Paclitaxel‐Loaded Stabilizer‐Free Poly(D,L‐lactide‐co‐glycolide) Nanoparticles Conjugated with Quantum Dots for Reversion of Anti‐Cancer Drug Resistance and Cancer Cellular Imaging

We prepared the PLGA‐loaded anti‐cancer drug and coated it with quantum dots to make it a dual‐function nanoparticles, and analyzed its potential use in cellular imaging and curing cancers. Two cancer cell lines, paclitaxel‐sensitive KB and paclitaxel‐resistant KB paclitaxel‐50 cervical carcinoma cells, were the relativistic models for analysis of the cytotoxicity of free paclitaxel and paclitaxel‐loaded PLGA conjugated with quantum‐dot nanoparticles. The paclitaxel‐loaded PLGA conjugated with quantum dots nanoparticles were significantly more cytotoxic than the free paclitaxel drug in paclitaxel‐resistant KB paclitaxel‐50 cells. This might have been because the cancer cells developed multi‐drug resistance (MDR), which hampered the action of free paclitaxel by pumping its molecules to extracellular areas. Addition of verapamil, a P‐glycoprotein inhibitor, reversed the MDR mechanism and significantly reduced KB paclitaxel‐50 cell viability. As a result, KB paclitaxel‐50 was highly associated with MDR on the cell membrane. The cytotoxicity results indicated that PLGA nanoparticles served as drug carriers and protected the drugs from MDR‐accelerated efflux. Combined quantum dots with PLGA nanoparticles allowed additional functionality for cellular imaging.     

Microtubule alterations and mutations induced by desoxyepothilone B: implications for drug-target interactions

Epothilones, like paclitaxel, bind to beta-tubulin and stabilize microtubules. We selected a series of four leukemia sublines that display increasing levels of resistance to the epothilone analog desoxyepothilone B (dEpoB). The dEpoB cells selected in 30-140 nM were approximately 15-fold cross-resistant to paclitaxel, while 300 nM selected cells were 467-fold resistant to this agent. The dEpoB-selected cells are hypersensitive to microtubule destabilizing agents, and express increased levels of class III beta-tubulin and MAP4. A novel class I beta-tubulin mutation, A231T, that affects microtubule stability but does not alter paclitaxel binding, was identified. The 300 nM selected cells acquired a second mutation, Q292E, situated near the M loop of class I beta-tubulin. These cells fail to undergo drug-induced tubulin polymerization due to dramatically reduced drug binding. The dEpoB-resistant leukemia cells provide novel insights into microtubule dynamics and, in particular, drug-target interactions.     

Cancer cells undergoing epigenetic transition show short-term resistance and are transformed into cells with medium-term resistance by drug treatment

To elucidate the epigenetic mechanisms of drug resistance, epigenetically reprogrammed H460 cancer cells (R-H460) were established by the transient introduction of reprogramming factors. Then, the R-H460 cells were induced to differentiate by the withdrawal of stem cell media for various durations, which resulted in differentiated R-H460 cells (dR-H460). Notably, dR-H460 cells differentiated for 13 days (13dR-H460 cells) formed a significantly greater number of colonies showing drug resistance to both cisplatin and paclitaxel, whereas the dR-H460 cells differentiated for 40 days (40dR-H460 cells) lost drug resistance; this suggests that 13dR-cancer cells present short-term resistance (less than a month). Similarly, increased drug resistance to both cisplatin and paclitaxel was observed in another R-cancer cell model prepared from N87 cells. The resistant phenotype of the cisplatin-resistant (CR) colonies obtained through cisplatin treatment was maintained for 2–3 months after drug treatment, suggesting that drug treatment transforms cells with short-term resistance into cells with medium-term resistance. In single-cell analyses, heterogeneity was not found to increase in 13dR-H460 cells, suggesting that cancer cells with short-term resistance, rather than heterogeneous cells, may confer epigenetically driven drug resistance in our reprogrammed cancer model. The epigenetically driven short-term and medium-term drug resistance mechanisms could provide new cancer-fighting strategies involving the control of cancer cells during epigenetic transition.Subject terms: Tumour heterogeneity, Epigenetics     

Chemistry and chemical biology of taxane anticancer agents

Taxol (paclitaxel) and Taxotere (docetaxel) are currently considered to be among the most important anticancer drugs in cancer chemotherapy. The anticancer activity of these drugs is ascribed to their unique mechanism of action, i.e., causing mitotic arrest in cancer cells, leading to apoptosis through inhibition of the depolymerization of microtubules. Although both paclitaxel and docetaxel possess potent antitumor activity, treatment with these drugs often results in a number of undesirable side effects, as well as multidrug resistance (MDR). Therefore, it has become essential to develop new anticancer agents with superior pharmacological properties, improved activity against various classes of tumors, and fewer side effects. This paper describes an account of our research on the chemistry of paclitaxel and taxoid anticancer agents at the biomedical interface, including: 1. The structure-activity relationship (SAR) study of taxoids leading to the development of the "second-generation" taxoids, which possess exceptional activity against drug-resistant cancer cells expressing the MDR phenotype. 2. Development of fluorinated taxoids to study the bioactive conformation of paclitaxel and photoaffinity labeling taxoids for mapping of the drug-binding domain on both microtubules and P-glycoprotein. 3. The synthesis of novel macrocyclic taxoids for the investigation into the common pharmacophore for microtubule stabilizing anticancer agents.     

In Vivo Evaluation of (−)-Zampanolide Demonstrates Potent and Persistent Antitumor Efficacy When Targeted to the Tumor Site

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (−)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (−)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but is significantly more persistent due to its covalent binding. We also provide the first reported in vivo antitumor evaluation of (−)-zampanolide where we determine that it has potent and persistent antitumor efficacy when delivered intratumorally. Future work on zampanolide to further evaluate its pharmacophore and determine ways to improve its systemic therapeutic window would make this compound a potential candidate for clinical development through its ability to circumvent taxane-resistance mechanisms.     

Synthesis of novel D-seco-taxoids derived from 1-deoxybaccatin VI

New D-seco-taxoids were synthesized from 1-deoxybaccatin VI and their structures were confirmed by 1HNMR, 13CNMR, ESIMS and X-ray crystallography. The key step of the synthesis involved the opening of the oxetane ring under acid and basic conditions in order to obtain new multidrug resistance (MDR) reversal agents and new synthetic precursors of paclitaxel analogues.     

Optimization of taxane binding to microtubules: binding affinity dissection and incremental construction of a high-affinity analog of paclitaxel

The microtubule binding affinities of a series of synthetic taxanes have been measured with the aims of dissecting individual group contributions and obtaining a rationale for the design of novel compounds with the ability to overcome drug resistance. As previously observed for epothilones, the positive and negative contributions of the different substituents to the binding free energies are cumulative. By combining the most favorable substitutions we increased the binding affinity of paclitaxel 500-fold. Insight into the structural basis for this improvement was gained with molecular modeling and NMR data obtained for microtubule-bound docetaxel. Taxanes with affinities for microtubules well above their affinities for P-glycoprotein are shown not to be affected by multidrug resistance. This finding strongly indicates that optimization of the ligand-target interaction is a good strategy to overcome multidrug resistance mediated by efflux pumps.     

Electrochemical Studies of Paclitaxel Interaction with Tubulin

Paclitaxel can stabilize microtubules and induce microtubule assembly, so people areinterested in the interaction of paclitaxel with tubulin. This interaction has been studiedby fluorometry'. but the electrochemical method has not been adopted to study it so far.Pig-brain tubulin was purified by temperature-depen4ent assemly(disassemblyprocedure=, protein concentrations were estimated by Bradford method and its purity wasdetermined by SDS-polyacrylamide gel electrophoresis (295 % purity). A …     

Synthesis, Conformational Analysis, and Biological Evaluation of Heteroaromatic Taxanes

The asymmetric syntheses of heteroaromatic 3-[(tert-butyldimethylsilyl)oxy]-2-azetidinones 12-16 via chiral ester enolate-imine cyclocondensation chemistry are described. The azetidinones contain heteroaromatic moieties which, in certain cases, contribute to a decrease in enantioselectivity due to possible alternate coordinations in the transition states. The (3R,4S)-3-[(tert-butyldimethylsilyl)oxy]-4-heteroaryl-2-azetidinones were subsequently converted to the heteroaromatic taxanes 31-36 and 43-45. Conformational analyses of the 3'-(2-pyridyl) analogue 31 and 3'-(2-furyl) analogue 43 indicate they have solution conformational preferences virtually identical to paclitaxel and docetaxel. Heteroaromatic N-acyl paclitaxel analogues 47-51 were prepared from N-debenzoylpaclitaxel via Schotten-Baumann acylation. The majority of the 14 analogues displayed good to excellent activity in a microtubule assembly assay in comparison to paclitaxel. The analogues were also tested for cytotoxicity against B16 melanoma cells. 3'-Dephenyl-3'-(2-pyridyl)paclitaxel (31), 3'-dephenyl-3'-(2-furyl)paclitaxel (34), N-BOC-3'-dephenyl-3'-(2-furyl)paclitaxel (43), 3'-dephenyl-3'-(2-furyl)-N-(hexanoyl)paclitaxel (44), and N-debenzoyl-N-(3-furoyl)paclitaxel (51) were found to be more cytotoxic than paclitaxel against this cell line. 3'-Dephenyl-3'-(4-pyridyl)paclitaxel (33) and N-debenzoyl-N-(2-furoyl)paclitaxel (50) displayed cytotoxicity against B16 melanoma cells similar to paclitaxel.     

Synthesis, modeling, and anti-tubulin activity of a D-seco paclitaxel analogue

[reaction: see text] We have previously described a model of paclitaxel-microtubule binding that led to the prediction that analogues of paclitaxel lacking any D ring could stabilize microtubules as well as paclitaxel if the substituent present at C4 did not have unfavorable steric interactions with the binding pocket. We report the synthesis of a 4-methyl paclitaxel analogue, compound 1, which bears this prediction out. Compound 1 is as potent as paclitaxel at microtubule stabilization in vitro; however, it has only about one-four-hundredth the cytotoxicity of paclitaxel.     

In vitro enhancement of anticancer activity of paclitaxel by a Cremophor free cyclodextrin-based nanosponge formulation

A severe limitation for cancer therapy is the poor water solubility of many important therapeutic anticancer drugs. The development of novel delivery systems is therefore currently ongoing. We propose the use of β-cyclodextrin based nanosponges to deliver paclitaxel as an alternative to classical formulation in Cremophor EL. They are solid nanoparticles with mean diameter lower than 500?nm and spherical shape. Nanosponges show a safe profile being non-hemolytic and non cytotoxic. Nanosponges dissolved and encapsulated paclitaxel up to 2?mg/ml. The paclitaxel-loaded nanosponges formed a water stable colloidal system avoiding the recrystallization of paclitaxel. The in vitro release studies showed an almost complete release in 2?h without initial burst effect. Our study demonstrates that delivery of paclitaxel via nanosponges increased the amount of paclitaxel entering cancer cells and lowers paclitaxel IC50, therefore enhancing its pharmacological effect. β-Cyclodextrin based nanosponges can therefore be considered an alternative system to solubilize and deliver the paclitaxel.     

Nanosized paclitaxel particles from supercritical carbon dioxide processing and their biological evaluation

The rapid expansion of a supercritical solution into a liquid solvent (RESOLV) technique with benign supercritical carbon dioxide was applied to obtain aqueous suspended nanoparticles of the highly potent anticancer drug paclitaxel. The paclitaxel nanoparticles were protected from agglomeration by using a known nontoxic stabilization agent. The aqueous suspended paclitaxel nanoparticles of different average particle sizes were evaluated in vitro against human breast cancer cells. The results suggest that the nanosized paclitaxel particles are effective, with an antineoplastic activity comparable to that of the commercial paclitaxel formulation. The technique should be generally applicable to the processing of nanoparticles from other important drugs with aqueous solubility problems.     

新型紫杉烷类衍生物的制备及生物活性评价

多药耐药性问题是导致第一代紫杉烷药物在临床化疗失败的主要原因。本文对紫杉醇C7、C10、C14、C3′多个位点的取代基进行改造,针对合成的6个新型的紫杉烷化合物,在体外考察其对多药耐药肿瘤细胞株以及人结肠癌HCT-116干细胞的增殖抑制活性,实验结果表明6个化合物的抗多药耐药活性均优于紫杉醇。采用P-gp高表达的犬肾细胞MDCK-MDR1进一步研究高活性候选化合物JT-3与P-gp的相互作用。以此研发抗多药耐药型的新一代紫杉烷类药物,对开发扩大抗癌新适应症的新一代紫杉烷类抗癌药意义重大。     

Synthesis of water-soluble dendrimers based on melamine bearing 16 paclitaxel groups

The design, synthesis, and characterization of triazine dendrimers derivatized with the anticancer agent paclitaxel are described. The precursor generation two dendrimer 1 is prepared in six linear steps in 64% overall yield and presents 16 amines and two groups for radioiodination. This macromolecule is subsequently derivatized with a paclitaxel conjugate to yield a generation three dendrimer, 2, which is then pegylated in two steps. The pegylated final products, 4a and 4b, with molecular weights of 46 and 77 kDa, respectively, solubilize paclitaxel in water. Pegylated dendrimer 4a is 30 wt % paclitaxel, 52 wt % PEG, and 18 wt % dendrimer. Target 4b is 18 wt % paclitaxel, 71 wt % PEG, and 11 wt % dendrimer.     

紫杉醇对柔红霉素与DNA作用影响的研究

采用紫外-可见光谱、荧光光谱、微分脉冲伏安法以及紫外-可见光谱电化学法等多种手段, 从分子水平研究了紫杉醇对柔红霉素与DNA作用的影响. 紫杉醇可以和柔红霉素形成分子间氢键; 紫杉醇的长链对柔红霉素糖环以及柔红霉素和DNA所形成加合物的外围有一定程度的缠绕作用, 最终使柔红霉素的药效增加毒副作用减小.     

Solubilization of Paclitaxel (taxol) by peptoad self-assemblies

A molecular dynamics simulation was carried out involving a paclitaxel molecule, 987 peptoad molecules, and 35 938 water molecules (conditions shown experimentally to effect paclitaxel solubilization in water). The peptoads are shown to form large clumps, the centers of which are dry and thus favorable to hydrogen bonding between paclitaxel and peptoads. Hydrogen-bonding equilibrium among the peptoads themselves in the developing clumps is achieved in 2 ns. The number and position of hydrogen bonds between the paclitaxel and peptoads fluctuate randomly from two to six within a 2-5 ns time frame. Hydrophobic association between the peptoad chains and the apolar paclitaxel groups does not seem to be an important element of the solubilization. Instead, the hydrophobic chains of the peptoads encasing the paclitaxel extend outward into the dry interior of the peptoad clump where other chains in the clump are located. One hopes that studies such as this will ultimately allow rational predictions when designing new and specific drug solubilizers.     

A New and Simply Available Class of Hydrosoluble Bioconjugates by Coupling Paclitaxel to Hyaluronic Acid through a 4‐Hydroxybutanoic Acid Derived Linker

A simple preparation of a new hydrosoluble paclitaxel bioconjugate 8 , representing a new class of paclitaxel derivatives, is described. Bioconjugate 8 was obtained by coupling hyaluronic acid ( 2 ) to paclitaxel ( 1 ) by means of a 4‐hydroxybutanoic acid derived linker (Scheme 2).     

In vitro and in vivo study of two types of long-circulating solid lipid nanoparticles containing paclitaxel.

Paclitaxel (Taxol), a diterpenoid isolated from Taxus brevifolia, is effective against several murine tumors, and is one of the most exciting anticancer molecules currently available. Due to its low solubility in water, it is clinically administered with polyethoxylated castor oil (Cremophor EL), which causes serious side effects. Inclusion of paclitaxel in solid lipid nanoparticles (SLNs) has proved to be a good approach to eliminate the need for Cremophor EL and improve the drug's antitumor efficacy. This paper describes the development of two types of long-circulating SLNs as colloidal carriers for paclitaxel. SLNs are constituted mainly of bioacceptable and biodegradable lipids. In vitro release kinetics showed that the release was very slow, the release of paclitaxel from F68-SLN is linear, and the release of paclitaxel from Brij78-SLN followed the Weibull equation. Pharmacokinetics was evaluated in KM mice after injection of paclitaxel formulated in Cremophor EL or in Brij78-SLN and F68-SLN. Encapsulation of paclitaxel in both SLNs produced marked differences compared with the free drug pharmacokinetics. F68-SLN and Brij78-SLN are long-circulating (t 1/2 beta, 10.06 and 4.88 h, respectively) compared with paclitaxel injection (t 1/2 beta, 1.36 h).     

Separation of 7-xylosyl-10-deacetyl paclitaxel and 10-deacetylbaccatin III from the remainder extracts free of paclitaxel using macroporous resins

The separation and enrichment of 10-deacetylbaccatin III (10-DAB III) and 7-xylosyl-10-deacetyl paclitaxel were studied on seven macroporous resins with special structures. The performance of 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III on macroporous resins including AB-8, ADS-17, ADS-21, ADS-31, ADS-8, H1020 and NKA-II was compared according to their adsorption and desorption properties. AB-8 provided a much higher adsorption capacity for 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III than other resins, and its adsorption data fitted well to the Langmuir and Freundlich isotherm. According to the adsorption and desorption capacities and the adsorption isotherms, AB-8 demonstrated a remarkable capability for the preparative separation of 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III from the remainder extracts free of paclitaxel. In order to optimize parameters of separation, dynamic adsorption and desorption experiments were carried out on the columns packed with AB-8 resin. The optimal conditions were: the processing volume 15 BV; concentrations of 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III in feed solution 0.0657 mg/mL and 0.1494 mg/mL; flow rate 1 mL/min; temperature 35 degrees C. The gradient elution program was as follows: 30% ethanol for 3 BV, then 80% of ethanol for 6 BV, flow rate 1 mL/min. After the AB-8 resin treatment, the contents of 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III in the product had increased from 0.053% and 0.2% to 3.34% and 1.69%, which were 62.43-fold and 8.54-fold of those in the untreated extracts, respectively, and the recoveries of 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III were 85.85% and 52.78%. The performance achieved good separation and higher recovery of 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III from remainder extracts free of paclitaxel by using AB-8 resin. It is a fast and effective method for the separation and enrichment of 7-xylosyl-10-deacetyl paclitaxel and 10-DAB III.     

Effects of lipid chain length on molecular interactions between paclitaxel and phospholipid within model biomembranes

Molecular interactions between an anticancer drug, paclitaxel, and phosphatidylcholine (PC) of various chain lengths were investigated in the present work by the Langmuir film balance technique and differential scanning calorimetry (DSC). Both the lipid monolayer at the air-water interface and lipid bilayer vesicles (liposomes) were employed as model biological cell membranes. Measurement and analysis of the surface pressure versus molecular area curves of the mixed monolayers of phospholipids and paclitaxel under various molar ratio showed that phospholipids and paclitaxel formed a nonideal miscible system at the interface. Paclitaxel exerted an area-condensing effect on the lipid monolayer at small molecular surface areas and an area-expanding effect at large molecular areas, which could be explained by the intermolecular forces and geometric accommodation between the two components. Paclitaxel and phospholipids could form thermodynamically stable monolayer systems: the stability increased with the chain length in the order DMPC (C14:0)>DPPC (C16:0)>DSPC (C18:0). Investigation of paclitaxel penetration into the pure lipid monolayer showed that DMPC had a higher ability to incorporate paclitaxel and the critical surface pressure for paclitaxel penetration also increased with the chain length in the order DMPC>DPPC>DSPC. A similar trend was testified by DSC studies on vesicles of the mixed paclitaxel/phospholipids bilayer. Paclitaxel showed the greatest interaction with DMPC while little interaction could be measured in the paclitaxel/DSPC liposomes. Paclitaxel caused broadening of the main phase transition without significant change at the peak melting temperature of the phospholipid bilayers, which demonstrated that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer. The interaction between paclitaxel and phospholipid was nonspecific and the dominant factor in this interaction was the van der Waals force or hydrophobic force. As the result of the lower net van der Waals interaction between hydrocarbon chains for the shorter acyl chains, paclitaxel interacted more readily with phospholipids of shorter chain length, which also increased the bilayer intermolecular spacing.