genotype 1 HCV NS3-4A protease (Ki = 7 nM)

Telaprevir inhibits the NS3-4A serine protease of the hepatitis C virus, which stops the virus's ability to process polyproteins. This, in turn, causes a time- and dose-dependent decrease in viral RNA replication, total HCV RNA levels, and protein levels in Con1 (genotype 1b) subgenomic HCV replicon cells. Telaprevir exhibits a noteworthy increase in inhibitory effect on HCV RNA replication over time, with IC50 values for 24, 48, 72, and 120 hours of incubation, respectively, of 0.574 μM, 0.488 μM, 0.210 μM, and 0.139 μM. Three separate experiments using a 48-hour incubation period show that telaprevir has an average IC50 of 0.354 μM and an average IC90 of 0.830 μM, respectively. Telaprevir has no appreciable cytotoxicity against HepG2, parental Huh-7, or HCV replicon cells after 48 hours of incubation. After incubating for 13 days without any rebound, replicon cells are completely free of HCV RNA when telaprevir (17.5 μM) is withdrawn. In comparison to treatment with each agent alone, telaprevir and IFN-α exhibit an additive to moderate synergistic effect on suppression of resistance mutations and reduction of HCV RNA replication without a discernible increase in cytotoxicity.[1]

In the mice model, oral telaprevir administration decreases HCV protease-dependent cleavage and subsequent liver-secreted SEAP into the blood to 18.7% and 18.4% at dosages of 10 and 25 mg/kg, respectively. [/2] In HCV-infected human hepatocyte chimeric mice with genotype 1b, administration of Telaprevir at 200 mg/kg for one week causes a 1.9 log reduction in HCV RNA; when treatment is combined with MK-0608 (50 mg/kg) for four weeks, viruses are eradicated from the mice.[3]

Stable Huh-7 cells containing the self-replicating, subgenomic HCV replicon, which is identical in sequence to the I377neo/NS3-3'/wt replicon are used for anti-HCV assays. Telaprevir serially diluted in DMEM plus 2% FBS and 0.5% dimethyl sulfoxide (DMSO) is incubated with replica cells at 37 °C for the specified amount of time. Using an RNeasy-96 kit, total cellular RNA is extracted, and a quantitative real-time polymerase chain reaction (QRT-PCR) assay is used to determine the copy number of HCV RNA in order to assess the 50% inhibitory concentration (IC50).

Evaluating Telaprevir (VX-950) or IFN-α in HCV replicon cells involves determining its IC50, IC90, and CC50. To sum up, 96-well plates are plated with 1×10⁴ replicon cells each well. Using antiviral agents serially diluted in DMEM plus 2% FBS and 0.5% DMSO, replicon cells are incubated at 37°C for the specified amount of time on the following day. With an RNeasy-96 kit, total cellular RNA is extracted, and a quantitative RT-PCR (QRT-PCR) assay is used to calculate the copy number of HCV RNA. The mean of five replicates in cell culture is represented by each datum point. A tetrazolium (MTS)-based cell viability assay is used to measure the cytotoxicity of Telaprevir under the same experimental conditions. One million parental Huh-7 cells or four million HepG2 cells per well are used in the cytotoxicity assay using human hepatocyte cell lines. In order to assess the cytotoxicity of Telaprevir against resting peripheral blood monoclonal cells, 1×10⁵ cells per well are cultured with Telaprevir in RPMI-1640 medium (without serum) for 48 hours, after which the MTS-based assay is used to determine the cell viability. Precoated with anti-human CD3 antibody, a 96-well plate is filled with 1×10⁵ cells per well in RPMI-1640 medium to test the cytotoxicity of VX-950 against proliferating PBMC. The cells are cultured for 72 hours at 37°C with Telaprevir and anti-human CD28 antibody. The [³H]thymidine update is used to measure the cell growth between the 48th and 72nd hours[1].

Mice: Recombinant adenovirus Ad-WT-HCVpro-SEAP, with 10⁹ IFU per mouse, is injected via the tail vein into five groups of six-week-old SCID mice (six animals per group). Two oral doses of Telaprevir (VX-950) at a dose of 10, 25, 75, 150, or 300 mg/kg are administered to each group of mice. First dose of Telaprevir is administered two hours prior to adenovirus injection; second dose is administered ten hours following injection. A second set of ten mice is given the vehicle on its own. Serum samples are taken 24 hours after injection, and the SEAP activity in each group administered with Telaprevir is contrasted with the vehicle group's. Rat and Canine Rats and dogs are used to assess the oral and intravenous pharmacokinetics of telaprevir (VX-950). One intravenous bolus dose of 0.95 mg/kg Telaprevir is given intravenously to three male Sprague-Dawley rats weighing 250–300 g. Heparinized tubes are used to collect serial blood samples prior to dosage administration and at intervals of 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours following the dose. Telaprevir in 10% ethanol, 40% polyethylene glycol 400, and 50% D5W is given intravenously as a bolus dose to three male beagle dogs (8–12 kg). Heparinized tubes are used to collect serial blood samples prior to dosage administration as well as at 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours later. Telaprevir is formulated in polyvinylpyrrolidone (PVP) K-30 plus 2% sodium lauryl sulfate and dosed as an oral gavage for oral studies in rats and dogs. Oral dosages of 40 mg/kg VX-950 are given to three male Sprague-Dawley rats (250–300 g) and 9.6 mg/kg VX-950 are given to four male Beagle dogs (10.9–12.0 kg). Blood samples are obtained before dosage administration and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours following dose administration in both oral studies. Plasma samples are obtained by centrifugation and kept at -70°C until analysis in both intravenous and oral studies. Samples from the oral studies are analyzed using an achiral LC/MS/MS method, while samples from the intravenous studies are analyzed using a chiral liquid chromatography followed by tandem mass spectrometry (LC/MS/MS) method.

Absorption
Telaprevir reaches peak plasma concentration 4-5hours after administration. Absolute bioavailability has not been determined. When taken with a normal fat meal (21g of fat), exposure increases by 235% compared to fasting conditions. With low (3.6g of fat) and high fat (56g of fat) meals, exposure increased 117% and 330% respectively.

Route of Elimination
Telaprevir is mainly eliminated in the feces (82%) with a smaller amount eliminated via expiration (9%) and very little in the urine (1%). 31.9% and 18.8% of drug in the feces was present as the parent compound and R-diastereomer of the parent compound respectively.

Volume of Distribution
The estimated apparent volume of distribution for Telapravir is 252 litres with an inter-individual variability of 72%.

Clearance
Telaprevir has an estimated aparent total body clearance of 32.4 liters per hour with an interindividual variability of 27.2%.

The pharmacokinetic properties of telaprevir have been evaluated in healthy adult subjects and in subjects with chronic hepatitis C. Following multiple doses of telaprevir (750 mg every 8 hr) in combination with peginterferon alfa and ribavirin in treatment-naive subjects with genotype 1 chronic hepatitis C, mean (SD) Cmax was 3510 (1280) ng/mL, Cmin was 2030 (930) ng/mL, and AUC8h was 22,300 (8650) ng.hr/mL.

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Metabolism / Metabolites
Telaprevir is extensively metabolized via hydrolysis, oxidation, and reduction. The major metabolites of Telaprevir are pyrazinoic acid, a metabolite that underwent reduction at the α-ketoamide bond, and the R-diastereomer of telaprevir which is 30-fold less active than the parent compound were found to be the predominant metabolites. The primary enzyme involved in the metabolism of Telaprevir is CYP3A4. Some metabolism is performed by aldo-keto reductases and other reductases.

Telaprevir is extensively metabolized in the liver, involving hydrolysis, oxidation, and reduction. Multiple metabolites were detected in feces, plasma, and urine. After repeated oral administration, the R-diastereomer of telaprevir (30-fold less active), pyrazinoic acid, and a metabolite that underwent reduction at the alpha-ketoamide bond of telaprevir (not active) were found to be the predominant metabolites of telaprevir.

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Biological Half-Life
Telaprevir has a half-life of elimination of 4.0-4.7 hours after a single dose and an effective half life of 9-11 hours at steady state.

The mean elimination half-life after single-dose oral administration of telaprevir 750 mg typically ranged from about 4.0 to 4.7 hours. At steady state, the effective half-life is about 9 to 11 hours.

Hepatotoxicity
In large randomized controlled trials, triple therapy with telaprevir, peginterferon and ribavirin was associated with a high rate of adverse events that often required dose adjustments and led to early discontinuation in 5% to 20% of patients. However, serum ALT elevations and clinically apparent liver injury were not generally mentioned as adverse events of therapy. Telaprevir, however, was associated with a high rate of rash, which was sometimes associated with features of hypersensitivity, including rare instances of DRESS and Stevens Johnson syndrome. These severe cutaneous reactions are often accompanied by laboratory evidence of hepatic injury (ALT and alkaline phosphatase elevations). In reported cases, however, the rash and other features of hypersensitivity typically overshadowed the hepatic injury and none were reported to be associated with jaundice.

Another rare but severe hepatic complications of telaprevir therapy occurs in patients with advanced fibrosis or cirrhosis, among whom de novo, seemingly spontaneous hepatic decompensation occurred in a proportion of treated subjects. Decompensation was particularly common in patients with advanced fibrosis or cirrhosis with a previous history of decompensation. The cause of the decompensation was not clear and the separate role of telaprevir in contrast to peginterferon and ribavirin could not be defined. Nevertheless, in postmarketing studies of triple therapy of chronic hepatitis C with cirrhosis, decompensation was reported in 2% to 8% of patients, and deaths from hepatic failure in 1% to 3%.
Likelihood score for the combination of telaprevir, peginterferon and ribavirin: B (likely cause of liver injury and hepatic decompensation in patients with preexisting cirrhosis or advanced fibrosis).

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Protein Binding
Telapravir is 59-76% bound to human plasma proteins following a single dose. It binds to both human serum albumin and α1-acid glycoprotein.

[1]. Antimicrob Agents Chemother . 2006 May;50(5):1813-22.

[2]. Antimicrob Agents Chemother . 2006 Mar;50(3):899-909.

[3]. J Hepatol . 2011 May;54(5):872-8.

The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log(10) was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log(10) reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.[1]

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Telaprevir is an oligopeptide consisting of N-(pyrazin-2-ylcarbonyl)cyclohexylalanyl, 3-methylvalyl, octahydrocyclopenta[c]pyrrole-1-carboxy, and 3-amino-N-cyclopropyl-2-oxohexanamide residues joined in sequence. Used for treatment of chronic hepatitis C virus genotype 1 infection. It has a role as a peptidomimetic, a hepatitis C protease inhibitor and an antiviral drug. It is an oligopeptide, a member of pyrazines, a cyclopentapyrrole and a member of cyclopropanes.

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Telaprevir is a direct acting antiviral medication used as part of combination therapy to treat chronic Hepatitis C, an infectious liver disease caused by infection with Hepatitis C Virus (HCV). HCV is a single-stranded RNA virus that is categorized into nine distinct genotypes, with genotype 1 being the most common in the United States, and affecting 72% of all chronic HCV patients. Treatment options for chronic Hepatitis C have advanced significantly since 2011, with the development of Direct Acting Antivirals (DAAs) such as Telaprevir. Telaprevir is an inhibitor of NS3/4A, a serine protease enzyme, encoded by HCV genotype 1. These enzymes are essential for viral replication and serve to cleave the virally encoded polyprotein into mature proteins like NS4A, NS4B, NS5A and NS5B. The barrier for develoment of resistance to NS3/4A inhibitors is lower than that of NS5B inhibitors, another class of DAAs. Subtitutions at amino acid positions 155, 156, or 168 are known to confer resistance. The substitutions of the enzyme's catalytic triad consisting of H58, D82, and S139 are also likely to alter the affinity of the drug for NS3/4A or the activity of the enzyme itself. Despite this disadvantage Telaprevir is still effective against HCV when paired with [DB00811], [DB00008], and [DB00022]. Telaprevir, [DB00811], [DB00008], and [DB00022] were used with the intent to cure, or achieve a sustained virologic response (SVR), after 12 weeks of daily administration of the combination therapy followed by 12 or 36 weeks of therapy with [DB00811], [DB00008], and [DB00022]. SVR and eradication of HCV infection is associated with significant long-term health benefits including reduced liver-related damage, improved quality of life, reduced incidence of Hepatocellular Carcinoma, and reduced all-cause mortality. Telaprevir was available as a fixed dose product (tradename Incivek) used for the treatment of chronic Hepatitis C. Approved in May 2011 by the FDA, Incivek was indicated for the treatment of HCV genotype 1 in combination with [DB00811], [DB00008], and [DB00022]. Incivek has since been withdrawn from the market.

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Telaprevir is a Hepatitis C Virus NS3/4A Protease Inhibitor. The mechanism of action of telaprevir is as a HCV NS3/4A Protease Inhibitor, and Cytochrome P450 3A Inhibitor, and P-Glycoprotein Inhibitor, and Organic Anion Transporting Polypeptide 1B1 Inhibitor, and Organic Anion Transporting Polypeptide 2B1 Inhibitor.
Telaprevir is an oral, direct acting hepatitis C virus (HCV) protease inhibitor that was used in combination with other antiviral agents in the treatment of chronic hepatitis C, genotype 1. Approved for use in the United States in 2012, it was withdrawn in 2015 when regimens of all oral direct acting agents with superior efficacy and better tolerance became available. Telaprevir was not linked to instances of acute liver injury during therapy, but was linked to cases of severe cutaneous reactions such as DRESS and Stevens Johnson syndrome which were associated with mild hepatic injury. In addition, when combined with peginterferon and ribavirin, telaprevir was associated with cases of hepatic decompensation in patients with preexisting cirrhosis.

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Telaprevir has been reported in Penicillium raistrickii with data available.
Telaprevir is an orally available peptidomimetic small molecule with activity against hepatitis C virus (HCV). Telaprivir is a selective protease inhibitor that targets the viral HCV NS3-4A serine protease and disrupts processing of viral proteins and formation of a viral replication complex.

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Drug Indication
Telaprevir, when used in combination with [DB00811], [DB00008], and [DB00022] is indicated for use in the treatment of chronic HCV genotype 1 infection in adults.

Incivo, in combination with peginterferon alfa and ribavirin, is indicated for the treatment of genotype-1 chronic hepatitis C in adult patients with compensated liver disease (including cirrhosis): who are treatment naïve; who have previously been treated with interferon alfa (pegylated or non-pegylated) alone or in combination with ribavirin, including relapsers, partial responders and null responders.

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Therapeutic Uses INCIVEK (telaprevir), in combination with peginterferon alfa and ribavirin, is indicated for the treatment of genotype 1 chronic hepatitis C in adult patients with compensated liver disease, including cirrhosis, who are treatment-naive or who have previously been treated with interferon-based treatment, including prior null responders, partial responders, and relapsers. /Included in US product label/

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Drug Warnings
Fatal and non-fatal serious skin reactions, including Stevens Johnson Syndrome (SJS), Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS), and Toxic Epidermal Necrolysis (TEN), have been reported in patients treated with INCIVEK combination treatment. Fatal cases have been reported in patients with progressive rash and systemic symptoms who continued to receive INCIVEK combination treatment after a serious skin reaction was identified. For serious skin reactions, including rash with systemic symptoms or a progressive severe rash, INCIVEK, peginterferon alfa, and ribavirin must be discontinued immediately. Discontinuing other medications known to be associated with serious skin reactions should be considered. Patients should be promptly referred for urgent medical care. US Natl Inst Health; DailyMed. Current Medication Information for INCIVEK (telaprevir) tablet, film coated (December 2012). Available from, as of March 12, 2013: https://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=ed0e4f33-cf21-4fe3-918d-1d5b3a23eee4 Hazardous Substances Data Bank (HSDB) Rash developed in 56% of patients receiving telaprevir during controlled clinical trials. Severe rash (e.g., generalized rash or rash with vesicles or bullae or ulcerations other than SJS) was reported in 4% of patients receiving telaprevir in conjunction with peginterferon alfa and ribavirin compared with less than 1% of patients receiving peginterferon alfa and ribavirin without telaprevir. Rash frequently was observed during the first 4 weeks of telaprevir treatment, but can occur at any time. Rash generally improves when telaprevir therapy is completed or discontinued; complete resolution may take weeks.

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Drug Tolerance
Certain amino acid substitutions (mutations) in the HCV NS3 protease domain (V36A/M, T54A/S, R155K/T, A156S, A156V/T, R155T with D168N, V36A with T54A, V36M/A with R155K/T, T54S/A with A156S/T) have been associated with reduced in vitro susceptibility to telaprevir. HCV with reduced susceptibility to telaprevir have been selected in vitro and have emerged during telaprevir treatment. The majority of HCV isolates from patients in phase 3 clinical studies who did not achieve sustained virologic response (SVR) had treatment-emergent resistance mutations. The clinical impact of prior exposure to HCV protease inhibitors (including telaprevir) is not known. HCV with telaprevir resistance-associated mutations have been isolated from some individuals who have not been exposed to HCV protease inhibitors. Telaprevir treatment-emergent resistance mutations can also result in decreased in vitro susceptibility or cross-resistance to other HCV NS3/4A protease inhibitors (e.g., boceprevir). Cross-resistance is not expected between telaprevir and interferons or ribavirin. American Society of Health-System Pharmacists 2012; Drug Information 2012. Bethesda, MD. 2012, p. 855

HCV genotype 1b replicons with reduced susceptibility to telaprevir have been selected in cell culture and characterized for telaprevir genotypic and phenotypic resistance. Additionally, resistance to telaprevir was evaluated in both biochemical and HCV genotype 1b replicon assays using site-directed mutants and recombinant NS3/4A from telaprevir Phase 2 clinical trials isolates. Variants V36A/M, T54A/S, R155K/T, A156S, R155T+D168N, and V36A+T54A conferred 3- to 25-fold reduced susceptibility to telaprevir; and A156V/T variants and the V36M/A+R155K/T and T54S/A+A156S/T double variants conferred greater than 62-fold reduced susceptibility to telaprevir. No amino acid substitutions were observed at the proteolytic cleavage sites.

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Pharmacodynamics
Telaprevir is classified as a direct-acting antiviral (DAA) and prevents viral replication in HCV genotype 1.

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Mechanism of Action
Telaprevir is a NS3/4a protease inhibitor used to inhibit viral HCV replication. NS3/4a protease is an integral part of viral replication and mediates the cleavage the virally encoded polyprotein to mature proteins (NS4A, NS4B, NS5A and NS5B). Telaprevir inhibits NS3/4A with an IC50 of 10nM.

Telaprevir is a peptidomimetic, selective hepatitis C virus (HCV) nonstructural 3/4A (NS3/4A) protease inhibitor. The drug is a direct-acting antiviral (DAA) agent with activity against HCV. Telaprevir contains an alpha-ketoamide functional group that covalently and reversibly binds the active serine site of HCV NS3/4 protease. By blocking proteolytic cleavage of NS4A, NS4B, NS5A, and NS5B from the HCV-encoded polyprotein, the drug inhibits HCV replication. Telaprevir has in vitro activity against HCV genotypes 1a, 1b, and 2, but is less active against genotypes 3a and 4a. American Society of Health-System Pharmacists 2012; Drug Information 2012. Bethesda, MD. 2012, p. 855

Telaprevir is an inhibitor of the HCV NS3/4A serine protease, necessary for the proteolytic cleavage of the HCV encoded polyprotein into mature forms of the NS4A, NS4B, NS5A and NS5B proteins and essential for viral replication. In a biochemical assay, telaprevir inhibited the proteolytic activity of the recombinant HCV NS3 protease domain with an IC50 value of 10 nM.

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Formulations / Preparations
INCIVEK (telaprevir) is supplied as purple film-coated capsule-shaped tablets containing 375 mg of telaprevir. Each tablet is debossed with the characters "V 375" on one side and is packaged as follows: 28-day packer contains 4 weekly cartons of 7 blister strips each (6 tablets per blister strip)

C36H53N7O6

679.85

679.405

C, 63.60; H, 7.86; N, 14.42; O, 14.12

402957-28-2

Telaprevir-d4

3010818

White to off-white solid powder

1.3±0.1 g/cm3

1.584

3.93

O=C(N([C@@H]1C(N[C@H](C(C(NC2CC2)=O)=O)CCC)=O)C[C@@]3(CCC[C@@]31[H])[H])[C@@H](NC([C@@H](NC(C4=NC=CN=C4)=O)C5CCCCC5)=O)C(C)(C)C

BBAWEDCPNXPBQM-GDEBMMAJSA-N

InChI=1S/C36H53N7O6/c1-5-10-25(29(44)34(48)39-23-15-16-23)40-33(47)28-24-14-9-13-22(24)20-43(28)35(49)30(36(2,3)4)42-32(46)27(21-11-7-6-8-12-21)41-31(45)26-19-37-17-18-38-26/h17-19,21-25,27-28,30H,5-16,20H2,1-4H3,(H,39,48)(H,40,47)(H,41,45)(H,42,46)/t22-,24-,25-,27-,28-,30+/m0/s1

(3S,3aS,6aR)-2-[(2S)-2-[[(2S)-2-cyclohexyl-2-(pyrazine-2-carbonylamino)acetyl]amino]-3,3-dimethylbutanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole-3-carboxamide

VX-950; LY-570310; MP-424; VX950; LY570310; MP424; VX 950; LY 570310; MP 424; trade names: Incivek; Incivo

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 (3.68 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 2: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL

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