ANA975, a 5-amino-3-beta -D-ribofuranosyl-3H-thiazolo[4,5-d]pyrimidin-2-one derivative, was synthesized in the search of an oral prodrug of isatoribine, a small molecule toll-like receptor 7 (TLR-7) agonist. Several strategies were studied to enable the kilogram-scale synthesis of ANA975. Three general total syntheses are described. In the phase I clinical study of ANA975 against hepatitis C virus (HCV), conversion to isatoribine in plasma was rapid and effective, delivering levels of isatoribine that have been shown to be clinically relevant.

The discovery of endosomal TLRs (Toll-like receptors) and their natural ligands has accelerated efforts to exploit them for therapeutic benefit. Importantly, this was preceded by clinical exploration of agents now known to be endosomal TLR agonists. Clinical effects in viral disease have been reported with agonists of TLR3, TLR7, TLR7/8 and TLR9, and the TLR7 agonist imiquimod is marketed for topical use against warts, a papillomavirus disease. The observed pre-clinical and clinical profiles of agonists of each of these TLRs suggest induction of a multifaceted innate immune response, with biomarker signatures indicative of type 1 interferon induction. However, these agents differ in both their pharmaceutical characteristics and the cellular distribution of their target TLRs, suggesting that drugs directed to these targets will display differences in their overall pharmacological profiles.

3556 The innate immune response provides the first line of defense against tumor cells by inducing a cascade of immunomodulatory events that includes the secretion of cytokines and the stimulation of natural killer (NK) cells. NK cells mediate anti-tumor activity by two different mechanisms. First, NK cells direct the lysis of tumor cells through the secretion of cytotoxic granules and promote apoptosis through the production of IFN-γ. The latter also increases the anti-tumor response of other immune cells. Second, NK cells also recognize and kill transformed cells through antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are activated by diverse signals, including type I interferons (IFN). Large amounts of type I IFN are produced by plasmacytoid dendritic cells (pDCs) upon engagement of the pattern recognition receptor toll-like receptor 7 (TLR7). The natural ligand for TLR7 is single stranded RNA, although a variety of small molecule agonists have also been discovered. This study investigated the effects of such a low molecular weight TLR7 agonist on human NK cell activity. ANA773 is an oral prodrug of a selective TLR7 agonist. This biologically active metabolite of ANA773 induced the secretion of IFN-α and various other cytokines from human peripheral blood mononuclear cells (PBMC) cultured in vitro. Cytokine levels were also elevated in the plasma of treated primates. Human PBMC stimulated with the active metabolite of ANA773 augmented NK cell cytotoxicity and cytokine secretion against both K562 erythroleukemic cells and transformed B cell lines. These effects were shown to be dependent on the presence of type I IFN, confirming the importance of IFN-α in mediating NK responses to TLR7 agonists. In addition to enhancing NK activation through the direct recognition of tumor cells, this small molecule TLR7 agonist also enhanced ADCC; treatment of human PBMC with the active metabolite augmented cytolysis of rituximab (anti-CD20) bound, CD20-expressing B cell tumor lines. Thus, the active metabolite of ANA773 promotes NK cell mediated anti-tumor response by promoting cytokine secretion, cytolysis of tumor cells, and antibody-dependent tumor cell lysis. These data indicate that TLR7 is an attractive therapeutic target for the treatment of tumor malignancies.

Toll-like receptor (TLR) 7 is a pattern-recognition receptor that activates the innate immune response. Stimulation of TLR7 induces type I interferons, pro- inflammatory cytokines, the upregulation of co-stimulatory molecules and leads to the development of an adaptive immune response. Small-molecule TLR7 agonists with broad-spectrum antiviral activities in animal models have been identified. Such compounds have been examined clinically for a number of different infectious disease indications, leading to marketing approval of one of these agents, imiquimod, for the topical treatment of external genital and perianal papillomavirus infections. In contrast with topical and intravenous routes of administration, compounds delivered orally have exhibited poor tolerability at desired doses, with substantial adverse events associated with gastrointestinal toxicity. However, dosing with masked oral prodrugs of TLR7 agonists, such as ANA-975, limits the adverse events associated with the activation of responsive gastrointestinal immune tissue. As a consequence, the treatment of systemic diseases, such as chronic hepatitis C, can now be explored with orally administered TLR7 agonists.

Immune‐based therapy is the mainstay treatment for chronic hepatitis C virus (HCV) infection but causes multiple side effects and achieves durable viral clearance in only approximately 50% of patients. Most new investigational anti‐HCV compounds are direct‐acting antivirals for which durability of response and risk of viral mutations and resistance are not yet known. Therefore, continuing discovery and development of new immune‐based treatments is desirable. Toll‐like receptors (TLRs) are pathogen recognition receptors that initiate the innate immune response. The responsiveness of HCV or other ongoing chronic systemic infections to treatment with a selective TLR agonist has not been reported. Isatoribine is a selective agonist of TLR7. In a proof‐of‐concept study, we found that once‐daily 7‐day treatment with intravenous isatoribine 800 mg caused a significant (P = .001) reduction of plasma HCV RNA (mean, −0.76; range, −2.85 to +0.21 log10 units) in otherwise untreated patients (n = 12) who were chronically infected with HCV. Viral load reduction occurred in patients infected with genotype 1 as well as non‐genotype 1 HCV. The reduction of viral load was correlated with induction of markers of a heightened immune antiviral state, including 2′‐, 5′‐ oligoadenylate synthetase levels in whole blood. This treatment was well tolerated, with a low frequency of mild to moderate adverse events. In conclusion, systemic administration of the selective TLR7 agonist isatoribine resulted in dose‐dependent changes in immunologic biomarkers and a statistically significant antiviral effect with relatively few and mild side effects. (HEPATOLOGY 2005;42:724–731.)

ABSTRACT The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-α), IFN-γ, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-α, IFN-γ, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-α and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.

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