HIV-1 capsid

Lenacapavir interferes with both early and late stages of HIV-1 replication, but is more effective against the early stages [2]. Lenacapavi (GS-6207) is a potent capsid inhibitor of HIV replication. Lenacapavi performs well in target cells (EC50=23 pM), full cycle assay (EC50=25 pM), and producer cells (EC50=439 PM).
In vitro, LEN showed potent antiviral activity against SHIV, as it did for HIV-1. In macaques, a single subcutaneous administration of LEN demonstrated dose proportional increases in and durability of drug plasma levels. [3]

A high-dose SHIV inoculum for the PrEP efficacy evaluation was identified via virus titration in untreated macaques. LEN-treated macaques were challenged with high-dose SHIV 7 weeks after drug administration, and the majority remained protected from infection, as confirmed by plasma PCR, cell-associated proviral DNA, and serology testing. Complete protection and superiority to the untreated group was observed among animals whose LEN plasma exposure exceeded its model-adjusted clinical efficacy target at the time of challenge. All infected animals had subprotective LEN concentrations and showed no emergent resistance. These data demonstrate effective SHIV prophylaxis in a stringent macaque model at clinically relevant LEN exposures and support the clinical evaluation of LEN for HIV PrEP in humans.[3]

MicroScale Thermophoresis Assays [1]
The binding affinities of CA with Pep-1 and PF74 were determined by measuring thermophoresis of fluorescently labeled CA-hexamers in the presence of increasing Pep-1 or PF74 concentrations. Peptide Pep-1 was synthesized in the Molecular Interaction Core and PF74 was purchased commercially. Fluorescent labeling of CA with Alexa Fluor 647 analog NT647 was performed according to the manufacturer’s instructions (MO-L004 Monolith Protein Labeling Kit). Briefly, 20 μM protein was incubated overnight with 3 M excess of dye at room temperature in a conjugation buffer provided with the labeling kit. The unreacted dye was removed by filtration through a gravity flow column provided with the kit. The elution fractions were collected in 2× MST buffer (40 mM MOPS, pH 7.2, 200 mM NaCl, and 0.2% pluronic F-127). Fluorescence intensity of each fraction was evaluated by MST, and fractions containing labeled protein were pooled. Protein concentration was determined by NanoDrop spectrophotometer. Aliquots were stored at −80°C until use. The reaction mixtures containing 200 nM labeled CA-hexamer and increasing concentrations of Pep-1 (1–2,000 nM) were loaded in the capillaries and the thermophoresis was monitored at 20% LED power, high MST power with 20 s MST-on time.

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In vitro HIV-1 CA assembly assay.[2]
The in vitro assembly of HIV-1 CA protein in the presence and absence of small molecule library compounds (10 μM) or 2-fold serially diluted GS-6207 was monitored by measuring changes in sample absorbance over time at 350 nm. Final assembly reactions contained 20 μM CA, 2 M NaCl, 50 mM sodium phosphate pH 7.5, 0.005% Antifoam 204 (Sigma-Aldrich) and 1% DMSO. Sample absorbance values at 350 nm were monitored over time at 25°C in 96-well or 384-well plates using an M5 plate reader, corrected for absorbance values in the absence of CA or NaCl, and the data analyzed using SoftMax Pro 6.3.1 as previously described38.

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GS-6207 binding assay.[2]
Surface plasmon resonance biosensor binding experiments were performed using the ProteOn XPR36 platform (CA hexamer and pentamer proteins) or the Biacore T100 platform (CA monomer and Gag proteins) as previously described21. Data were analyzed using ProteOn Manager 3.1.0 or Scrubber 2.0 and fit with a simple kinetic model with a term for mass transport added when necessary.

Cytotoxicity assays.[2]
For cytotoxicity assessment in MT-4 cells, PBMCs, primary human CD4+ T-cells and monocyte-derived macrophages, the protocol was identical to that of the respective antiviral assay, including assay duration, except that no virus was added to the plates. Protocols for cytotoxicity assessments in Huh-7, Gal-HepG2, Gal-PC-3 and MRC-5 cell lines, as well as in primary human hepatocytes, have been previously described37. The effect of test compounds on cell viability was measured using CellTiter-Glo. Data analysis was performed using GraphPad Prism 7.0 to calculate CC50 values.

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GS-6207 resistance analysis.[2]
Dose-escalation selections for drug-resistant HIV-1 variants were performed in MT-2 cells infected with HIV-1HXB2D using twofold incremental increases in GS-6207 concentration as previously described31. The resistance profile of each emergent virus passage was then assessed in the 5-day cytoprotection antiviral MT-2 assay after titrating virus inoculums to normalize the m.o.i. across all samples. Viral breakthrough selections were conducted under conditions of fixed, constant drug concentrations over a period of 35 days in human PBMCs independently infected with six different HIV-1 isolates (BaL, 92US657, 91US0006, 7406, 7467 and 7576) as previously described21. GS-6207 was tested at fixed drug concentrations equal to 4-fold, 8-fold, and 16-fold its EC95 value of 0.23 nM (0.92 nM, 1.9 nM, and 3.7 nM GS-6207, respectively), using six replicate cell cultures per experimental condition. Viruses that emerged in the presence of GS-6207 were genotyped by population sequencing. Total RNA was isolated from mock- and GS-6207-selected virus-containing supernatants using the QiaAMP Viral RNA Mini Kit. A 986-bp fragment encoding HIV-1 capsid and the adjacent p2 spacer peptide was amplified by RT-PCR using the Qiagen OneStep RT-PCR Kit in combination with primers 5’-CAGTAGCAACCCTCTATTGTGTGC-3’ and 5’-CCTAGGGGCCCTGCAATTT-3’. RT-PCR products were sequenced by Elim Biopharmaceuticals. To identify codon changes, gene sequences from selected HIV-1 variants were aligned using DNA Sequencher 4.9 Software with that of the input virus and virus passaged in the absence of GS-6207. For samples containing > 1 codon change, PCR products were subcloned, DNA was isolated from individual bacterial colonies, and the CA gene was sequenced to assess the linkage of all observed substitutions.

Drug and formulation.[3]
LEN and the liquid chromatography–mass spectrometry internal standard GS-224337 were synthesized internally. and subjected to a standard quality control analysis. For antiviral assays, LEN was dissolved in DMSO to produce a 10 mM stock concentration and stored frozen at –0°C. For animal dosing studies, LEN was dissolved in vehicle (58.03% polyethylene glycol 300, 27.1% water, 6.78% ethanol, 6.61% poloxamer 188, 1.48% sodium hydroxide) at 300 mg/mL, stored at ambient temperature, and protected from light until dosing. The formulation contained additional excipients absent from the clinical formulation in order to tailor the pharmacokinetic profile in macaques.

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Animal studies.[3]
All animals were housed at Bioqual Inc. For the in vivo SHIV stock titration study, 8 untreated outbred Indian-origin male rhesus macaques aged 3–5 years were challenged intrarectally per round for a total of 5 challenge rounds using increasing virus doses ranging from 0.625 to 100 TCID50, with the 100 TCID50 round performed twice for increased resolution (Supplemental Table 2). Plasma viral load was measured to confirm the infection status. For LEN pharmacokinetics and PrEP efficacy determination, 20 outbred Indian-origin male rhesus macaques aged 3–5 years were assigned to 5 study groups with an even weight distribution (Supplemental Table 2). On study week 0, 4 animals per group were administered LEN at 5, 10, 20, 50, or 75 mg/kg in the scapular region by subcutaneous injection. LEN was prepared as a 300 mg/mL stock solution, and no more than 2 mL solution was injected into a single subcutaneous site. Injection sites were monitored daily by veterinary staff for 2 weeks and then weekly through the end of study. On week 7, 11 animals were challenged by the intrarectal route with 1 mL RPMI containing 100 TCID50 SHIV-SF162P3. Whole blood was collected and processed into plasma and PBMCs as necessary for the assessment of routine hematology and clinical chemistry, viral load analysis, serology, and the bioanalysis of drug levels. Animals were considered protected if they remained SHIV negative by a plasma PCR assay and seronegative by enzyme immunoassay through week 10 after challenge.Animals confirmed as SHIV positive in both the virus titration and the PrEP efficacy studies were placed on a daily subcutaneous ART regimen between weeks 4 and 10 after infection to prevent AIDS disease progression. The formulated ART cocktail contained tenofovir disoproxil fumarate (5.1 mg/mL), emtricitabine (40 mg/mL), and dolutegravir (2.5 mg/mL) and was administered subcutaneously once daily at 1 mL/kg.

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Bioanalysis of LEN in macaque plasma.[3]
Rhesus plasma samples were stored frozen at –80°C and thawed, and a 50 μL aliquot of each was treated with 200 μL acetonitrile containing an internal standard. After precipitation of the protein component, a 100 μL aliquot of the supernatant was transferred to a clean 96-well plate and mixed with 200 μL water. A 10 μL aliquot of the above solution was then injected into a Q-Exactive high resolution mass spectrometer from Thermo Electron with electrospray ionization in positive mode. Quantification was performed using an accurate mass ([M+H]+) of 968.1508 for LEN and 756.3289 for the internal standard. The lower and upper limits of quantitation for LEN were 1 nM and 10,000 nM, respectively.

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Plasma viral load assay.[3]
A QIAsymphony SP (Qiagen) automated sample preparation platform along with a Virus/Pathogen DSP midi kit and the cellfree500 protocol were used to extract viral RNA from 500 μL plasma. A reverse primer specific to the gag gene of SIVmac251 (5′-CACTAGGTGTCTCTGCACTATCTGTTTTG-3′) was annealed to the extracted RNA and then reverse transcribed into cDNA using SuperScript III Reverse Transcriptase along with RNAse Out (Thermo Fisher Scientific). The resulting cDNA was treated with RNase H (Thermo Fisher Scientific) and then added (2 replicates) to a custom 4× TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) containing primers and a fluorescently labeled hydrolysis probe specific for the gag gene of SIVmac251 (forward primer 5′-GTCTGCGTCATCTGGTGCATTC-3′, reverse primer 5′-CACTAGGTGTCTCTGCACTATCTGTTTTG-3′, probe 5′-/56-FAM/CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG/3BHQ_1/-3′). The qPCR was then carried out on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Mean SIV gag RNA copies per reaction were interpolated using quantification cycle data and a serial dilution of a highly characterized custom RNA transcript containing a 730 bp sequence of the SIV gag gene. The assay limit of quantification is approximately 62 RNA copies per milliliter of sample.

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ELISA.[3]
Rhesus serum samples from viremic study animals were tested for the presence of antibodies against HIV-1 by ELISA using the GS HIV-1/HIV-2 PLUS O EIA assay kit from Bio-Rad. Individual macaque sera (150 μL) mixed with 50 μL specimen diluent supplied in the kit were added to assay plates precoated with recombinant purified HIV-1 capsid (p24) protein and transmembrane glycoprotein (gp160) and incubated for 1 hour at room temperature. The plates were then washed 3 times with a sodium chloride and Tween 20–containing wash buffer from the kit and incubated for 1 hour with a HRP-conjugated antigen solution containing peptides mimicking various immunodominant epitopes of HIV-1 gp160 and p24 proteins. Wells with antibody against HIV-1 bound to the antigen coating the wells and to the peroxidase-conjugated antigens in the conjugate solution to form immobilized stable antigen-antibody-antigen complexes. The plates were washed 3 more times with the above wash buffer, developed with a working solution of tetramethylbenzidine, stopped by the addition of 1 N sulfuric acid, and analyzed at 450 nm using a Versamax microplate reader using the Softmax Pro 6.5.1 software. Samples with an OD450 nm absorbance value of more than 0.2 were considered positive.

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IPDA.[3]
The SHIV-adapted version of IPDA (SHIV-IPDA) was used to determine the number of intact SHIV proviruses. Total genomic DNA was extracted from unfractionated PBMCs using a QIAamp DNA Mini kit. DNA quality and quantity were evaluated by spectrophotometry and fluorometry, respectively, and SHIV-IPDA was then performed on the isolated DNA. In brief, SHIV-IPDA consists of a 3-component multiplex droplet-digital PCR (ddPCR) reaction. The first is a SHIV proviral discrimination reaction targeting two conserved, frequently deleted regions of the SHIV genome to determine the intact provirus count; the second is a 2-long terminal repeat (2-LTR) DNA circle reaction to determine 2-LTR circle counts; and the third is a copy reference/DNA-shearing reaction targeting ribonuclease P/MRP subunit P30 (RPP30) to determine assay input cell equivalents and the DNA shearing index. All ddPCR reactions were performed using a Bio-Rad QX200 AutoDG ddPCR system with Bio-Rad ddPCR supermix for probes with no dUTP. After DNA shearing index correction and subtraction of intact 2-LTR circles, the intact proviral frequencies were reported per million input cells. The endpoint ddPCR data were collected using Bio-Rad QuantaSoft version 1.7.4.0917.

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Plasma virus genotypic analysis.[3]
Total RNA was extracted from 50 μL plasma aliquots obtained from each viremic monkey using the MagMAX-96 Viral RNA Isolation Kit (Life Technologies) in conjunction with the Thermo Fisher Scientific KingFisher Flex automated extraction platform and eluted in 60 μL AVE buffer. The capsid coding area of gag in each sample was then individually amplified by RT-PCR using the SuperScript IV One-Step RT-PCR System (Life Technologies) and the Qiagen OneStep RT-PCR Kit according to the manufacturers’ recommended protocols. Amplification of the SHIV capsid coding region in each sample was performed using primers (SIV-CA-F [5′-CCAAAAACAAGTAGACCAACAG-3′] and SIV-CA-R [5′-TGCAAAAGGGATTGGCAC-3′]) and the products subjected to population-level bulk sequencing at Elim Biopharmaceuticals Inc. using the same primer set. To identify codon changes, capsid encoding sequences for each sample were aligned using DNA Sequencher Software (Gene Codes Corporation) with that of the parent challenge virus stock. A sequence alignment for major consensus HIV-1 subtype, HIV-2, and SHIV-SF162P3 capsid amino acid sequences was performed using BioEdit Sequence Alignment Editor version 7.2.6.

Absorption, Distribution and Excretion
Following subcutaneous injection, lenakapavir is slowly released but completely absorbed, with peak plasma concentrations occurring at 84 days post-administration. Absolute bioavailability after oral administration is low, ranging from approximately 6% to 10%. The time to peak concentration (Tmax) after oral administration is approximately 4 hours. The mean steady-state peak plasma concentration (Cmax) (%CV) after oral and subcutaneous administration is 97.2 (70.3) ng/mL. Based on population pharmacokinetic analysis, HIV-1-infected patients who had previously received extensive treatment had lenakapavir exposures (AUCtau, Cmax, and Ctrough) that were 29% to 84% higher than those who were not infected with HIV-1. The effect of a low-fat meal on drug absorption is negligible. Following a single intravenous injection of radiolabeled lenakapavir in healthy subjects, 76% of the total radioactivity was recovered in feces, and less than 1% was recovered in urine. The major components in plasma (69%) and feces (33%) are unmetabolized lenakapavir. The steady-state volume of distribution is 976 L in heavily treated HIV-1 infected patients. The clearance rate of lenakapavir in heavily treated HIV-1 infected patients is 3.62 L/h. Metabolism/Metabolites: Metabolism plays a minor role in the clearance of lenakapavir. It is metabolized via CYP3A4 and UGT1A1-mediated oxidation, N-dealkylation, hydrogenation, amide hydrolysis, glucuronidation, hexose conjugation, pentose conjugation, and glutathione conjugation. The metabolites of lenakapavir have not been fully elucidated. No single circulating metabolite accounts for more than 10% of plasma drug exposure. Biological Half-Life: The median half-life after oral administration is 10 to 12 days; the median half-life after subcutaneous administration is 8 to 12 weeks.

Hepatotoxicity
In a small, open-label clinical trial conducted before market launch, 10% of patients experienced elevated serum transaminases, with two cases (3%) exceeding five times the upper limit of normal. Both patients presented with jaundice. However, other causes of liver injury were identified in both cases: one attributable to alcoholic hepatitis, and the other to “reconstruction syndrome” resulting from the recovery of the immune response after successful HIV replication control. Both patients continued lenacapavir treatment and recovered successfully. Since lenacapavir’s approval and widespread use, no published cases of lenacapavir-related liver injury have been reported. One drawback of long-acting drugs like lenacapavir is that treatment cannot be immediately discontinued if toxicity or intolerance occurs. Finally, in patients with a history of chronic hepatitis B or C, the recovery of the immune response following the addition of lenacapavir to an ineffective antiretroviral therapy regimen may lead to reconstruction syndrome and relapse of chronic viral hepatitis.
Probability Score: E (Unlikely to be the cause of liver injury with specific clinical manifestations).
Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the use of lenakapavir during lactation. Because the drug has a protein binding rate exceeding 98.5%, its concentration in breast milk is likely to be low. Achieving and maintaining viral suppression through antiretroviral therapy can reduce the risk of breast milk transmission to below 1%, but not zero. For HIV-infected individuals receiving antiretroviral therapy with a persistently low viral load below the detection limit, breastfeeding should be supported if they choose to do so. If viral load is not suppressed, pasteurized donor breast milk or formula is recommended.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
In vitro studies have shown that lenakapavir binds to plasma proteins at a rate of approximately 99.8%.

[1]. GS-CA Compounds: First-In-Class HIV-1 Capsid Inhibitors Covering Multiple Grounds. Front Microbiol. 2019 Jun 20;10:1227.

[2]. Clinical targeting of HIV capsid protein with a long-acting small molecule

[3]. Long-acting lenacapavir acts as an effective preexposure prophylaxis in a rectal SHIV challenge macaque model. J Clin Invest. 2023 Aug 15; 133(16): e167818.

Lenacapaver is a prescription drug approved by the U.S. Food and Drug Administration (FDA). It is approved under two different brand names for the following uses: Lenacapaver oral tablets and injections (brand name: Sunlenca) For the treatment of HIV-infected adults who are unresponsive to other anti-HIV drugs and meet specific criteria determined by a healthcare provider. Lenacapaver must be used in combination with other anti-HIV drugs when used for HIV treatment. Lenacapaver oral tablets and injections (brand name: Yeztugo) For HIV pre-exposure prophylaxis (PrEP) to reduce the risk of HIV infection in HIV-negative adults and adolescents who weigh at least 77 pounds (35 kg) and are at risk of sexually transmitted HIV transmission. Lenacapaver used for PrEP must always be used in conjunction with safe sex practices (such as condom use) to reduce the risk of contracting other sexually transmitted infections. Lenapavir sodium is the sodium salt of lenapavir, a human immunodeficiency virus type 1 (HIV-1) capsid function inhibitor with anti-HIV activity. After administration, lenapavir targets and binds to the interface between the hexameric subunits of the HIV capsid protein (p24). This inhibits capsid function, including capsid-mediated nuclear uptake of the pre-integration complex, viral particle production, and proper capsid core formation, thereby inhibiting HIV-1 replication. Lenapavir sodium is a small molecule drug that has reached Phase IV clinical trials (covering all indications) and was first approved in 2022 for the treatment of HIV-1 infection and HIV infection.

C39H31CLF10N7NAO5S2

990.26

989.12546

C, 47.30; H, 3.16; Cl, 3.58; F, 19.19; N, 9.90; Na, 2.32; O, 8.08; S, 6.48

2283356-12-5

Lenacapavir;2189684-44-2; 2283356-18-1 (HCl); 2937414-47-4 (Lenacapavir pacfosacil); 2283356-12-5 (sodium)

153435888

White to off-white solids at room temperature

1

19

13

65

2050

3

C(C1=NN(CC(=O)N[C@H](C2N=C(C#CC(C)(C)S(=O)(=O)C)C=CC=2C2C=CC(Cl)=C3C(NS(=O)(=O)C)=NN(CC(F)(F)F)C=23)CC2C=C(F)C=C(F)C=2)C2C(F)(F)[C@@H]3C[C@@H]3C1=2)(F)(F)F.[Na+]

SSXPGMNGIORJAQ-PZNXWHLTSA-M

InChI=1S/C39H32ClF10N7O5S2.Na/c1-36(2,63(3,59)60)10-9-21-5-6-22(23-7-8-26(40)30-32(23)57(17-37(43,44)45)54-35(30)55-64(4,61)62)31(51-21)27(13-18-11-19(41)14-20(42)12-18)52-28(58)16-56-34-29(33(53-56)39(48,49)50)24-15-25(24)38(34,46)47;/h5-8,11-12,14,24-25,27H,13,15-17H2,1-4H3,(H2,52,54,55,58);/q;+1/p-1/t24-,25+,27-;/m0./s1

sodium [4-chloro-7-[2-[(1S)-2-(3,5-difluorophenyl)-1-[[2-[(2S,4R)-5,5-difluoro-9-(trifluoromethyl)-7,8-diazatricyclo[4.3.0.02,4]nona-1(6),8-dien-7-yl]acetyl]amino]ethyl]-6-(3-methyl-3-methylsulfonylbut-1-ynyl)-3-pyridinyl]-1-(2,2,2-trifluoroethyl)indazol-3-yl]-methylsulfonylazanide

GS-6207 sodium; Lenacapavir sodium; GS-HIV Sodium; 2283356-12-5; BDT58WJ9WE; UNII-BDT58WJ9WE;

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.)