Antihelminthic; STAT3 (IC50 = 0.25 μM in HeLa cells); ROS; NF-κB; mTORC1; Wnt/β-catenin; Notch;

Treatment with niclosamide monohydrate (0.6 nM-46 µM) reduces adrenocortical cancer cell proliferation in BD140A, SW-13 and NCI-H295R cells [3]. Niclosamide monohydrate (0.05-5 μM, 24 hours) treatment suppresses STAT3-mediated luciferase reporter activity in HeLa cells [4]. Treatment with niclosamide monohydrate (10 μM) reduces viral replication in Vero E6 cells [5].

The oral gavage form of niclosamide (100 mg/kg, 200 mg/kg; once weekly; 8 weeks) suppresses the formation of adrenocortical carcinoma tumors in vivo [3].

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Niclosamide inhibits ACC tumor growth in vivo[3]
To confirm our in vitro observations, the effect of niclosamide treatment was evaluated in ACC xenografts. Niclosamide treatments, at both doses (100 mg/kg and 200 mg/kg), were well tolerated, with no observed toxicity or side effects in the mice. There were no significant weight differences among the groups (Fig. 5). Four weeks after treatment, mice treated with niclosamide at 100 mg/kg and 200 gm/kg showed a 60% and 80% inhibition in tumor growth, respectively, as compared to the vehicle control group (P < 0.01 for both groups) (Fig. 5). The same treatment schedule was maintained for 8 weeks, at which time, more than 90% tumor growth inhibition was observed for the two treated groups, as compared to the control group.

Protein Kinase profiling assay (Table S1): Assay for 22 different proteins kinases was carried out by a CRO. All of the protein kinases were expressed either in Sf9 insect cells or in E.coli as recombinant GST-fusion proteins or His-tagged proteins. Protein kinases were purified by affinity chromatography using either GSH-agarose or Ni_NTH-agarose. A radiometric protein kinase assay was used for measuring the kinase activity of the 22 protein kinases. Briefly, for each protein kinase, 50 μl reaction cocktail containing 60 mM HEPES-NaOH, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 0.02 0.2 0 10 20 30 40 50 60 70 80 90 100 110 Drug Conc.(μM) Relative colony number (% of control) IC50 : 0.1μM S9 μg/ml PEG20000, 1 μM [γ-33P]-ATP(appox.6×1005cpm), test compound, adequate amount of enzyme and its substrate. The PKC-alpha assay additionally contained 1 mM Cacl2, 4 mM EDTA, 5 μg/ml phosphatidylserine and 1 μg/ml 1, 2-Dioleyl-glycerol). The reaction cocktails were incubated at 37o C for 60 minutes and stopped with 50 μl 2% (v/v) H3PO4. Incorporation of 33Pi was determined with a microplate scintillation counter. The activities and the IC50 values were calculated using Quattro Workflow V2.28[4].

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In summary, niclosamide, an FDA-approved anthelmintic drug, was identified as a new small-molecule inhibitor of the STAT3 signaling pathway. This drug potently inhibited the activation, nuclear translocation, and transactivation of STAT3 but had no obvious effects on the closely related STAT1 and STAT5 proteins, the upstream JAK1, JAK2, and Src kinases, or other receptor tyrosine kinases. Furthermore, niclosamide inhibited the transcription of STAT3 target genes and induced cell growth inhibition, apoptosis, and cell cycle arrest of cancer cells with constitutively active STAT3. Although niclosamide does not have an ideal pharmarcokinetic profile (i.e., poor oral bioavailability) in humans as an anticestodal drug, it represents a new potent lead compound with salicylic amide scaffold for development of STAT3 pathway inhibitors as new molecularly targeted anticancer drugs. The further structural optimization and extensive mechanism study on niclosamide are undergoing and will be reported in due course.[4]

Cell viability assay [4]
Cell Types: Hela Cell
Tested Concentrations: 0.05-5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibits STAT3-mediated luciferase reporter gene activity, IC50 is 0.25 μM.

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Cell viability assay [5]
Cell Types: Vero E6 Cell
Tested Concentrations: 10 μM
Incubation Duration: 2 days
Experimental Results: Inhibition of the synthesis of SARS-CoV viral antigen in Vero E6 cells.

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Cellular proliferation assay[3]
3 × 103 and 6 × 103 cells were plated in 96-well plates depending on the cell line. 100 µL of fresh culture medium containing the drug or vehicle was added. Cell count was determined using the CyQuant kit, according to the manufacturer’s instructions, and cell number was measured using a SpectraMax M5 microplate reader (ex485/em538). Assays were performed in quadruplicate and the experiments were repeated three times.

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NCI-H295R and SW-13 cells, which form multicellular aggregates (MCA) or spheroids, were plated in Ultra Low Cluster 24-well plates at 1 × 105 cells/0.5 mL or 6 × 104 cells/0.5 mL depending on the cell line. Spheroids were allowed to develop for one or two weeks at 37°C in 5% CO2, and media was exchanged twice a week. Spheroids were treated with niclosamide or the vehicle at varying concentrations, and imaged weekly.

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Caspase 3/7 activity assay[3]
Cells were plated in 96-well plates and treated with niclosamide or the vehicle. Caspase 3/7 activity was measured using the Caspase-Glo 3/7 assay, according to manufacturer’s instructions.

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Cell cycle analysis[3]
Cells plated in six-well plates were treated with niclosamide or the vehicle. At 48 hours, cells were fixed for 30 minutes in 70% ethanol at 4°C, and stained with 50 µg/mL of propidium iodide containing 100 mg/mL of ribonuclease A. Flow cytometry was performed on a Canto I flow cytometer using CellQuest software. Data was generated for at least 20,000 events per sample and analyzed using Modfit software.

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Cellular migration assays[3]
NCI-H295R and SW-13 cells were plated in six-well plates and treated with varying concentrations of niclosamide or the vehicle for 24 hours. Cells were trypsinized and plated in transwell chambers at a density of 1 × 105 cells per 0.5 mL. The lower chamber was filled with DMEM supplemented with 10% FBS as a chemoattractant. Cells were allowed to migrate for 24 hours or 48 hours depending on the cell line, and were fixed and stained with Diff-Quik. Cells were imaged and counted in three random fields per well, and the experiments performed in triplicate. For the wound-healing assay in BD140A cells, which do not migrate in the Boyden chamber model, cells were plated in six-well plates until confluent and treated with niclosamide or the vehicle. The cells were scratched using a sterile pipette tip and photographed at various time points.

Animal/Disease Models: Nu+/Nu+ mice injected with NCI-H295R cells [3]
Doses: 100 mg/kg, 200 mg/kg
Route of Administration: po (oral gavage); 100 mg/kg, 200 mg/kg; once a week ; 8-week
Experimental Results: Tumor growth was inhibited by 60%-80% compared to the control group.

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In vivo mouse studies[3] Animal studies were approved by the National Cancer Institute Animal Care and Use Committee. Mice were maintained according to National Institutes of Health (NIH) Animal Research Advisory Committee (ARAC) guidelines. 5 × 106 NCI-H295R cells were injected into the flank of Nu+/Nu+ mice. Tumors were allowed to grow and mice were randomized into three treatment groups (8 mice per treatment group). Mice were treated with 100 mg/kg of niclosamide, 200 mg/kg of niclosamide, or the vehicle (PEG500) everyday by oral gavage. Tumor sizes were measured in two dimensions every week with calipers and recorded.

[1]. The biology and toxicology of molluscicides, Bayluscide. Pharmacol Ther. 1982;19(2):245-95.

[2]. Niclosamide: Beyond an antihelminthic drug. Cell Signal. 2018 Jan;41:89-96.

[3]. Identification of Niclosamide as a Novel Anticancer Agent for Adrenocortical Carcinoma. Clin Cancer Res. 2016 Jul 15;22(14):3458-66.

[4]. Identification of Niclosamide as a New Small-Molecule Inhibitor of the STAT3 Signaling Pathway. ACS Med Chem Lett. 2010 Sep 7;1(9):454-9.

[5]. Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide. Antimicrob Agents Chemother. 2004 Jul;48(7):2693-6.

Niclosamide is a secondary carboxamide resulting from the formal condensation of the carboxy group of 5-chlorosalicylic acid with the amino group of 2-chloro-4-nitroaniline. It is an oral anthelmintic drug approved for use against tapeworm infections. It has a role as a piscicide, a molluscicide, an antiparasitic agent, an anticoronaviral agent, an anthelminthic drug, an apoptosis inducer and a STAT3 inhibitor. It is a member of monochlorobenzenes, a member of salicylanilides, a C-nitro compound, a secondary carboxamide and a member of benzamides. It is functionally related to a 5-chlorosalicylic acid.

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Niclosamide is an antihelminthic used for the treatment of tapeworm infections. Helminths (worms) are multicellular organisms that infect very large numbers of humans and cause a broad range of diseases. Over 1 billion people are infected with intestinal nematodes, and many millions are infected with filarial nematodes, flukes, and tapeworms. They are an even greater problem in domestic animals. Niclosamide, once marketed in the US under the brand name Niclocide, was voluntarily withdrawn from market by Bayer in 1996.

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Niclosamide is an orally bioavailable chlorinated salicylanilide, with anthelmintic and potential antineoplastic activity. Upon oral administration, niclosamide specifically induces degradation of the androgen receptor (AR) variant V7 (AR-V7) through the proteasome-mediated pathway. This downregulates the expression of the AR variant, inhibits AR-V7-mediated transcriptional activity, and reduces AR-V7 recruitment to the prostate-specific antigen (PSA) gene promoter. Niclosamide also prevents AR-V7-mediated STAT3 phosphorylation and activation. This inhibits AR/STAT3-mediated signaling and prevents expression of STAT3 target genes. Altogether, this may inhibit growth of AR-V7-overexpressing cancer cells. The AR-V7 variant, which is encoded by contiguous splicing of AR exons 1/2/3/CE3, is upregulated in a variety of cancer cell types, and is associated with both cancer progression and resistance to AR-targeted therapies.

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Niclosamide is used for the treatment of most tapeworm infections. Helminths (worms) are multicellular organisms that infect very large numbers of humans and cause a broad range of diseases. Over 1 billion people are infected with intestinal nematodes, and many millions are infected with filarial nematodes, flukes, and tapeworms. They are an even greater problem in domestic animals. An antihelmintic that is active against most tapeworms.

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Drug Indication
For the treatment of tapeworm and intestinal fluke infections: Taenia saginata (Beef Tapeworm), Taenia solium (Pork Tapeworm), Diphyllobothrium latum (Fish Tapeworm), Fasciolopsis buski (large intestinal fluke). Niclosamide is also used as a molluscicide in the control of schistosomiasis.

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Therapeutic Use Niclosamide in the free-base form only is used primarily as a cestocide and to a lesser extent as a trematocide. The drug is formulated as a chewable tablet containing 500 mg of niclosamide. It is very effective against tapeworrn infections caused by Taenia saginata, Taenia solium, and Diphyllobothrium latum; tapeworms such as Hymenolepis diminuta, Hymenolepis nana, and Dipylidium caninum are somewhat more recalcitrant. For infections of Taenia saginata, Taenia solium, and Diphyllobothrium latum, single oral dosages of 2 gm (adult), 1.5 gm (child > 34 kg), and 1.0 gm (child 11-34 kg) are recommended. 0ther tapeworms may require repeated treatment for example, 2 gm/day in single daily doses for 7 days (adults), 1.5 gm given in a single dose on the first day followed by 1 gm/day for the next 6 days (child > 34 kg), and 1 gm given in a single dose on the first day followed by 500 mg/day for next 6 days (child 11-34 kg). Safety for use in children under 2 years of age has not been established. Since niclosamide is active only against intestinal cestodes, it is not effective for treatment of cysticercosis. As a trematocide, niclosamide is active mainly against flukes such as Fasciolopsis buski in the intestines.

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Pharmacodynamics
Niclosamide is an antihelminth used against tapeworm infections. It may act by the uncoupling of the electron transport chain to ATP synthase. The disturbance of this crucial metabolic pathway prevents creation of adenosine tri-phosphate (ATP), an essential molecule that supplies energy for metabolism.

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Absorption
Niclosamide appears to be minimally absorbed from the gastrointestinal tract—neither the drug nor its metabolites have been recovered from the blood or urine.

VERY LITTLE IS ABSORBED FROM THE GI TRACT ... . Gilman, A.G., T.W. Rall, A.S. Nies and P. Taylor (eds.). Goodman and Gilman's The Pharmacological Basis of Therapeutics. 8th ed. New York, NY. Pergamon Press, 1990., p. 965
EXPOSING RAINBOW TROUT TO (14)C-BAYER 73 CAUSED BILE TO WATER (14)C RATIO OF 10,000:1. AFTER 24 HR OF EXPOSURE, THIN LAYER CHROMATOGRAPHY OF UNFRACTIONATED BILE FROM FISH SHOWED 1 MAJOR RADIOACTIVE PEAK. UNCHANGED BAYER 73 WAS FOUND IN BILE.

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Metabolism / Metabolites
ANTHELMINTIC NICLOSAMIDE...REDUCED TO CORRESPONDING AMINO DERIVATIVE BY BOTH MOUSE- & SHEEP-LIVER ENZYME PREPARATIONS & BY ENZYMES FROM CESTODES & NEMATODES. ...NICLOSAMIDE WAS NOT HYDROLYZED EITHER BY MAMMALIAN & HELMINTH ENZYME PREPN OR BY WHOLE HELMINTHS.

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Mechanism of Action
Niclosamide works by killing tapeworms on contact. Adult worms (but not ova) are rapidly killed, presumably due to uncoupling of oxidative phosphorylation or stimulation of ATPase activity. The killed worms are then passed in the stool or sometimes destroyed in the intestine. Niclosamide may work as a molluscicide by binding to and damaging DNA.

Niclosamide has prominent activity against most of the cestodes that infect man; Enterobius (Oxyuris) vermicularis is also susceptible. At low concn, niclosamide stimulates oxygen uptake by Hymenolepis diminuta, but at higher concn respiration is inhibited and glucose uptake is blocked. The principal action of the drug may be to inhibit anaerobic phosphorylation of adenosine diphosphate (ADP) by the mitochondria of the parasite, an energy producing process that is dependent on CO2 fixation ... . Gilman, A.G., T.W. Rall, A.S. Nies and P. Taylor (eds.). Goodman and Gilman's The Pharmacological Basis of Therapeutics. 8th ed. New York, NY. Pergamon Press, 1990., p. 965

Cestocidal activity is due to inhibition of absorption of glucose by the tapeworm and uncoupling of the oxidative phosphorylation process in the mitochondria of cestodes. Resultant blocking of the Krebs cycle leads to accumulation of lactic acid, which kills the tapeworm. ... overstimulation of adenosine triphosphatase (ATPase) activity of the mitochondria may be related to cestodal action of niclosamide.

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Toxicity Summary
Niclosamide works by killing tapeworms on contact. Adult worms (but not ova) are rapidly killed, presumably due to uncoupling of oxidative phosphorylation or stimulation of ATPase activity. The killed worms are then passed in the stool or sometimes destroyed in the intestine. Niclosamide may work as a molluscicide by binding to and damaging DNA.

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Twenty-one active compounds were identified, with an efficacy of >80% in all three cell lines. Of these, niclosamide showed higher efficacy and lower IC50 than established anti-ACC drugs. We then validated niclosamide-inhibited cellular proliferation in all three ACC cell lines. Next, we investigated the mechanism by which niclosamide inhibited ACC cell proliferation, and found that it induced caspase-dependent apoptosis and G1 cell-cycle arrest. Niclosamide also decreased cellular migration and reduced the level of mediators of epithelial-to-mesenchymal transition, such as N-cadherin and vimentin. Furthermore, niclosamide treatment resulted in decreased expression of β-catenin. We also evaluated the effect of niclosamide on energy metabolism in ACC cell lines and found it resulted in mitochondrial uncoupling. Niclosamide treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo Conclusions: Our findings suggest that niclosamide has anti-ACC activity through its inhibition of multiple altered cellular pathways and cellular metabolism in ACC. Our results provide a preclinical rationale for evaluating niclosamide therapy in a clinical trial for ACC.[3]

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Inhibition of the signal transducer and activator of transcription 3 (STAT3) signaling pathway has been considered a novel therapeutic strategy to treat human cancers with constitutively active STAT3. In this study, we report the identification of niclosamide, an FDA-approved anthelmintic drug, as a new small-molecule inhibitor of the STAT3 signaling pathway. This compound potently inhibited the activation and transcriptional function of STAT3 and consequently induced cell growth inhibition, apoptosis, and cell cycle arrest of cancer cells with constitutively active STAT3. Our study provides a new promising lead compound with a salicylic amide scaffold for the development of STAT3 pathway inhibitors as novel molecularly targeted anticancer drugs.[4]

C13H8N2O4CL2.H2O

345.1349

343.997

C, 45.24; H, 2.92; Cl, 20.54; N, 8.12; O, 23.18

73360-56-2

Niclosamide;50-65-7;Niclosamide olamine;1420-04-8;Niclosamide sodium;40321-86-6;Niclosamide-13C6 monohydrate;1325559-12-3;Niclosamide;50-65-7; 36466-48-5 (piperazine); 73360-56-2 (hydrate);

12296604

Typically exists as solid at room temperature

224-229ºC

4.702

3

5

2

22

404

0

O=C(NC1=CC=C([N+]([O-])=O)C=C1Cl)C2=CC(Cl)=CC=C2O.O

ZBXRPLQCPHTHLM-UHFFFAOYSA-N

InChI=1S/C13H8Cl2N2O4.H2O/c14-7-1-4-12(18)9(5-7)13(19)16-11-3-2-8(17(20)21)6-10(11)15;/h1-6,18H,(H,16,19);1H2

5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide;hydrate

5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide monohydrate; Niclosamide (monohydrate); BAY2353 monohydrate; UNII-20Z25R1145; 2',5-Dichloro-4'-nitrosalicylanilide monohydrate; Niclosamide monohydrate; 20Z25R1145;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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