| It is well known that protein is the essencially structural material for organisms. One of the fundamental functions of protein is the capability of reversible binding with different small molecules, especially with drugs which can greatly influence drugs’ in vivo processes. Thus, the knowledge about protein-drug interaction, involving how do different drugs bind to protein and how strong are the bindings, is critical to provide information for pharmacodynamics elucidation, pharmacokinetics investigation and new drug designation.Saliva is the ultrafiltration of blood, and many specimens including drugs in saliva are closely related to those in blood. Saliva drug concentrations, which generally equal to blood levels, can well reflect effective therapeutic drug concentrations. Thus, the development of highly sensitive approach for saliva drug determination is of pivotal importance in non-invasive therapeutic drug monitoring and saliva pharmacokinetics.Herein, the interaction behaviors of model protein bovine serum albumin (BSA) with19small molecules (macrolides, tetracyclines, sulfonamides, and diphacinone DPN, etc.) were studied by flow injection chemiluminescence (FI-CL) analysis and molecular docking, giving binding parameters (K, n), and thermodynamic parameters (ΔH, ΔS,ΔG). The correlations of the binding affinities of antibiotics to BSA vs structural properties of antibiotics were also extensively investigated. By using non-invasive sampling, saliva pharmacokinetics for roxithromycin ROX and clarithromycin CLA were studied by FI-CL analysis, with absorption rate constant ka, elimination rate constant ke, elimination half-life time t1/2and total apparent clearance Cltotal obtained. This dissertation contained three parts:Part1IntroductionChapter1:254references mainly from recent five years were cited. The significance of interaction investigation of protein and small molecules (especially drugs) with common utilized model proteins, small molecules, researching technologies and theoretic models were reviewed; the importance of saliva drug pharmacokinetics with current applications of saliva matrix were summarized; the backgrounds and experimental designation of this dissertation were given with full contents outlined.Part2Research reportsChapter2:Interaction study of BSA with antibiotics by FI-CL analysis1. It was found that BSA could accelerate the electron transfer rate of excited3-aminophthalate leading to remarkable enhanced CL intensity from luminol, based on which the CL system of luminol-BSA was studied. Results showed that the CL intensity from luminol-BSA system could be quenched by macrolides (erythromycin ERY, kitasamycin KIT, midecamycin MID, josamycin JOS, erythromycin ethylsuccinate ERE, josamycin propionate JOP and midecamycin acetate MIA), tetracyclines (tetracycline TET, oxytetracycline OXY and chlortetracycline CHL) and sulfonamides (sulfanilamide SNM, sulfaguanidine SGD, sulfadizine SDZ and sulfamethazine SMZ). The CL intensity decrements were logarithm over antibiotics’ concentrations, yielding a general calibration equation of ΔI=AlogCantibiotic+B (r>0.99, Cantibiotie:1.0～5000pmol·L-1), with the quenching proportionalities in the monotonic increasing order of ERY<KIT<MID<JOS<ERE<JOP<MIA<TET<OXY<CHL<SNM<SGD<SDZ<SMZ.2. By homemade FI-CL model of protein-small molecule interaction, the bindingparameters (K:103～105L·mol-1, n:approximately1.0) of antibiotics to BSA were obtained, with K values generally obeying the sequence:macrolides<tetracyclines<sulfonamides. Results showed that the ester groups substituting for-OH at R1～R4in macrolides,-Cl or-OH at R1～R2in tetracyclines, and the additional-C(=NH)NH2at R1in sulfonamides could increase the K values. Thermodynamic parameters (ΔH:-40.67～28.04kJ·mol-1, AS:-33.34～189.40J·mol·L-1, ΔG:-33.82～-18.35kJ·mol-1) revealed the main stable force for the spontaneously formed complexes of BSA with mcrolides, tetracyclines and sulfonamides was hydrophobic interaction, hydrogen bond and van der Waals force, and electrostatic interaction, respectively.3. It was found that the logK values increased with increasing determination sensitivityfactor A and intercept B (r>0.98), with B first defined as quenching efficiency factor. Further investigation revealed that antibiotics’ basic physicochemical descriptors including molecular size (molecular weight MW, molecular volume MV), steric property (molar refractivity MR, polarizability POL) and hydrophobicity (partition coefficient logP,bioconcentration factor logBCF, distribution coefficient logD) were essential factors for their binding affinities to BSA.Chapter3:Interaction study of BSA with antibiotics by molecular dockingThe molecular docking study revealed antibiotics located in the pocket at subdommain IIA of BSA. Results showed that the functional ester groups at R1～R4in macrolides enhanced hydrophobic contacts with BSA, the-Cl and-OH at R1～R2in tetracyclines generated additional hydrogen bonds with BSA, and the differences of R1group in sulfonamides obviously influenced their steric conformations at binding sites of BSA. The derived values of ΔG (-33.24～-18.75kJ·mol-1) and K (1.93×103～6.68×105L·mol-1) followed the order of ERY<KIT<MID<JOS<ERE<JOP<MIA<TET<OXY<CHL<SGD<SDZ<SMZ, well agreeing with results from FI-CL analysis.The work consisting of chapter2and chapter3was published in RSC Advances (2013, doi:10.1039/C3RA45885G).Chapter4:Saliva ROX/CLA pharmacokinetic study1. The interactions of BSA with ROX and CLA were studied based on their quenching effect on luminol-BSA CL system, giving K (106L·mol-1) and n (≈1.0), with hydrophobic interaction as the main stable force. Molecular docking analysis showed that ROX/CLA could enter into the pocket at subdommain IIA of BSA.2. Human saliva ROX/CLA in24h after a single oral intake of150/250mg were continuously monitored by FI-CL analysis, with recoveries of90.9%～110.1%(RSDs<6.4%, n=7). The pharmacokinetic parameters for ROX/CLA (ka:0.97±0.18/0.058±0.006h-1, ke:0.082±0.010/0.15±0.01h-1,t1/2:8.56±1.11/4.66±0.08h, and Cltotal:1.90±0.22/8.26±0.18L·h-1, etc.) were successfully obtained, according well with results from blood testing in literature. This work was published in Applied Spectroscopy (2013,67:54-58), Applied Biochemistry and Biotechnology (2013, doi:10.1007/s12010-013-0605-4).Chapter5:Applications of luminol-BSA CL system1. The interaction behaviors of BSA with azithromycin AZM and DPN were studied by FI-CL and molecular docking, showing AZM/DPN entered into subdommain ⅡA of BSA via hydrophobic interaction, with K of9.50×106/8.85×105L-mol-1and n of0.97/0.77, respectively. This proposed approach was applied to AZM quantification in pharmaceutical and spiked human serum samples (recoveries:91.3%～103.9%, RSDs<2.5%, n=5), DPN determination in human serum and gastric juice samples, and DPN monitoring in water samples by sunlight during43h (recoveries:91.8%～114.3%, RSDs<5.0%, n=5). This works were published in Spectrochimica Acta Part A (2011,79:232-235) and Journal of AOACINTERNATIONAL (2013, in press).2. Based on that fomaldehyde remarkably enhanced the CL intensity from luminol-BSA system, the modified homemade FI-CL model of log[(I-I0)/I0]=nlog[D]+logK was deduced, with the K (1.89×106L·mol-1) and n (0.86) for BSA-formaldehyde complex successfully obtained. The binding site and key residues involving in BSA-formaldehyde interaction were given by molecular docking. The present approach was employed to determine formaldehyde vapor pressure in liquid, formaldehyde content in real air and beer samples (recoveries:90.7%～109.3%, RSDs<4.5%, n=7). This work was published in Journal of the Chinese Chemical Society (2013, doi:10.1002/jccs.201300231).Part3ConclusionChapter6:The features and novelties of this dissertation were summarized... |
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