CYP3A4
Hepatitis B virus (HBV) capsid formation inhibitor [1]
.

HBV Antigen Reduction: In HBV-infected HepatoPac cocultures (using patient-derived HBV at 40 vge/cell, genotype D), treatment with RO6889678 was administered. The levels of secreted HBsAg and HBeAg in the culture media were measured using chemiluminescent immunoassays (CLIA). However, the specific reduction values or IC50 for HBsAg/HBeAg were not detailed in the provided text for this experimental setup [1]
.
Intracellular Enrichment: RO6889678 showed significant intracellular accumulation in human HepatoPac hepatocyte cocultures. After a single dose, the intracellular concentration was dramatically higher than the extracellular concentration, resulting in a hepatocyte enrichment ratio (tissue partition coefficient, Kp) of 78 ± 6. This indicates active uptake by hepatocytes [1]
.
Metabolite Identification (MetID): In human HepatoPac cocultures incubated with 10 μM RO6889678 for 96 hours, unchanged parent drug was the most abundant component (~24% of drug-related material). The major metabolic pathway was glucuronidation (~60% of drug-related material), forming multiple acyl glucuronide conjugate isomers due to acyl migration. Minor oxidative metabolites and a glucuronide of an ester hydrolysis metabolite were also detected [1]
.
CYP450 Enzyme Induction: In a multiple-dosing study (every second day for 10 days) in HepatoPac, RO6889678 induced the mRNA expression and activity of several cytochrome P450 enzymes. After 10 days of treatment, the maximal fold induction (Emax) and concentration for half-maximal induction (EC50, in μM) were: for CYP3A4 (Emax: 9.1±0.4 for mRNA, 4.7±0.1 for activity; EC50: 0.05±0.01 for mRNA, 0.023±0.003 for activity); CYP2B6 (Emax: 13±0.5 for mRNA, 16±1.1 for activity; EC50: 0.23±0.05 for mRNA, 0.22±0.10 for activity); CYP2C8 (Emax: 3.9±0.4 for mRNA, 4.8±0.1 for activity; EC50: 0.06±0.03 for mRNA, 0.13±0.03 for activity); CYP2C9 (Emax: 4.8±0.3 for mRNA, 2.5±0.2 for activity; EC50: 0.06±0.03 for mRNA, 0.06±0.047 for activity); and CYP1A2 (Emax: 2.7±0.1 for mRNA, 3.8±0.2 for activity; EC50: 1.7±0.5 for mRNA, 0.05±0.03 for activity). No significant induction (<2-fold) was observed for CYP2D6, UGT1A1, and UGT1A3 mRNA under these conditions [1]
.
Transporter Induction: After 10 days of multiple dosing with RO6889678 in HepatoPac, mRNA levels of transporters were also induced. The Emax and EC50 values were: for OATP1B1 (Emax: 3.6±0.2; EC50: 0.06±0.03 μM), OATP1B3 (Emax: 2.8±0.1; EC50: 0.16±0.02 μM), and ABCG2 (BCRP) (Emax: 2.9±0.2; EC50: 0.09±0.04 μM). No induction was observed for NTCP and ABCB1 (P-gp) [1]
.
Disease State ADME: In HepatoPac cultures infected with HBV (~30% hepatocytes infected), the ADME profile of RO6889678 was compared to healthy controls. No significant differences were found in hepatocyte enrichment (76±14 vs. 78±6 in healthy) or apparent intrinsic clearance (5.5±0.3 vs. 5.2±0.2 μL/min/mg protein in healthy). Furthermore, the induction potential of RO6889678 on metabolic enzymes and transporters was unchanged in the diseased state [1]
.

Human Pharmacokinetics: Following single oral doses of RO6889678 (30–2000 mg as tablets) to healthy volunteers under fasted conditions, plasma concentrations were measured. At a low dose of 100 mg, the observed oral clearance was 90 L/h. However, at the highest dose of 2000 mg, the observed oral clearance was reduced to 12 L/h, indicating a more than proportional (non-linear) increase in exposure with dose. The observed maximal concentration (Cmax) at 300 mg and above was higher than predicted from low-dose data, with an approximately 17-fold higher exposure at the highest dose compared to predictions [1]
.

Metabolic Stability & MetID: RO6889678 (10 μM) was incubated in 96-well human HepatoPac cocultures at 37°C in a 5% CO2 atmosphere for up to 96 hours to assess metabolism. Samples were analyzed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (LC-MS/MS) using a Q-Exactive Orbitrap mass spectrometer. Metabolites were separated on a C18 column with a water/acetonitrile gradient containing 0.1% formic acid. Metabolites were identified by comparing product ion spectra with the parent compound and by interpreting the spectra of the found metabolites, matching the elemental composition of product ions to proposed fragment structures. The semi-quantitative abundance of metabolites was assessed based on peak areas of extracted ion chromatograms [1]
.

HBV Infection and Treatment: Human HepatoPac cocultures (24-well format) were infected with patient-derived HBV (genotype D) at 40 viral genome equivalents (vge)/cell. The infection was carried out overnight in maintenance medium supplemented with 10% fetal calf serum. The next day, the virus inoculum was removed, and fresh medium was added. After another 24 hours, the cultures were treated with RO6889678 at days 0, 2, and 4. The levels of secreted HBV antigens (HBsAg and HBeAg) in the supernatant were quantified at day 10 using chemiluminescent immunoassays (CLIA) with standards for quantification. Cell viability was monitored by measuring albumin production [1]
.
Cellular Uptake Assay: To determine active uptake, RO6889678 (at various concentrations) was incubated in 96-well human HepatoPac cocultures at 37°C in a 5% CO2 atmosphere, with and without 2 μM rifamycin (an OATP inhibitor). At time points up to 60 minutes, cells were lysed, and compound concentrations were quantified by LC-MS/MS. Uptake rates (pmol/min/mg protein) were derived from the initial linear portion of the concentration-time curve after correcting for non-specific binding using fibroblast-only control plates [1]
.
Biliary Efflux Assay: To assess biliary efflux, HepatoPac cocultures were pre-incubated for 10 minutes in either Ca²⁺/Mg²⁺-containing buffer (HBSS+/+, which maintains intact bile canaliculi) or Ca²⁺/Mg²⁺-free buffer (HBSS-/-, which disrupts tight junctions and opens bile pockets). After a wash with HBSS+/+, RO6889678 (1, 3, and 10 μM) was applied. At time points up to 20 minutes, samples were taken, and compound concentrations were quantified by LC-MS/MS. The biliary efflux rate was calculated as the difference in uptake rate between the HBSS+/+ and HBSS-/- conditions [1]
.
CYP450 Enzyme Activity Assay: The activity of specific CYP450 enzymes was measured in HepatoPac cultures after treatment with RO6889678. Cultures were incubated with probe substrates: midazolam (1 μM, for CYP3A4), bupropion (1 μM, for CYP2B6), diclofenac (1 μM, for CYP2C9), tacrine (1 μM, for CYP1A2), amodiaquine (20 μM, for CYP2C8), and SN-38 (50 μM, for UGT1A1). Metabolite formation (e.g., 1'-hydroxymidazolam, hydroxybupropion, 4-hydroxydiclofenac, hydroxytacrine, N-desethylamodiaquine, and SN-38 glucuronide) was quantified over time by LC-MS/MS. Metabolite formation rates (pmol/min/mg protein) were derived from the initial linear rate of the concentration-time curve [1]
.

General Properties: RO6889678 is a highly soluble (>1 mg/mL) and low passive permeability (0.2 x 10⁻⁶ cm/s) compound. Its logD7.4 is 0.07, and it has pKa values of 2.3 (acid) and 5.8 (base) [1]
.
Plasma Protein Binding & Blood Partition: The plasma protein binding of RO6889678 is 0.93 (i.e., 93% bound), with albumin being the major binding protein. The blood-to-plasma ratio is 0.7 [1]
.
Hepatic Uptake: RO6889678 is a substrate of organic anion transporting polypeptides OATP1B1 and OATP1B3, which mediate its active uptake into hepatocytes. In HepatoPac, the active uptake clearance was measured as 2.6 ± 0.1 μL/min/mg protein [1]
.
Metabolism: RO6889678 is metabolized via two main pathways: direct glucuronidation by UGT1A1 and UGT1A3 (the major route, ~60% of metabolism) and CYP3A4-mediated oxidation (a minor route). The glucuronide metabolites show acyl migration. The half-life of the synthesized RO6889678-glucuronide in pH 7.4 phosphate buffer was 6.6 hours, suggesting low potential for clinical reactivity [1]
.
Biliary Efflux: RO6889678 is a substrate of the efflux transporter ABCG2 (BCRP), which contributes to its biliary excretion. In HepatoPac, the biliary clearance was measured as 1.6 ± 0.1 μL/min/mg protein [1]
.
Intrinsic Clearance: In the HepatoPac long-term liver model, the apparent intrinsic clearance of RO6889678 from the extracellular medium was 5.2 ± 0.2 μL/min/mg protein. The half-life in the system was approximately 1.9 days [1]
.
Human PK Prediction: A PBPK model, integrating the HepatoPac-derived apparent intrinsic clearance and the 78-fold hepatocyte enrichment (Kp), predicted a total hepatic plasma clearance of 7.4 mL/min/kg and a volume of distribution (Vss) of 1 L/kg (compared to 0.15 L/kg predicted without accounting for active uptake). The model predicted an oral bioavailability of 41% [1]
.

Drug-Drug Interaction (DDI) as Perpetrator (Induction): RO6889678 is a potent inducer of several CYP enzymes (CYP3A4, CYP2B6, CYP2C8, CYP2C9, CYP1A2) and transporters (OATP1B1, OATP1B3, BCRP) in human hepatocytes, indicating potential for DDIs when co-administered with drugs that are substrates of these proteins [1]
.
Drug-Drug Interaction (DDI) as Victim (Inhibition): Co-incubation of RO6889678 (1 μM) with the potent CYP3A4 inhibitor ritonavir (2 μM) in HepatoPac decreased the intrinsic clearance of RO6889678 from 5.2 ± 0.2 to 0.9 ± 0.1 μL/min/mg protein, demonstrating a significant metabolic DDI where ritonavir inhibits its clearance. The hepatocyte enrichment factor remained similar (64 vs. 78) [1]
.
Complex DDI with Ritonavir: Co-treatment of RO6889678 with ritonavir revealed a complex interaction. While ritonavir inhibited CYP3A4 activity (reducing RO6889678's clearance), it concurrently induced UGT1A1 activity by 2-fold, highlighting the potential for multiple, opposing DDI mechanisms [1]
.

[1]. Simultaneous Assessment of Clearance, Metabolism, Induction, and Drug-Drug Interaction Potential Using a Long-Term In Vitro Liver Model for a Novel Hepatitis B Virus Inhibitor. J Pharmacol Exp Ther. 2018 May;365(2):237-248.

Drug Candidate Background: RO6889678 (chemical name: (3S)-4-[(4R)-4-(2-Chloro-4-fluoroheptyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic acid) is a novel and highly potent inhibitor of hepatitis B virus (HBV) capsid formation. It was designed to target the liver while maintaining moderate peripheral exposure [1]
.
Biopharmaceutics Classification: Based on its high solubility and low passive permeability, coupled with active hepatic uptake, RO6889678 is classified as a Biopharmaceutics Drug Disposition Classification System (BDDCS) class 3 drug [1]
.
Mechanism of Action: As an HBV capsid inhibitor, RO6889678 disrupts the formation of the viral capsid, a critical step in the HBV replication cycle [1]
.
Study System: The research extensively utilized the HepatoPac platform, a long-term, micropatterned coculture of human hepatocytes with murine fibroblasts, to assess multiple ADME parameters of RO6889678 in a single, physiologically relevant in vitro system, including in a model of HBV infection [1]
.

C21H20CLFN4O5S

494.9237

494.082

C, 50.96; H, 4.07; Cl, 7.16; F, 3.84; N, 11.32; O, 16.16; S, 6.48

1578153-27-1

89996234

Solid powder

-0.5

2

10

7

33

831

2

COC(=O)C1=C(NC(=N[C@H]1C2=C(C=C(C=C2)F)Cl)C3=NC=CS3)CN4CCOC[C@H]4C(=O)O

QJLRUAVQNXTCMO-RDJZCZTQSA-N

InChI=1S/C21H20ClFN4O5S/c1-31-21(30)16-14(9-27-5-6-32-10-15(27)20(28)29)25-18(19-24-4-7-33-19)26-17(16)12-3-2-11(23)8-13(12)22/h2-4,7-8,15,17H,5-6,9-10H2,1H3,(H,25,26)(H,28,29)/t15-,17-/m0/s1

(S)-4-(((R)-6-(2-Chloro-4-fluorophenyl)-5-(methoxycarbonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)morpholine-3-carboxylic acid

RO-6889678; RO 6889678;RO6889678;

2934.99.03.00

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.)