Eï¬ƒcient Drug Metabolism Strategy Based on Microsome ... Eï¬ƒcient Drug...
Embed Size (px)
Transcript of Eï¬ƒcient Drug Metabolism Strategy Based on Microsome ... Eï¬ƒcient Drug...
Efficient Drug Metabolism Strategy Based on MicrosomeMesoporous Organosilica NanoreactorsXiaoni Fang, Peng Zhang, Liang Qiao, Xiaoyan Feng, Xiangmin Zhang, Hubert H. Girault,
and Baohong Liu*,
Department of Chemistry, Institutes of Biomedical Sciences, and State Key Laboratory of Molecular Engineering of Polymers, FudanUniversity, Shanghai 200433, ChinaLaboratoire dElectrochimie Physique et Analytique, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland
*S Supporting Information
ABSTRACT: A rapid and accurate in vitro drug metabolism strategy hasbeen proposed based on the design of a biomimetic nanoreactor composedof amino-functionalized periodic mesoporous organosilica (NH2-PMO) andmicrosomes. The amphiphilic nature and positive charge of NH2-PMOmake it highly suited for the immobilization of hydrophobic and negativelycharged microsomes to form nanoreactors, which can in turn extractsubstrates from solutions. Such nanoreactors provide a suitable environ-ment to confine multiple enzymes and substrates with high localconcentrations, as well as to maintain their catalytic activities for rapidand highly effective drug metabolic reactions. Coupled with high-performance liquid chromatographymass spectrometry analysis, themetabolites of nifedipine and testosterone were quantitatively characterized,and the reaction kinetics was evaluated. Both the metabolism conversionand reaction rate were significantly improved with the NH2PMOnanoreactors compared to bulk reactions. This strategy is simple and cost-effective for promising advances in biomimeticmetabolism study.
In modern drug development, assessment of safety alwaysconsists of designed bioassays for toxicology and pharma-cokinetics, animal toxicity studies, and finally human clinicaltrials.1 Approximately 40% of drug candidates fail at the clinicaltesting stages owing to unanticipated toxicity, which drives updrug development cost significantly.2,3 Screening of toxicity atearly stages of the drug discovery process can minimize suchfailures by selecting drug candidates with acceptable levels ofboth bioactivity and toxicity.4,5 In the past decade, the numberof screenable drug targets increased dramatically with the rapiddevelopment of combinatorial chemistry and advances inbioinformatics, genomics, and proteomics.6,7 However, thegap between the number of screenable drug candidates and theapproved new drugs is widening. Therefore, development oflow-cost and high-throughput toxicity screening methods,which can address early-stage toxicology and mimic humanmetabolism of drug candidates to predict the likelihood of drugcandidate toxicity, is the major challenge in drug discovery.Human liver microsomes are enriched sources of metabolic
enzymes, such as cytochrome P450 (CYP450) and uridinediphosphoglucuronosyl transferase (UGT), which together areresponsible for nearly 80% of the metabolism of currentlymarketed drugs and are therefore widely used for in vitrometabolism and toxicity studies.8 Carlson et al.9,10 developed a384-well plate assay system for high-throughput metabolism,where microsomal enzymes were dispersed in each well of the
384-well plate to form a series of parallel assays forsimultaneous metabolism of many drug candidates. Theautomation facilitated parallel sample processing, thus enablinghigh-throughput drug metabolism screening. However, as aresult of the relatively low concentration of microsomes anddrugs, the in-solution metabolic reaction, e.g., in a well of the384-well plate, exhibited slow kinetics.To address this issue, microsome-immobilized bioreactors
have attracted wide interest and offered distinct advantages,including fast reaction kinetics, minimal consumption ofmicrosomes, good stability, long storage time, and rapidseparation of products from microsomal enzymes. Rusling etal.11,12 designed a silica microbead bioreactor coated with DNAand enzymes to measure reactive metabolites and DNA adductformation rates relevant to genotoxicity screening. Althoughthis bioreactor can contribute to the efficient analysis of majorand minor DNA adducts as well as the metabolites in less than5 min of reaction time, there is still a potential to achieve highermetabolism efficiency by simultaneously concentrating enzymesand drugs in a nanoreactor. It is then desirable to design ananoreactor with a good affinity for both multiple enzymes andmultiple drugs for their enrichment, while the motion of
Received: August 14, 2014Accepted: October 14, 2014Published: October 14, 2014
2014 American Chemical Society 10870 dx.doi.org/10.1021/ac503024h | Anal. Chem. 2014, 86, 1087010876
molecules and the contact between enzymes and drugs is notrestricted.1315
On the basis of these considerations, mesoporous materialsare considered as potential candidates to form such ananoreactor. Mesoporous materials have been reported asexcellent hosts for the immobilization of proteins and enzymesdue to the highly ordered mesoporous structure and sufficientfunctional groups for enzyme immobilization.1621Additionally,with the surface modification of mesoporous materials, selectiveenrichment of proteins, such as phosphorylated protein,22
glycosidoprotein,23 and membrane proteins, can be realized.24
Furthermore, since the local environment inside the meso-porous materials is mainly regulated by the surfacemodification, fast enzymatic reactions can occur despite thebulk buffer conditions,25,26 making it possible to performmesoporous-material-assisted enzymatic reaction in a buffercondition optimized for substrate extraction instead ofenzymatic reaction itself.It is a challenge to design a host material for the
simultaneous enrichment of different enzymes and drugs withvaried physical and chemical properties to realize complexmultistep metabolic reactions. During the in vitro drugmetabolism studies, microsomes and NADPH are normallyused, where NADPH acts as an electron donor while themicrosomal enzymes act as the catalysts of oxidativemetabolites of drug molecules by oxygen. The catalytic cycleof the CYP450 enzyme in microsomes features delivery ofelectrons from NADPH to CYP450 reductase (CPR) forsubsequent delivery to the P450 heme. The reduced heme isfurther involved in the oxidation of substrate molecules byoxygen. The microsomes are hydrophobic, while NADPH ishydrophilic.27,28 In addition, both microsomes and NADPH arenegatively charged in normal reaction solutions.29,30 Therefore,an amphiphilic periodic mesoporous organosilica (PMO) witha surface modification of amino groups to have positive surfacecharges (NH2-PMO) is designed and synthesized in this workas a suitable candidate for the coenrichment of microsomes,NADPH, and drugs.Highly efficient metabolic reactions are demonstrated with
the assistance of NH2-PMO to form a biomimetic nanoreactor.Not only does it enrich multiple enzymes and substrates toform high local concentrations, the NH2-PMO nanoreactor canalso provide a suitable microenvironment to maintain thestability and catalytic activity of the enzymes. Both a highconversion ratio and a large reaction rate constant wereachieved when using nifedipine and testosterone as targetdrugs. The quantification and structural characterization of themetabolites were well achieved by high-performance liquidchromatographymass spectrometry (HPLCMS) analysis.The NH2-PMO-based biomimetic nanoreactor strategy issimple, cost-effective, rapid, and accurate for drug metabolismanalysis. It is expected to accelerate the drug screening and leadto promising advances in drug discovery and development.
EXPERIMENTAL SECTIONChemicals. Cytochrome P450 CYP3A4 isozyme micro-
somes expressed in baculovirus-infected insect cells (human,recombinant) with cytochrome P450 reductase and cyto-chrome b5, reduced nicotinamide adenine dinucleotidephosphate (NADPH), nifedipine (98%, powder), testoster-one (98%, powder), caffeine (99%, powder), a triblockcopolymer, EO106PO70EO106 (Pluronic F127), bis-(trimethoxysilyl)ethane (BTME), 1,3,5-trimethylbenzene
(TMB), dry toluene, (3-aminopropyl)triethoxysilane(APTES), potassium chloride, and ethanol were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Methanol,acetonitrile, and methylene chloride were obtained fromDikma Technologies Inc. (Lake Forest, CA). Nifedipinesustained-release tablets were obtained from the YangtzeRiver Pharmaccutical Group (Jiangsu, China). The phosphatebuffer (PB) solution (0.1 M, pH 7.4) was prepared byKH2PO4 and K2HPO4. The stock solutions of drugs andcaffeine were prepared in methanol. NADPH was prepared inPB solution. All reagents were used as received without furtherpurification. Deionized water (18.4 M/cm) used for allexperiments was obtained from a Milli-Q system (Millipore,Bedford, MA).
Synthesis and Characterization of NH2-PMO Materi-als. PMO materials were synthesized according to a previouslyreported method.31 Briefly, 0.5 g of F127, 2.5 g of KCl, and 0.5g of TMB were dissolved in 30 g of HCl (0.1 M). After themixture was stirred for 6 h at 0 C, 2.0 g of BTME was added.The resultant mixture was hydrothermally treated at 100 C foranother 24 h after being stirred for 24 h at room temperature.The resulting powders were filtered and washed thoroughly bythe mixture of 60 mL of ethanol and 5 mL of HCl (2 M) at 60C to remove the template. The final PMO products wereobtained by filtration and dried at room temperature in air. In astandard modification process,32 PMO materials were first driedand degassed at 110 C and then dispersed in dry toluene (0.2g of PMO in 30 mL). An excess of APTES (2 mL) was addedunder stirring, and the mixture was stirred and refluxed for 24 hat 110 C. The resulting solid was filtered and washed bytoluene, dichloromethane, and ethanol three times. The finalNH2-PMO products were obtained through drying at 7