HBsAg(IC50: 2.8 nM);HBeAg(IC50: 2.6 nM);HBV DNA(IC50: 3.2 nM); TENT; PAPD5 and PAPD7

Besides the potent activity in inhibition of HBsAg expression, RG7834 also displayed a very potent activity in inhibition of HBV DNA production in HepaG2.2.15 cells with an IC50 < 0.13 nM while the IC50 of the nucleoside agent lamivudine (3-TC) was around 25 nM.[1]
In HBV-infected dHepaRG cells, RG7834 inhibited HBsAg, HBeAg, and HBV DNA at a single digital nM concentration, while (R)-64, the enantiomer of RG7834, did not show significant inhibition against all the three viral markers at concentration up to 1 μM. The nucleoside drug ETV showed very potent activity in inhibition of HBV DNA production with an IC50 = 0.06 nM but no activity against HBsAg and HBeAg. The different antiviral profile between RG7834 and ETV indicated a differentiated MOA of RG7834 compared with nucleoside drugs.[1]
To evaluate the antiviral selectivity of RG7834, the compound was tested in a panel of 15 different DNA and RNA viruses. The IC50 of RG7834 against all these viruses was higher than 10 μM, which demonstrated the high specificity of RG7834 against HBV.[1]
To get better understanding of the MOA, we tried different approaches to identify the molecular target(s) for DHQ chemical series. Finally, the noncanonical poly(A) RNA polymerases, PAP-associated domain-containing proteins 5 and 7 (PAPD5 and PAPD7) were identified as protein targets for DHQ compounds. [1]
With IC50s of 2.8, 2.6, and 3.2 nM, respectively, RG7834 ((S)-(+)-64) is a potent and highly selective oral bioavailable HBV inhibitor that potently inhibits HBV DNA and antigens (HBsAg and HBeAg) in dHepaRG Cells[1].
CYP3A4, CYP2D6, CYP2C9 (IC50s >50 μM), and the hERG channel are not inhibited by RG7834[1].

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RG7834-induced HBV mRNA decay had two catalytic phases[2].
RG7834 suppressed poly(A) polymerase function of PAPD5/7[2].
RG7834-induced HBV RNA 3′ trimming occurred in both the nucleus and cytoplasm[2].

HBV-infected human liver chimeric uPA-SCID mice demonstrate anti-HBV efficacy in response to RG7834 (4 mg/kg, twice daily for 21 days)[1].
In mice, RG7834 (2, 14.5 mg/kg, p.o.) has a half-life of 4.9 hours and demonstrates good oral bioavailability[1].

Microsomal Stability Assay[1]
To determine the microsomal stability, microsomes were preincubated with test compound for 10 min at 37 °C in potassium phosphate buffer (100 mM, pH 7.4). The final incubation mixtures consisted of 0.5 mg of microsomal protein/mL liver microsomes, 1 mM NADP, 3 mM glucose 6-phosphate, 3 mM MgCl2, and 0.05 mg/mL glucose 6-phosphate dehydrogenase in a total volume of 400 μL of potassium phosphate buffer (100 mM, pH 7.4). The reactions were initiated with the addition of NADPH regenerating system. At different time points (0, 3, 6, 9, 15, and 30 min), an aliquot (50 μL) sample was taken and quenched with 150 μL of acetonitrile containing internal standard. Following precipitation and centrifugation, the supernatants were analyzed by LC-MS/MS.

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Plasma Protein Binding Assay[1]
The unbound compound was determined using a 96-well Micro-Equilibrium dialysis device with molecular weight cutoff membrane of 12–14 kDa. Diazepam was used as positive control. Pooled mouse and human plasma were purchased from Biopredic. Compounds were measured in a cassette of 2–5 with an initial total concentration of 1 μM, and one of the cassette compounds is the positive control. The integrity of membranes was tested by determining the unbound fraction values of the positive control. Equal volumes of blank dialysis buffer (Soerensen buffer at pH 7.4) and matrix samples containing substances were loaded into the acceptor and donor compartment, respectively. The HTD dialysis block was then sealed and kept in an incubator at 37 °C for 5 h under 5% CO2 environment. Then the drug concentrations were quantified by LC-MS/MS.

HBsAg Assay[1]
HepG2.2.15 cells were seeded in duplicate into white, 96-well plates at 1.5 × 104 cells/well. The cells were treated with a 3-fold serial dilution series of the compounds in DMSO. The final DMSO concentration in all wells was 1%, and DMSO was used as no drug control. The HBsAg chemiluminescence immunoassay (CLIA) kit was used to measure the levels of secreted HBV antigens semiquantitatively. For the detection, 50 μL/well culture supernatant was used and the procedure conducted as directed by manufacturer’s instructions. The cytotoxicity was measured using CellTiter-Glo. Using the E-WorkBook Suite, dose–response curves were generated and the IC50 and CC50 values extrapolated. The IC50 and CC50 are defined as the compound concentration (or conditioned media log dilution) at which HBsAg secrection and cytotoxicity, respectively, are reduced by 50% compared to the no drug control.

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HBV DNA Assay[1]
The assay employs real-time qPCR to directly measure extracellular HBV DNA copy number. HepG2.2.15 cells were plated in 96-well microtiter plates. Only the interior wells were utilized to reduce “edge effects” observed during cell culture, the exterior wells were filled with complete medium to help minimize sample evaporation. On the following day, the HepG2.2.15 cells were washed and the medium was replaced with complete medium containing various concentrations of a test compound in triplicate. 3TC was used as the positive control, while media alone was added to cells as a negative control. Three days later, the culture medium was replaced with fresh medium containing the appropriately diluted drug. Six days following the initial administration of the test compound, the cell culture supernatant was collected, treated with Pronase, and then used in a real-time qPCR/TaqMan assay to determine HBV DNA copy numbers. Antiviral activity was calculated from the reduction in HBV DNA levels (IC50).

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Caco-2 Assay[1]
The drug was prepared with 10 μM input drug solution in pH 7.4 HBSS. For apical to basolateral direction, 200 μL of input drug solution was added to the apical side and 700 μL of pH 7.4 HBSS (1% DMSO) to the basolateral side of the Caco-2 cells. For the basolateral to apical side direction, 700 μL of input drug solution was added to the basolateral side and 200 μL of pH 7.4 HBSS (1% DMSO) to the apical side of the Caco-2 cells. The plate was incubated for 1 h in 5% CO2 at 37 °C, 95% humidity condition. The amounts of the drugs at the apical side and basolateral side were determined, and the permeability from A to B and B to A direction was calculated.

Animal Model: HBV-infected human liver chimeric uPA-SCID mice[1]
Dosage: 4 mg/kg
Administration: Twice daily for 21 days
Result:demonstrates good oral bioavailability[1]. reduced serum HBV DNA in mice by 0.6 log10 and lowered both HBsAg and HBeAg.

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Pharmacokinetic (PK) Analysis in Mice[1]
Compound was evaluated in mice at iv 1 mg/kg, po 2 mg/kg, and po 14.5 mg/kg. Compound solutions were prepared by dissolving the solid in 5% DMSO, 40% PEG400, and 55% saline for the iv dose and 1% RC-591 in water for the oral dose. Blood samples were collected at predetermined times into sodium heparin containing tubes, and plasma was separated via centrifugation (4 °C, 8000 rpm, 6 min) and stored frozen at −80 °C pending bioanalysis. Liver samples in the po group were harvested immediately after the collection of blood. The liver samples were then rinsed with saline, dried with filter paper, and stored at −80 °C until bioanalysis. Compound concentrations in the plasma and liver samples were determined by LC–MS/MS. The data were analyzed using a noncompartmental module of WinNonlin Professional 5.2. This PK study was approved by the Institutional Animal Care and Use Committee (IACUC) of Roche Pharma Research and Early Development China.

:J Med Chem. 2018 Oct 4.

C22H27NO6

401.452886819839

401.18

C, 65.82; H, 6.78; N, 3.49; O, 23.91

2072057-18-0

199482-36-3 (S-isomer hydrate);2072057-17-9 (S-isomer);2072057-18-0 (R-isomer);1802407-46-0 (racemic);

118261819

Solid powder

3.8

1

7

8

29

685

1

CC(C)[C@H]1CC2=CC(=C(C=C2C3=CC(=O)C(=CN13)C(=O)O)OC)OCCCOC

KBXLMOYQNDMHQT-QGZVFWFLSA-N

InChI=1S/C22H27NO6/c1-13(2)17-8-14-9-21(29-7-5-6-27-3)20(28-4)10-15(14)18-11-19(24)16(22(25)26)12-23(17)18/h9-13,17H,5-8H2,1-4H3,(H,25,26)/t17-/m1/s1

(6R)-6-Isopropyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid

RG7834 R-isomer; RG 7834 R-isomer; RG-7834 R-isomer; RO7020322 R-isomer; RO 7020322 R-isomer; RO-7020322 R-isomer; RO0321; RO-0321; RO 0321;

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.)