Study On Interactions Of Docetaxel With Protein And Cyclodextrins Inclusion Complexes

Docetaxel((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester,13-ester with 50, 20-epoxy-1,2α,4,7b,10βand 13a-hexahydroxytax-11-en-9-one 4-acetate 2-benzoate, trihydrate, DTX) is a new taxoid, structurally similar to paclitaxel. DTX binds to microtubules reversibly with high affinity, and this binding stabilizes microtubules and prevents depolymerisation from calcium ions. DTX has more effective than paclitaxel as an inhibitor of microtubule depolymerization. It blocks mitosis by inhibiting mitotic spindle assembly and is an effective anti-tumor substance. It is clinically effective against advanced breast, ovarian and non-small cell lung cancer. Some chemotherapeutic drugs, which were used with DTX, show higher anticancer efficacy for inpatients with breast, pancreatic, gastric and urothelial carcinomas than they were used alone.Human serum albumin (HSA) is the most abundant protein of blood plasma, and has high Solubility. It has an important role in maintaining the colloidal osmotic pressure in blood. It also transports and distributes exogenous and endogenous substances, such as nutrients, hormones, fatty acids, antineoplastic and dyes. Therefore, HSA play a key role in pharmacological mechanisms and pharmacodynamics of drug.Human hemoglobin (HHb) is a major protein component in erythrocytes. It is an important functional protein for reversible oxygen carrying. Besides the carrier of oxygen, HHb can also reversibly bind with many kinds of endogenous and exogenous agents such as many drugs (hormones, benzidine, caffeine, mitomycin), herbicides, heteropolyacid, dyes, flavonoids.In our paper, the binding of DTX to HSA and HHb have investigated systematically by different spectroscopy methods and molecular modeling technology. The results could be help for understanding the transport and toxicity of DTX in vivo.In addition, we investigated the inclusion complexes of DTX with two kinds of cyclodextrin derivatives systematically in order to provide important information on new preparations research.1. Interaction of the DTX with HSA using optical spectroscopy methodsThe interaction between DTX and HSA has been investigated systematically by the fluorescence quenching technique, synchronous fluorescence spectroscopy, ultraviolet (UV)-vis absorption spectroscopy, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) under physiological conditions. Our fluorescence data showed that HAS had only one DTX binding site and the binding process was a static quenching procedure. According to the Van’t Hoff equation, the thermodynamic parameters standard enthalpy (ΔH) and standard entropy (ΔS) were calculated to be-41.07KJ mol-1 and-49.72Jmol-1 K-1. These results suggested that the binding process was spontaneous and an exothermic reaction, the hydrogen bond was the predominant intermolecular force stabling the DTX-HAS complex. 1-anilino-8-naphthalenesulfonate (ANS) is often used to detect the presence of hydrophobic regions of protein. DTX can quench the fluorescence of HSA-ANS indicated that DTX replaced ANS and connected with HSA. DTX and ANS probably had same bonding site with HSA. Because ANS bound to the hydrophobic cavity of HSA, DTX also bound to the hydrophobic cavity of HSA. These results were in according with the results of our Binding parameters. Through the research of Forster’s non-radiative energy transfer, the distance between DTX and HSA was 4.72nm. The results indicated the energy transfer from HSA to DTX occurred with high probability, but not the major reasons of fluorescence quenching of protein. The data from the CD, FT-IR and UV-vis spectroscopy supported the change in the secondary structure of protein caused by the interaction of DTX with HSA, and the a-helical structure of HSA was increased with the addition of DTX. The study of molecular modeling showed that DTX was located in the hydrophobic cavity, the site I of protein. Through the study we found that the binding constants between DTX and HSA was decreased with the increased of pH.2. Studies on the interaction between DTX and HHb by spectroscopic analysis and molecular dockingThe interaction of DTX to human hemoglobin (HHb) has also been investigated systematically by the fluorescence quenching spectroscopy, UV-vis absorption spectroscopy, synchronous fluorescence, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) under physiological conditions. The results obtained from analysis of fluorescence data showed that the binding of DTX to HHb was a dynamic quenching procedure which was different from HSA. Each HHb had only one binding site for the drug. The thermodynamic parameters, the enthalpy change and the entropy change were calculated to be 9.18 KJ mol-1 and 0.116 Jmol-1 K-1, respectively, which suggested that hydrophobic interaction played a major role in the reaction between DTX and HHb. Through the research of Forster’s non-radiative energy transfer, the distance between DTX and HHb was 3.77 nm. The results of UV-vis absorption spectroscopy, synchronous fluorescence spectra, CD and FT-IR shown that DTX could induce conformational changes in HHb, the a-helical structure of HHb was decreased with the addition of DTX which is counter to HSA. The study of molecular docking showed that DTX was located in the central cavity of HHb, partial to the subunitα1. There were many hydrophobic amino residues around DTX, therefore, the interaction between HHb and DTX was dominated by hydrophobic force, which was in agreement with the thermodynamic analysis. There were three hydrogen bonds between HHb and DTX. Hydrogen bond acted as an "anchor", intensely determining the 3D space position of DTX in the binding pocket and facilitating the hydrophobic interaction. So the H-bonds interaction between DTX and HHb was not exclusive.3. Study on solubility and preparation of DTX inclusion complexes using hydroxypropyl-β-cyclodextrinCyclodextrins (CD) make up a family of cyclic oligosaccharides. The common types of CD isα-CD、β-CD、γ-CD which composed of 6,7,8α-D-glucopyranoside units linked 1�'4 glycosidic bond. The CD has a hydrophobic cavity, while the rims of the surrounding walls are hydrophilic. This structure is widely used in the formation of the inclusion compounds greatly modifies the physical and chemical properties of the guest molecule, mostly in terms of water solubility. This is the reason why cyclodextrins have attracted much interest in many fields, especially pharmaceutical applications.Because the low solubility and safety of CD, it can’t be used in injection. Hydroxypropyl-β-cyclodextrin (HP-β-CD) is derivatives of CD, the solubility of it was more than 500g/L. HP-β-CD can’t be metabolized, and also not be accumulated. It also has lower skin irritation, renal toxicity and hemolytic. HP-β-CD had been approved as pharmaceutical excipients for injection in USA, Europe and the Far East.The solid inclusion complexes of DTX/HP-β-CD were prepared in different mole ratios, reaction time and temperature by freezed-drying method. The characteristics of which were studied preliminarily by in vitro. Through HPLC methods, some specifications such as the dissolution test, encapsulation efficiency and drug loading of inclusion complexes were investigated. The results showed that total drug solubility was evidently increased 86.6 times; drug loading was 50.3% under mole ratios 1:55,2h reaction time at 25℃. The differential scanning calorimetry (DSC), X-ray diffraction (XRD), infrared spectroscopy (IR),1H-NMR data indicated that the solid inclusion complexes were formed according to structure verification test. The solubility of DTX was improved by HP-P-CD complexes obviously.4. Study on solubility and preparation of DTX inclusion complexes using sulfobutyl-etherl-β-cyclodextrinIn this part, we choose sulfobutyl-etherl-β-cyclodextrin (SBE-β-CD) as the inclusion material. SBE-β-CD has been one of the most popularβ-CD derivatives used as a pharmaceutical excipient due to an improved toxicity profile and ability to solubilize several poorly water-soluble drugs. SBE-β-CD is a negatively chargedβ-CD derivative prepared by the addition of sulfobutylether (SBE) groups toβ-CD.The solid inclusion complexes of DTX/SBE7-β-CD were prepared also at different mole ratios, reaction time and temperature by freezed-drying method. The HPLC methods were applied to investigate the solubility, encapsulation efficiency and drug loading of inclusion complexes. The results showed that total drug solubility was evidently increased from 2.903μg/mL to 443.4μg/mL; drug loading was 88.7% under mole ratios 1:70,2h reaction time at 25℃. The DSC, XRD, IR and 1H-NMR data also indicated that the solid inclusion complexes were formed according to structure verification test. The solubility of DTX was improved by SBE7-β-CD complexes, the inclusion effect was better than HP-β-CD obviously.5. Investigation on the antitumor activity of DTX on different cancer cell linesBy exploit method of MTT, the cytotoxicity of DTX in Hela、ECA-109、PC-3、HepG2 had been studied. We compared the growth inhibition rate in at different concentration range from 100-400 nmol-1 under 24h exposure time. The results showed that DTX appeared strong antineoplastic activity in different cell lines. Hela was the most sensitive and HepG2 was least sensitive. Through the investigation of cell morphology, the great change had been found between before and after addition of DTX.