Hepatocyte Concentrations of Imaging Compounds Associated with Transporter Inhibition: Evidence in Perfused Rat Livers.

In the liver, several approaches are used to investigate and predict the complex issue of drug-induced transporter inhibition. These approaches include in vitro assays and pharmacokinetic models that predict how inhibitors modify the systemic and liver concentrations of the victim drugs. Imaging is another approach that shows how inhibitors might alter liver concentrations stronger than systemic concentrations. In perfused rat livers associated with a gamma counter that measures liver concentrations continuously, we previously showed how fluxes across transporters generate the hepatocyte concentrations of two clinical imaging compounds, one with a low extraction ratio [gadobenate dimeglumine (BOPTA)] and one with a high extraction ratio [mebrofenin (MEB)]. BOPTA and MEB are transported by rat organic anion transporting polypeptide and multiple resistance-associated protein 2, which are both inhibited by rifampicin. The aim of the study is to measure how rifampicin modifies the hepatocyte concentrations and membrane clearances of BOPTA and MEB and to determine whether these compounds might be used to investigate transporter-mediated drug-drug interactions in clinical studies. We show that rifampicin coperfusion greatly decreases BOPTA hepatocyte concentrations, but increases those of MEB. Rifampicin strongly decreases BOPTA hepatic clearance. In contrast, rifampicin decreases moderately MEB hepatic clearance and blocks the biliary intrinsic clearance, increasing MEB hepatocyte concentrations. In conclusion, low concentrations prevent the quantification of BOPTA biliary intrinsic clearance, while MEB is a promising imaging probe substrate to evidence transporter-mediated drug-drug interactions when inhibitors act on influx and efflux transporters.

Hepatocyte Concentrations of Indocyanine Green Reflect Transfer Rates Across Membrane Transporters.

Perioperative imaging with indocyanine green (ICG) is developing to increase safety in dissecting anatomical structures during hepatobiliary surgery. Images obtained with the fluorescence camera rely on concentrations measured in liver regions of interest. However, how ICG sinusoidal uptake and hepatocyte elimination rates generate ICG hepatocyte concentrations is largely unknown. To investigate such issue and better understand the role of membrane transporters in generating ICG hepatocyte concentrations, we perfused ICG in livers isolated from normal livers. Whether the well-known transporter inhibitor rifampicin modifies hepatocyte ICG concentrations was also studied. The dye has a very high and constant extraction ratio (96%) into hepatocytes. This persistent high extraction ratio generates a huge uphill concentration gradient across the sinusoidal membrane: from 5 µM (sinusoids) to 1600 µM (liver). When inside hepatocytes, ICG has low hepatocyte elimination (7 nmol/min.) and liver concentrations do not decrease much over time. Moreover, the tiny hepatocyte ICG efflux is mainly due to ICG return back to sinusoids (90%). Rifampicin slightly inhibits ICG uptake into hepatocytes and when inside hepatocytes blocks ICG efflux into bile canaliculi. In contrast, it increases ICG efflux back to sinusoids with significant decrease in ICG liver concentrations. Imaging with ICG in the perioperative period reflects the high hepatocyte concentrations and relies on the high extraction ratio across hepatocyte sinusoidal membrane. Although ICG concentrations are low in bile ducts, they are adequate for a good visualization and avoid bile duct injury.

Quantification of drug transport function across the multiple resistance-associated protein 2 (Mrp2) in rat livers.

To understand the transport function of drugs across the canalicular membrane of hepatocytes, it would be important to measure concentrations in hepatocytes and bile. However, these concentration gradients are rarely provided. The aim of the study is then to measure these concentrations and define parameters to quantify the canalicular transport of drugs through the multiple resistance associated-protein 2 (Mrp2) in entire rat livers. Besides drug bile excretion rates, we measured additional parameters to better define transport function across Mrp2: (1) Concentration gradients between hepatocyte and bile concentrations over time; and (2) a unique parameter (canalicular concentration ratio) that represents the slope of the non-linear regression curve between hepatocyte and bile concentrations. This information was obtained in isolated rat livers perfused with gadobenate dimeglumine (BOPTA) and mebrofenin (MEB), two hepatobiliary drugs used in clinical liver imaging. Interestingly, despite different transport characteristics including excretion rates into bile and hepatocyte clearance into bile, BOPTA and MEB have a similar canalicular concentration ratio. In contrast, the ratio was null when BOPTA was not excreted in bile in hepatocytes lacking Mrp2. The canalicular concentration ratio is more informative than bile excretion rates because it is independent of time, bile flows, and concentrations perfused in portal veins. It would be interesting to apply such information in human liver imaging where hepatobiliary compounds are increasingly investigated.

IL-2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes.

Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.

Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection.

Ideal vaccines should be stable, safe, molecularly defined, and out-of-shelf reagents efficient at triggering effector and memory Ag-specific T cell-based immune responses. Dendritic cell-derived exosomes could be considered as novel peptide-based vaccines because exosomes harbor a discrete set of proteins, bear functional MHC class I and II molecules that can be loaded with synthetic peptides of choice, and are stable reagents that were safely used in pioneering phase I studies. However, we showed in part I that exosomes are efficient to promote primary MHC class I-restricted effector CD8(+) T cell responses only when transferred onto mature DC in vivo. In this work, we bring evidence that among the clinically available reagents, Toll-like receptor 3 and 9 ligands are elective adjuvants capable of triggering efficient MHC-restricted CD8(+) T cell responses when combined to exosomes. Exosome immunogenicity across species allowed to verify the efficacy of good manufactory procedures-manufactured human exosomes admixed with CpG oligonucleotides in prophylactic and therapeutic settings of melanoma in HLA-A2 transgenic mice. CpG adjuvants appear to be ideal adjuvants for exosome-based cancer vaccines.