Bingfang Yan

Anti-Influenza Prodrug Oseltamivir Is Activated by Carboxylesterase Human Carboxylesterase 1, and the Activation Is Inhibited by Antiplatelet Agent Clopidogrel

Oseltamivir is the main medicine recommended by the World Health Organization in anticipation of next influenza pandemic. This anti-influenza viral agent is an ester prodrug, and the antiviral activity is achieved by its hydrolytic metabolite: oseltamivir carboxylate. In this study, we report that the hydrolytic activation is catalyzed by carboxylesterase human carboxylesterase (HCE) 1. Liver microsomes rapidly hydrolyzed oseltamivir, but no hydrolysis was detected with intestinal microsomes or plasma. The overall rate of the hydrolysis varied among individual liver samples and was correlated well with the level of HCE1. Recombinant HCE1 but not HCE2 hydrolyzed this prodrug and produced similar kinetic parameters as the liver microsomes. Several HCE1 natural variants differed from the wild-type enzyme on the hydrolysis of oseltamivir. In the presence of antiplatelet agent clopidogrel, the hydrolysis of oseltamivir was inhibited by as much as 90% when the equal concentration was assayed. Given the fact that hydrolysis of oseltamivir is required for its therapeutic activity, concurrent use of both drugs would inhibit the activation of oseltamivir, thus making this antiviral agent therapeutically inactive. This is epidemiologically of significance because people who receive oseltamivir and clopidogrel simultaneously may maintain susceptibility to influenza infection or a source of spreading influenza virus if already infected.

Antiplatelet Agents Aspirin and Clopidogrel Are Hydrolyzed by Distinct Carboxylesterases, and Clopidogrel Is Transesterificated in the Presence of Ethyl Alcohol

Aspirin (acetylsalicylic acid) and clopidogrel are two major antithrombogenic agents that are widely used for the treatment and prevention of cerebro- and cardiovascular conditions such as stroke. Combined use produces enhanced therapeutic effect. Aspirin and clopidogrel both are esters, and hydrolysis leads to decreased or inactivated therapeutic activity. The aim of the study was to determine whether aspirin and clopidogrel are hydrolyzed by the same enzyme(s), thus reciprocally prolonging the antithrombogenic activity. To test this possibility, microsomes from the liver and intestine were assayed for the hydrolysis of aspirin and clopidogrel. In contrary to the hypothesis, aspirin and clopidogrel were hydrolyzed in a tissue-differential manner. Liver microsomes hydrolyzed both drugs, whereas intestinal microsomes hydrolyzed aspirin only. Consistent with the tissue distribution of two carboxylesterases human carboxylesterase (HCE) 1 and HCE2, recombinant HCE1 hydrolyzed clopidogrel, whereas recombinant HCE2 hydrolyzed aspirin. In addition, hydrolysis of clopidogrel among liver samples was correlated well with the level of HCE1, and hydrolysis of aspirin with HCE2. Certain natural variants differed from the wild-type enzymes on the hydrolysis of aspirin or clopidogrel. In the presence of ethyl alcohol, clopidogrel is converted to ethyl clopidogrel. Carboxylesterases are important pharmacological determinants for drugs containing ester linkages and exhibit a large interindividual variation. The isoform-specific hydrolysis of aspirin and clopidogrel suggests that these two antithrombogenic agents may have pharmacokinetic interactions with different sets of ester drugs, and the altered hydrolysis by polymorphic mutants provides a molecular explanation to the interindividual variation.

Combinatorial Photothermal and Immuno Cancer Therapy Using Chitosan-Coated Hollow Copper Sulfide Nanoparticles

Near-infrared light-responsive inorganic nanoparticles have been shown to enhance the efficacy of cancer photothermal ablation therapy. However, current nanoparticle-mediated photothermal ablation is more effective in treating local cancer at the primary site than metastatic cancer. Here, we report the design of a near-infrared light-induced transformative nanoparticle platform that combines photothermal ablation with immunotherapy. The design is based on chitosan-coated hollow CuS nanoparticles that assemble the immunoadjuvants oligodeoxynucleotides containing the cytosine-guanine (CpG) motifs. Interestingly, these structures break down after laser excitation, reassemble, and transform into polymer complexes that improve tumor retention of the immunotherapy. In this “photothermal immunotherapy” approach, photothermal ablation-induced tumor cell death reduces tumor growth and releases tumor antigens into the surrounding milieu, while the immunoadjuvants potentiate host antitumor immunity. Our results indicated that combined photothermal immunotherapy is more effective than either immunotherapy or photothermal therapy alone against primary treated and distant untreated tumors in a mouse breast cancer model. These hollow CuS nanoparticles are biodegradable and can be eliminated from the body after laser excitation.

Human carboxylesterases HCE1 and HCE2: Ontogenic expression, inter-individual variability and differential hydrolysis of oseltamivir, aspirin, deltamethrin and permethrin

Carboxylesterases hydrolyze chemicals containing such functional groups as a carboxylic acid ester, amide and thioester. The liver contains the highest carboxylesterase activity and expresses two major carboxylesterases: HCE1 and HCE2. In this study, we analyzed 104 individual liver samples for the expression patterns of both carboxylesterases. These samples were divided into three age groups: adults (>or= 18 years of age), children (0 days-10 years) and fetuses (82-224 gestation days). In general, the adult group expressed significantly higher HCE1 and HCE2 than the child group, which expressed significantly higher than the fetal group. The age-related expression was confirmed by RT-qPCR and Western immunoblotting. To determine whether the expression patterns reflected the hydrolytic activity, liver microsomes were pooled from each group and tested for the hydrolysis of drugs such as oseltamivir and insecticides such as deltamethrin. Consistent with the expression patterns, adult microsomes were approximately 4 times as active as child microsomes and 10 times as active as fetal microsomes in hydrolyzing these chemicals. Within the same age group, particularly in the fetal and child groups, a large inter-individual variability was detected in mRNA (430-fold), protein (100-fold) and hydrolytic activity (127-fold). Carboxylesterases are recognized to play critical roles in drug metabolism and insecticide detoxication. The findings on the large variability among different age groups or even within the same age group have important pharmacological and toxicological implications, particularly in relation to pharmacokinetic alterations of ester drugs in children and vulnerability of fetuses and children to pyrethroid insecticides.

A Comparative Study of Hollow Copper Sulfide Nanoparticles and Hollow Gold Nanospheres on Degradability and Toxicity

Gold and copper nanoparticles have been widely investigated for photothermal therapy of cancer. However, degradability and toxicity of these nanoparticles remain concerns. Here, we compare hollow CuS nanoparticles (HCuSNPs) with hollow gold nanospheres (HAuNS) in similar particle sizes and morphology following intravenous administration to mice. The injected pegylated HCuSNPs (PEG-HCuSNPs) are eliminated through both hepatobiliary (67 percentage of injected dose, %ID) and renal (23 %ID) excretion within one month postinjection. By contrast, 3.98 %ID of Au is excreted from liver and kidney within one month after iv injection of pegylated HAuNS (PEG-HAuNS). Comparatively, PEG-HAuNS are almost nonmetabolizable, while PEG-HCuSNPs are considered biodegradable nanoparticles. PEG-HCuSNPs do not show significant toxicity by histological or blood chemistry analysis. Principal component analysis and 2-D peak distribution plots of data from matrix-assisted laser desorption ionization-time-of-flight imaging mass spectrometry (MALDI-TOF IMS) of liver tissues demonstrated a reversible change in the proteomic profile in mice receiving PEG-HCuSNPs. This is attributed to slow dissociation of Cu ion from CuS nanoparticles along with effective Cu elimination for maintaining homeostasis. Nonetheless, an irreversible change in the proteomic profile is observed in the liver from mice receiving PEG-HAuNS by analysis of MALDI-TOF IMS data, probably due to the nonmetabolizability of Au. This finding correlates with the elevated serum lactate dehydrogenase at 3 months after PEG-HAuNS injection, indicating potential long-term toxicity. The comparative results between the two types of nanoparticles will advance the development of HCuSNPs as a new class of biodegradable inorganic nanomaterials for photothermal therapy.

DEC1, a basic helix-loop-helix transcription factor and a novel target gene of the p53 family, mediates p53-dependent premature senescence.

Cellular senescence plays an important role in tumor suppression. p53 tumor suppressor has been reported to be crucial in cellular senescence. However, the underlying mechanism is poorly understood. In this regard, a cDNA microarray assay was performed to identify p53 targets involved in senescence. Among the many candidates is DEC1, a basic helix-loop-helix transcription factor that has been recently shown to be up-regulated in K-ras-induced premature senescence. However, it is not clear whether DEC1 is capable of inducing senescence. Here, we found that DEC1 is a novel target gene of the p53 family and mediates p53-dependent premature senescence. Specifically, we showed that DEC1 is induced by the p53 family and DNA damage in a p53-dependent manner. We also found that the p53 family proteins bind to, and activate, the promoter of the DEC1 gene. In addition, we showed that overexpression of DEC1 induces G1 arrest and promotes senescence. Moreover, we found that targeting endogenous DEC1 attenuates p53-mediated premature senescence in response to DNA damage. Furthermore, overexpression of DEC1 induces cellular senescence in p53-knockdown cells, albeit to a lesser extent. Finally, we showed that DEC1-induced senescence is p21-independent. Taken together, our data provided strong evidence that DEC1 is one of the effectors downstream of p53 to promote premature senescence.

The expression of antiapoptotic protein survivin is transcriptionally upregulated by DEC1 primarily through multiple sp1 binding sites in the proximal promoter.

Human differentially expressed in chondrocytes (DEC), mouse stimulated with retinoic acid and rat split and hairy related proteins constitute a structurally distinct class of the basic helix-loop-helix proteins. DEC1 is abundantly expressed in tumors and protects against apoptosis induced by serum starvation. In this study, we report that DEC1 antiapoptosis is achieved by inducing survivin, an antiapoptotic protein. In paired tumor–normal tissues, survivin and DEC1 exhibited a paralleled expression pattern. Tetracycline-induced expression of DEC1 in stable lines proportionally increased the expression of survivin. In reporter assays, DEC1 transactivated the survivin promoter but repressed the DEC2 promoter. In contrast to the repression, the activation was delayed and varied depending on serum concentrations and cycle blockers. Studies with reporter mutants located, in the survivin promoter, two Sp1 sites that supported DEC1 transactivation. Electrophoretic mobility shift assay and chromatin immunoprecipitation detected the presence of DEC1 in the survivin promoter. These findings establish that the survivin gene is a transcription target of DEC1, and induction of survivin is at least in part responsible for DEC1 antiapoptosis.

Hollow Copper Sulfide Nanoparticle-Mediated Transdermal Drug Delivery

A photothermal ablation-enhanced transdermal drug delivery methodology is developed based on hollow copper sulfide nanoparticles (HCuSNPs) with intense photothermal coupling effects. Application of nanosecond-pulsed near-infrared laser allows rapid heating of the nanoparticles and instantaneous heat conduction. This provides very short periods of time but extremely high temperatures in local regions, with focused thermal ablation of the stratum corneum. The depth of skin perforations can be controlled by adjusting the laser power. Skin disruption by HCuSNP-mediated photothermal ablation significantly increases the permeability of human growth hormone. This technique offers compelling opportunities for macromolecular drug and vaccine delivery.

Significantly reduced cytochrome P450 3A4 expression and activity in liver from humans with diabetes mellitus.

BACKGROUND AND PURPOSE Patients with diabetes mellitus require pharmacotherapy with numerous medications. However, the effect of diabetes on drug biotransformation is not well understood. Our goal was to investigate the effect of diabetes on liver cytochrome P450 3As, the most abundant phase I drug‐metabolizing enzymes in humans.

Interleukin-6 Alters the Cellular Responsiveness to Clopidogrel, Irinotecan, and Oseltamivir by Suppressing the Expression of Carboxylesterases HCE1 and HCE2

Carboxylesterases constitute a class of enzymes that play important roles in the hydrolytic metabolism of drugs and other xenobiotics. Patients with liver conditions such as cirrhosis show increased secretion of proinflammatory cytokines [e.g., interleukin-6 (IL-6)] and decreased capacity of hydrolysis. In this study, we provide a molecular explanation linking cytokine secretion directly to the decreased capacity of hydrolytic biotransformation. In both primary hepatocytes and HepG2 cells, treatment with IL-6 decreased the expression of human carboxyl-esterases HCE1 and HCE2 by as much as 60%. The decreased expression occurred at both mRNA and protein levels, and it was confirmed by enzymatic assay. In cotransfection experiments, both HCE1 and HCE2 promoters were significantly repressed, and the repression was comparable with the decrease in HCE1 and HCE2 mRNA, suggesting that transrepression is responsible for the suppressed expression. In addition, pretreatment with IL-6 altered the cellular responsiveness in an opposite manner of overexpression of HCE1 and HCE2 toward various ester therapeutic agents (e.g., clopidogrel). Transfection of HCE1, for example, decreased the cytotoxicity induced by antithrombogenic agent clopidogrel, whereas pretreatment with IL-6 increased the cytotoxicity. Such a reversal was observed with other ester drugs, including anticancer agent irinotecan and anti-influenza agent oseltamivir. The altered cellular responsiveness was observed when drugs were assayed at sub- and low-micromolar concentrations, suggesting that suppressed expression of carboxylesterases by IL-6 has profound pharmacological consequences, particularly with those that are hydrolyzed in an isoform-specific manner.