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].

---

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.

---

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.

---

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).

---

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.

---

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.

[1]. Discovery of RG7834: The First-in-Class Selective and Orally Available Small Molecule Hepatitis B Virus Expression Inhibitor with Novel Mechanism of Action. J Med Chem. 2018 Oct 4.

[2]. The Dihydroquinolizinone Compound RG7834 Inhibits the Polyadenylase Function of PAPD5 and PAPD7 and Accelerates the Degradation of Matured Hepatitis B Virus Surface Protein mRNA. Antimicrob Agents Chemother . 2020 Dec 16;65(1):e00640-20.

Chronic hepatitis B virus (HBV) infection is a serious public health burden, and current therapies cannot achieve satisfactory cure rate. There are high unmet medical needs of novel therapeutic agents with differentiated mechanism of action (MOA) from the current standard of care. RG7834, a compound from the dihydroquinolizinone (DHQ) chemical series, is a first-in-class highly selective and orally bioavailable HBV inhibitor which can reduce both viral antigens and viral DNA with a novel mechanism of action. Here we report the discovery of RG7834 from a phenotypic screening and the structure-activity relationship (SAR) of the DHQ chemical series. RG7834 can selectively inhibit HBV but not other DNA or RNA viruses in a virus panel screening. Both in vitro and in vivo profiles of RG7834 are described herein, and the data support further development of this compound as a chronic HBV therapy.[1]

---

Hepatitis B virus (HBV) mRNA metabolism is dependent upon host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 are cellular, noncanonical, poly(A) polymerases (PAPs) whose main function is to oligoadenylate the 3' end of noncoding RNA (ncRNA) for exosome degradation. HBV seems to exploit these two ncRNA quality-control factors for viral mRNA stabilization, rather than degradation. RG7834 is a small-molecule compound that binds PAPD5/7 and inhibits HBV gene production in both tissue culture and animal study. We reported that RG7834 was able to destabilize multiple HBV mRNA species, ranging from the 3.5-kb pregenomic/precore mRNAs to the 2.4/2.1-kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7-kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization was initiated by a shortening of the poly(A) tail, followed by an accelerated degradation process in both the nucleus and cytoplasm. In cells expressing HBV mRNA, both PAPD5/7 were found to be physically associated with the viral RNA, and the polyadenylating activities of PAPD5/7 were susceptible to RG7834 repression in a biochemical assay. Moreover, in PAPD5/7 double-knockout cells, viral transcripts with a regular length of the poly(A) sequence could be initially synthesized but became shortened in hours, suggesting that participation of PAPD5/7 in RNA 3' end processing, either during adenosine oligomerization or afterward, is crucial for RNA stabilization.[2]

C22H27NO6

401.452886819839

401.18

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

2072057-17-9

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

118261815

White to off-white solid powder.

3.8

85.3Ų

O=C(C1=CN2[C@H](C(C)C)CC3=CC(OCCCOC)=C(OC)C=C3C2=CC1=O)O

KBXLMOYQNDMHQT-KRWDZBQOSA-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-/m0/s1

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

RG7834; RG 7834; RG-7834; RO7020322; RO 7020322; RO-7020322

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 : ~125 mg/mL (~311.37 mM)

Solubility in Formulation 1: ≥ 2.87 mg/mL (7.15 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (5.18 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.18 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (5.18 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

---

Solubility in Formulation 5: 5% DMSO+95% (20% SBE-β-CD in Saline): ≥ 2.87 mg/mL (7.15 mM)

---

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