Background: This study aimed to investigate the antitumor efficacy of Pien Tze Huang (PZH) in colorectal cancer (CRC) and elucidate its immunomodulatory mechanism, with a focus on tumor-associated macrophages (TAMs) and colony-stimulating factor 1 receptor (CSF-1R) signaling. Methods: Antitumor effects were evaluated in subcutaneous MC38 allograft and azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CRC mouse models. TAMs infiltration was analyzed via immunohistochemistry and flow cytometry. Macrophage proliferation and migration were assessed by using MTT and Transwell assays. RNA sequencing, RT-qPCR, Western blotting, cycloheximide chase, inhibitor, polysome profiling, and gene silencing assays were performed to determine the mechanism underlying CSF-1R regulation. Results: PZH suppressed mouse CRC growth in both subcutaneous MC38 allograft and AOM/DSS-induced models, with its efficacy being partly dependent on reducing TAMs infiltration. In vitro assays further revealed that PZH impaired macrophage proliferation and migration. Mechanistically, PZH selectively downregulated CSF-1R via a multilayered program involving rapid posttranslational degradation through proteasomal and lysosomal pathways, as well as receptor proteolytic cleavage mediated by the TLR2-iRhom2-ADAM17 axis. Conclusion: Our experiments identify TAMs and CSF-1R as key cellular and molecular targets of PZH, thereby highlighting its ability to modulate TAMs within the tumor microenvironment (TME) and inhibit CRC progression. These results provide mechanistic insights into the immunomodulatory actions of PZH and identify CSF-1R as a promising target for macrophage-targeted therapies in CRC.

Programmed death-ligand 1 (PD-L1) is overexpressed in multiple cancers and critical for their immune escape. It has previously shown that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability, yet its role in CRC immune escape is unclear. Here, we demonstrate that SRC-1 is positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibits PD-L1 expression in CRC cells and retards murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration of CD8+ T cells. Genetic ablation of SRC-1 in mice also decreases PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, these findings highlight a crucial role of SRC-1 in regulating PD-L1 expression and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment.

Histone demethylase JMJD2D (also known as KDM4D) can specifically demethylate H3K9me2/3 to activate its target gene expression. Our previous study has demonstrated that JMJD2D can protect intestine from dextran sulfate sodium (DSS)-induced colitis by activating Hedgehog signaling; however, its involvement in host defense against enteric attaching and effacing bacterial infection remains unclear. The present study was aimed to investigate the role of JMJD2D in host defense against enteric bacteria and its underlying mechanisms. The enteric pathogen Citrobacter rodentium (C. rodentium) model was used to mimic clinical colonic infection. The responses of wild-type and JMJD2D-/- mice to oral infection of C. rodentium were investigated. Bone marrow chimeric mice were infected with C. rodentium. JMJD2D expression was knocked down in CMT93 cells by using small hairpin RNAs, and Western blot and real-time PCR assays were performed in these cells. The relationship between JMJD2D and STAT3 was studied by co-immunoprecipitation and chromatin immunoprecipitation. JMJD2D was significantly up-regulated in colonic epithelial cells of mice in response to Citrobacter rodentium infection. JMJD2D-/- mice displayed an impaired clearance of C. rodentium, more body weight loss, and more severe colonic tissue pathology compared with wild-type mice. JMJD2D-/- mice exhibited an impaired expression of IL-17F in the colonic epithelial cells, which restricts C. rodentium infection by inducing the expression of antimicrobial peptides. Accordingly, JMJD2D-/- mice showed a decreased expression of β-defensin-1, β-defensin-3, and β-defensin-4 in the colonic epithelial cells. Mechanistically, JMJD2D activated STAT3 signaling by inducing STAT3 phosphorylation and cooperated with STAT3 to induce IL-17F expression by interacting with STAT3 and been recruited to the IL-17F promoter to demethylate H3K9me3. Our study demonstrates that JMJD2D contributes to host defense against enteric bacteria through up-regulating IL-17F to induce β-defensin expression.

Abstract Programmed death-ligand 1 (PD-L1), one of the immunosuppressive molecules, overexpresses in multiple cancers and is critical for their immune escape. We previously showed that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability; however, the role of SRC-1 in CRC immune escape is unclear. Here, we demonstrated that SRC-1 was positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibited PD-L1 expression in both human and murine CRC cells and retarded murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration and antitumor activity of effector CD8+ T cells. Genetic ablation of SRC-1 in mice also decreased PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, our findings highlight a crucial role of SRC-1 in facilitating CRC immune escape and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment. Citation Format: Yilin Hong, Qiang Chen, Zinan Wang, Yong Zhang, Bei Li, Xu Kong, Pingli Mo, Nengming Xiao, Jianming Xu, Yunbin Ye, Chundong Yu. Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1688.

Pien Tze Huang (PZH), a Class I nationally protected Traditional Chinese Medicine (TCM), has been used to treat liver diseases such as hepatitis; however, the effect of PZH on the progression of sepsis is unknown. Here, we reported that PZH attenuated lipopolysaccharide (LPS)-induced sepsis in mice and reduced LPS-induced production of proinflammatory cytokines in macrophages by inhibiting the activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalling. Mechanistically, PZH stimulated signal transducer and activator of transcription 3 (STAT3) phosphorylation to induce the expression of A20, which could inhibit the activation of NF-κB and MAPK signalling. Knockdown of the bile acid (BA) receptor G protein-coupled bile acid receptor 1 (TGR5) in macrophages abolished the effects of PZH on STAT3 phosphorylation and A20 induction, as well as the LPS-induced inflammatory response, suggesting that BAs in PZH may mediate its anti-inflammatory effects by activating TGR5. Consistently, deprivation of BAs in PZH by cholestyramine resin reduced the effects of PZH on the expression of phosphorylated-STAT3 and A20, the activation of NF-κB and MAPK signalling, and the production of proinflammatory cytokines, whereas the addition of BAs to cholestyramine resin-treated PZH partially restored the inhibitory effects on the production of proinflammatory cytokines. Overall, our study identifies BAs as the effective components in PZH that activate TGR5-STAT3-A20 signalling to ameliorate LPS-induced sepsis.

Background & Aims: HBV infection is a global health burden. Covalently closed circular DNA (cccDNA) transcriptional regulation is a major cause of poor cure rates of chronic hepatitis B (CHB) infection. Herein, we evaluated whether targeting host factors to achieve functional silencing of cccDNA may represent a novel strategy for the treatment of HBV infection.Methods: To evaluate the effects of Jumonji C domain-containing (JMJD2) protein subfamily JMJD2A-2D proteins on HBV replication, we used lentivirus-based RNA interference to suppress the expression of isoforms JMJD2A-2D in HBV-infected cells. JMJD2D-knockout mice were generated to obtain an HBV-injected model for in vivo experiments. Co-immunoprecipitation and ubiquitylation assays were used to detect JMJD2D-HBx interactions and HBx stability modulated by JMJD2D. Chromatin immunoprecipitation assays were performed to investigate JMJD2D-cccDNA and HBx-cccDNA interactions.Results: Among the JMJD2 family members, JMJD2D was significantly upregulated in mouse livers and human hepatoma cells. Downregulation of JMJD2D inhibited cccDNA transcription and HBV replication. Molecularly, JMJD2D sustained HBx stability by suppressing the TRIM14-mediated ubiquitin-proteasome degradation pathway and acted as a key co-activator of HBx to augment HBV replication. The JMJD2D-targeting inhibitor, 5C-8-HQ, suppressed cccDNA transcription and HBV replication.Conclusion: Our study clarified the mechanism by which JMJD2D regulates HBV transcription and replication and identified JMJD2D as a potential diagnostic biomarker and promising drug target against CHB, and HBV-associated hepatocarcinoma.Impact and implications: HBV cccDNA is central to persistent infection and is a major obstacle to healing CHB. In this study, using cellular and animal HBV models, JMJD2D was found to stabilise and cooperate with HBx to augment HBV transcription and replication. This study reveals a potential novel translational target for intervention in the treatment of chronic hepatitis B infection.(c) 2023 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Pien Tze Huang (PZH) is a valuable traditional Chinese medicine, which has a variety of biological activities such as clearing heat-toxin, resolving blood stasis, detoxifying, relieving pain, and anti-inflammation. PZH has a partial role in suppressing the progression of CRC, while the underlying mechanism is a pending mystery; especially whether PZH mediates the immune escape of CRC remains unclear. Our study reported that PZH suppressed the proliferative activity of CRC by inhibiting Wnt/β-catenin signaling to down-regulate the expression of PCNA and Cyclin D1. In addition, PZH suppressed the immune escape of CRC and elevated the infiltration of CD8+ T cells in tumor tissues, which depends on the suppression of PD-L1 levels via inhibiting IFNGR1-JAK1-STAT3-IRF1 signaling. More importantly, PZH pharmacologically elevated the antitumor efficacy of anti-PD-1/PD-L1 immunotherapy as demonstrated by slower tumor growth, higher infiltration and function of CD8+ T cells in the combination of PZH and PD-1/PD-L1 antibody compared with monotherapy with either agent. These results demonstrate that PZH has the potential role in inhibiting CRC proliferation and immune evasion, especially the synergistic enhancement effect of PZH on immunotherapy.

GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log 10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

Background and Aims GS‐9688 (selgantolimod) is an oral selective small molecule agonist of toll‐like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS‐9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. Approach and Results WHV‐infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS‐9688, or 3 mg/kg GS‐9688. Vehicle and 1 mg/kg GS‐9688 had no antiviral effect, whereas 3 mg/kg GS‐9688 induced a >5 log 10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS‐9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti‐WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS‐9688 was confirmed in a second woodchuck study. The antiviral response to GS‐9688 did not correlate with systemic GS‐9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS‐9688. Conclusions Finite, short‐duration treatment with a clinically relevant dose of GS‐9688 is well tolerated and can induce a sustained antiviral response in WHV‐infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS‐9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

Purpose: GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG), was designed as a cytotoxic agent that preferentially targets lymphoid cells. Our objective was to characterize the antiproliferative activity, pharmacokinetics, pharmacodynamics, and safety of GS-9219.Experimental Design: GS-9219 was selected through screening in proliferation assays and through pharmacokinetic screening. The activation pathway of GS-9219 was characterized in lymphocytes, and its cytotoxic activity was evaluated against a panel of hematopoietic and nonhematopoietic cell types. To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo, the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEG were evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with spontaneous, advanced-stage non-Hodgkin's lymphoma (NHL) following a single i.v. administration of GS-9219 as monotherapy.Results: In lymphocytes, GS-9219 is converted to its active metabolite, PMEG diphosphate, via enzymatic hydrolysis, deamination, and phosphorylation. GS-9219 has substantial antiproliferative activity against activated lymphocytes and hematopoietic tumor cell lines. In contrast, resting lymphocytes and solid tumor lines were less sensitive to GS-9219. GS-9219, but not PMEG, depleted the germinal centers in lymphoid tissues of normal beagle dogs at doses that were tolerated. In addition, GS-9219 displayed significant in vivo efficacy in five dogs with spontaneous NHL after a single administration, with either no or low-grade adverse events.Conclusion: GS-9219 may have utility for the treatment of NHL.

Purpose: GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG), was designed as a cytotoxic agent that preferentially targets lymphoid cells. Our objectivewas to characterize the antiproliferative activity, pharmacokinetics,pharmacodynamics, and safety of GS-9219. Experimental Design: GS-9219 was selected through screening in proliferation assays and through pharmacokinetic screening. The activation pathway of GS-9219 was characterized in lymphocytes, and its cytotoxic activity was evaluated against a panel of hematopoietic and nonhematopoietic cell types.To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo, the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEGwere evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with spontaneous, advanced-stage non ^ Hodgkin’s lymphoma (NHL) following a single i.v. administration of GS-9219 as monotherapy. Results: In lymphocytes, GS-9219 is converted to its active metabolite, PMEG diphosphate, via enzymatic hydrolysis, deamination, and phosphorylation. GS-9219 has substantial antiproliferative activity against activated lymphocytes andhematopoietic tumor cell lines. In contrast, resting lymphocytes and solid tumor lines were less sensitive to GS-9219. GS-9219, but not PMEG, depleted the germinal centers in lymphoid tissues of normal beagle dogs at doses that were tolerated. In addition, GS-9219 displayed significant in vivo efficacy in five dogs with spontaneous NHL after a single administration, with either no or low-grade adverse events. Conclusion:GS-9219 may have utility for the treatment of NHL. Non–Hodgkin’s lymphoma (NHL) is the second fastest growing form of cancer and the fifth leading cause of cancer deaths in the United States. The American Cancer Society estimates that the annual incidence of all forms of NHL in the United States in 2006 was 58,870 cases. In 2006, the estimated number of deaths due to NHL in the United States was 18,840 (1). Despite the introduction of rituximab in 1997, the latest Surveillance Epidemiology and End Results 5-year relative survival data (1999-2003) show a 5-year survival rate of 63% in NHL (indolent NHL ranges from 71% to 89%, depending on subtype; aggressive NHL ranges from 34% to 54%; ref. 1). Therefore, there is still a major unmet medical need in NHL patients for novel agents with improved efficacy compared with existing treatment modalities, especially in NHL patients who have failed frontline therapy. The acyclic nucleotide 9-(2-phosphonylmethoxyethyl)guanine (PMEG) forms an active phosphorylated metabolite, PMEG diphosphate (PMEGpp), in cells and causes cytotoxicity in dividing cells due to potent inhibition of the nuclear DNA polymerases a, y, and q, resulting in inhibition of DNA Cancer Therapy: Preclinical Authors’ Affiliations: Department of Research and Development, Gilead Sciences, Inc., Foster City, California; Department of Experimental Therapeutics, M. D. Anderson Cancer Center, Houston, Texas; Center for Clinical Trials and Research, School of Veterinary Medicine and Paul P. Carbone Comprehensive Cancer Center, University of Wisconsin, Madison,Wisconsin; and College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado Received 8/21/07; revised11/16/07; accepted12/13/07. Grant support:Gilead Sciences, Inc. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Conflict of interest: H. Reiser, J.Wang,W.J.Watkins, A.S. Ray, R. Shibata, G. Birkus,T. Cihlar, S.Wu, B. Li, X. Liu, I.N. Henne, G.H.I.Wolfgang, M. Desai, G.R. Rhodes, A. Fridland,W.A. Lee, and D.B. Tumas are employees and/or shareholders of Gilead Sciences, Inc. Requests for reprints: Hans Reiser, Department of Research and Development, Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404. Phone: 650522-6327; E-mail: hans.reiser@gilead.com. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-2061 www.aacrjournals.org Clin Cancer Res 2008;14(9)May1, 2008 2824 Research. on August 29, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from synthesis and/or DNA repair (2). In rodent models, PMEG has activity against leukemia and melanoma. However, the utility of PMEG as an anticancer agent is limited by its poor cellular permeability and toxicity, especially for the kidney and gastrointestinal tract (3–5). We synthesized prodrug analogues of PMEG to improve permeability and selectivity, with the hypothesis that this would lead to better efficacy and a more favorable therapeutic window. As lymphoid malignancies were the primary target of this effort, our strategy was to identify a PMEG prodrug that effectively loaded peripheral blood mononuclear cells (PBMC) with PMEGpp and resulted in minimal plasma levels of PMEG. Compounds were analyzed for their antiproliferative activity in vitro and for their cell and tissue distribution in vivo following i.v. administration. As a starting point for prodrug design, we used the N-substituted prodrug of PMEG, 9-(2-phosphonylmethoxyethyl)-N-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), due to its specific intracellular activation and ability to limit plasma exposure to the nephrotoxic agent PMEG (6, 7). Phosphonoamidate prodrugmoieties were added to increase the efficiency of lymphoid cell and tissue loading. Here, we describe a novel compound, GS-9219 (diethyl N,N ¶-[({2-[2-amino-6(cyclopropylamino)-9H-purin-9-yl]ethoxy}methyl)phosphonoyl]di-L-alaninate), which met the pharmacokinetic and potency selection criteria. The cytotoxic activity of GS-9219 was evaluated in vitro against a panel of hematopoietic and nonhematopoietic cell lines. To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo , the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEG were evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with naturally occurring, advanced-stage NHL following a single i.v. administration of GS-9219 as monotherapy. Materials andMethods Compounds. GS-9219 (succinate salt), cPrPMEDAP, PMEG, PMEGp, PMEGpp, and 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine (PMEDAP) were synthesized at Gilead Sciences, Inc. Cytarabine, cladribine, and fludarabine desphosphate were purchased from Sigma. Clofarabine was synthesized by Acme Biosciences. Deoxycoformycin was purchased from SuperGen. Cell culture and proliferation assays. All cell lines were purchased from the American Type Culture Collection and cultured in RPMI 1640 (Life Technologies/Invitrogen) supplemented with 15% fetal bovine serum (Hyclone), 2mmol/L L-glutamine (Life Technologies/Invitrogen), and antibiotics. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation using standard procedures. T and B cells were purified using antibody-conjugated magnetic beads. T lymphoblasts were generated by stimulation of CD3 T cells with 1 Ag/mL phytohemagglutinin (PHA-P; Sigma) and 10 units/mL interleukin-2 (Roche Applied Science; ref. 8). B lymphoblasts were generated by stimulation of CD19 B cells with 20 Ag/mL pokeweedmitogen (Sigma). Cell proliferation was quantified either by bromodeoxyuridine (BrdUrd) incorporation assay or by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche Diagnostics). Metabolite analysis following in vitro cell loading with [C]GS9219. Twenty million PHA-stimulated T cells were incubated with 10 Amol/L [C]GS-9219 for 24 h. Cells were washed once with tissue culture medium and twice with PBS, incubated in 80% methanol overnight, and centrifuged at 14,000 rpm for 15 min to remove denatured proteins. The methanol extracts were lyophilized and dissolved in the high-performance liquid chromatography (HPLC) loading buffer. A portion of each sample was used for scintillation counting to calculate the total pmoles, and the rest was used for HPLC analysis to calculate the ratio of metabolites. HPLC analysis was done by gradient elution using a Phenomenex Prodigy column (5 Am, ODS3 150 4 mm), where buffer A is 25 mmol/L K2HPO4 (pH 6.0) and 5 mmol/L tetrabutylammonium bromide and buffer B is 25 mmol/L K2HPO4 (pH 6.0), 70% acetonitrile, and 5 mmol/L tetrabutylammonium bromide. Pharmacokinetic studies. The plasma and PBMC pharmacokinetic profiles of GS-9219 and select metabolites were determined following a 30-min i.v. infusion of 3 mg/kg GS-9219 (formulated as a 5% dextrose solution) to three male beagle dogs. At predefined time points, blood was drawn for plasma and PBMC isolation. Isolated PBMCs were resuspended in PBS. A small aliquot of cells was used for cell counting to determine the concentration of cells. The remaining cell suspension was centrifuged to pellet cells and the pellet was resuspended in 70% methanol lyses buffer. Plasma levels of GS-9219 and cPrPMEDAP, or PBMC levels of cPrPMEDAP were determined by reverse-phase liquid chromatography using 0.2% formic acid, an acetonitrile gradient, and a Synergi Fusion-RP 80A column (150 2.1 mm, 4 Am) or a Synergi Hydro-RP 80A column (50 2 mm, 4 Am), respectively (columns purchased from Phenomenex, Inc.). Detection of analytes was accomplished by mass spectrometry using an API 4000 triple quadruple instrument (Applied Biosystems/MDS Sciex) operating in multiple reaction monitoring an