibition of the upstream effector molecule Rac can eliminate oncogene-induced Pak growth and activity. Although both Rac1 and Rac2 are constitutively activated by the guanine nucleotide exchange factor (GEF) Vav1, the deletion of Rac1 or Rac2 alone can only moderately correct the growth of KIT-positive leukemia cells, while the simultaneous deletion of both leads to 75% growth inhibition. In vivo experiments have shown that inhibition of Vav, Rac or Pak can delay the occurrence of myeloproliferative neoplasms (MPN) in mice and correct related pathological changes. To evaluate the role of Rac GEF in oncogene-induced transformation, we used a Rac inhibitor, EHop-016, which specifically targets Vav1. EHop-016 was found to be a potent inhibitor of human and mouse leukemia cell growth. These studies identified Pak and Rac GTPases (including Vav1) as potential therapeutic targets for oncogenic KIT in MPN and AML. [2]

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EHop-016 is a potent dual-pathway (oral/intraperitoneal) selective Rac1 inhibitor that exerts antiproliferative, antimigration, anti-invasive and pro-apoptotic effects by specifically blocking Vav2 and Tiam1-mediated Rac1 activation and inhibiting the downstream PAK/ERK signaling pathway. [1]
Preclinical studies have shown that EHop-016 has a significant inhibitory effect on various solid tumors (lung cancer, prostate cancer, breast cancer) and their metastases, especially suitable for tumors with high Rac1 activity or Vav2/Tiam1 overexpression. [2]
EHop-016 has low toxicity and good bioavailability, and is expected to become a candidate drug for targeted therapy of tumor metastases. It is currently in the preclinical development stage.[1][2]

C25H30N6O

430.55

430.248

C, 69.74; H, 7.02; N, 19.52; O, 3.72

1380432-32-5

51031035

Light yellow to yellow solid powder

1.27

3.263

2

6

8

32

573

0

O1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C([H])([H])C([H])([H])C([H])([H])N([H])C1=NC([H])=C([H])C(=N1)N([H])C1C([H])=C([H])C2=C(C=1[H])C1=C([H])C([H])=C([H])C([H])=C1N2C([H])([H])C([H])([H])[H]

AFTZZRFCMOAFCR-UHFFFAOYSA-N

InChI=1S/C25H30N6O/c1-2-31-22-7-4-3-6-20(22)21-18-19(8-9-23(21)31)28-24-10-12-27-25(29-24)26-11-5-13-30-14-16-32-17-15-30/h3-4,6-10,12,18H,2,5,11,13-17H2,1H3,(H2,26,27,28,29)

4-N-(9-ethylcarbazol-3-yl)-2-N-(3-morpholin-4-ylpropyl)pyrimidine-2,4-diamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.81 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 (5.81 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 (5.81 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: 2% DMSO +30% PEG 300 +5% Tween 80 +ddH2O: 5mg/mL

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Solubility in Formulation 5: 10 mg/mL (23.23 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.)