Pharmaceutical Research (v.31, #4)

Current Challenges in Bioequivalence, Quality, and Novel Assessment Technologies for Topical Products by Avraham Yacobi; Vinod P. Shah; Edward D. Bashaw; Eva Benfeldt; Barbara Davit; Derek Ganes; Tapash Ghosh; Isadore Kanfer; Gerald B. Kasting; Lindsey Katz; Robert Lionberger; Guang Wei Lu; Howard I. Maibach; Lynn K. Pershing; Russell J. Rackley; Andre Raw; Chinmay G. Shukla; Kailas Thakker; Nathalie Wagner; Elizabeta Zovko; Majella E. Lane (837-846).
This paper summarises the proceedings of a recent workshop which brought together pharmaceutical scientists and dermatologists from academia, industry and regulatory agencies to discuss current regulatory issues and industry practices for establishing therapeutic bioequivalence (BE) of dermatologic topical products. The methods currently available for assessment of BE were reviewed as well as alternatives and the advantages and disadvantages of each method were considered. Guidance on quality and performance of topical products was reviewed and a framework to categorise existing and alternative methods for evaluation of BE was discussed. The outcome of the workshop emphasized both a need for greater attention to quality, possibly, via a Quality-By-Design (QBD) approach and a need to develop a “whole toolkit” approach towards the problem of determination of rate and extent in the assessment of topical bioavailability. The discussion on the BE and clinical equivalence of topical products revealed considerable concerns about the variability present in the current methodologies utilized by the industry and regulatory agencies. It was proposed that academicians, researchers, the pharmaceutical industry and regulators work together to evaluate and validate alternative methods that are based on both the underlying science and are adapted to the drug product itself instead of single “universal” method.
Keywords: bioequivalence; dermatologic topical product; generic; in vitro ; in vivo ; quality-by-design

Breast Cancer Resistance Protein-Mediated Efflux of Luteolin Glucuronides in HeLa Cells Overexpressing UDP-Glucuronosyltransferase 1A9 by Lan Tang; Ye Li; Wei-Ying Chen; Shan Zeng; Ling-Na Dong; Xiao-Juan Peng; Wen Jiang; Ming Hu; Zhong-Qiu Liu (847-860).
UDP-glucuronosyltransferases (UGTs) are responsible for the formation of glucuronides of polyphenolic flavonoids. This study investigated the UGT1A9-mediated glucuronidation of luteolin and the kinetics of luteolin glucuronide efflux.HeLa cells overexpressing UGT1A9 (HeLa-UGT1A9) were used to determine the kinetics of breast cancer resistance protein (BCRP)-mediated transport of luteolin glucuronides. Human UGT isoforms were used to determine glucuronidation rates.UGT1A9 was found to catalyze the production of four luteolin glucuronides, including three known monoglucuronides and a novel 3′, 4′-diglucuronide. Ko143, a potent specific inhibitor of BCRP, significantly inhibited efflux of luteolin monoglucuronides from HeLa1A9 cells and increased their intracellular levels in a dose-dependent manner. The formation of luteolin diglucuronide was observed when intracellular concentration of total monoglucuronides went above 0.07 nM.Intracellular accumulation of diglucuronide was detected at high monoglucuronide concentrations (>0.07 nM). Diglucuronide production is speculated to be a compensatory pathway for luteolin disposition.
Keywords: BCRP; glucuronidation; HeLa cells; luteolin; UGT1A9

To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood–brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker.Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis.P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury.Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters.
Keywords: biliary excretion; blood–brain barrier; hepatic ischemia-reperfusion; P-glycoprotein; pharmacokinetics

Electrosprayed Microparticles with Loaded pDNA-Calcium Phosphate Nanoparticles to Promote the Regeneration of Mature Blood Vessels by Xueqin Guo; Tian Xia; Huan Wang; Fang Chen; Rong Cheng; Xiaoming Luo; Xiaohong Li (874-886).
The lack of control over microvasculature formation remains a key roadblock to the therapeutic vascularization and regeneration of functional tissues. In the current study, the integration of plasmid DNA (pDNA) condensation and electrospraying technologies was proposed to promote the regeneration of mature blood vessels through injectable or infusible administration of microparticles.Calcium phosphate (CP) nanoparticles with encapsulated plasmids encoding vascular endothelial growth factors (pVEGF) and basic fibroblast growth factor (pbFGF) were synthesized using reverse microemulsions. Electrosprayed microparticles with the loading of CP-pDNA nanoparticles were evaluated on both endothelial cells and smooth muscle cells and after subcutaneous infusion into animals.CP-pDNA nanoparticles was obtained with an average size of around 110 nm and electrosprayed into microparticles, resulting in high loading efficiency and extended protection on pDNA from external DNase environment. The inoculation of poly(ethylene glycol) into microparticle matrices realized a gradual release for 4 weeks of CP-pDNA nanoparticles, leading to an incremental transfection efficiency and strong secretion of extracellular matrices. After subcutaneous infusion of microparticles with encapsulated both CP-pVEGF and CP-pbFGF nanoparticles, significantly higher densities of blood vessels were achieved than those containing individual nanoparticles, and induced a rapid generation of mature blood vessels with few cytotoxicity and inflammation reactions.Electrosprayed microparticle with CP-pDNA nanoparticles encapsulated promoted the formation of vascular networks, providing clinical relevance for therapeutic vascularization and regeneration of functional tissues after injection to ischemic sites or entrapment into tissue engineering scaffolds.
Keywords: electrosprayed microparticle; extracellular matrix secretion; mature blood vessel; pDNA-loaded calcium phosphate nanoparticle; therapeutic vascularization

Effect of Rhamnolipids on Permeability Across Caco-2 Cell Monolayers by Charity J. Wallace; Scott H. Medina; Mohamed E. H. ElSayed (887-894).
This report describes the effect of rhamnolipids (RLs), an amphiphilic biosurfactant produced by the bacterium Pseudomonas aeruginosa, on the integrity and permeability across Caco-2 cell monolayers.We measured the trans-epithelial electrical resistance (TEER) and permeability of [14C]mannitol across Caco-2 cell monolayers upon incubation with 0.01–5.0% v/v RLs as a function of incubation time (30, 60, 90, and 120 min). We also studied the recovery of RL-treated Caco-2 cell monolayers upon incubation with Kaempferol, which is a natural flavonoid that promotes the assembly of the tight junctions.TEER of Caco-2 cell monolayers incubated with 0.01–5.0% v/v RLs solution dropped to 80–28% of that of untreated cells. Decline in TEER was associated with an increase in [14C]mannitol permeability as a function of RLs concentration and incubation time with Caco-2 cells. Incubation of RLs-treated Caco-2 cell monolayers with normal culture medium for 48 h did not restore barrier integrity. Whereas, incubation of a RLs-treated Caco-2 cells with culture medium containing Kaempferol for 24 h restored barrier function indicated by the higher TEER and lower [14C]mannitol permeability values.These results show the ability of RLs to modulate the integrity and permeability of Caco-2 cell monolayers in a concentration- and time-dependent fashion, which suggest their potential to function as a non-toxic permeation enhancer.
Keywords: Caco-2 cell monolayers; oral drug delivery; paracellular permeability; rhamnolipids; transpethelial electrical resistance

The structure–function relationship and mechanism of polycations as gene carriers have attracted considerable research interest in recent years. The present study was to investigate the relationship between polycation chain length and transfection efficiency (RCL-TE), and the corresponding mechanism by O-methyl-free N,N,N-trimethyl chitosans (TMCs) as gene carriers.Four TMCs with various chain lengths were synthesized and used to evaluate the RCL-TE. To investigate the details of RCL-TE, a number of factors such as cytotoxicity, cellular uptake efficiency, cellular uptake pathway and intracellular trafficking, were evaluated.In comparison to short chain length TMCs (S-TMCs), long chain length ones (L-TMCs) mediated higher gene expression. The polyplexes formed by L-TMCs and pDNA showed higher stability. The cellular uptake pathway and intracellular trafficking of these TMC/pDNA polyplexes were different. These above factors are probably the key ones in RCL-TE rather than polycation–DNA binding affinity, polyplex particle size in water, zeta potential, serum, cytotoxicity, and cellular uptake efficiency.For rational design of chitosan-based polycations as gene carriers, polycations with relative long chain lengths are more favorable and more attention should be paid to polyplex stability, function of uncomplexed polycation chains, cellular uptake pathway, and intracellular trafficking.
Keywords: gene carrier; mechanism; polycation chain length; transfection efficiency; trimethyl chitosan

Human and Non-Human Primate Intestinal FcRn Expression and Immunoglobulin G Transcytosis by Pamela J. Hornby; Philip R. Cooper; Connie Kliwinski; Edwin Ragwan; John R. Mabus; Benjamin Harman; Suzanne Thompson; Amanda L. Kauffman; Zhengyin Yan; Susan H. Tam; Haimanti Dorai; Gordon D. Powers; Jill Giles-Komar (908-922).
To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs).Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys.In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1–5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing).In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.
Keywords: drug delivery; epithelial cell; Fc receptor; monoclonal antibody; oral bioavailability

The present work reports rationalized development and characterization of solidified self-nanoemulsifying drug delivery system for oral delivery of combinatorial (tamoxifen and quercetin) therapeutic regimen.Suitable oil for the preparation of liquid SNEDDS was selected based on the maximum saturation solubility of both the drugs while surfactant and co-surfactant were selected based on their emulsification ability. Extreme vertices mixture design and 32 full factorial design were implemented for optimization of liquid SNEDDS and concentration of solid carrier in lyophilization mixture. Finally, extensive characterization of the developed formulation was performed and in vitro cellular uptake was evaluated in Caco-2 cell culture model.Extreme vertices mixture design indicated the desirability of 0.663, corresponded to 40:30:30 w/w as optimum ratio of oil (Capmul® MCM), surfactant (Cremophor RH 40) and co-surfactant (Labrafil 1944CS) in liquid SNEDDS, which solubilized high amount of tamoxifen (10 mg/g) and quercetin (19.44 mg/g). A, 32 full factorial design revealed the optimum concentration of the selected solid carrier (Aerosil 200) of 5.24% w/w and 1.61, when measured in terms of total solid content and liquid SNEDDS: Aerosil 200 ratio, respectively. The developed formulation revealed instantaneous emulsification (in < 2 min), while maintaning all the quality attributes even after storage at accelerated stability condition for 6 months. Finally, the developed formulation revealed 9.63-fold and 8.44-fold higher Caco-2 uptake of tamoxifen and quercetin, respectively in comparison with free drug counterparts.The developed formulation strategy revealed a great potential for oral delivery of combination drugs having utmost clinical relevance.
Keywords: dual drug loading; lyophilization; quercetin; SNEDDS; tamoxifen

The present work focuses on the in vivo evaluation of tamoxifen and quercetin combination loaded into solid self-nanoemulsifying drug delivery system (s-Tmx-QT-SNEDDS).Lyophilization was employed to prepare s-Tmx-QT-SNEDDS using Aerosil 200 as carrier. The developed formulation was evaluated for in vitro cell cytotoxicity, in vivo pharmacokinetics, antitumor efficacy and toxicity studies. In vivo pharmacokinetics revealed ~8-fold and ~4-fold increase in oral bioavailability of tamoxifen and quercetin, respectively as compared to free counterparts. s-Tmx-QT-SNEDDS exhibited significantly higher cell cytotoxicity, as compared to free drug combination revealing ~32-fold and ~22-fold higher dose reduction index for tamoxifen and quercetin, respectively estimated using median effect dose analysis. s-Tmx-QT-SNEDDS could suppress tumor growth in DMBA induced tumor bearing animals by ~80% in contrast to ~35% observed with tamoxifen citrate. The significant appreciation in antitumor efficacy was further supported by normalized levels of tumor angiogenesis markers (MMP-2 and MMP-9). Finally, complete obliteration in tamoxifen induced hepatotoxicity was observed upon administration of developed formulation in contrast to that of clinically available tamoxifen citrate when measured as function of hepatotoxicity markers and histopathological changes.In nutshell, co-encapsulation of quercetin with tamoxifen in solid SNEDDS poses great potential in improving the therapeutic efficacy and safety of tamoxifen.
Keywords: DMBA induced breast tumor; hepatotoxicity; quercetin; SNEDDS; tamoxifen

Topical Anti-Inflammatory Potential of Quercetin in Lipid-Based Nanosystems: In Vivo and In Vitro Evaluation by Carla Caddeo; Octavio Díez-Sales; Ramon Pons; Xavier Fernàndez-Busquets; Anna Maria Fadda; Maria Manconi (959-968).
To develop quercetin-loaded phospholipid vesicles, namely liposomes and PEVs (Penetration Enhancer-containing Vesicles), and to investigate their efficacy on TPA-induced skin inflammation.Vesicles were made from a mixture of phospholipids, quercetin and polyethylene glycol 400 (PEG), specifically added to increase drug solubility and penetration through the skin. Vesicle morphology and self-assembly were probed by Cryo-Transmission Electron Microscopy and Small/Wide Angle X-ray Scattering, as well as the main physico-chemical features by Light Scattering. The anti-inflammatory efficacy of quercetin nanovesicles was assessed in vivo on TPA-treated mice dorsal skin by the determination of two biomarkers: oedema formation and myeloperoxidase activity. The uptake of vesicles by 3T3 fibroblasts was also evaluated.Small spherical vesicles were produced. Their size and lamellarity was strongly influenced by the PEG content (0%, 5%, 10% v/v). The administration of vesicular quercetin on TPA-inflamed skin resulted in an amelioration of the tissue damage, with a noticeable attenuation of oedema and leukocyte infiltration, especially using 5% PEG-PEVs, as also confirmed by confocal microscopy. In vitro studies disclosed a massive uptake and diffusion of PEVs in dermal fibroblasts.The proposed approach based on quercetin vesicular formulations may be of value in the treatment of inflammatory skin disorders.
Keywords: dermal fibroblasts; mice; quercetin; skin inflammation; vesicles

Classification of the Crystallization Behavior of Amorphous Active Pharmaceutical Ingredients in Aqueous Environments by Bernard Van Eerdenbrugh; Shweta Raina; Yi-Ling Hsieh; Patrick Augustijns; Lynne S. Taylor (969-982).
To classify the crystallization behavior of amorphous active pharmaceutical ingredients (API) exposed to aqueous environments.A set of approximately 50 chemically and physically diverse active pharmaceutical ingredients (APIs) was selected for this study. Two experimental setups were employed to characterize the crystallization behavior of the amorphous API in an aqueous environment. For the first approach, precipitation, as evidenced by the development of turbidity, was induced using the solvent shift method, by mixing concentrated API solutions in DMSO with an aqueous buffer in a capillary. Subsequently, crystallization was monitored in situ over time using synchrotron radiation (simultaneous SAXS/WAXS beamline 12-ID-B at the Advanced Photon Source, Argonne National Laboratories, Argonne, IL). In the second approach, amorphous films were prepared by melt quenching; after adding buffer, crystallization was monitored with time using polarized light microscopy.In general, the crystallization behavior of a given compound was similar irrespective of the experimental method employed. However, the crystallization behavior among different compounds varied significantly, ranging from immediate and complete crystallization to no observable crystallization over biorelevant time scales. Comparison of the observed behavior with previous studies of crystallization tendency in non-aqueous environments revealed that the crystallization tendency of individual APIs was somewhat similar regardless of the crystallization environment.API properties, rather than the method by which amorphous materials are generated, tend to dictate crystallization behavior in aqueous media.
Keywords: active pharmaceutical ingredient; amorphous; crystallization; polarized light microscopy; synchrotron radiation

Nano-Encapsulation of Plitidepsin: In Vivo Pharmacokinetics, Biodistribution, and Efficacy in a Renal Xenograft Tumor Model by Hugo Oliveira; Julie Thevenot; Elisabeth Garanger; Emmanuel Ibarboure; Pilar Calvo; Pablo Aviles; Maria Jose Guillen; Sébastien Lecommandoux (983-991).
Plitidepsin is an antineoplasic currently in clinical evaluation in a phase III trial in multiple myeloma (ADMYRE). Presently, the hydrophobic drug plitidepsin is formulated using Cremophor®, an adjuvant associated with unwanted hypersensitivity reactions. In search of alternatives, we developed and tested two nanoparticle-based formulations of plitidepsin, aiming to modify/improve drug biodistribution and efficacy.Using nanoprecipitation, plitidepsin was loaded in polymer nanoparticles made of amphiphilic block copolymers (i.e. PEG-b-PBLG or PTMC-b-PGA). The pharmacokinetics, biodistribution and therapeutic efficacy was assessed using a xenograft renal cancer mouse model (MRI-H-121 xenograft) upon administration of the different plitidepsin formulations at maximum tolerated multiple doses (0.20 and 0.25 mg/kg for Cremophor® and copolymer formulations, respectively).High plitidepsin loading efficiencies were obtained for both copolymer formulations. Considering pharmacokinetics, PEG-b-PBLG formulation showed lower plasma clearance, associated with higher AUC and Cmax than Cremophor® or PTMC-b-PGA formulations. Additionally, the PEG-b-PBLG formulation presented lower liver and kidney accumulation compared with the other two formulations, associated with an equivalent tumor distribution. Regarding the anticancer activity, all formulations elicited similar efficacy profiles, as compared to the Cremophor® formulation, successfully reducing tumor growth rate.Although the nanoparticle formulations present equivalent anticancer activity, compared to the Cremophor® formulation, they show improved biodistribution profiles, presenting novel tools for future plitidepsin-based therapies.
Keywords: cancer therapy; drug delivery; nanomedicine; plitidepsin; polymersomes

Thermodynamic and Fluorescence Analyses to Determine Mechanisms of IgG1 Stabilization and Destabilization by Arginine by Masakazu Fukuda; Daisuke Kameoka; Takuya Torizawa; Satoshi Saitoh; Masaya Yasutake; Yoshimi Imaeda; Akiko Koga; Akihiko Mizutani (992-1001).
To investigate mechanisms governing the stabilization and destabilization of immunoglobulin (IgG1) by arginine (Arg).The effects of Arg on the aggregation/degradation, thermodynamic stability, hydrophobicity, and aromatic residues of IgG1 were respectively investigated by size-exclusion chromatography, differential scanning calorimetry, probe fluorescence, and intrinsic fluorescence.Arg monohydrochloride (Arg–HCl) suppressed IgG1 aggregation at near-neutral pH, but facilitated aggregation and degradation at acidic pH or at high storage temperature. Equimolar mixtures of Arg and aspartic acid (Asp) or glutamic acid (Glu) suppressed aggregation without facilitating degradation even at high temperature. Arg–HCl decreased the thermodynamic stability of IgG1 by enthalpic loss, which was counteracted by using Asp or Glu as a counterion for Arg. The suppression of aggregation by Arg–HCl was well correlated with the decrease in hydrophobicity of IgG1. The intrinsic fluorescence of IgG1 was unaffected by Arg–HCl.Suppression of IgG1 aggregation can be attributed to the interaction between Arg and hydrophobic residues; on the other hand, facilitation of aggregation and degradation is presumably due to the interaction between Arg and some acidic residues, which could be competitively inhibited by simultaneously adding either Asp or Glu.
Keywords: aggregation; arginine; degradation; DSC; IgG

Critical Evaluation of Human Oral Bioavailability for Pharmaceutical Drugs by Using Various Cheminformatics Approaches by Marlene T. Kim; Alexander Sedykh; Suman K. Chakravarti; Roustem D. Saiakhov; Hao Zhu (1002-1014).
Oral bioavailability (%F) is a key factor that determines the fate of a new drug in clinical trials. Traditionally, %F is measured using costly and time-consuming experimental tests. Developing computational models to evaluate the %F of new drugs before they are synthesized would be beneficial in the drug discovery process.We employed Combinatorial Quantitative Structure-Activity Relationship approach to develop several computational %F models. We compiled a %F dataset of 995 drugs from public sources. After generating chemical descriptors for each compound, we used random forest, support vector machine, k nearest neighbor, and CASE Ultra to develop the relevant QSAR models. The resulting models were validated using five-fold cross-validation.The external predictivity of %F values was poor (R2 = 0.28, n = 995, MAE = 24), but was improved (R2 = 0.40, n = 362, MAE = 21) by filtering unreliable predictions that had a high probability of interacting with MDR1 and MRP2 transporters. Furthermore, classifying the compounds according to the %F values (%F < 50% as “low”, %F ≥ 50% as ‘high”) and developing category QSAR models resulted in an external accuracy of 76%.In this study, we developed predictive %F QSAR models that could be used to evaluate new drug compounds, and integrating drug-transporter interactions data greatly benefits the resulting models.
Keywords: drugs; intestinal membrane transporter; oral bioavailability; QSAR

Comparison of PLA Microparticles and Alum as Adjuvants for H5N1 Influenza Split Vaccine: Adjuvanticity Evaluation and Preliminary Action Mode Analysis by Weifeng Zhang; Lianyan Wang; Yuan Liu; Xiaoming Chen; Jiahui Li; Tingyuan Yang; Wenqi An; Xiaowei Ma; Ruowen Pan; Guanghui Ma (1015-1031).
To compare the adjuvanticity of polymeric particles (new-generation adjuvant) and alum (the traditional and FDA-approved adjuvant) for H5N1 influenza split vaccine, and to investigate respective action mode.Vaccine formulations were prepared by incubating lyophilized poly(lactic acid) (PLA) microparticles or alum within antigen solution. Antigen-specific immune responses in mice were evaluated using ELISA, ELISpot, and flow cytometry assay. Adjuvants’ action modes were investigated by determining antigen persistence at injection sites, local inflammation response, antigen transport into draining lymph node, and activation of DCs in secondary lymphoid organs (SLOs).Alum promoted antigen-specific humoral immune response. PLA microparticles augmented both humoral immune response and cell-mediated-immunity which might enhance cross-protection of influenza vaccine. With regard to action mode, alum adjuvant functions by improving antigen persistence at injection sites, inducing severe local inflammation, slightly improving antigen transport into draining lymph nodes, and improving the expression of MHC II on DCs in SLOs. PLA microparticles function by slightly improving antigen transport into draining lymph nodes, and promoting the expression of both MHC molecules and co-stimulatory molecules on DCs in SLOs.Considering the adjuvanticity and side effects (local inflammation) of both adjuvants, we conclude that PLA microparticles are promising alternative adjuvant for H5N1 influenza split vaccine.
Keywords: adjuvanticity; alum; influenza vaccine; microparticles; mode of action

Multifunctional Tumor-Targeting Nanocarriers Based on Hyaluronic Acid-Mediated and pH-Sensitive Properties for Efficient Delivery of Docetaxel by Shuangshuang Song; Fen Chen; Huan Qi; Fei Li; Tiegang Xin; Jingwen Xu; Tiantian Ye; Naicheng Sheng; Xinggang Yang; Weisan Pan (1032-1045).
The objective of this work was to develop a multifunctional tumor-targeting nanocarrier based on the mechanism of CD44-mediated endocytosis and pH-induced drug release to improve the therapeutic efficacy of docetaxel (DTX).Hyaluronic acid-coated docetaxel-loaded cholesteryl hemisuccinate vesicles (HA-CHEMS vesicles) were prepared. The physiochemical properties and pH-dependent drug release of HA-CHEMS vesicles were evaluated. The HA-CHEMS vesicles were investigated for CD44-mediated internalization and in vitro cell viability using MCF-7,A549 and L929 cells.In addition,tissue distribution as well as antitumor efficacy was also evaluated in MCF-7 tumor-bearing mouse model.The particle size and zeta potential of HA-CHEMS vesicles were 131.4 ± 6.2 nm and −13.3 ± 0.04 mV,respectively. The in vitro drug release results demonstrated a pH-responsive drug release under different pH conditions. In vitro cell viability tests suggested that the encapsulation of DTX in HA-CHEMS vesicles led to more than 51.6-fold and 46.3-fold improved growth inhibition in MCF-7 and A549 cell lines,respectively compared to Taxotere®. From the cell uptake studies,the coumarin 6-loaded HA-CHEMS vesicles enhanced intracellular fluorescent intensity in the CD44-overexpressing cell line (MCF-7). Biodistribution studies revealed selective accumulation of HA-CHEMS vesicles in the MCF-7 bearing BalB/c nude mice as a result of passive accumulation and active targeting (CD44-mediated endocytosis). Compared to Taxotere®,HA-CHEMS vesicles exhibited higher antitumor activity by reducing tumor volume (P < 0.05) and drug toxicity,demonstrating the success of the multifunctional targeting delivery.This work corresponds to the preparation of a multifunctional tumor-targeted delivery system. Our investigation shows that hyaluronan-bearing docetaxel-loaded cholesteryl hemisuccinate vesicles (HA-CHEMS vesicles) is a highly promising therapeutic system,leading to tumor regression after intravenous administration without visible toxicity.
Keywords: CD44; docetaxel; hyaluronic acid; multifunctional targeting; pH-sensitivity

Ocular Delivery of pRNA Nanoparticles: Distribution and Clearance After Subconjunctival Injection by Liang Feng; S. Kevin Li; Hongshan Liu; Chia-Yang Liu; Kathleen LaSance; Farzin Haque; Dan Shu; Peixuan Guo (1046-1058).
RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA of bacteriophage phi29 DNA packaging motor were previously found to be thermodynamically stable. As the nanoparticles could have potential in ocular drug delivery, the objectives in the present study were to investigate the distribution of pRNA nanoparticles after subconjunctival injection and examine the feasibility to deliver the nanoparticles to the cells of cornea and retina.Alexa647-labeled pRNA nanoparticles (pRNA-3WJ and pRNA-X) and double-stranded RNA (dsRNA) were administered via subconjunctival injection in mice. Alexa647 dye was a control. Topical administration was performed for comparison. Ocular clearance of pRNA nanoparticles and dsRNA after the injection was assessed using whole-body fluorescence imaging of the eyes. The numbers of cells in the ocular tissues with nanoparticle cell internalization were determined in fluorescence microscopy of dissected eye tissues.After subconjunctival injection, pRNA nanoparticles and dsRNA were observed to distribute into the eyes and cleared through the lymph. pRNA-3WJ, pRNA-X, and dsRNA were found in the cells of the conjunctiva, cornea, and sclera, but only pRNA-X was in the cells of the retina. Topical administration was not effective in delivering the nanoparticles to the eye.The pRNA nanoparticles were delivered to the cells in the eye via subconjunctival injection, and cell internalization was achieved in the cornea with pRNA-3WJ and pRNA-X and in the retina with pRNA-X. Only the X-shape pRNA-X could enter the retina.
Keywords: double-stranded RNA; ocular delivery; pRNA nanoparticle; subconjunctival; topical

A Novel Three-Dimensional Large-Pore Mesoporous Carbon Matrix as a Potential Nanovehicle for the Fast Release of the Poorly Water-soluble Drug, Celecoxib by Yanzhuo Zhang; Hong Wang; Chuanjun Li; Baoxiang Sun; Yu Wang; Siling Wang; Cunqiang Gao (1059-1070).
A novel mesocellular carbon foam (MSU-FC) with a large pore size and a three-dimensional porous structure for the oral delivery of poorly water-soluble drugs was prepared. The goal of this study was to improve in vitro dissolution and in vivo absorption of celecoxib (CEB), a model drug, by means of novel carbon-based nanoparticles prepared from the MSU-FC matrix.The MSU-FC matrix was synthesized by an inverse replica templating method using mesocellular silica template. A solvent immersion/evaporation method was used to load the drug molecules. The drug-loaded nanoparticles were characterized for morphology, surface area, particle size, mesoporous structure, crystallinity, solubility and dissolution. The effect of MSU-FC on cell viability was measured using the MTT conversion assay. Furthermore, the oral bioavailability of CEB-loaded MSU-FC in fasted rats was compared with that of the marketed product.Our results demonstrate that CEB incorporation into the prepared MSU-FC resulted in an approximately 9-fold increase in aqueous solubility in comparison with crystalline CEB. MSU-FC produced accelerated immediate release of CEB in comparison with crystalline CEB (pure CEB powder or marketed formulation) and the drug-loaded conventional mesoporous carbon particles. The relative bioavailability of CEB for CEB-loaded MSU-FC was 172%. In addition, MSU-FC nanoparticles exhibited very low toxicity.The MSU-FC nanomatrix has been shown to be a promising drug delivery vehicle for improving the dissolution and biopharmaceutical characteristics of poorly water-soluble drugs.
Keywords: carbon matrix; crystallinity; cytotoxicity; dissolution; drug absorption

Transdermal Delivery and Cutaneous Targeting of Antivirals using a Penetration Enhancer and Lysolipid Prodrugs by Denisa Diblíková; Monika Kopečná; Barbora Školová; Marcela Krečmerová; Jaroslav Roh; Alexandr Hrabálek; Kateřina Vávrová (1071-1081).
In this work, we investigate prodrug and enhancer approaches for transdermal and topical delivery of antiviral drugs belonging to the 2,6-diaminopurine acyclic nucleoside phosphonate (ANP) group. Our question was whether we can differentiate between transdermal and topical delivery, i.e., to control the delivery of a given drug towards either systemic absorption or retention in the skin.The in vitro transdermal delivery and skin concentrations of seven antivirals, including (R)- and (S)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP), (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine ((S)-HPMPDAP), its 8-aza analog, and their cyclic and hexadecyloxypropyl (HDP) prodrugs, was investigated with and without the penetration enhancer dodecyl-6-(dimethylamino)hexanoate (DDAK) using human skin.The ability of ANPs to cross the human skin barrier was very low (0.5–1.4 nmol/cm2/h), and the majority of the compounds were found in the stratum corneum, the uppermost skin layer. The combination of antivirals and the penetration enhancer DDAK proved to be a viable approach for transdermal delivery, especially in case of (R)-PMPDAP, an anti-HIV effective drug (30.2 ± 2.3 nmol/cm2/h). On the other hand, lysophospholipid-like HDP prodrugs, e.g., HDP-(S)-HPMPDAP, reached high concentrations in viable epidermis without significant systemic absorption.By using penetration enhancers or lysolipid prodrugs, it is possible to effectively target systemic diseases by the transdermal route or to target cutaneous pathologies by topical delivery.
Keywords: acyclic nucleoside phosphonate antivirals; lysolipid prodrug; penetration enhancer; skin absorption; transdermal drug delivery

Comparison of the Accuracy of Experimental and Predicted pKa Values of Basic and Acidic Compounds by Luca Settimo; Krista Bellman; Ronald M. A. Knegtel (1082-1095).
Assessment of the accuracy of experimental and theoretical methods of pKa determination for acids and bases as separate classes.Four literature pKa datasets were checked for errors and pKa values assigned unambiguously to a single acidic and/or basic ionisation centre. A new chemically diverse and drug-like dataset was compiled from high-throughput UV–vis spectrophotometry pKa data. Measured pKa values were compared with data obtained by alternative methods and predictions by the Epik, Chemaxon and ACD pKa DB software packages.The pKa values of bases were considerably less accurately predicted than those of acids, in particular for structurally complex bases. Several new chemical motifs were identified for which pKa values were particularly poorly predicted. Comparison of pKa values obtained by UV–vis spectrophotometry and different literature sources revealed that low aqueous solubility and chromophore strength can affect the accuracy of experimental pKa determination for certain bases but not acids.The pKa prediction tools Epik, Chemaxon and ACD pKa DB provide significantly less accurate predictions for bases compared to acids. Certain chemical features are underrepresented in currently available pKa data sets and as a result poorly predicted. Acids and bases need to be considered as separate classes during pKa predictor development and validation.
Keywords: bases; pKa measurement; pKa prediction; solubility; UV–vis spectrophotometry

AAPS Connection (1096-1098).