Longfa Kou

Enhanced Oral Delivery of Paclitaxel Using Acetylcysteine Functionalized Chitosan-Vitamin E Succinate Nanomicelles Based on a Mucus Bioadhesion and Penetration Mechanism

In addition to being a physiological protective barrier, the gastrointestinal mucosal membrane is also a primary obstacle that hinders the oral absorption of many therapeutic compounds, especially drugs with a poor permeability. In order to resolve this impasse, we have designed multifunctional nanomicelles based on the acetylcysteine functionalized chitosan-vitamin E succinate copolymer (CS-VES-NAC, CVN), which exhibit marked bioadhesion, possess the ability to penetrate mucus, and enhance the oral absorption of a hydrophobic drug with a poor penetrative profile, paclitaxel. The intestinal absorption (Ka = 0.38 ± 0.04 min(-1), Papp = 0.059 cm · min(-1)) of CVN nanomicelles was greatly improved (4.5-fold) in comparison with paclitaxel solution, and CLSM (confocal laser scanning microscope) pictures also showed not only enhanced adhesion to the intestinal surface but improved accumulation within intestinal villi. The in vivo pharmacokinetics indicated that the AUC0-t (586.37 ng/mL · h) of CVN nanomicelles was markedly enhanced compared with PTX solution. In summary, the novel multifunctional CVN nanomicelles appear to be a promising nanocarrier for insoluble and poorly permeable drugs due to their high bioadhesion and permeation-enhancing capability.

Enteric Polymer Based on pH-Responsive Aliphatic Polycarbonate Functionalized with Vitamin E To Facilitate Oral Delivery of Tacrolimus

To improve the bioavailability of orally administered drugs, we synthesized a pH-sensitive polymer (poly(ethylene glycol)-poly(2-methyl-2-carboxyl-propylene carbonate)-vitamin E, mPEG-PCC-VE) attempting to integrate the advantages of enteric coating and P-glycoprotein (P-gp) inhibition. The aliphatic polycarbonate chain was functionalized with carboxyl groups and vitamin E via postpolymerization modification. Optimized by comparison and central composite design, mPEG113-PCC32-VE4 exhibited low critical micelle concentration of 1.7 × 10(-6) mg/mL and high drug loading ability for tacrolimus (21.2% ± 2.7%, w/w). The pH-responsive profile was demonstrated by pH-dependent swelling and in vitro drug release. Less than 4.0% tacrolimus was released under simulated gastric fluid after 2.5 h, whereas an immediate release was observed under simulated intestinal fluid. The mPEG113-PCC32-VE4 micelles significantly increased the absorption of P-gp substrate tacrolimus in the whole intestine. The oral bioavailability of tacrolimus micelles was 6-fold higher than that of tacrolimus solution in rats. This enteric polymer therefore has the potential to become a useful nanoscale carrier for oral delivery of drugs.

Relationship between dissolution and bioavailability for nimodipine colloidal dispersions: the critical size in improving bioavailability.

To compare the dissolution and bioavailability for nimodipine microcrystals and nanocrystals, and to determine the critical size range in improving the oral absorption of nimodipine. Nimodipine microcrystals and nanocrystals were prepared using a microprecipitation method. The particle size was determined with a laser diffraction method. X-ray powder diffraction was applied to inspect the potential crystal form transition. The aqueous solubility was determined by shaking flasks, and the dissolution behavior was evaluated using the paddle method. The pharmacokinetics was performed in beagle dogs in a crossover experimental design. Three nimodipine colloidal dispersions (16296.7, 4060.0 and 833.3 nm) were prepared, respectively. Nimodipine had undergone crystal form transition during microprecipitation process, but experienced no conversion under the high-pressure homogenization. The colloidal dispersions did not show any difference in aqueous equilibrium solubility. Additionally, the three formulations also displayed similar dissolution curves in purified water and 0.05% SDS. The AUC for dispersions of 4060.0 and 833.3 nm sizes was 1.69 and 2.59-fold higher than that for 16296.7 nm system in dogs. To sum up, the critical particle size was found to be within the range of 833.3-4060.0 nm (average volume-weighted particle size) in improving the bioavailability of nimodipine, and dissolution performance was not an effective index in evaluating the bioavailability for nimodipine colloidal dispersions.

Combination of l-Carnitine with Lipophilic Linkage-Donating Gemcitabine Derivatives as Intestinal Novel Organic Cation Transporter 2-Targeting Oral Prodrugs

Novel organic cation transporter 2 (OCTN2, SLC22A5) is responsible for the uptake of carnitine through the intestine and, therefore, might be a promising molecular target for designing oral prodrugs. Poor permeability and rapid metabolism have greatly restricted the oral absorption of gemcitabine. We here describe the design of intestinal OCTN2-targeting prodrugs of gemcitabine by covalent coupling of l-carnitine to its N4-amino group via different lipophilic linkages. Because of the high OCTN2 affinity, the hexane diacid-linked prodrug demonstrated significantly improved stability (3-fold), cellular permeability (15-fold), and oral bioavailability (5-fold), while causing no toxicity as compared to gemcitabine. In addition, OCTN2-targeting prodrugs can simultaneously improve the permeability, solubility, and metabolic stability of gemcitabine. In summary, we present the first evidence that OCTN2 can act as a new molecular target for oral prodrug delivery and, importantly, the linkage carbon chain length is a key factor in modifying the affinity of the substrate for OCTN2.

Transporter occluded-state conformation-induced endocytosis: Amino acid transporter ATB0,+-mediated tumor targeting of liposomes for docetaxel delivery for hepatocarcinoma therapy

Rapidly proliferating tumor cells upregulate specific amino acid transporters, which hold great potential for tumor-selective drug delivery. Published reports have focused primarily on blocking these transporters as a means of starving the tumor cells of amino acids, but their potential in drug delivery remains understudied. In the present study, we developed liposomes functionalized with lysine and polyoxyethylene stearate conjugate (LPS) to interact with ATB0,+, an amino acid transporter overexpressed in hepatocarcinoma and the liver cancer cell line HepG2. The LPS modified liposomes (LPS-Lips) were ~100nm in size and exhibited high drug encapsulation efficiency as 94.7%. The uptake of LPS-Lips in HepG2 cells was dependent on Na+ and Cl-. Molecular dynamic simulation showed that a sustained occluded state of the transporter upon binding to co-transported ions was formed and LPS-Lips triggered the cellular internalization of liposomes. We loaded these LPS-Lips with docetaxel and evaluated the potential of ATB0,+-mediated endocytosis of the drug-loaded LPS-Lips in HepG2 cells in vitro and in syngeneic mouse transplants in vivo. Compared with unmodified liposomes, which did not interact with ATB0,+, LPS-Lips exhibited the ability to deliver docetaxel more efficiently into tumor cells with consequent greater antitumor efficacy and less systemic toxicity. These studies provide first evidences that ATB0,+ can be used as a novel and effective target for drug delivery system in tumor cells using chemically modified liposomes for loading with chemotherapeutics and targeting them for the transporter-mediated endocytosis. As ATB0,+ is highly upregulated in several cancers, this approach holds potential for tumor-selective delivery of drugs to treat these cancer types.

Interspecies prediction of oral pharmacokinetics of different lacidipine formulations from dogs to human: physiologically based pharmacokinetic modelling combined with biorelevant dissolution

The aim of the present study was to use physiologically based pharmacokinetic (PBPK) modelling combined with biorelevant dissolution to quantitatively predict dog oral drug pharmacokinetic (PK) of different formulations, and to make an interspecies extrapolation to human. Lacidipine, a typical BCS class II drug, was chosen as a model drug. The three lacidipine formulations involved were immediate release tablets including two commercial products and one self-made micronized tablets. First of all, the PBPK models in rat and dog after intravenous administration of lacidipine solutions and oral administration of lacidipine suspensions were developed and validated based on physicochemical properties and the literature data, which were used to verify the accuracy of the PBPK model in prediction drug PK in animals. The solubility and dissolution behaviour of lacidipine formulations were determined in the media simulating the fasted state intestinal fluid of human (FaSSIF/FaSSIF-V2). Then the Z-factor values obtained by fitting the dissolution profiles of the three formulations were utilized to evaluate the effect of in vitro dissolution behaviours on PK performance. At last, the verified PBPK model in dog was extrapolated to predict human PK of the three formulations. The results showed that PBPK models from dog to human were successfully developed. By integrating biorelevant dissolution with PBPK model, it is achievable to quantitatively and accurately predict the plasma concentration–time profiles for various formulations after oral administration.

Development of a Supercritical Fluid Chromatography-Tandem Mass Spectrometry Method for the Determination of Azacitidine in Rat Plasma and Its Application to a Bioavailability Study

Azacitidine is widely used for the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukaemia (AML). The analysis of azacitidine in biological samples is subject to interference by endogenous compounds. Previously reported high-performance liquid chromatography/tandem mass spectrometric (HPLC-MS/MS) bioanalytical assays for azacitidine suffer from expensive sample preparation procedures or from long separation times to achieve the required selectivity. Herein, supercritical fluid chromatography with tandem mass spectrometry (SFC-MS/MS) was explored as a more promising technique for the selective analysis of structure-like or chiral drugs in biological matrices. In this study, a simple, rapid and specific SFC/MS/MS analytical method was developed for the determination of azacitidine levels in rat plasma. Azacitidine was completely separated from the endogenous compounds on an ACQUITY UPLC™ BEH C18 column (100 mm × 3.0 mm, 1.7 μm; Waters Corp., Milford, MA, USA) using isocratic elution with CO2/methanol as the mobile phase. The single-run analysis time was as short as 3.5 min. The sample preparation for protein removal was accomplished using a simple methanol precipitation method. The lower limit of quantification (LLOQ) of azacitidine was 20 ng/mL. The intra-day and inter-day precisions were less than 15%, and the relative error (RE) was within ±15% for the medium- and high-concentration quality control (QC) samples and within ±20% for the low-concentration QC samples. Finally, the developed method was successfully applied to a pharmacokinetic study in rats following the intravenous administration of azacitidine.

Novel nanostructured enoxaparin sodium-PLGA hybrid carriers overcome tumor multidrug resistance of doxorubicin hydrochloride

Novel enoxaparin sodium-PLGA hybrid nanocarries (EPNs) were successfully designed for sustained delivery of hydrophilic cationic doxorubicin hydrochloride (DOX) and to overcome multidrug resistance (MDR). By incorporation of the negative polymer of enoxaparin sodium (ES), DOX was highly encapsulated into EPNs with an encapsulation efficiency of 92.49%, and ES effectively inhibited the proliferation of HUVEC cell lines. The in vivo pharmacokinetics study after intravenous injection indicated that DOX-loaded EPNs (DOX-EPNs) exhibited a higher area under the curve (AUC) and a longer half-life (t1/2) in comparison with DOX solution (DOX-Sol). The biodistribution study demonstrated that DOX-EPNs increased the DOX level in plasma and decreased the accumulation of DOX in liver and spleen. Compared with DOX-Sol, DOX-EPNs increased the cytotoxicity in P-gp over-expressing MCF-7/Adr cells, attributed to the higher intracellular efficiency of DOX produced by the EPNs. DOX-EPNs entered into resistant tumor cells by multiple endocytosis pathways, which resulted in overcoming the multidrug resistance of MCF-7/Adr cells by escaping the efflux induced by P-gp transporters.

Development and validation of a UPLC–MS/MS assay for the determination of gemcitabine and its L-carnitine ester derivative in rat plasma and its application in oral pharmacokinetics

A simple and rapid UPLC–MS/MS method to simultaneously determine gemcitabine and its L-carnitine ester derivative (2-deoxy-2, 2-difluoro-N-((4-amino-4-oxobutanoyl) oxy)-4-(trimethyl amm-onio) butanoate-cytidine, JDR) in rat plasma was developed and validated. The conventional plasma sample preparation method of nucleoside analogues is solid-phase extraction (SPE) which is time-consuming and cost-expensive. In this study, gradient elution with small particles size solid phase was applied to effectively separate gemcitabine and JDR, and protein precipitation pretreatment was adopted to remove plasma protein and extract the analytes with high recovery(>81%). Method validation was performed as per the FDA guidelines, and the standard curves were found to be linear in the range of 5–4000u2009ng/ml for JDR and 4–4000u2009ng/ml for gemcitabine, respectively. The lower limit of quantitation (LLOQ) of gemcitabine and JDR was 4 and 5u2009ng/ml, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Finally, the developed method was successfully applied to investigate the pharmacokinetic studies of JDR and gemcitabine after oral administration to rats.

Ascorbate-conjugated nanoparticles for promoted oral delivery of therapeutic drugs via sodium-dependent vitamin C transporter 1 (SVCT1)

Abstract Oral administration is the most convenient route for most patients. However, many therapeutics in clinical applications are limited due to the poor solubility and permeability as well as low stability in the intestinal tract. It still remains a significant challenge of how to improve the oral drug bioavailability. In this study, we have designed functional ascorbate-conjugated NPs (As-PLGA NPs) to offer the interaction with sodium-dependent vitamin C transporter 1 (SVCT1) on the epithelial cells. Cellular uptake study indicated that conjugation of 20% ascorbate to the surface of PLGA NPs might achieve a maximum efficacy in Caco-2 cells. Besides, the majority of As-PLGA NPs were predominantly internalized into cells via caveolae-mediated pathway and thereby circumnavigated the lysosomal compartment. Furthermore, the competitive inhibition test and Na+-dependent study provide strong evidence that SVCT1 was involved in the internalization of As-PLGA NPs. Finally, the biodistribution and perfusion study demonstrated that As-PLGA NPs accumulated in the villi and penetrated to the basolateral side, thus significantly enhanced the intestinal absorption. In summary, it showed that SVCT1 expressed in the apical side of epithelial cells might be conceivably exploited as a potential target for oral delivery of therapeutic drug-loaded pharmaceutical nanocarriers. By means of the interaction of SVCT1 and ascorbate on the surface of PLGA NPs, the nanoparticles and transporters accumulated in coated pits, internalizing as ligand-transporter complexes and switched the subcellular sorting of NPs. Graphical Abstract