Pharmaceutical Research (v.32, #5)

Solvent-Free Melting Techniques for the Preparation of Lipid-Based Solid Oral Formulations by Karin Becker; Sharareh Salar-Behzadi; Andreas Zimmer (1519-1545).
Lipid excipients are applied for numerous purposes such as taste masking, controlled release, improvement of swallowability and moisture protection. Several melting techniques have evolved in the last decades. Common examples are melt coating, melt granulation and melt extrusion. The required equipment ranges from ordinary glass beakers for lab scale up to large machines such as fluid bed coaters, spray dryers or extruders. This allows for upscaling to pilot or production scale. Solvent free melt processing provides a cost-effective, time-saving and eco-friendly method for the food and pharmaceutical industries. This review intends to give a critical overview of the published literature on experiences, formulations and challenges and to show possibilities for future developments in this promising field. Moreover, it should serve as a guide for selecting the best excipients and manufacturing techniques for the development of a product with specific properties using solvent free melt processing.
Keywords: agglomeration; coating; extrusion; lipid; melt; solvent-free

Cell-Penetrating Antimicrobial Peptides – Prospectives for Targeting Intracellular Infections by Jesper S. Bahnsen; Henrik Franzyk; Edward J. Sayers; Arwyn T. Jones; Hanne M. Nielsen (1546-1556).
To investigate the suitability of three antimicrobial peptides (AMPs) as cell-penetrating antimicrobial peptides.Cellular uptake of three AMPs (PK-12-KKP, SA-3 and TPk) and a cell-penetrating peptide (penetratin), all 5(6)-carboxytetramethylrhodamine-labeled, were tested in HeLa WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effects of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the disrupting ability of the peptides in the presence of bilayer membranes of different composition were analyzed.AMP uptake relative to penetratin was ~13% (PK-12-KKP), ~66% (SA-3) and ~50% (TPk). All four peptides displayed a punctate uptake pattern in HeLa WT cells with co-localization to lysosomes and no indication that clathrin-mediated endocytosis was the predominant uptake mechanism. TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes.PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development.
Keywords: antibiotics; antimicrobial peptides; cell-penetrating peptides; peptide-membrane interaction; Staphylococcus aureus

Polyaspartamide-Doxorubicin Conjugate as Potential Prodrug for Anticancer Therapy by Chiara Di Meo; Felisa Cilurzo; Mariano Licciardi; Cinzia Scialabba; Rocchina Sabia; Donatella Paolino; Donatella Capitani; Massimo Fresta; Gaetano Giammona; Claudio Villani; Pietro Matricardi (1557-1569).
To synthesize a new polymeric prodrug based on α,β-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-d,l-aspartamide copolymer bearing amine groups in the side chain (PHEA-EDA), covalently linked to the anticancer drug doxorubicin and to test its potential application in anticancer therapy.The drug was previously derivatized with a biocompatible and hydrophilic linker, leading to a doxorubicin derivative highly reactive with amino groups of PHEA-EDA. The PHEA-EDA-DOXO prodrug was characterized in terms of chemical stability. The pharmacokinetics, biodistribution and cytotoxicity of the product was investigated in vitro and in vivo on human breast cancer MCF-7 and T47D cell lines and NOD-SCID mice bearing a MCF-7 human breast carcinoma xenograft. Data collected were compared to those obtained using free doxorubicin.The final polymeric product is water soluble and easily hydrolysable in vivo, due to the presence of ester and amide bonds along the spacer between the drug and the polymeric backbone. In vitro tests showed a retarded cytotoxic effect on tumor cells, whereas a significant improvement of the in vivo antitumor activity of PHEA-EDA-DOXO and a survival advantage of the treated NOD-SCID mice was evidenced, compared to that of free doxorubicin.The features of the PHEA-EDA-DOXO provide a potential protection of the drug from the plasmatic enzymatic degradation and clearance, an improvement of the blood pharmacokinetic parameters and a suitable body biodistribution. The data collected support the promising rationale of the proposed macromolecular prodrug PHEA-EDA-DOXO for further potential development and application in the treatment of solid cancer diseases.
Keywords: antitumor activity; biodistribution; doxorubicin; PHEA-EDA; polymeric prodrug

Stability, Permeability and Growth-Inhibitory Properties of Gonadotropin-Releasing Hormone Liposaccharides by Daryn Goodwin; Pegah Varamini; Pavla Simerska; Istvan Toth (1570-1584).
In this study we aimed to address the poor drug-like properties of Gonadotropin-Releasing Hormone (GnRH) peptide through modification with lipids and carbohydrates.GnRH peptide was conjugated to 2-amino-D,L-octanoic acid (C8) and 2-amino-D,L-dodecanoic acid (C12) in monomer and dimer, along with (69) or without (25 and 11) a glucose moiety. Peptides were tested for their biological activity using different tumour cell lines. The toxicity of the constructs was evaluated in peripheral blood mononuclear cells (PBMC).All (glyco)lipopeptides showed improved metabolic stability in Caco-2 cell homogenates. Those with single lipid moiety (2, 4 and 8) exhibited prodrug-like properties. Permeability across Caco-2 cell monolayers was enhanced in the dimer C8-modified (glyco)lipopeptide (3) and the lipopeptide with C12 inserted mid-sequence (11). Most of the constructs showed moderate-to-high antiproliferative activity against GnRH-receptor positive DU145 and OVCAR-3 cells (up to 60%). Compound 11 was the most effective with IC50 = 26.4 ± 1.07 μ−1, which was comparable to triptorelin (25.1 ± 1.14 μg.mL−1). The sensitivity of OVCAR-3 cells to the effect of all analogues except for 11 decreased significantly in estrogen-reconstituted media. Only compounds 2, 4, 5 and 8 showed a steroid-dependent effect in DU145 cells. No compounds exhibited significant toxicity on PBMCs.These results indicated lipidation and glycosylation improves the druggability of GnRH and could lead to an increased direct antitumour activity in some hormone dependent and independent reproductive cancers.
Keywords: antiproliferative activity; Gonadotropin-Releasing Hormone; lipoamino acid; liposaccharide; peptide delivery

Formulation and Pharmacokinetics of Thermosensitive Stealth® Liposomes Encapsulating 5-Fluorouracil by Chantal Al Sabbagh; Nicolas Tsapis; Anthony Novell; Patricia Calleja-Gonzalez; Jean-Michel Escoffre; Ayache Bouakaz; Hélène Chacun; Stéphanie Denis; Juliette Vergnaud; Claire Gueutin; Elias Fattal (1585-1603).
We optimize the encapsulation and investigate the pharmacokinetics of 5-Fluorouracil (5-FU) delivered by thermosensitive stealth® liposomes (TSLs) designed to trigger drug release upon hyperthermia using focused ultrasound (FUS).5-FU was encapsulated into liposomes made of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 either as a free molecule or complexed with copper-polyethylenimine. Heat-triggered drug release was evaluated using either a water bath or FUS. Formulation cytotoxicity was assessed on HT-29 cell line by MTS assay. Pharmacokinetics and biodistribution of 5-FU were evaluated in HT-29-tumor bearing mice.5-FU was easily encapsulated using the lipid hydration method (encapsulation efficacy of 13%) but poorly retained upon dilution. 5-FU complexation with copper-polyethylenimine improved 5-FU retention into liposomes and allowed to obtain an encapsulation efficacy of 37%. At 42°C, heat-triggered 5-FU release from TSLs was 63% using a water bath and 68% using FUS, within 10 min, whereas it remained below 20% for the non-thermosensitive formulation. The MTS assay revealed that formulation toxicity arose from 5-FU and not from the excipients. In addition, 5-FU complex encapsulation into TSLs induces a reduction of the IC50 from 115 down to 49 μM. Pharmacokinetics reveals a longer circulation of encapsulated 5-FU and a more important body exposure, although tumor passive targeting is not significantly higher than free 5-FU.Complexation of 5-FU with copper-polyethylenimine appears an interesting strategy to improve 5-FU retention into TSLs in vitro and in vivo. TSLs allow heat-triggered release of the drug within 10 min at 42°C, a reasonable time for future in vivo experiments.
Keywords: 5-Fluorouracil; copper-polyethylenimine complex; focused ultrasound; pharmacokinetics; thermosensitive stealth® liposomes

The focus of this research was to formulate and evaluate a theranostic liposomal delivery system using indocyanine green (ICG) as a photosensitizer, triggered by near infrared (NIR) irradiation, for in vivo photodynamic therapy (PDT) of breast cancer.Cytotoxicity of PDT using liposomal ICG (LPICG) as well as free ICG (FRICG) was evaluated in the human MDA-MB-468 triple-negative breast cancer (TNBC) cell line. NIR irradiation-induced increase in temperature was also monitored both in vitro and in vivo. Quantitative pharmacokinetic profile and fluorescence imaging-based biodistribution patterns of both formulations were obtained using the human TNBC xenograft model in nude mice. Overall safety, tolerability, and long-term anti-tumor efficacy of LPICG versus FRICG-mediated PDT was evaluated.Significant loss of cell viability was achieved following photoactivation of LPICG via NIR irradiation. Temperatures of irradiated LPICG increased with increasing concentrations of loaded ICG, which correlated with significant rise of temperature compared to PBS in vivo (p < 0.01). Pharmacokinetic assessment revealed a significant increase in systemic distribution and circulation half-life of LPICG, and NIR fluorescence imaging demonstrated enhanced accumulation of liposomes within the tumor region. Tumor growth in mice treated with LPICG followed by NIR irradiation was significantly reduced compared to those treated with FRICG, saline, and irradiation alone. In vivo photodynamic therapy using LPICG demonstrated targeted biodistribution and superior anti-tumor efficacy in a human TNBC xenograft model compared to FRICG. In addition, this unique delivery system exhibited a promising role in NIR image-guided delivery and real-time biodistribution monitoring of formulation with ICG serving as the fluorescent probe.
Keywords: indocyanine green; liposomes; near-infrared florescence imaging; photodynamic therapy; triple-negative breast cancer

Superparamagnetic iron oxide nanoparticles (IONPs) are being investigated for brain cancer therapy because alternating magnetic field (AMF) activates them to produce hyperthermia. For central nervous system applications, brain entry of diagnostic and therapeutic agents is usually essential. We hypothesized that AMF-induced hyperthermia significantly increases IONP blood–brain barrier (BBB) association/uptake and flux.Cross-linked nanoassemblies loaded with IONPs (CNA-IONPs) and conventional citrate-coated IONPs (citrate-IONPs) were synthesized and characterized in house. CNA-IONP and citrate-IONP BBB cell association/uptake and flux were studied using two BBB Transwell® models (bEnd.3 and MDCKII cells) after conventional and AMF-induced hyperthermia exposure.AMF-induced hyperthermia for 0.5 h did not alter CNA-IONP size but accelerated citrate-IONP agglomeration. AMF-induced hyperthermia for 0.5 h enhanced CNA-IONP and citrate-IONP BBB cell association/uptake. It also enhanced the flux of CNA-IONPs across the two in vitro BBB models compared to conventional hyperthermia and normothermia, in the absence of cell death. Citrate-IONP flux was not observed under these conditions. AMF-induced hyperthermia also significantly enhanced paracellular pathway flux. The mechanism appears to involve more than the increased temperature surrounding the CNA-IONPs.Hyperthermia induced by AMF activation of CNA-IONPs has potential to increase the BBB permeability of therapeutics for the diagnosis and therapy of various brain diseases.
Keywords: alternating magnetic field; blood–brain barrier permeability; cross-linked nanoassemblies; flux; superparamagnetic iron oxide nanoparticles

Human Nail Plate Modifications Induced by Onychomycosis: Implications for Topical Therapy by A. Baraldi; S. A. Jones; S. Guesné; M. J. Traynor; W. J. McAuley; M. B. Brown; S. Murdan (1626-1633).
Through the characterisation of the human onchomycotic nail plate this study aimed to inform the design of new topical ungual formulations.The mechanical properties of the human nail were characterised using a Lloyd tensile strength tester. The nail’s density was determined via pycnometry and the nail’s ultrastructure by electron microscopy. Raman spectroscopy analysed the keratin disulphide bonds within the nail and its permeability properties were assessed by quantifying water and rhodamine uptake.Chronic in vivo nail plate infection increased human nailplate thickness (healthy 0.49 ± 0.15 mm; diseased 1.20 ± 0.67 mm), but reduced its tensile strength (healthy 63.7 ± 13.4 MPa; diseased 41.7 ± 5.0 MPa) and density (healthy 1.34 ± 0.01 g/cm3; diseased 1.29 ± 0.00 g/cm3). Onchomycosis caused cell-cell separation, without disrupting the nail disulfide bonds or desmosomes. The diseased and healthy nails showed equivalent water uptake profiles, but the rhodamine penetration was 4-fold higher in the diseased nails using a PBS vehicle and 3 -fold higher in an ethanol/PBS vehicle.Onchomycotic nails presented a thicker but more porous barrier, and its eroded intracellular matrix rendered the tissue more permeable to topically applied chemicals when an aqueous vehicle was used.
Keywords: barrier; fungal; nail; onchomycosis; topical drug delivery

Evaluation of the Usefulness of Breast Cancer Resistance Protein (BCRP) Knockout Mice and BCRP Inhibitor-Treated Monkeys to Estimate the Clinical Impact of BCRP Modulation on the Pharmacokinetics of BCRP Substrates by Tsuyoshi Karibe; Rie Hagihara-Nakagomi; Koji Abe; Tomoki Imaoka; Tsuyoshi Mikkaichi; Satoru Yasuda; Masakazu Hirouchi; Nobuaki Watanabe; Noriko Okudaira; Takashi Izumi (1634-1647).
To evaluate whether the impact of functional modulation of the breast cancer resistance protein (BCRP, ABCG2 421C>A) on human pharmacokinetics after oral administration is predictable using Bcrp knockout mice and cynomolgus monkeys pretreated with a BCRP inhibitor, elacridar.The correlation of the changes of the area under the plasma concentration-time curve (AUC) caused by ABCG2 421C>A with those caused by the Bcrp knockout in mice, or BCRP inhibition in monkeys, was investigated using well-known BCRP substrates (rosuvastatin, pitavastatin, fluvastatin, and sulfasalazine).In mice, the bioavailability changes, which corrected the effect of systemic clearance by Bcrp knockout, correlated well with the AUC changes in humans, whereas the correlation was weak when AUC changes were directly compared. In monkeys, the AUC changes pretreated with elacridar resulted in a good estimation of those in humans within approximately 2-fold ranges.This study suggests that pharmacokinetics studies that use the correction of the bioavailability changes in Bcrp knockout mice are effective for estimating clinical AUC changes in ABCG2 421C>A variants for BCRP substrate drugs and those studies in monkeys that use a BCRP inhibitor serve for the assessment of BCRP impact on the gastrointestinal absorption in a non-rodent model.
Keywords: ABCG2 421C>A; BCRP; Cynomolgus monkey; Gastrointestinal absorption; Knockout mouse

Human ALDH1B1 Polymorphisms may Affect the Metabolism of Acetaldehyde and All-trans retinaldehyde—In Vitro Studies and Computational Modeling by Brian C. Jackson; Philip Reigan; Bettina Miller; David C. Thompson; Vasilis Vasiliou (1648-1662).
To elucidate additional substrate specificities of ALDH1B1 and determine the effect that human ALDH1B1 polymorphisms will have on substrate specificity.Computational-based molecular modeling was used to predict the binding of the substrates propionaldehyde, 4-hydroxynonenal, nitroglycerin, and all-trans retinaldehyde to ALDH1B1. Based on positive in silico results, the capacity of purified human recombinant ALDH1B1 to metabolize nitroglycerin and all-trans retinaldehyde was explored. Additionally, metabolism of 4-HNE by ALDH1B1 was revisited. Databases queried to find human polymorphisms of ALDH1B1 identified three major variants: ALDH1B1*2 (A86V), ALDH1B1*3 (L107R), and ALDH1B1*5 (M253V). Computational modeling was used to predict the binding of substrates and of cofactor (NAD+) to the variants. These human polymorphisms were created and expressed in a bacterial system and specific activity was determined.ALDH1B1 metabolizes (and appears to be inhibited by) nitroglycerin and has favorable kinetics for the metabolism of all-trans retinaldehyde. ALDH1B1 metabolizes 4-HNE with higher apparent affinity than previously described, but with low throughput. Recombinant ALDH1B1*2 is catalytically inactive, whereas both ALDH1B1*3 and ALDH1B1*5 are catalytically active. Modeling indicated that the lack of activity in ALDH1B1*2 is likely due to poor NAD+ binding. Modeling also suggests that ALDH1B1*3 may be less able to metabolize all-trans retinaldehyde and that ALDH1B1*5 may bind NAD+ poorly.ALDH1B1 metabolizes nitroglycerin and all-trans-retinaldehyde. One of the three human polymorphisms, ALDH1B1*2, is catalytically inactive, likely due to poor NAD+ binding. Expression of this variant may affect ALDH1B1-dependent metabolic functions in stem cells and ethanol metabolism.
Keywords: aldehyde dehydrogenase; ALDH1B1; computational modeling; polymorphism; substrate specificity

Phospho-NSAIDs Have Enhanced Efficacy in Mice Lacking Plasma Carboxylesterase: Implications for their Clinical Pharmacology by Chi C. Wong; Ka-Wing Cheng; Ioannis Papayannis; George Mattheolabakis; Liqun Huang; Gang Xie; Nengtai Ouyang; Basil Rigas (1663-1675).
The purpose of the study was to evaluate the metabolism, pharmacokinetics and efficacy of phospho-NSAIDs in Ces1c-knockout mice.Hydrolysis of phospho-NSAIDs by Ces1c was investigated using Ces1c-overexpressing cells. The rate of phospho-NSAID hydrolysis was compared between wild-type, Ces1c+/− and Ces1c−/− mouse plasma in vitro, and the effect of plasma Ces1c on the cytotoxicity of phospho-NSAIDs was evaluated. Pharmacokinetics of phospho-sulindac was examined in wild-type and Ces1c−/− mice. The impact of Ces1c on the efficacy of phospho-sulindac was investigated using lung and pancreatic cancer models in vivo.Phospho-NSAIDs were extensively hydrolyzed in Ces1c-overexpressing cells. Phospho-NSAID hydrolysis in wild-type mouse plasma was 6–530-fold higher than that in the plasma of Ces1c−/− mice. Ces1c-expressing wild-type mouse serum attenuated the in vitro cytotoxicity of phospho-NSAIDs towards cancer cells. Pharmacokinetic studies of phospho-sulindac using wild-type and Ces1c−/− mice demonstrated 2-fold less inactivation of phospho-sulindac in the latter. Phospho-sulindac was 2-fold more efficacious in inhibiting the growth of lung and pancreatic carcinoma in Ces1c −/− mice, as compared to wild-type mice.Our results indicate that intact phospho-NSAIDs are the pharmacologically active entities and phospho-NSAIDs are expected to be more efficacious in humans than in rodents due to their differential expression of carboxylesterases.
Keywords: carboxylesterase; non-steroidal anti-inflammatory drugs; pharmacokinetics; xenografts

Synthesis and Εvaluation of Αnticancer Αctivity in Cells of Novel Stoichiometric Pegylated Fullerene-Doxorubicin Conjugates by George E. Magoulas; Marina Bantzi; Danai Messari; Efstathia Voulgari; Chrisostomi Gialeli; Despoina Barbouri; Athanassios Giannis; Nikos K. Karamanos; Dionissios Papaioannou; Konstantinos Avgoustakis (1676-1693).
To synthesize pegylated stoichiometrically and structurally well-defined conjugates of fullerene (C60) with doxorubicin (DOX) and investigate their antiproliferative effect against cancer cell lines.Stoichiometric (1:1 and 1:2) pegylated conjugates of C60 with DOX were synthesized using the Prato reaction to create fulleropyrrolidines equipped with a carboxyl function for anchoring a polyethylene glycol (PEG) moiety and either a hydroxyl group for attaching one molecule of DOX or a terminal alkyne group for attaching two molecules of DOX through a click reaction. In both conjugates, the DOX moieties are held through a urethane-type bond. Drug release was studied in phosphate buffer (PBS, pH 7.4) and MCF-7 cancer cells lysate. The uptake of the conjugates by MCF-7 cancer cells and their intracellular localization were studied with fluorescence microscopy. The antiproliferative activity of the conjugates was investigated using the WST-1 test.One or two DOX molecules were anchored on pegylated C60 particles to form DOX-C60-PEG conjugates. Drug liberation from the conjugates was significantly accelerated in the presence of tumor cell lysate compared to PBS. The conjugates could be internalized by MCF-7 cells. DOX from the conjugates exhibited much delayed, compared to free DOX, localization in the nucleus and antiproliferative activity.Pegylated DOX-C60 conjugates (1:1) and (2:1) with well-defined structure were successfully synthesized and found to exhibit comparable, but with a delayed onset, antiproliferative activity with free DOX against MCF-7 cancer cells. The results obtained justify further investigation of the potential of these conjugates as anticancer nanomedicines.
Keywords: antiproliferative activity; conjugates; doxorubicin; fullerenes; nanoparticles

Formulation and Performance of Danazol Nano-crystalline Suspensions and Spray Dried Powders by Sumit Kumar; Rajan Jog; Jie Shen; Banu Zolnik; Nakissa Sadrieh; Diane J. Burgess (1694-1703).
This study focuses on the formulation optimization, in vitro and in vivo performance of differently sized nano-crystalline liquid suspensions and spray-dried powders of a poorly soluble BCS class II compound i.e. Danazol.A DoE approach was utilized to optimize stabilizer concentration and formulate danazol (BCS class II) nano-crystalline suspensions and dry powders via wet milling followed by spray drying. Solubility studies were performed to select best stabilizers. Particle size, PXRD, contact angle measurement and in vitro dissolution were utilized in characterization of the liquid and spray-dried powder formulations.The liquid nano-crystalline suspensions followed particle size-dependent dissolution rates i.e. faster dissolution for smaller crystals. The spray-dried nano-crystal powders did not show fast dissolution profiles compared to the liquid nano-crystalline suspension. The poor dissolution of the spray-dried powder correlated to its high LogP value (i.e. LogP 4.53) and poor wetting (or polar surface-area). In vivo bioavailability studies showed superior performance of the liquid nano-crystalline suspensions compared to other milled and un-milled formulations.Wet-milling and spray-drying optimization for danazol nano-crystalline suspension was performed. This study indicates that drug candidates with high LogP values and low polar surface area may not be suitable for formulation as dry nano-crystals.
Keywords: danazol nano-crystals; DoE; in vivo oral bioavailability; nano-crystalline; spray-drying

Fractional Thermolysis by Bipolar Radiofrequency Facilitates Cutaneous Delivery of Peptide and siRNA with Minor Loss of Barrier Function by Woan-Ruoh Lee; Shing-Chuan Shen; Chi-Kuang Sun; Ibrahim A. Aljuffali; Shih-Yun Suen; Yin-Ku Lin; Jhi-Joung Wang; Jia-You Fang (1704-1713).
In this study, we aimed to illustrate the utility of fractional radiofrequency (RF) that generated microchannels in the skin, allowing delivery of peptide and siRNA via the skin. The mechanisms involved in the correlation between macromolecule permeation and skin structure were also elucidated.The morphology of the skin was examined by transmission electron microscopy (TEM), higher harmonic generation microscopy (HGM), and physiological factors. In vivo skin distribution of macromolecules was assessed by fluorescence and confocal microscopies.RF thermolysis selectively created an array of micropores deep into the epidermis without significant removal of the stratum corneum (SC). With energy of 30 mJ, a pore depth of 35 μm was achieved. The bipolar RF resulted in a 3-fold increase of transepidermal water loss (TEWL) compared with intact skin. The respective skin accumulation and flux of the peptide with a molecular weight (MW) of 2335 Da was 3- and 23-fold greater for the RF-treated group than for the non-treatment group. RF enhanced skin accumulation of siRNAs with MW of 10 and 15 kDa by 6.2- and 2.6-fold, respectively. Cutaneous penetration of the macromolecules with an MW of at least 40 kDa could be accomplished by RF. Confocal microscopy imaging revealed that RF could effectively deliver the peptide up to at least a 74-μm depth. The penetration depth of siRNA by RF irradiation was about 50 μm.The novel RF device efficiently delivered macromolecules into the skin while reserving SC layers to support some barrier functions. In this work, for the first time the assistance of fractional RF on peptide and siRNA transport was demonstrated.
Keywords: fractional ablation; peptide; radiofrequency; siRNA; skin permeation

Bridging Laboratory and Large Scale Production: Preparation and In Vitro-Evaluation of Photosensitizer-Loaded Nanocarrier Devices for Targeted Drug Delivery by Susanne Beyer; Li Xie; Susanna Gräfe; Vitali Vogel; Kerstin Dietrich; Arno Wiehe; Volker Albrecht; Werner Mäntele; Matthias G. Wacker (1714-1726).
Industrial production of nanosized drug delivery devices is still an obstacle to the commercialization of nanomedicines. This study encompasses the development of nanoparticles for peroral application in photodynamic therapy, optimization according to the selected product specifications, and the translation into a continuous flow process.Polymeric nanoparticles were prepared by nanoprecipitation of Eudragit® RS 100 in presence and in absence of glycofurol. The photosensitizer temoporfin has been encapsulated into these carrier devices. Process parameters were optimized by means of a Design of Experiments approach and nanoparticles with optimal characteristics were manufactured by using microreactor technology. The efficacy was determined by means of cell culture models in A-253 cells.Physicochemical properties of nanoparticles achieved by nanoprecipitation from ethanolic solutions were superior to those obtained from a method based upon glycofurol. Nanoencapsulation of temoporfin into the matrix significantly reduced toxicity of this compound, while the efficacy was maintained. The release profiles assured a sustained release at the site of action. Finally, the transfer to continuous flow technology was achieved.By adjusting all process parameters, a potent formulation for application in the GI tract was obtained. The essential steps of process development and scale-up were part of this formulation development.
Keywords: Design of Experiments; Drug targeting; Eudragit® RS 100; Nanoparticles; Photodynamic therapy

Self Assembled Ionically Sodium Alginate Cross-Linked Amphotericin B Encapsulated Glycol Chitosan Stearate Nanoparticles: Applicability in Better Chemotherapy and Non-Toxic Delivery in Visceral Leishmaniasis by Pramod K. Gupta; Anil K. Jaiswal; Shalini Asthana; Ashwni Verma; Vivek Kumar; Prashant Shukla; Pankaj Dwivedi; Anuradha Dube; Prabhat R. Mishra (1727-1740).
To investigate the applicability, localization, biodistribution and toxicity of self assembled ionically sodium alginate cross-linked AmB loaded glycol chitosan stearate nanoparticles for effective management of visceral leishmaniasis.Here, we fabricated Amphotericin B (AmB) encapsulated sodium alginate-glycol chitosan stearate nanoparticles (AmB-SA-GCS-NP) using strong electrostatic interaction between oppositely charged polymer and copolymer by ionotropic complexation method. The tagged FAmB-SA-GCS-NP was compared with tagged FAmB for in vitro macrophagic uptake in J774A macrophages and in vivo localization in liver, spleen, lung and kidney tissues. The AmB-SA-GCS-NP and plain AmB were compared for in vitro and in vivo antileishmanial activity, pharmacokinetics, organ distribution and toxicity profiling.The morphology of SA-GCS-NP revealed as nanocrystal (size, 196.3 ± 17.2 nm; PDI, 0.216 ± 0.078; zeta potential, (−) 32.4 ± 5.1 mV) by field emission scanning electron microscopy and high resolution transmission electron microscopy. The macrophage uptake and in vivo tissue localization studies shows tagged FAmB-SA-GCS-NP has significantly higher (~1.7) uptake compared to tagged FAmB. The biodistribution study of AmB-SA-GCS-NP showed more localized distribution towards Leishmania infected organs i.e. spleen and liver while lesser towards kidney. The in vitro (IC50, 0.128 ± 0.024 μg AmB/ml) and in vivo (parasite inhibition, 70.21 ± 3.46%) results of AmB-SA-GCS-NP illustrated significantly higher (P < 0.05) efficacy over plain AmB. The monomeric form of AmB within SA-GCS-NP, observed by UV-visible spectroscopy, favored very less in vitro and in vivo toxicities compared to plain AmB.The molecular organization, toxicity studies, desired localization and biodistribution of cost effective AmB-SA-GCS-NP was found to be highly effective and can be proved as practical delivery platform for better management of leishmaniasis.
Keywords: glycol chitosan; molecular organization; stearic acid; sodium alginate; tissue localization; toxicity

Rifampicin Loaded Mannosylated Cationic Nanostructured Lipid Carriers for Alveolar Macrophage-specific Delivery by Xu Song; Qing Lin; Ling Guo; Yao Fu; Jianfeng Han; Huan Ke; Xun Sun; Tao Gong; Zhirong Zhang (1741-1751).
In this study, cationic mannosylated nanostructured lipid carriers (Man-NLCs) were developed for the targeted delivery of rifampicin to alveolar macrophages.Rifampicin loaded Man-NLCs (RFP-Man-NLCs) and rifampicin loaded unmodified nanostructured lipid carriers (REP-NLCs) were prepared using thin film homogenization method and characterized by particle size, polydispersity index, zeta potential, transmission electron microscopy, encapsulation efficiency, pharmacokinetics, biodistribution, cell specific targeting, cytotoxicity and inflammatory response.RFP-Man-NLCs and REP-NLCs obtained displayed a size distribution around 160 nm (PDI <0.30) with positive charges of approximately 30 mV. The encapsulation efficiency of RFP was above 90%. In the biodistribution study, both RFP-Man-NLCs and RFP-NLCs, compared with the commercially available rifampicin solution, displayed superior lung-targeting ability. Compared to REP-NLCs, RFP-Man-NLCs exhibited significantly higher uptake efficiency in NR8383 cells and alveolar macrophages, which achieved cell-specific targeting. In addition, RFP-Man-NLCs were demonstrated to be a safe formulation with minimum toxicity and no inflammatory response.RFP-Man-NLCs provided an alternative strategy for selectively delivering rifampicin to alveolar macrophages.
Keywords: alveolar macrophages; cationic nanostructured lipid carriers; mannosylation; rifampicin

Release, Partitioning, and Conjugation Stability of Doxorubicin in Polymer Micelles Determined by Mechanistic Modeling by Andrei Ponta; Kyle D. Fugit; Bradley D. Anderson; Younsoo Bae (1752-1763).
To better understand the mechanistic parameters that govern drug release from polymer micelles with acid-labile linkers.A mathematical model was developed to describe drug release from block copolymer micelles composed of a poly(ethylene glycol) shell and a poly(aspartate) core, modified with drug binding linkers for pH-controlled release [hydrazide (HYD), aminobenzoate-hydrazide (ABZ), or glycine-hydrazide (GLY)]. Doxorubicin (Dox) was conjugated to the block copolymers through acid-labile hydrazone bonds. The polymer drug conjugates were used to prepare three polymer micelles (HYD-M, ABZ-M, and GLY-M). Drug release studies were performed to identify the factors governing pH-sensitive release of Dox. The effect of prolonged storage of copolymer material on release kinetics was also observed.Biphasic drug release kinetics were observed for all three micelle formulations. The developed model was able to quantify observed release kinetics upon the inclusion of terms for unconjugated Dox and two populations of conjugated Dox. Micelle/water partitioning of Dox was also incorporated into the model and found significant in all micelles under neutral conditions but reduced under acidic conditions. The drug binding linker played a major role in drug release as the extent of Dox release at specific time intervals was greater at pH 5.0 than at pH 7.4 (HYD-M > ABZ-M > GLY-M). Mathematical modeling was also able to correlate changes in release kinetics with the instability of the hydrazone conjugation of Dox during prolonged storage.These results illustrate the potential utility of mechanistic modeling to better assess release characteristics intrinsic to a particular drug/nanoparticle system.
Keywords: controlled drug release; doxorubicin; hydrazone; mechanistic modeling; nanoparticles

Model-Based Evaluation of the Impact of Formulation and Food Intake on the Complex Oral Absorption of Mavoglurant in Healthy Subjects by Thierry Wendling; Kayode Ogungbenro; Etienne Pigeolet; Swati Dumitras; Ralph Woessner; Leon Aarons (1764-1778).
To compare the pharmacokinetics of intravenous (IV), oral immediate-release (IR) and oral modified-release (MR) formulations of mavoglurant in healthy subjects, and to assess the food effect on the MR formulation’s input characteristics.Plasma concentration-time data from two clinical studies in healthy volunteers were pooled and analysed using NONMEM®. Drug entry into the systemic circulation was modelled using a sum of inverse Gaussian (IG) functions as an input rate function, which was estimated specifically for each formulation and food state.Mavoglurant pharmacokinetics was best described by a two-compartment model with a sum of either two or three IG functions as input function. The mean absolute bioavailability from the MR formulation (0.387) was less than from the IR formulation (0.436). The MR formulation pharmacokinetics were significantly impacted by food: bioavailability was higher (0.508) and the input process was shorter (complete in approximately 36 versus 12 h for the fasted and fed states, respectively).Modelling and simulation of mavoglurant pharmacokinetics indicate that the MR formulation might provide a slightly lower steady-state concentration range with lower peaks (possibly better drug tolerance) than the IR formulation, and that the MR formulation’s input properties strongly depend on the food conditions at drug administration.
Keywords: dose superimposition; food effect; input rate function; mavoglurant population pharmacokinetics; modified-release formulation

In Silico Prediction of Percutaneous Absorption and Disposition Kinetics of Chemicals by Longjian Chen; Lujia Han; Ouarda Saib; Guoping Lian (1779-1793).
To develop in-silico model for predicting percutaneous absorption and disposition kinetics of chemicals in skin layers so as to facilitate the design of transdermal drug delivery systems and skin care products, and risk assessment of occupational or consumer exposure.A general-purpose computer model for simulating skin permeation, absorption and disposition kinetics in the stratum corneum, viable dermis and dermis has been developed. Equations have been proposed for determining the partition and diffusion properties of chemicals by considering molecular partition, binding and mobility in skin layers. In vitro skin penetration data of 12 chemicals was used to validate the model.The observed and simulated permeation and disposition in skin layers were compared for 12 tested chemicals. For most tested chemicals, the experimental and model results are in good agreement with the coefficient of determination >0.80 and relative root mean squared error <1.20. The disposition kinetic parameters of the maximum concentration and the area under the curve in the viable epidermis and dermis initially increased with hydrophobicity, but reached maxima and then decreased with further increase of hydrophobicity.By considering skin physiological structure and composition, the partition and diffusion properties of chemicals in skin layers are determined. This allows in-silico simulation of percutaneous permeation, absorption and disposition kinetics of wide chemical space. The model produced results in good agreement with experimental data of 12 chemicals, suggesting a much improved framework to support transdermal delivery of drug and cosmetic actives as well as integrated risk assessment.
Keywords: diffusion; disposition; model; percutaneous absorption; skin

Osteotropic Polypeptide Nanoparticles with Dual hydroxyapatite Binding Properties and Controlled Cisplatin Delivery by Laura de Miguel; Iuliana Popa; Magali Noiray; Eric Caudron; Ludovica Arpinati; Didier Desmaele; Gerardo Cebrián-Torrejón; Antonio Doménech-Carbó; Gilles Ponchel (1794-1803).
Nanoparticles with prolonged residence time in bone constitute a valuable strategy for bone disease treatments. The aim of this work was to synthesise a simple nanoparticulate system exhibiting both anticancer and hydroxyapatite binding properties for potential bone cancer applications.The amphiphilic copolymer poly(γ-benzyl-glutamate)-block-poly(glutamic acid) (PBLG-b-PGlu) was synthetised by ring opening polymerization and nanoparticles were obtained by a simple nanoprecipitation method. Nanoparticles were characterized in terms of cisplatin interaction, association, and release as well as interaction with hydroxyapatite and their cytoxicity was studied in three prostate cancer cell lines.PBLG-b-PGlu nanoparticles of ~50 nm in size were successfully prepared. They could display for the first time dual hydroxyapatite binding and anticancer properties mediated by the PGlu moiety. They could complex cisplatin at a drug loading content of 6.2% (w/w). Cisplatin release was triggered by physiological concentrations of chloride ions according to an almost zero order kinetics during 14 days. Simultaneously, these nanoparticles showed in vitro hydroxyapatite binding. Finally, they were shown to exert a cytotoxic effect in three prostate cancer cell lines that potentially metastasize to bone.These properties suggest the potential utility of cisplatin-loaded PBLG-b-PGlu nanoparticles as carrier systems for the treatment of bone metastases.
Keywords: cisplatin; hydroxyapatite; nanoparticles; poly(benzylglutamate); poly(glutamic acid)

The Secondary Drying and the Fate of Organic Solvents for Spray Dried Dispersion Drug Product by Daniel S. Hsieh; Hongfei Yue; Sarah J. Nicholson; Daniel Roberts; Richard Schild; John F. Gamble; Mark Lindrud (1804-1816).
To understand the mechanisms of secondary drying of spray-dried dispersion (SDD) drug product and establish a model to describe the fate of organic solvents in such a product.The experimental approach includes characterization of the SDD particles, drying studies of SDD using an integrated weighing balance and mass spectrometer, and the subsequent generation of the drying curve. The theoretical approach includes the establishment of a Fickian diffusion model.The kinetics of solvent removal during secondary drying from the lab scale to a bench scale follows Fickian diffusion model. Excellent agreement is obtained between the experimental data and the prediction from the modeling.The diffusion process is dependent upon temperature. The key to a successful scale up of the secondary drying is to control the drying temperature. The fate of primary solvents including methanol and acetone, and their potential impurity such as benzene can be described by the Fickian diffusion model. A mathematical relationship based upon the ratio of diffusion coefficient was established to predict the benzene concentration from the fate of the primary solvent during the secondary drying process.
Keywords: amorphous; Fickian diffusion; modeling; secondary drying; spray dried dispersion

Exploring the Impact of Drug Properties on the Extent of Intestinal Lymphatic Transport - In Vitro and In Vivo Studies by Emma Lawless; Brendan T. Griffin; Aoife O’Mahony; Caitriona M. O’Driscoll (1817-1829).
Intestinal lymphatic transport of specific lipophilic drugs offers therapeutic advantages and maximises oral bioavailability. The aims of this study were; to compare intestinal lymphatic transport of a range of drugs and to investigate the influence of cyclosporine A on the mechanism/extent of lymphatic transport.Caco2 cells and an anaesthetised mesenteric lymphatic cannulated rat model were used for in vitro and in vivo studies. Lymphatic transport of three lipophilic drugs was directly compared in a long chain fatty acid formulation. In addition, the impact of cyclosporine A on triglyceride turnover was evaluated in vivo and in vitro.The extent of intestinal lymphatic transport in rats was positively correlated with drug solubility in triglyceride and negatively correlated with drug aqueous solubility. Cyclosporine A displayed non-linear lymphatic transport kinetics and reduced intestinal lymph triglyceride. In vitro experiments indicated that the cellular processes affected were intracellular lipid processing and/or lipid secretion.The linear correlations obtained using a range of lipophilic drugs confirm that the simplified approach of determining aqueous or triglyceride drug solubility is useful in predicting the extent of lymphatic transport. In vitro experiments correlated with in vivo observations, demonstrating the usefulness of the Caco-2 model for mechanistic investigations.
Keywords: cyclosporine A; in vitro-in vivo correlations; lipid formulations; lymphatic transport

Profiling the Role of Deacylation-Reacylation in the Lymphatic Transport of a Triglyceride-Mimetic Prodrug by Sifei Han; Luojuan Hu; Tim Quach; Jamie S. Simpson; Natalie L. Trevaskis; Christopher J. H. Porter (1830-1844).
Recent studies have demonstrated the potential for a triglyceride (TG) mimetic prodrug to promote the delivery of mycophenolic acid (MPA) to the lymphatic system. Here, the metabolic pathways that facilitate the lymphatic transport of the TG prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) were examined to better inform the design of next generation prodrugs. In vitro hydrolysis experiments in simulated intestinal conditions and in vivo rat lymphatic transport experiments were conducted in the presence and absence of orlistat and A922500 (inhibitors of lipolysis and TG re-esterification, respectively), to evaluate the importance of 2-MPA-TG digestion and re-esterification of 2-MPA-MG (the 2-monoglyceride derivative) in promoting lymphatic transport.2-MPA-TG was rapidly hydrolysed to 2-MPA-MG on incubation with fresh bile and pancreatic fluid (BPF), but not in simulated gastric fluid, heat-inactivated BPF or BPF + orlistat. Orlistat markedly decreased lymphatic transport and systemic exposure of 2-MPA-TG derivatives suggesting that inhibition of pancreatic lipase hindered luminal digestion and absorption of the prodrug. A922500 also significantly decreased lymphatic transport of 2-MPA-TG but redirected MPA to the portal blood, suggesting that hindered re-acylation of 2-MPA-MG resulted in intracellular degradation.Incorporation into TG deacylation-reacylation pathways is a critical determinant of the utility of lymph directed TG-mimetic prodrugs.
Keywords: DGAT; lipase; lymphatic transport; prodrug; triglyceride mimetic

Impedance Spectroscopy for the Non-Destructive Evaluation of In Vitro Epidermal Models by F. Groeber; L. Engelhardt; S. Egger; H. Werthmann; M. Monaghan; H. Walles; J. Hansmann (1845-1854).
Reconstructed human epidermis (RHE) is standardly used for the risk assessment of chemical compounds. However, analysis is dependent on invasive methods such as histological processing or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining.As an alternative, we have developed a non-destructive technology to analyze the integrity of epidermal equivalents based on impedance spectroscopy. RHEs were generated and impedance spectra were recorded. from these spectra, we extrapolated electrical characteristics such as the capacitance and the ohmic resistance. Furthermore, the measurable electrical parameters were used to quantify the effects of mechanical and chemical disruption of the epidermal integrity.A fully matured RHE exhibits typical impedance spectra in a frequency ranging between 1 Hz and 100 kHz, which is comparable to the spectra of freshly isolated human epidermal biopsies. We could show that, during RHE maturation, these characteristics change significantly. Thus, capacitance and ohmic resistance can be employed as a criterion for the quality control of skin equivalents. Additionally, our application of impedance spectroscopy reveals sufficient sensitivity to detect a transient decreased ohmic resistance caused by 2-propanol, which is classified as a non-irritant by MTT assays.These results indicate that impedance spectroscopy can be employed as a non-destructive complementary method to assess mild irritative effects, which is currently not possible.
Keywords: alternative test method; impedance spectroscopy; non-destructive testing; reconstructed human epidermis

AAPS Connection (1855-1857).