Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results have demonstrated that capsaicinoids contained in pepper spray products produce airway inflammation and cause respiratory epithelial cell death. The mechanisms of these cellular responses to capsaicinoids appear to proceed via distinct cellular pathways, but both pathways are initiated by TRPV1.
ABSTRACT:Fa2N-4 cells have been proposed as a tool to identify CYP3A4 inducers. To evaluate whether Fa2N-4 cells are a reliable surrogate for cryopreserved human hepatocytes, we assessed the basal mRNA expression of 64 drug disposition genes in Fa2N-4 cells. Significant differences were found in the expression of major drugmetabolizing enzymes, nuclear receptors, and transporters between both cell types. Importantly, the expression of constitutive androstane receptor (CAR) and several hepatic uptake transporters was significantly lower (>50-fold) in Fa2N-4 cells, whereas the expression of pregnane X-receptor (PXR) and aryl hydrocarbon receptor (
ABSTRACT:The rhesus monkey (Macaca mulatta) is a primate species used extensively as a preclinical safety species in drug development. In this report, we describe the cloning, expression, and characterization of CYP3A64 (AY334551), a CYP3A4 homolog expressed in rhesus liver. The deduced amino acid sequence was found to be 93% similar to human CYP3A4, 83% similar to human CYP3A5, and identical to the previously reported cynomolgus monkey CYP3A8 Cytochromes P450 (P450s) are a superfamily of enzymes involved in the elimination of a wide variety of chemical xenobiotics including pharmaceuticals, carcinogens, and environmental pollutants (Wrighton and Stevens, 1992). CYP3A4 is the most abundant of these enzymes in humans and is responsible for the biotransformation of nearly 50% of all pharmaceuticals (Guengerich, 1995). Substrates for CYP3A4 include such structurally distinct molecules as testosterone, nifedipine, lidocaine, lovastatin, erythromycin, cyclosporine, diazepam, midazolam, and coumarins.Rhesus monkeys (Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis) are widely used throughout the pharmaceutical industry as preclinical safety species. Much emphasis is placed on the overall drug safety profile and the extrapolated CYP3A metabolic activities of monkeys to corresponding human drug metabolism variables, even though very little information is known about monkey CYP3A enzymes or their metabolic capabilities. There are relatively few reports, compared with rat and human, regarding the specific activity of rhesus and cynomolgus monkey CYP3A enzyme activities, and most of these reports are from purified regenerated systems or liver homogenates and not from recombinantly expressed enzyme systems (Ohta et al., 1989;Ohmori et al., 1993;Ramana and Kohli, 1999;Matsunaga et al., 2002). Although it is believed that monkey CYP3A metabolic capabilities should be similar to those of human CYP3A, there has not been an in-depth investigation of the enzymatic properties of the individual monkey CYP3A isoforms. Therefore, we sought to clone, express, and characterize the major CYP3A4-like drug-metabolizing enzyme from rhesus monkey liver.Based on its predicted homology to human CYP3A4, we cloned the CYP3A4 homolog from rhesus monkeys. The cloned cDNA was expressed using a commonly used insect cell expression system and characterized with multiple probe substrates. Insect cells offer several advantages over other expression systems: 1) they do not require alteration of the P450 coding sequence for expression, 2) they lack endogenous cytochromes P450, and 3) they can be used to make microsomes or coexpressed with NADPH-P450 oxidoreductase (OR) to produce Supersomes (Gonzalez and Korzekwa, 1995;Crespi and Miller, 1999).It is valuable to understand the metabolic capabilities of preclinical animal models to accurately predict safety and clearance profiles of pharmaceutical candidates in development. Therefore, in addition to the evaluation of the rhesus CYP3A64 enzyme activity in relationship Article, publication date, ...
Efficient siRNA delivery is dependent not only on the ability of the delivery vehicle to target a specific organ but also on its ability to enable siRNA entry into the cytoplasm of the target cells. Polymers with endosomolytic properties are increasingly being used as siRNA delivery vehicles due to their potential to facilitate endosomal escape and intracellular delivery. Addition of disulfide bonds in the backbone of these polymers was expected to provide degradability through reduction by glutathione in cytosol. This paper describes the synthesis of new endosomolytic bioreducible poly(amido amine disulfide) polymers whose lytic potential can be masked at physiological pH, but can be restored at acidic endosomal pH. These polymer conjugates gave good in vitro knockdown (KD) and did not demonstrate cytotoxicity in a MTS assay. Efficient mRNA KD for apolipoprotein B in mouse liver was observed with these polyconjugates following intravenous dosing.
Antiretroviral therapy (ART) limits human immunodeficiency virus 1 (HIV-1) replication but does not eliminate the long-lived reservoir established shortly after viral acquisition. A successful HIV cure intervention necessitates either elimination or generation of long-term immune control of the persistent viral reservoir. Immune modulating strategies in conjunction with ART hold promise for achieving cure by inducing viral antigen expression and augmenting infected cell killing.
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