HIV

Amprenavir promotes the specific interactions between the nuclear receptor pregnane X receptor (PXR) and the coactivators SRC-1 and PBP. Amprenavir is docked into the high-resolution crystal structure of human PXR in complex with SR12813. Amprenavir occupies all four subpockets, and its hydroxyl group forms a hydrogen bond with Ser247, which is located in the connection region of PXR, to help to position the drug in the optimal orientation inside the receptor. Amprenavir forms direct contacts with one residue on αAF of the PXR activation function-2 (AF-2) surface, Phe429, which may stabilize the active AF-2 conformation of the receptor and contribute to the agonist activity of amprenavir on PXR. Amprenavir induces the expression of bona fide PXR target genes involved in phase I (CYP3A4), phase II (UGT1A1), and phase III (MDR1) metabolism in both HepaRG cells and LS180 cells.

Amprenavir increases atherogenic LDL cholesterol fractions in WT mice, but not in PXR−/− mice. Amprenavir stimulates expression of known PXR target genes, including CYP3A11, glutathione transferase A1, and MDR1a, in the intestine of WT mice but not in PXR−/− mice. Amprenavir-mediated PXR activation stimulates the expression of both LipF and LipA in the intestine of WT mice, but not in PXR−/− mice, indicating a possible role of intestinal PXR in mediating dietary lipid breakdown and absorption in mammals.

Correlation between true concentration and concentration in the perfusate: extraction-efficiency correction factor [2]
Standards were prepared in beakers containing increasing concentrations of amprenavir (0.94, 2.44, 9.76, 24.4, and 48.8 µM) and fosamprenavir (5.6, 14.2, 56.9, 142, and 284 µM) together in HBSS. The beakers were stirred with a magnetic stirbar and heated to 37 °C on a heating plate. The microdialysis probes were consecutively placed in the respective beakers to determine the extraction-efficiency correction factor. The perfusate contained 0.2% (w/V) Vitamin E TPGS in HBSS and was pumped through the tubes and probes with a syringe pump at a constant rate of 10 µL/min. Samples were collected in time intervals of 15-20, 20-25, and 25-30 min and analyzed with UHPLC-UV.

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Microdialysis extraction-efficiency correction factor in side-by-side cells for bioconversion/permeation studies [2]
Solutions containing 69.8 µM fosamprenavir and 53.9 µM amprenavir were filled into both the donor and acceptor compartments of side-by-side cells separated by PermeaPad®. The water jackets of the side-by-side cells were kept at 37 °C while the microdialysis was running for 30 min to equilibrate. Afterwards, the correction factor was determined by measuring inside the side-by-side cells as described in Section 2.5.1.

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Dynamic bioconversion/permeation studies [2]
Bioconversion/permeation studies [2]
The bioconversion/permeation studies were carried out in side-by-side cells attached to a circulating water heating system at 37 °C and with the biomimetic barrier PermeaPad® at a surface area of 1.77 cm2 separating donor and acceptor compartments. The acceptor compartments were filled with 7 mL of 0.2% Vitamin E TPGS in HBSS, while the donor volume was 5.0 mL (setup 1), 6.0 mL (setup 2) and 5.5 mL (setup 3); the slight variation between the three replicates was due to minor differences in the geometry of the side-by-side cells as the volume had to be enough to fully cover both the membrane and the microdialysis probe. A microdialysis probe attached to a syringe pump filled with 0.2% Vitamin E TPGS in HBSS was immersed into the donor compartment (Images of the full setup can be found in the supplementary material). Both compartments were stirred at 500 rpm by cross-type magnetic stir bars.

The donor compartments were filled with either a 500 µM amprenavir suspension, which had equilibrated overnight in a shaking water bath, or a 437 µM fosamprenavir solution. 20 min prior to all dissolution/bioconversion experiments, the syringe pump was started with a flowrate of 10 µL/min. In the case of fosamprenavir, the donor was filled at this point and the microdialysis probe was immersed into the fosamprenavir solution to equilibrate. After 20 min, bovine alkaline phosphatase was added to the donor to initiate the experiment at concentrations corresponding to 5.3 U/mL or 53 U/mL, respectively. In the case of amprenavir suspension, the microdialysis probe was immersed into the donor compartment immediately after adding the amprenavir suspension to start the experiments. Dialysate samples were collected in 10 min intervals for a total of 180 min. Afterwards, the dialysate concentrations were quantified with UHPLC-UV and converted to donor concentrations using the probe-specific extraction-efficiency correction factors described in Section 2.5.2. Conventional 200 µL acceptor samples were withdrawn after 30, 60, 90, 120, and 180 min and quantified with UHPLC-UV.

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Dissolution/bioconversion/permeation study with a human dose [2]
The dissolution step was added to the dynamic bioconversion/permeation experiments by placing an amount of fosamprenavir in the donor chambers equal to a final concentration of 4500 µM. These experiments were carried out the same way as the bioconversion/permeation experiments in Section 2.7.1. However, like with the amprenavir suspension, the microdialysis was calibrated for 20 min with the probe outside the donor. The microdialysis probe was added immediately to the donor after adding HBSS and 53 U/mL enzyme at the start of the experiment.

Permeation experiments in MDCK-wt cells [2]
Marbine-Darby Canine kidney wild-type (MDCK-wt) cells were maintained in culture medium composed of Dulbecco's Modified Eagle Medium with high glucose supplemented with 10% foetal bovine serum and 500U/mL penicillin/streptomycin. Cells were seeded on 6 well transwell-plates (0.4 µm polycarbonate membrane) at the cell density of 1.8 × 106 cells/transwell and differentiated for three days in cell culture media. After the differentiation, the cell culture media was aspirated, and the cells were washed three times with HBSS. Then, the filter supports with MDCK-wt cells were transferred into 6-well plates, and HBSS was added to the wells’ apical (1.5 mL) and basolateral (2.6 mL) sides. Before the bioconversion/permeation experiments, the plates were pre-incubated in HBSS for 20 min at 37 °C.

The bioconversion/permeation experiments were initiated by replacing the HBSS on the apical side with either 385 µM fosamprenavir solutions or 80 µM amprenavir solutions. The cells were incubated in Heidolph Titramax incubator at 400 rpm and 37 °C. Samples of 500 µL were withdrawn from the basolateral side at 10, 20, 30, 45, 60, 90, 120, 150, and 180 min. After sampling, 500 µL HBSS was added to the basolateral side as a replacement for the sample. The drug concentrations were quantified from the samples with UHPLC-UV.

The ntegrity of the MDCK-wt cells was investigated in a similar experiment with 1:1000 diluted radiolabeled 3H mannitol (15.9 Ci/mmol) in HBSS under the same conditions as in the bioconversion/permeation experiments with MDCK-wt cells. The 3H mannitol was quantified by mixing a 100 µL acceptor sample and 400 µL scintillation medium and measured on a radioactivity counter.

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Amprenavir induced PXR target gene expression in both HepaRG hepatoma cells and LS180 intestinal cells.

These two Phase I, open-label, single-dose, randomized, crossoverstudies in 40 healthymale subjects investigated the pharmacokinetic and safety profiles of various formulations of the amprenavir prodrug GW433908 in the presence and absence of food compared with amprenavir capsules. GW433908 is a phosphate ester prodrug of the antiretroviral protease inhibitor amprenavir, with improved solubility over the parent molecule and a potential for reduced pill burden on current dosing regimens. The calcium salt of the prodrug, GW433908G, was selected for further investigation, as it appeared to offer the greatest potential for the development of new drug formulations. In the fasting state, (1) GW433908G tablet and suspension were bioequivalent in terms of both AUC and Cmax, and (2) GW433908G tablet and suspension were bioequivalent to amprenavir capsules for AUC; however, Cmax was lower with GW433908G. After a high-fat meal compared with fasting, (1) the bioavailability of GW433908G suspension was decreased by 20% and Cmax by 41%, and (2) for GW433908G tablets, there was no influence on AUC(12% lower Cmax). After a low-fat meal compared with fasting, (1) there was bioequivalence for GW433908G tablets, but (2) bioavailability was decreased by 23% for amprenavir capsules (Cmax was also lower, by 46%). Overall, for GW433908G and amprenavir capsules, food had a negligible influence on plasma concentration at 12 hours postdose (C12). Whether administered as tablets or suspension, GW433908G pharmacokinetics was only slightly affected by food. GW433908G tablets were well tolerated and delivered plasma amprenavir concentrations equivalent to the recommended therapeutic amprenavir dose but with fewer tablets. The possibility of a lower pill burden offered by GW433908 may be of clinical benefit in the treatment of HIV infection.[1]

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10 mg/kg; p.o.

WT and PXR-/- mice

Absorption, Distribution and Excretion
The absolute oral bioavailability of amprenavir after administration of fosamprenavir in humans has not been established. After administration of a single 1,400 mg dose in the fasted state, fosamprenavir oral suspension (50 mg/mL) and fosamprenavir tablets (700 mg) provided similar amprenavir exposures (AUC), however, the Cmax of amprenavir after administration of the suspension formulation was 14.5 % higher compared with the tablet.
Excretion of unchanged amprenavir in urine and feces is minimal. The renal elimination of unchanged amprenavir represents approximately 1% of the administered dose; therefore, renal impairment is not expected to significantly impact the elimination of amprenavir. Amprenavir, the active metabolite of fosamprenavir, is metabolized in the liver by the cytochrome P450 enzyme system.
Fosamprenavir is a prodrug that is rapidly hydrolyzed to amprenavir by enzymes in the gut epithelium as it is absorbed. After administration of a single dose of fosamprenavir to HIV-1-infected patients, the time to peak amprenavir concentration (Tmax) occurred between 1.5 and 4 hours (median 2.5 hours). The absolute oral bioavailability of amprenavir after administration of fosamprenavir in humans has not been established.
Administration of a single 1400 mg dose of fosamprenavir in the fed state compared with the fasted state was associated with no significant changes in amprenavir Cmax, Tmax or AUC.
The following are areas under the curve (AUC) values of amprenavir based on the drug therapy regimen administered: Fosamprenavir 1400 mg twice per day: 27.6 to 39.2 ug/hr/mL (median 33 ug/hr/mL; Fosamprenavir 1400 mg once per day plus ritonavir 200 mg once per day: 59.7 to 80.8 ug/hr/mL (median 69.4 ug/hr/mL);Fosamprenavir 700 mg twice per day plus ritonavir 100 mg twice per day: 69 to 90.6 ug/hr/mL (median 79.2 ug/hr/mL). Fosamprenavir may be taken with or without food.
Peak plasma concentration (Cmax): The following Cmax values of amprenavir based on the drug therapy regimen administered: Fosamprenavir 1400 mg once per day plus ritonavir 200 mg once per day: 6.32 to 8.28 ug/mL (median 7.24 ug/mL); Fosamprenavir 1400 mg twice per day: 4.06 to 5.72 ug/mL (median 4.82 ug/mL); Fosamprenavir 700 mg twice per day plus ritonavir 100 mg twice per day: 5.38 to 6.86 ug/mL (median 6.08 ug/mL)...
For more Absorption, Distribution and Excretion (Complete) data for FOSAMPRENAVIR (10 total), please visit the HSDB record page.

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Metabolism / Metabolites
In the gut epithelium during absorption, fosamprenavir is rapidly and almost completely hydrolyzed to amprenavir and inorganic phosphate prior to reaching the systemic circulation. Amprenavir is metabolized in the liver by the cytochrome P450 3A4 (CYP3A4) enzyme system.
After oral administration, fosamprenavir is rapidly and almost completely hydrolyzed to amprenavir and inorganic phosphate prior to reaching the systemic circulation. This occurs in the gut epithelium during absorption. Amprenavir is metabolized in the liver by the cytochrome P450 and 3A4 (CYP3A4) enzyme system. The 2 major metabolites result from oxidation of the tetrahydrofuran and aniline moieties. Glucuronide conjugates of oxidized metabolites have been identified as minor metabolites in urine and feces.

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Biological Half-Life
The plasma elimination half-life of amprenavir (the active metabolite) is approximately 7.7 hours.
The plasma elimination half-life of amprenavir is approximately 7.7 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No published information is available on the use of fosamprenavir during breastfeeding. Fosamprenavir is not recommended during breastfeeding. Achieving and maintaining viral suppression with antiretroviral therapy decreases breastfeeding transmission risk to less than 1%, but not zero. Individuals with HIV who are on antiretroviral therapy with a sustained undetectable viral load and who choose to breastfeed should be supported in this decision. If a viral load is not suppressed, banked pasteurized donor milk or formula is recommended.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Gynecomastia has been reported among men receiving highly active antiretroviral therapy. Gynecomastia is unilateral initially, but progresses to bilateral in about half of cases. No alterations in serum prolactin were noted and spontaneous resolution usually occurred within one year, even with continuation of the regimen. Some case reports and in vitro studies have suggested that protease inhibitors might cause hyperprolactinemia and galactorrhea in some male patients, although this has been disputed. The relevance of these findings to nursing mothers is not known. The prolactin level in a mother with established lactation may not affect her ability to breastfeed.

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Protein Binding
Very high (approximately 90%); primarily bound to alpha 1 -acid glycoprotein. Higher amounts of unbound amprenavir present as amprenavir serum concentrations increase.

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Interactions
Amprenavir inhibits and induces cytochrome P-450 (CYP) isoenzyme 3A4; potential pharmacokinetic interactions (altered metabolism of the other drug). Caution is advised if fosamprenavir is used concomitantly with substrates, inhibitors, or inducers of CYP3A4. Concomitant use with drugs with a narrow therapeutic index that are CYP3A4 substrates is not recommended. Amprenavir does not inhibit CYP2D6, CYP1A2, CYP2C9, CYP2C19, or CYP2E1.
Pharmacokinetic interaction with antacids (decreased amprenavir peak concentrations and AUC). ... This pharmacokinetic interaction is not clinically important and there are no restrictions for concomitant use of fosamprenavir and antacids.
Pharmacokinetic interaction following concomitant use of ritonavir-boosted fosamprenavir with flecainide or propafenone (increased plasma concentrations of the antiarrhythmic agent). Potential for serious and/or life-threatening adverse effects (eg, cardiac arrhythmias). Concomitant use of ritonavir-boosted fosamprenavir with flecainide or propafenone is contraindicated.
Pharmacokinetic interaction with amiodarone, bepridil (no longer commercially available in US), systemic lidocaine, or quinidine (increased concentrations of the antiarrhythmic). Potential for serious and/or life-threatening adverse effects. Use concomitantly with caution and, if possible, monitor plasma concentrations of the antiarrhythmic agents.
For more Interactions (Complete) data for FOSAMPRENAVIR (37 total), please visit the HSDB record page.

[1]. Pharmacokinetics of GW433908, a prodrug of amprenavir, in healthy male volunteers. Mol Pharmacol.2013 Jun;83(6):1190-9.

[2]. In-vitro dynamic dissolution/bioconversion/permeation of fosamprenavir using a novel tool with an artificial biomimetic permeation barrier and microdialysis-sampling. Eur J Pharm Sci. 2023 Feb 1:181:106366.

Fosamprenavir is a sulfonamide with a structure based on that of sulfanilamide substituted on the sulfonamide nitrogen by a (2R,3S)-4-phenyl-2-(phosphonooxy)-3-({[(3S)-tetrahydrofuran-3-yloxy]carbonyl}amino)butyl group. It is a pro-drug of the HIV protease inhibitor and antiretroviral drug amprenavir. It has a role as a prodrug. It is functionally related to a sulfanilamide.
Fosamprenavir is a prodrug of amprenavir, an inhibitor of human immunodeficiency virus (HIV) protease.
Fosamprenavir is a Protease Inhibitor. The mechanism of action of fosamprenavir is as a HIV Protease Inhibitor.
Fosamprenavir is a prodrug form of amprenavir. In the body fosamprenavir is metabolized to amprenavir, a synthetic derivative of hydroxyethylamine sulfonamide that selectively binds to and inhibits human immunodeficiency virus (HIV) protease.
See also: Fosamprenavir Calcium (annotation moved to).

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Drug Indication
Indicated in combination with other antiretroviral agents for the treatment of human immunodeficiency virus (HIV-1) infection, as well as postexposure prophylaxis of HIV infection in individuals who have had occupational or nonoccupational exposure to potentially infectious body fluids of a person known to be infected with HIV when that exposure represents a substantial risk for HIV transmission. The use of fosamprenavir is pending revision due to a potential association between the drug and myocardial infarction and dyslipidemia in HIV infected adults.
FDA Label

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Mechanism of Action
Fosamprenavir is a prodrug that is rapidly hydrolyzed to amprenavir by cellular phosphatases in the gut epithelium as it is absorbed. Amprenavir is an inhibitor of HIV-1 protease. During HIV replication, HIV protease cleaves viral polypeptide products of the Gag and Gag-Pol genes to form structural proteins of the virion core and essential viral enzymes. Amprenavir interferes with this process by binding to the active site of HIV-1 protease, thereby preventing the processing of viral Gag and Gag-Pol polyprotein precursors, resulting in the formation of immature non-infectious viral particles.
Fosamprenavir is a prodrug of amprenavir, an inhibitor of HIV protease. ...Fosamprenavir is rapidly converted to amprenavir by cellular phosphatases in vivo. Amprenavir is an inhibitor of HIV-1 protease. Amprenavir binds to the active site of HIV-1 protease and thereby prevents the processing of viral Gag and Gag-Pol polyprotein precursors, resulting in the formation of immature noninfectious viral particles.

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Therapeutic Uses
Fosamprenavir is indicated in combination with other antiretroviral agents for the treatment of HIV infections in adults. /Included in US product labeling/

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Drug Warnings
Rash (usually maculopapular and of mild or moderate intensity, with or without pruritus) has been reported in about 19% of patients receiving fosamprenavir in clinical studies; manifestations occurred approximately 11 days after initiation of fosamprenavir and persisted for a median of 13 days. Severe or life-threatening skin reactions, including Stevens-Johnson syndrome, were reported in less than 1% of patients receiving fosamprenavir in clinical studies.
Fosamprenavir contains a sulfonamide moiety, which may cause allergic-type reactions (e.g., rash) in certain susceptible individuals. The potential for cross-sensitivity between drugs with sulfonamide moieties and fosamprenavir is unknown. Use fosamprenavir with caution in patients with known hypersensitivity to sulfonamide-containing drugs.
Patients with chronic hepatitis B or C virus infection and those with marked increases in AST or ALT concentrations prior to fosamprenavir therapy may be at increased risk for further elevations in hepatic enzyme concentrations. Liver function tests should be performed prior to initiating therapy with fosamprenavir, and patients should be monitored closely during treatment. ... Increases in serum AST (SGOT) and/or ALT (SGPT) concentrations (more than 5 times the upper limit of normal) have been reported in approximately 4-8% of patients receiving fosamprenavir in clinical studies.
Possible amprenavir resistance. The possible effect of fosamprenavir therapy on subsequent therapy with other HIV protease inhibitors in unknown.
For more Drug Warnings (Complete) data for FOSAMPRENAVIR (14 total), please visit the HSDB record page.

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Pharmacodynamics
Fosamprenavir is hydrolyzed by cellular phosphatases to the antiretroviral protease inhibitor amprenavir. This hydrolysis allows for the slow release of amprenavir, reducing the number of pills a patient must take.

C25H34N3NA2O9PS

629.57

226700-80-7

226700-79-4; 226700-81-8; 226700-80-7

Typically exists as solids at room temperature

[Na+].[Na+].CC(CN(S(C1=CC=C(N)C=C1)(=O)=O)C[C@H]([C@H](CC1=CC=CC=C1)NC(O[C@H]1CCOC1)=O)OP([O-])(=O)[O-])C

Amprenavir phosphate sodium; Fosamprenavir sodium; 226700-80-7; Fosamprenavir sodium [USAN]; GW 433908A; UNII-XSG28FSA0W; XSG28FSA0W; GW-433908A; Fosamprenavir sodium (USAN); GW 433908 sodium

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.)