Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.

The role of serotonin in contributing to viral clearance has not been investigated in patients with chronic hepatitis C (HCV). In this prospective study we collected blood samples from 39 patients prior to and during antiviral therapy. Participants completed mood rating scales to monitor psychiatric symptoms, and serum serotonin and tryptophan levels were measured. Significant differences in serotonin levels were found between patients who achieved sustained viral responses (SVRs) and those who did not. Regression analysis revealed that serotonin was the only variable with a statistically significant relationship with antiviral therapy outcomes, even after controlling for other variables known to be associated with outcomes. Baseline serum serotonin levels, in combination with other variables such as degree of liver fibrosis, may be clinically useful for identifying patients in whom HCV can be cleared by antiviral therapy. Additional clinical predictors that could forecast treatment success are needed so that interventions to improve SVR rates, and reduce side effects, can be developed.

Activation of the immune system via administration of cytokines is used for the treatment of chronic viral infections such as hepatitis C and for cancers resistant to radiotherapy. Cytokine-based treatments induce a range of “sickness” behaviors (e.g. depression, anxiety, pain, anorexia, and fatigue). Activation of the hypothalamic pituitary-adrenal axis via the induction of corticotropin releasing factor (CRF) may underlie these unwanted side effects. This study used repeated systemic injections of the pro-inflammatory cytokine interleukin-1β (IL-1β) to model the sickness behaviors and biochemical effects of immune system activation. We assessed the ability of CRF type I receptor (CRF1) antagonism to reduce biochemical and behavioral signs of sickness induced by IL-1β treatment. Forty Wistar rats were assigned to one of four groups: 1) saline + vehicle; 2) saline + DMP904 (CRF1 antagonist); 3) IL-1β + vehicle; 4) IL-1β + DMP904. Rats received intraperitoneal injections of either DMP904 or vehicle and of IL-1β or saline for six days. Sickness behavior was evaluated using body weight assessments and forced swim testing (FST). Blood and brain samples were collected to measure cytokine, p38 mitogen activated protein kinase (MAPK), and phospho-p38 MAPK levels using multiplex techniques. There were significant reductions in body weights and FST immobility times associated with IL-1β administration. Rats administered IL-1β had significantly higher serum levels of IL-10, but not interferon-γ. Within the hippocampus, IL-1β reduced levels of p38 MAPK, but had no impact on levels of phospho-p38 MAPK except in the presence of DMP904. When administered alone, DMP904 had no significant effect on p38 MAPK or phospho-p38 MAPK in the hippocampus, but when given with IL-1β led to increased phosphorylation of p38 MAPK. IL-1β and DMP904 reduced levels of p38 MAPK within the hypothalamus, while co-administration of IL-1β and DMP904 abolished the effects of either drug alone. IL-1β decreased immobility time in the FST, and led to reductions in body weight, changes in serum cytokine levels and p38 MAPK regulation within the hippocampus and hypothalamus. DMP904 blocked some of the neurochemical effects of IL-1β, but did not impact the behavioral measures, or serum cytokines. Thus, additional studies will be needed to determine whether CRF1 antagonism is an effective treatment for cytokine-induced sickness.

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