Inducing a complex interpretation network, PF-4878691 (10 μM, 4 h) maximizes responses to RNA viruses, boosts costimulatory capacity, and increases the innate antiviral immune response of plasmacytoid dendritic cells. Neurite cell death rate [2].

Inducing a complex interpretation network, PF-4878691 (10 μM, 4 h) maximizes responses to RNA viruses, boosts costimulatory capacity, and increases the innate antiviral immune response of plasmacytoid dendritic cells. Neurite cell death rate [2].

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In vitro peripheral blood mononuclear cell (PBMC)-stimulation experiments with PF-4878691 were conducted, and the antiviral efficacy of the corresponding supernatants was evaluated in a Hepatitis C Virus (HCV) replicon assay. Based on these experiments, a pharmacokinetic/pharmacodynamic (PK/PD) model suggested a separation of the antiviral response from the induction of proinflammatory cytokines. [1]
An ex vivo HCV replicon bioassay was used to assess the antiviral activity of serum samples from subjects dosed with PF-4878691. Serum from subjects receiving 6 mg and 9 mg doses showed significant, dose- and frequency-dependent inhibition of HCV replicon activity. After the fourth dose, mean inhibition reached 97% and 99% in the 6 mg and 9 mg groups, respectively. Serum from the 3 mg dose group showed minimal activity similar to placebo. [1]
When human whole blood was challenged ex vivo with a range of commercially available TLR7 agonists, PF-4878691 and other agonists were shown to upregulate TLR7 expression, indicating this is a common mechanism of action for the class. [1]

In BALB/c and C57bl/6j mice, PF-4878691 (10–150 mg, oral gavage, single dose) causes pharmacological effects [1].

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In a multiple-dose clinical study in healthy volunteers, oral PF-4878691 (3, 6, and 9 mg twice weekly for 2 weeks) induced a dose-dependent and dose-frequency-related innate immune and interferon (IFN) response. Biomarkers such as 2’5’-oligoadenylate synthetase (OAS) isoforms 1 & 2 mRNA, interferon-γ-inducible protein 10 (IP-10), and neopterin were significantly elevated in the 6 mg and 9 mg groups. [1]
A novel finding was the in vivo autoinduction of TLR7 expression in response to PF-4878691. TLR7 mRNA levels increased significantly with dose and repeated dosing, reaching mean maximal levels of 2.28-fold (6 mg) and 3.20-fold (9 mg) relative to placebo after the fourth dose. This autoinduction likely created a positive-feedback loop, amplifying the pharmacological response. [1]
Serum from subjects in the 6 mg and 9 mg groups demonstrated robust antiviral activity in the ex vivo HCV replicon assay, supporting the hypothesis that these doses are in an efficacious range. PK/PD modeling suggested that twice-weekly 6 mg doses of PF-4878691 might deliver a >1 log drop in HCV viral load after 2 weeks. [1]
The study was terminated prematurely due to serious adverse events (SAEs) at the 9 mg dose (and significant AEs at 6 mg), indicating a lack of therapeutic window. The 3 mg dose was well-tolerated but did not induce sufficient biomarker response or ex vivo antiviral activity to meet the predefined proof-of-mechanism criteria. [1]

Whole-blood transcriptional profiling: Blood was collected in specialized RNA stabilization tubes. mRNA was isolated and used for transcriptional profiling by quantitative real-time PCR (TaqMan assays). Assays were run in triplicate. The fold increase in gene expression (e.g., for TLR7, OAS-1, OAS-2) was calculated using the 2^(-ΔΔCt) method, normalized to the housekeeping gene β-actin. [1]
Ex vivo HCV replicon bioassay: Serum samples from dosed subjects were incubated with HCV replicon cells in 96-well plates for 72 hours. Luciferase assay reagents were then used to quantify replicon activity. The assay was performed in triplicate, with positive (IFN-α) and negative controls on each plate. Antiviral efficacy was expressed as percentage inhibition of HCV replicon activity. [1]
Immunophenotyping (Flow Cytometry): Blood samples were stained with fluorescent antibody cocktails targeting specific cell surface markers (e.g., CD3, CD4, CD8, CD69, CD16, CD56, HLA-DR) and their respective isotype controls. After lysing red blood cells, samples were analyzed by flow cytometry within 4 hours to quantify immune cell subsets and activation markers (e.g., CD69+ natural killer cells). [1]

Animal/Disease Models: balb/c (Bagg ALBino) mouse, C57bl/6 J mice [3]
Doses: 30 mg/kg, 60 mg/kg, 90 mg/kg, 150 mg/kg
Route of Administration: po (oral gavage)
Experimental Results: Induces dose- and time-dependent lymphopenia and 2,5-oligoadenylate synthetase (2,5, OAS). Causes cardiovascular changes. TLR7 receptor RNA was Dramatically increased.

In healthy volunteers, the median time to peak plasma concentration (Tmax) after a single oral dose of PF-4878691 (3, 6, 9 mg) ranged from 3 to 8 hours, indicating a moderate to slow rate of absorption. The terminal half-life was approximately 12 to 16 hours and was dose-independent. [1] Drug exposure (Cmax and AUC0-48) increased proportionally to the dose after multiple doses. Moderate inter-individual variability in AUC0-48 was observed. [1] One subject who experienced a serious adverse event (SAE) after a 9 mg dose had a relatively high Cmax value (10.41 ng/mL) on day 11. However, drug exposure in other subjects who experienced adverse events did not differ significantly from that in their cohort. [1]

In clinical studies, administration of PF-4878691 was associated with dose- and time-dependent adverse events (AEs), primarily in the 6 mg and 9 mg dose groups. The most consistent and clinically significant laboratory finding was acute reversible lymphopenia (decreased total lymphocyte and CD4+ T cell counts). [1] Two subjects receiving the 9 mg dose experienced serious adverse events (SAEs) characterized by flu-like symptoms (fever, chills, general pain), hypotension (one severe requiring intervention), nausea, vomiting, and severe lymphopenia. These subjects also experienced transient elevations in liver transaminases. [1] Other reported adverse events possibly associated with TLR7 agonists included fever, headache, flu-like illness, diarrhea, nausea, vomiting, and dizziness. [1] The study was terminated early due to the occurrence of serious adverse events (SAEs), indicating that the doses (6 mg and 9 mg) required to induce a potent antiviral response were not tolerated. No significant adverse events were observed at the 3 mg dose. [1]

[1]. The innate immune response, clinical outcomes, and ex vivo HCV antiviral efficacy of a TLR7 agonist (PF-4878691). Clin Pharmacol Ther. 2011 Jun;89(6):821-9.

[2]. Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists . BMC immunology, 2007, 8: 1-19.

[3]. Antiviral applications of Toll-like receptor agonists. J Antimicrob Chemother. 2012 Apr;67(4):789-801.

852A has been used in therapeutic trials for various cancers, including melanoma, tumors, breast cancer, ovarian cancer, and cervical cancer.
The TLR7 agonist 852A is a synthetic imidazoquinone Toll-like receptor 7 (TLR7) agonist with immunostimulatory and potential antitumor activity. TLR7 agonist 852A binds to and activates TLR7, thereby stimulating plasmacytoid dendritic cells (pDCs) through the TLR7-MyD88-dependent signaling pathway. Activation of pDCs leads to the secretion of interferon-α, the production of pro-inflammatory cytokines, the upregulation of co-stimulatory molecules, and enhanced T-cell and B-cell stimulation responses.
PF-4878691 (formerly known as 852A) is a potent and selective small-molecule Toll-like receptor 7 (TLR7) agonist being developed as a potential oral treatment for chronic hepatitis C virus (HCV) infection. [1]
This drug is designed to induce an endogenous antiviral interferon response with effects comparable to pegylated interferon α (Peg-IFN-α) with fewer side effects. [1]
A key mechanistic finding of this study is that TLR7 receptor expression can be self-induced in vivo after agonist stimulation. This positive feedback loop amplifies immune and biomarker responses, potentially leading to increased severity of adverse reactions after repeated dosing and narrowing of the therapeutic window. [1]
This study identified a pharmacogenomics factor: a homozygous polymorphism (Val168Leu) in the interferon-α receptor 1 (IFNAR1) gene in one subject who was unresponsive to a 9 mg dose and did not experience any adverse reactions. The functional implications of this polymorphism were not elucidated in this study. [1]
The authors concluded that although PF-4878691 successfully activated the TLR7 pathway at the predicted antiviral dose, its associated mechanistic toxicities (especially hypotension and severe lymphopenia) resulted in an insufficient therapeutic window for systemic treatment of hepatitis C virus. They suggested that future studies could explore whether very low non-systemic active doses could provide local antiviral benefits in the liver. [1]

C17H23N5O2S

361.46

361.157

532959-63-0

10309114

White to off-white solid powder

3.46

2

6

7

25

538

0

YZOQZEXYFLXNKA-UHFFFAOYSA-N

InChI=1S/C17H23N5O2S/c1-3-14-21-15-16(12-8-4-5-9-13(12)20-17(15)18)22(14)11-7-6-10-19-25(2,23)24/h4-5,8-9,19H,3,6-7,10-11H2,1-2H3,(H2,18,20)

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: ≥ 1.92 mg/mL (5.31 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 19.2 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.

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Solubility in Formulation 2: ≥ 1.92 mg/mL (5.31 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 19.2 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.

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Solubility in Formulation 3: ≥ 1.92 mg/mL (5.31 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 19.2 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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