Effect of dipterinyl calcium pentahydrate on hepatitis B virus replication in transgenic mice

Phillip Moheno 0 John Morrey Dietmar Fuchs 0 SanRx Pharmaceuticals, Inc. , 603 Colima Street, La Jolla, California 92037- 8032 , USA Background: Dipterinyl calcium pentahydrate (DCP) has previously been shown to inhibit MDA-MB-231 human breast cancer xenographs in nude mice in a manner correlated with increases in plasma IL-12 and IL-4 concentrations, and decreases in plasma IL-6 levels. DCP also inhibits indoleamine 2,3-dioxygenase (IDO), an immuno-inhibitory enzyme, in human PBMCs (Peripheral Blood Mononuclear Cells). Methods: In the present study, DCP was administered per os, once daily for 14 days to hepatitis B virus (HBV) transgenic mice at 23, 7.3, and 2.3 mg/(kg d). Multivariate stepwise regression and MANOVA analyses, by gender and treatment, of liver HBV DNA and RNA measures, liver core and serum HBe antigen assays, serum cytokine/ chemokine profiles, and IDO metabolite measurements were performed. Results: DCP caused a significant dose-response reduction of log liver HBV DNA as measured by PCR in the female HBV mice. The gender dependence of the anti-HBV DNA activity was explained by the DCP Effects Model (DCPEM) (p = .001) which includes three serum biomarker changes caused by DCP: 1) decreased MCP-1; 2) decreased Kyn/Trp (an estimation of IDO activity); and 3) increased GM-CSF. Conclusions: Immunomodulation via IDO or TDO (tryptophan 2,3-dioxygenase) pathways, along with serum MCP1 and GM-CSF are proposed to play roles in the anti-HBV mechanism of DCP based upon their coordinated modulation in the reduction of viral DNA replication in HBV mice. - Background Hepatitis B virus (HBV) causes both transient and persistent infections of the liver in humans. The number of chronic HBV carriers is estimated to be 400 million worldwide; nearly 25% of which are projected to succumb to liver failure or liver cancer [1]. Additionally, HBV infection remains an important cause of acute and chronic liver disease in the United States [2]. Dipterinyl calcium pentahydrate (DCP), shown in Figure 1, has demonstrated significant antitumor activity associated with plasma IL-12 concentration increases in MDA-MB231 (human breast cancer) xenographs in nude mice [3,4]. This finding, along with previous work demonstrating IL-12 suppression of HBV replication in transgenic mice [5], prompted us to investigate the activities of DCP in the HBV transgenic mouse model. DCP is a stable, sparingly soluble compound that can be solubilized in aqueous solutions to 440 M with sonication. For this study, an orally administered suspension was used. It was hypothesized that because of the antitumor changes elicited by DCP, as well as the antiand pro-inflammatory plasma cytokine/chemokine concentration changes reported previously, DCP would reduce liver HBV DNA levels and possibly other HBV parameters in transgenic mice carrying an infectious clone of HBV. The investigators anticipated that DCP might work via cytokine/chemokine modulatory mechanisms similar to those described by others [5-9]. Evidence for immunomodulation by DCP was further investigated by measurement and analysis of the enzyme indoleamine 2,3-dioxygenase (IDO) serum metabolites, tryptophan (Trp) and kynurenine (Kyn). Tryptophan (Trp) is the substrate for IDO, a key immunological inhibitor of T cells, and an identified tumor escape mechanism [10]. Recent studies have demonstrated that IDOmediated immune suppression is prevalent in hepatitis B infection [11]. The IDO enzymatic product, kynurenine (Kyn), and its downstream metabolites, kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, are H N H N Ca N N Figure 1 The structure of dipterinyl calcium pentahydrate, (C6H4N5O)2Ca5H2O (MW 454.4). The X-ray crystallographic structure was given previously [3]. directly involved in the regulation of T cells and other lymphocytic cell types, i.e., NK cells and B cells [12]. We have shown previously that DCP inhibits IDO activity in human PBMCs (Peripheral Blood Mononuclear Cells) [3]. Certain neurotoxic end-products of the tryptophankynurenine pathway, such as quinolinic acid, produced under chronic inflammatory conditions (e.g., cardiovascular disease, multiple sclerosis, diabetes, cancer, and major depression) may contribute to the brain damage seen in depression and dementia [13]. For the study described here, the calculation of the serum Kyn-to-Trp ratio (Kyn/ Trp) allowed us to estimate the extent of IDO activity in the serum of the HBV mice [14]. Materials and methods Materials Compounds DCP was suspended in 0.4% carboxymethylcellulose (CMC) at concentrations sufficient to deliver the desired dose by oral gavage in a volume of 0.1 mL per 20 g mouse. The solution was stored at 4C during the course of the experiment. The volume was adjusted for the weight of each mouse. The structure of DCP [3] is given in Figure 1. The positive control, adefovir dipivoxil (ADV) (Gilead Pharmaceuticals), was prepared in the same manner as the DCP for the appropriate dosages. Methods In vivo testing Animals Homozygous adult female and male transgenic HBV mice were used (20.6 2.8 g). The mice were originally obtained from Dr. Frank Chisari (Scripps Research Institute, La Jolla, CA) [15] and were subsequently raised in the Biosafety Level 3 (BL-3) area of the AAALAC-accredited USU Laboratory Animal Research Center (LARC). The animals were derived from founder 1.3.32 [15]. This study was conducted in accordance with the approval of the Institutional Animal Care and Use Committee of Utah State University. Experimental design DCP was administered per os, once daily for 14 days to 94 randomly assigned HBV transgenic mice at 23 mg/(kg d) (10 females/10 males), 7.3 mg/(kg d) (10 females/10 males), and 2.3 mg/(kg d) (10 females/10 males). DCP dosages were based upon previously reported efficacious levels for antitumor activity [3]. ADV was used as a positive control at 10 mg/(kg d) (10 females/5 males) using the same treatment schedule and vehicle (0.4% CMC). 10 female/9 male control vehicle mice were used. This efficacious ADV dosage used was previously demonstrated in HBV mice [9]. On day 14, mice were euthanized to collect serum and liver samples to perform liver HBV DNA, liver HBV RNA, liver core and serum HBe antigen assays, serum cytokine/chemokine profiles, and IDO metabolite measurements. No plasma level measurements of DCP were performed. Virology Liver HBV DNA assays Southern blot hybridization and quantitative PCR (qPCR) were performed on liver tissues [9]. For Southern blot hybridization, the ratio of the viral DNA bands to the transgene band was used to determine the concentration of viral DNA per host DNA. This calculation was based upon the knowledge that there were 1.3 copies of the transgene present per host cell with this line of transgenic mice. The transgene was used as an internal indicator to calculate the pg of HBV DNA per g of homozygous cellular host DNA. For qPCR, the assay was run with a series of 10-fold dil (...truncated)