Faldaprevir (BI 201335) targets the HCV NS3 protease (genotype 1b). In an enzymatic assay using genotype 1b NS3-NS4A enzyme and a fluorogenic depsipeptide substrate, the compound exhibited an IC50 of 3 nM. [1]
NS3 protease (genotype 1) (IC50 = 3 nM; EC50 = 3 nM) [1]

In a cell-based bicistronic luciferase reporter replicon assay (genotype 1b), Faldaprevir (BI 201335) demonstrated an EC50 of 3 nM. [1]

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- Enzymatic Activity: As a noncovalent inhibitor, Faldaprevir potently binds and inhibits the HCV NS3/4A protease, with Ki values of 2.0 nM (GT1b) and 2.6 nM (GT1a).
- Antiviral Activity: In Huh-7 cells, Faldaprevir effectively inhibits HCV RNA replication. The half-maximal effective concentrations (EC50) are 3.1 nM for genotype 1b and 6.5 nM for genotype 1a. It also shows activity against genotype 2a, with EC50 values of approximately 43 to 50 nM.
- Cytotoxicity: The half-maximal cytotoxic concentration (CC50) in Huh-7 cells is 30,000 nM, indicating a high selectivity index.
- Metabolic Stability & Transport: Faldaprevir is metabolized in vitro primarily by CYP3A4/5 in the liver to form monohydroxylated metabolites M2a and M2b. These metabolites are substrates for the efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP).

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Faldaprevir (BI 201335) is a potent and selective noncovalent linear inhibitor of the HCV NS3 protease, exhibiting IC50 of 3 nM in enzymatic assay and EC50 of 3 nM in cell‑based replicon assay (genotype 1b). [1]
The compound shows high stability in human liver microsomes (T1/2 > 100 min) and rat liver microsomes (T1/2 > 300 min). [1]

In a Phase Ib clinical trial (SOUND-C1) in treatment-naive patients with chronic HCV genotype 1 infection, a regimen consisting of Faldaprevir (BI 201335) 120 mg once daily, deleobuvir (BI 207127) 600 mg three times daily, and weight-based ribavirin for 4 weeks, followed by response-guided faldaprevir plus pegylated interferon-α2a/ribavirin, achieved a sustained virological response 24 weeks after completion of treatment (SVR24) rate of 94% (16/17 patients). [2]

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- Clinical Efficacy: In a Phase 2 trial (SILEN-C3) for treatment-naive HCV GT1 patients, Faldaprevir (120 mg once daily) combined with PegIFN/RBV achieved high sustained virologic response (SVR) rates of 67% (12-week faldaprevir group) and 74% (24-week faldaprevir group).
- Hyperbilirubinemia Mechanism: In monkeys and clinical studies, faldaprevir causes reversible, unconjugated hyperbilirubinemia, primarily through competitive inhibition of UGT1A1 (bilirubin conjugation) and OATP1B1/1B3 (bilirubin uptake).

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In a Phase Ib clinical trial (SOUND‑C1) of interferon‑free oral combination therapy, treatment‑naive HCV genotype‑1 patients receiving Faldaprevir (BI 201335) 120 mg once daily plus deleobuvir (400 or 600 mg three times daily) and ribavirin for 4 weeks, followed by response‑guided faldaprevir plus pegylated interferon‑α2a/ribavirin to week 24 or 48, achieved sustained virological response 24 weeks after treatment (SVR24) rates of 73% (11/15) in the deleobuvir 400 mg group and 94% (16/17) in the 600 mg group. [2]

Biochemical assays were carried out using 0.5 µM genotype 1b NS3-NS4A enzyme and a fluorogenic depsipeptide substrate (anthranilyl-DDIVPAbu[CO-O]AMY(3-NO2)-TW-OH). The reaction buffer consisted of 50 mM Tris-HCl (pH 8.0), 0.25 M sodium citrate, 0.01% n-dodecyl-β-D-maltoside, 1 mM TCEP, and 5% DMSO. Mixtures were incubated at 23°C for 60-70 minutes and terminated by the addition of 1 M MES (pH 5.8). Fluorescence of the N-terminal product (anthranilyl-DDIVP-Abu) was measured using a plate reader. Percent inhibition at each inhibitor concentration was used to determine the median effective concentration (IC50). [1]

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- Protease Inhibition Assay: Recombinant HCV NS3/4A protease is incubated with a fluorogenic peptide substrate and serial dilutions of faldaprevir. The inhibition of protease activity is measured by a decrease in fluorescence signal, and the Ki value is calculated.
- Reaction Phenotyping: Faldaprevir is incubated with recombinant human CYP450 enzymes (e.g., CYP3A4/5) or human hepatocytes. The formation of metabolites (M2a and M2b) is analyzed by LC-MS to identify the primary enzymes responsible for its metabolism.
- Transporter Studies: Transport assays are performed using cell lines overexpressing specific transporters like P-gp, BCRP, OATP1B1, OATP1B3, or MRP2. The uptake or efflux of faldaprevir or its metabolites is measured to assess interaction and calculate IC50 values.

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The biochemical assay for NS3 protease activity was performed using 0.5 mM genotype 1b NS3‑NS4A enzyme and a fluorogenic depsipeptide substrate (antranilyl‑DDIVPAbu[CO‑O]AMY(3‑NO2)‑TW‑OH) in a reaction buffer containing 50 mM Tris‑HCl (pH 8.0), 0.25 M sodium citrate, 0.01% n‑dodecyl‑β‑D‑maltoside, 1 mM TCEP, and 5% DMSO. Mixtures were incubated at 23 °C for 60‑70 minutes, and the reaction was terminated by adding 1 M MES (pH 5.8). Fluorescence of the N‑terminal product (antranilyl‑DDIVP‑Abu) was measured using a plate reader. Percent inhibition at each inhibitor concentration was calculated, and the IC50 value was determined by nonlinear regression. [1]

The cell-based replicon assay used a bicistronic luciferase reporter replicon encoding the Con1 genotype 1b NS2-NS5B coding region. Compounds were incubated with cells for 72 hours. The relative levels of luciferase present were determined using a luciferase substrate on a Top-count instrument. EC50 values were determined by nonlinear regression. [1]

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- Antiviral Activity (Replicon Assay): Huh-7 cells harboring an HCV subgenomic replicon are incubated with various concentrations of faldaprevir for 72 hours. HCV RNA levels are measured by RT-PCR, or luciferase activity is measured, to determine the EC50 for inhibition of viral replication.
- Cytotoxicity Assay: Huh-7 cells are treated with a range of faldaprevir concentrations. Cell viability is assessed using an MTT assay to calculate the CC50.

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The cell‑based replicon assay used a bicistronic luciferase reporter replicon encoding the Con1 genotype 1b NS2‑NS5B coding region. Compounds were incubated with cells for 72 hours, and the relative levels of luciferase were measured using a luciferase substrate on a top‑count instrument. EC50 values were determined by nonlinear regression. [1]

For oral pharmacokinetic studies, male Sprague-Dawley rats (275-300 g) were fasted overnight with access to 10% dextrose in water. An oral dose of 5 mg/kg of Faldaprevir (BI 201335) was administered in a dosing volume of 10 mL/kg of a vehicle consisting of 0.5% Methocel and 0.3% Tween-80. For intravenous pharmacokinetic experiments, a dose of 2 mg/kg was administered. Blood samples were collected from the cannulated right carotid at various time points post-dosing (0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours for oral; with an additional 5-minute sample for intravenous). Plasma samples from three rats were pooled at each time point. [1]

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- Rat PK and Distribution Study: Rats receive a single oral (10 mg/kg) or intravenous (2 mg/kg) dose of [¹⁴C]-labeled faldaprevir. Blood, tissues, urine, and feces are collected at predetermined time points. Radioactivity is measured by liquid scintillation counting, and metabolites are identified by LC-MS.
- Monkey Hyperbilirubinemia Study: Monkeys are administered faldaprevir orally (≥20 mg/kg per day). Blood samples are drawn periodically to measure serum levels of total, direct, and indirect bilirubin to assess the drug's effect on bilirubin clearance.

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For rat pharmacokinetic studies, male Sprague‑Dawley rats (275‑300 g) were fasted overnight with access to 10% dextrose in water. Oral administration was performed at a dose of 5 mg/kg in a dosing volume of 10 mL/kg using a vehicle consisting of 0.5% Methocel and 0.3% Tween‑80. Blood samples were collected from the cannulated right carotid at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours post‑dosing, and plasma samples from three rats were pooled at each time point. [1]
Intravenous pharmacokinetic experiments were performed at a dose of 2 mg/kg, with an additional sample collection at 5 minutes. Plasma samples were extracted by solid‑phase extraction and analyzed by LC‑MS. Non‑compartmental analysis was performed using WinNonlin software. [1]

Faldaprevir (BI 201335) (compound 29) was evaluated in rats. After an oral dose of 5 mg/kg, it achieved a maximum plasma concentration (Cmax) of 0.60 µM and an AUC0→∞ of 1.7 µM·h. After an intravenous dose of 2 mg/kg, it exhibited a half-life (T1/2) of 1.2 hours, a clearance (Cl) of 20 (mL/min)/kg, and a volume of distribution (Vss) of 1.9 L/kg. The overall oral bioavailability (F) in rats was 40%. The compound partitions favorably into the liver in rats with a 40-fold increase in liver versus plasma concentration after oral dosing. It was stable in human (T1/2 > 100 min) and rat liver microsomes (T1/2 > 300 min). [1]

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- Human PK: In healthy subjects, faldaprevir is slowly absorbed. After single doses (4-1200 mg), the median tmax ranges from 4.0 to 14.0 hours, and the geometric mean terminal t1/2 ranges from 15.5 to 39.2 hours. Exposure (Cmax and AUC) increases in a more than dose-proportional manner. Urinary excretion of unchanged drug is less than 0.1% of the dose. A high-fat meal increases its relative bioavailability by 14%.
- Rat PK: Following oral administration in rats, radioactivity is rapidly distributed to tissues, with the highest levels in the liver, lung, kidney, and adrenal gland. Plasma clearance (CL) is approximately 18 mL/min/kg.
- Metabolism & Excretion: Faldaprevir is primarily eliminated via the hepatobiliary route. In rats, > 90% of the radioactive dose is recovered in feces, with <1.1% in urine. In humans, excretion is almost exclusively fecal (98.7%), with negligible urinary excretion (0.11%). Unchanged faldaprevir accounts for 52% of fecal radioactivity, while monohydroxylated metabolites M2a and M2b account for 41%.

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In rats, after oral administration of 5 mg/kg, Faldaprevir (BI 201335) achieved Cmax = 0.60 μM, AUC0→∞ = 1.7 μM·h, and oral bioavailability (F) = 40%. [1]
After intravenous administration of 2 mg/kg, the half‑life (T1/2) was 1.2 h, volume of distribution at steady state (Vss) was 1.9 L/kg, and clearance (Cl) was 20 mL/min/kg. [1]
The compound partitions favorably into the liver in rats, with a 40‑fold higher concentration in liver versus plasma after oral dosing. [1]

In the SOUND-C1 clinical trial, during the 4-week interferon-free treatment phase with Faldaprevir (BI 201335), deleobuvir, and ribavirin, there were no severe or serious adverse events and no premature treatment discontinuations due to adverse events. The most common adverse events during this period were mild-to-moderate nausea (47%), vomiting (38%), diarrhoea (22%), asthenia (28%), pruritus (28%), rash (16%), and photosensitivity (19%). One patient experienced anaemia requiring ribavirin dose reduction. During the subsequent faldaprevir plus pegylated interferon-α2a/ribavirin treatment phase, the most common adverse events were pruritus (38%), rash (31%), and asthenia (31%); these were severe in approximately 3% of patients. Three patients discontinued all drugs due to severe adverse events: pancytopenia; eczema and pruritus; and maculopapular rash, pruritus, and eye swelling. Effects on bilirubin were mainly via an increase in the indirect fraction and were not associated with increases in alanine aminotransferase or clinical signs of liver dysfunction. [2]

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- Hyperbilirubinemia: The most notable adverse effect is reversible, unconjugated hyperbilirubinemia, which is clinically benign and not associated with hepatotoxicity. It results from competitive inhibition of UGT1A1, OATP1B1, and OATP1B3.
- Safety Summary: Faldaprevir is generally well-tolerated. In clinical trials combining it with PegIFN/RBV, approximately 6% of patients discontinued treatment due to adverse events. Most adverse events were mild or moderate in severity.

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In the Phase Ib SOUND‑C1 clinical trial, during the 4‑week interferon‑free phase (faldaprevir + deleobuvir + ribavirin), the most common adverse events were mild‑to‑moderate nausea (47%), vomiting (38%), diarrhoea (22%), asthenia (28%), pruritus (28%), rash (16%) and photosensitivity (19%). No severe or serious adverse events and no premature treatment discontinuations due to adverse events occurred during this period. One patient in the deleobuvir 400 mg group developed anaemia on day 22 requiring ribavirin dose reduction. [2]
During the subsequent faldaprevir plus pegylated interferon‑α2a/ribavirin treatment period (week 5 to end of treatment), the most common adverse events were pruritus (38%), rash (31%) and asthenia (31%); these were severe in approximately 3% of patients. Three patients discontinued all drugs due to severe adverse events: pancytopenia; eczema and pruritus; maculopapular rash, pruritus and eye swelling. One patient discontinued faldaprevir due to moderate rash. Two patients discontinued pegylated interferon‑α2a/ribavirin due to moderate pustular rash and severe interstitial pneumopathy, respectively. [2]
Laboratory changes from baseline to day 28 in the faldaprevir + deleobuvir + ribavirin groups (pooled data) included median increases in total bilirubin (0.58‑1.04 mg/dl) and indirect bilirubin (0.26‑0.47 mg/dl), median decreases in haemoglobin (‑1.4 to ‑2.6 g/dl) and median increases in platelets (68‑89 × 10^9/L). From day 1 to week 24 during faldaprevir + pegylated interferon‑α2a/ribavirin treatment, median changes included: ALT ‑22 U/l, total bilirubin +0.37 mg/dl, indirect bilirubin +0.17 mg/dl, haemoglobin ‑2.3 g/dl, platelets ‑48 × 10^9/L, white blood cells ‑2.9 × 10^9/L. [2]

[1]. Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335). J Med Chem. 2010 Sep 9;53(17):6466-76.

[2]. Faldaprevir (BI 201335), deleobuvir (BI 207127) and ribavirin oral therapy for treatment-naive HCV genotype 1: SOUND-C1 final results. Antivir Ther. 2013;18(8):1015-9.

Faldaprevir (BI 201335) is a noncovalent linear inhibitor of the HCV NS3 protease, a class of inhibitors that have only ionic interactions with the catalytic site on the NS3 protease. This mechanism is unusual among serine proteases and imparts selectivity. The compound was discovered through optimization of linear tripeptide inhibitors, where the introduction of a C8-bromo substituent on the quinoline moiety was found to be optimal for improving both cell-based potency and pharmacokinetic profile. In preclinical studies, it was stable in human and rat liver microsomes. In the SOUND-C1 trial, the combination of faldaprevir, deleobuvir, and ribavirin for 4 weeks followed by response-guided therapy demonstrated potent antiviral activity, with a 94% SVR24 rate in the deleobuvir 600 mg group. Patients who experienced viral breakthrough during the interferon-free phase were successfully treated with the subsequent interferon-containing regimen. [1][2]

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Faldaprevir has been investigated for the treatment of chronic hepatitis C.

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Drug Indications
Treatment of chronic viral hepatitis C

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Faldaprevir (BI 201335) is a noncovalent linear tripeptide inhibitor of the HCV NS3 protease, containing a C‑terminal carboxylic acid and a vinyl‑ACCA moiety at P1. It was developed for the treatment of chronic hepatitis C virus (HCV) genotype 1 infection. [1]
At the time of the 2010 publication, the compound was being evaluated in Phase IIb clinical trials. [1]
In the SOUND‑C1 study (clinicaltrials.gov NCT01132313), the interferon‑free regimen of faldaprevir, deleobuvir and ribavirin demonstrated potent antiviral activity and favourable tolerability in treatment‑naive patients with HCV genotype 1. Patients who experienced viral breakthrough or re‑increase during interferon‑free treatment could be successfully rescued with pegylated interferon‑α2a/ribavirin‑based therapy. [2]

C40H49BRN6O9S

869.829

868.246

C, 55.23; H, 5.68; Br, 9.19; N, 9.66; O, 16.55; S, 3.69

801283-95-4

Faldaprevir-d6;2750534-88-2; 1215856-44-2; Faldaprevir-d7;1613250-18-2

42601552

Solid powder

1.5±0.1 g/cm3

1.647

5.34

4

12

15

57

1530

5

N([C@@]1(C[C@H]1C=C)C(=O)O)C([C@@H]1C[C@@H](OC2C=C(C3=CSC(NC(=O)C(C)C)=N3)N=C3C(=C(C=CC=23)OC)Br)CN1C(=O)[C@H](C(C)(C)C)NC(=O)OC1CCCC1)=O

LLGDPTDZOVKFDU-XUHJSTDZSA-N

InChI=1S/C40H49BrN6O9S/c1-8-21-17-40(21,36(51)52)46-34(49)27-15-23(18-47(27)35(50)32(39(4,5)6)44-38(53)56-22-11-9-10-12-22)55-29-16-25(26-19-57-37(43-26)45-33(48)20(2)3)42-31-24(29)13-14-28(54-7)30(31)41/h8,13-14,16,19-23,27,32H,1,9-12,15,17-18H2,2-7H3,(H,44,53)(H,46,49)(H,51,52)(H,43,45,48)/t21-,23-,27+,32-,40-/m1/s1

(1R,2S)-1-((2S,4R)-4-((8-bromo-2-(2-isobutyramidothiazol-4-yl)-7-methoxyquinolin-4-yl)oxy)-1-((S)-2-(((cyclopentyloxy)carbonyl)amino)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamido)-2-vinylcyclopropane-1-carboxylic acid

BI 201335; BI201335; Faldaprevir; 801283-95-4; BI-201,335; 958X4J301A; (1R,2S)-1-((2S,4R)-4-(8-bromo-2-(2-isobutyramidothiazol-4-yl)-7-methoxyquinolin-4-yloxy)-1-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamido)-2-vinylcyclopropanecarboxylic acid; BI-201335;

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)

DMSO: ~2 mg/mL (2.3 mM)

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.)