Pharmaceutical Research (v.26, #2)

Antisense Makes Sense in Engineered Regenerative Medicine by Yongchang Yao; Chunming Wang; Rohan R. Varshney; Dong-An Wang (263-275).
The use of antisense strategies such as ribozymes, oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) in gene therapy, in conjunction with the use of stem cells and tissue engineering, has opened up possibilities in curing degenerative diseases and injuries to non-regenerating organs and tissues. With their unique ability to down-regulate or silence gene expression, antisense oligonucleotides are uniquely suited in turning down the production of pathogenic or undesirable proteins and cytokines. Here, we review the antisense strategies and their applications in regenerative medicine with a focus on their efficacies in promoting cell viability, regulating cell functionalities as well as shaping an optimal microenvironment for therapeutic purposes.
Keywords: antisense; oligodeoxynucleotides; regenerative medicine; ribozyme; RNA interference (RNAi)

Design of a Liposomal Candidate Vaccine Against Pseudomonas aeruginosa and its Evaluation in Triggering Systemic and Lung Mucosal Immunity by Béatrice Heurtault; Philippe Gentine; Jean-Sébastien Thomann; Corinne Baehr; Benoît Frisch; Françoise Pons (276-285).
To design and evaluate liposomal constructs capable of inducing a potent systemic and airway humoral response to Pseudomonas aeruginosa Liposomes contained a peptide derived from P. aeruginosa pilin protein as B epitope, a peptide derived from Influenza hemagglutinin protein as Th epitope, the TLR agonist Pam3CAG or Pam2CAG as adjuvant, and a mannosylated lipid as dendritic cell targeting agent. These constructions were administered to mice intraperitoneally (i.p.) or intranasally (i.n.). Their immunogenicity was evaluated by measuring B epitope-specific immunoglobulins in the serum and the airways by ELISA.The B epitope, in its native form or after substitution of a cysteine by a serine, induced high systemic IgG titers when formulated in the presence of Pam3CAG or Pam2CAG and administered i.p.. No IgA response was observed in the airways upon injection of candidate vaccines by i.p. route, whatever the B epitope or the adjuvant. However, i.n. vaccination resulted in a significant local production of IgA. Finally, the production of IgG was more rapid when mannose was incorporated.All liposomal candidate vaccines tested induced the production of IgG and/or IgA directed against an immunogenic peptide from P. aeruginosa. Liposomal constructs could be attractive in the vaccination against P. aeruginosa.
Keywords: airway mucosal immunity; liposomes; Pseudomonas aeruginosa ; vaccination

Design and Synthesis of N 4,N 9-Disubstituted Spermines for Non-viral siRNA Delivery – Structure-Activity Relationship Studies of siFection Efficiency Versus Toxicity by Moustafa K. Soltan; Hassan M. Ghonaim; Mohamed El Sadek; M. Abou Kull; Lubna Abd El-aziz; Ian S. Blagbrough (286-295).
To study the effect of sequentially changing the chain length, oxidation level, and charge distribution in N 4,N 9-diacyl and N 4,N 9-dialkyl spermines on siRNA formulation, and then to compare their lipoplex transfection efficiency in cell lines.Eight N 4,N 9-diacyl polyamines: N 4,N 9-[didecanoyl, dilauroyl, dimyristoyl, dimyristoleoyl, dipalmitoyl, distearoyl, dioleoyl and diretinoyl]-1,12-diamino-4,9-diazadodecane were synthesized. Their abilities to bind to siRNA and form nanoparticles were studied using a RiboGreen intercalation assay and particle sizing. Two diamides were also reduced to afford tetraamines N 4,N 9-distearyl- and N 4,N 9-dioleyl-1,12-diamino-4,9-diazadodecane. Delivery of fluorescein-labelled Label IT® RNAi Delivery Control was studied in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA), and compared with TransIT-TKO.The design, synthesis, and structure-activity relationship studies of a series of N 4,N 9-disubstituted spermines as efficient vectors for non-viral siRNA delivery to primary skin and cancer cell lines is reported. These non-liposomal cationic lipids are promising siRNA carriers based on the naturally occurring polyamine spermine showing that C-18 is a better chain length as shorter chains are more toxic. N 4,N 9-Distearoyl spermine and N 4,N 9-dioleoyl spermine are efficient siRNA formulation and delivery vectors, even in the presence of serum, comparable to TransIT-TKO. However, four positive charges distributed as in spermine was significantly more toxic.
Keywords: lipopolyamines; N 4,N 9-dioleoyl spermine; NVGT; primary skin cells; siRNA delivery

High-Throughput Self-Interaction Chromatography: Applications in Protein Formulation Prediction by David H. Johnson; Arun Parupudi; W. William Wilson; Lawrence J. DeLucas (296-305).
Demonstrate the ability of an artificial neural network (ANN), trained on a formulation screen of measured second virial coefficients to predict protein self-interactions for untested formulation conditions.Protein self-interactions, quantified by the second virial coefficient, B 22, were measured by self-interaction chromatography (SIC). The B 22 values of lysozyme were measured for an incomplete factorial distribution of 81 formulation conditions of the screen components. The influence of screen parameters (pH, salt and additives) on B 22 value was modeled by training an ANN using B 22 value measurements. After training, the ANN was asked to predict the B 22 value for the complete factorial of parameters screened (12,636 conditions). Twenty of these predicted values (distributed throughout the range of predictions) were experimentally measured for comparison.The ANN was able to predict lysozyme B 22 values with a significance of p < 0.0001 and RMSE of 2.6 × 10−4 mol ml/g2.The results indicate that an ANN trained on measured B 22 values for a small set of formulation conditions can accurately predict B 22 values for untested formulation conditions. As a measure of protein–protein interactions correlated with solubility, B 22 value predictions based on a small screen may enable rapid determination of high solubility formulations.
Keywords: artificial neural network; formulation development; physical protein stability; self-interaction chromatography; systematic screening

Neutralization of IL-13 is an attractive approach for treatment of asthma. In this report, we developed a novel PK–PD model that described the relationship between the circulating concentrations of total IL-13 and a neutralizing anti-IL-13 antibody (Ab-02) in the model of acute airway inflammation induced by Ascaris challenge to cynomolgus monkeys, as well as in naive monkeys.Cynomolgus monkeys were administered a single intravenous or subcutaneous dose of Ab-02. Total IL-13 and Ab-02 concentrations were measured by immunoassays.Modeling and simulations indicated that: (1) Ascaris challenge induced ∼ three-fold increase in circulating IL-13 concentrations, when compared to naive animals, consistent with the notion that Ascaris-induced airway inflammation was IL-13-mediated; (2) the transient increase in total IL-13 concentrations observed in both naive and Ascaris-challenged monkeys following Ab-02 administration was due to the increase in Ab-02-bound IL-13, while free IL-13 was decreased; and (3) the extent and duration of neutralization of circulating IL-13 were different in naive and Ascaris-challenged monkeys for the same Ab-02 dose regimen.The PK–PD model presented in this report may be applied to study drug–ligand interactions when a free ligand cannot be directly assayed but total ligand concentrations are modulated by the drug administration.
Keywords: Asthma; IL-13; Monoclonal antibody; Pharmacodynamics; Pharmacokinetics

Improved Bioequivalence Assessment of Topical Dermatological Drug Products Using Dermatopharmacokinetics by Berthe N’Dri-Stempfer; William C. Navidi; Richard H. Guy; Annette L. Bunge (316-328).
A dermatopharmacokinetic (DPK) approach, in which drug levels in the stratum corneum (SC) are measured as a function of time post-application and post-removal of the product using tape-strip sampling in vivo in humans, has been considered for the comparative assessment of topical bioavailability. Its application to-date has been limited by contradictory results and concerns that variability in the method necessitates large numbers of treatment sites and volunteers. The objective of this study was to test whether a revised protocol could better assess bioequivalence.A blinded study of three 1% econazole nitrate cream products, for which the SC is the site of action, was conducted to examine several modifications to the DPK methodology. In addition to protocol changes designed to reduce experimental variability, bioequivalence was assessed at a single uptake time and a single clearance time measured in duplicate in each subject.Conclusive determinations of bioequivalence were achieved with only four treatment sites per product in each of 14 volunteers, which was less than one-third the number required in a previous DPK investigation.Comparative bioequivalence can be assessed conclusively with fewer treatment sites in fewer subjects with robust methods that should be less sensitive to inter-laboratory differences.
Keywords: dermatopharmacokinetics; econazole; skin; stratum corneum; tape stripping; topical drug bioequivalence

To investigate the movement of intravitreally injected human serum albumin nanoparticles (HSA-NP) with respect to nanoparticle surface charge and retinal injury.HSA-NPs were developed by a desolvation technique. HSA-NPs were cationized by covalent coupling of hexamethylenediamine on the particle surface. Either anionic or cationic HSA-NPs were injected to determine the effect of surface charge on intravitreal nanoparticle movement. HSA-NPs were injected intravitreally into both normal and laser photocoagulated eyes to examine the effect of the integrity of retinal tissue on the retinal penetration. The retinal penetration of fluorescence labeled anionic HSA-NPs was investigated by confocal microscopy.Anionic particles (−33.3 ± 6.1 mV) more easily diffused through the 3-dimensional vitreal network of collagen fibrils than did their cationic counterparts (11.7 ± 7.2 mV). In the laser photocoagulated retina, more HSA-NPs were detected in the choroidal space, compared to the normal retina. The immunohistochemical studies indicated that HSA-NPs were taken up into Müller cells.The movement of intravitreal nanoparticles depended on both nanoparticles surface charge and retinal injury. The Müller cells might play an important role in the retinal penetration of nanoparticles. The anionic HSA-NP is a promising drug or gene delivery carrier to the sub-retinal space and RPE.
Keywords: human serum albumin nanoparticles; Müller cells; trans-retinal penetration

Gelatin–methotrexate conjugates (G-MTX) with known molecular weight (MW), drug load, and charge were prepared and evaluated for growth inhibition on leukemia cells.Gelatin (34 to 171 kDa) was reacted with a carbodiimide to prepare G-MTX with high (G-MTX-H) and low (G-MTX-L) drug loads. Cationic conjugates were prepared by ethylenediamine modification. MTX:gelatin molar ratios were determined spectrophotometricaly. Isoelectric focusing electrophoresis (IEF) and turbidity were used to measure isoelectric points (IEP). Growth inhibition profiles and IC50 values were determined on HL-60 cells using a modified MTT assay.IC50 values of anionic G-MTX-L (drug loads 0.5:1 to 2.2:1) increased linearly from 46 to 180 nM with MW. But, IC50 values for anionic G-MTX-H (drug loads 7.4:1 to 25:1) showed little, if any, MW dependence and were about two times higher. IC50 values for cationic G-MTX-L ranged from 770 to 2,900 nM and the relationship with MW was non-linear.The growth inhibition ranking was MTX > anionic G-MTX-L > anionic G-MTX-H > cationic G-MTX-L. High drug load may hinder lysosomal enzyme degradation and drug release and contribute to suppression of the MW effect observed with G-MTX-L. A mechanism change is suggested as the cationic conjugates increase to the highest MW.
Keywords: charge; drug load; HL-60 growth inhibition; macromolecular drug conjugates; molecular weight

Cannabinoids have received renewed interest due to their antitumorigenic effects. Using human cervical carcinoma cells (HeLa), this study investigates the role of cyclooxygenase-2 (COX-2) in apoptosis elicited by the endocannabinoid analog R(+)-methanandamide (MA).COX-2 expression was assessed by RT-PCR and Western blotting. PGE2/PGD2 levels in cell culture supernatants and DNA fragmentation were measured by ELISA.MA led to an induction of COX-2 expression, PGD2 and PGE2 synthesis. Cells were significantly less sensitive to MA-induced apoptosis when COX-2 was suppressed by siRNA or the selective COX-2 inhibitor NS-398. COX-2 expression and apoptosis by MA was also prevented by the ceramide synthase inhibitor fumonisin B1, but not by antagonists to cannabinoid receptors and TRPV1. In line with the established role of peroxisome proliferator-activated receptor γ (PPARγ) in the proapoptotic action of PGs of the D and J series, inhibition of MA-induced apoptosis was also achieved by siRNA targeting lipocalin-type PGD synthase (L-PGDS) or PPARγ. A role of COX-2 and PPARγ in MA-induced apoptosis was confirmed in another human cervical cancer cell line (C33A) and in human lung carcinoma cells (A549).This study demonstrates COX-2 induction and synthesis of L-PGDS-derived, PPARγ-activating PGs as a possible mechanism of apoptosis by MA.
Keywords: Apoptosis; cyclooxygenase-2; lipocalin-type prostaglandin D synthase; peroxisome proliferator-activated receptor γ; R(+)-methanandamide

Meal-Induced Acceleration of Tablet Transit Through the Human Small Intestine by Hala M. Fadda; Emma L. McConnell; Michael D. Short; Abdul W. Basit (356-360).
The transit of dosage forms through the small intestine is considered to be constant at around 3 h, and unaffected by the presence of food. Here we address this assumption and examine how the timing of tablet and food administration can influence small intestine transit time.A non-disintegrating, radiolabelled tablet was given to ten healthy volunteers in a three-way crossover study using three different feeding regimens (1) fasted (tablet administered on an empty stomach and food withheld for four hours) (2) fed (tablet administered after food) and (3) pre-feed (tablet administered 45 min before food). Tablet transit through the gastrointestinal tract was followed using gamma scintigraphy.The small intestinal transit times of tablets after fasted and fed dosing regimens were similar, median 204 and 210 min respectively. With the pre-feed dose, small intestinal transit time was significantly shorter than in the fasted or fed state at 141 min. With this dosing regimen, in six of the volunteers tablets were in the upper small intestine when food arrived and these had a median small intestinal transit time of 100 min.The timing of food ingestion has a clear effect on small intestinal transit of single-unit formulations and this has implications for drug bioavailability.
Keywords: gamma scintigraphy; gastrointestinal transit; intestinal flow; migrating myoelectric complex; modified release; motility

Colloidal Structures in Media Simulating Intestinal Fed State Conditions with and Without Lipolysis Products by Dimitrios G. Fatouros; Isabelle Walrand; Bjorn Bergenstahl; Anette Müllertz (361-374).
To study the ultrastructure of biorelevant media and digestion products of self-nanoemulsifying drug delivery system (SNEDDS) at high level BS/PL conditions.Cryogenic transmission electron microscopy (Cryo-TEM) was employed to visualize the colloid structures in the biorelevant media and lipolytic products generated during hydrolysis of a SNEDDS formulation. Their electrical properties were investigated by measuring their ζ-potential values.In the biorelevant media, vesicles (either unilamellar or multilamellar) and bilayer fragments are visualized. Occasionally, vesicles with an internal deformed structure are recognized, suggesting surface tension or uneven lateral stress. Visualization studies of the intermediate colloidal phases produced during digestion of a SNEDDS using the in vitro lipolysis model revealed the formation of similar structures as previously reported. The ζ-potential of the media was negatively charged and decreased from −23 to −35 mV with increasing surfactant/lipid load. Lower ζ-potential values (−16 mV) obtained for the structures formed during the lipid hydrolysis of the SNEDDS were probably due to the presence of calcium, which shields the surface, thereby reducing the charge.The diversity of these vesicles in terms of size, lamellarity, and internal organization advocate their important role during lipid digestion in the gastrointestinal milieu.
Keywords: ζ-potential; biorelevant media; cryogenic transmission electron microscopy; in vitro digestion lipolysis model; lipolytic products; micelles; multilamellar vesicles; self-nanoemulsifying drug delivery systems; unilamellar

Blocking Effect of an Immuno-Suppressive Agent, Cynarin, on CD28 of T-Cell Receptor by Guo-Chung Dong; Ping-Hsien Chuang; Kai-chun Chang; Pey-shynan Jan; Pei-Ing Hwang; Huan-Bin Wu; Myunggi Yi; Huan-Xiang Zhou; Hueih Min Chen (375-381).
Cynarin, a potential immunosuppressant that blocks the interaction between the CD28 of T-cell receptor and CD80 of antigen presenting cells, was found in Echinacea purpurea by a new pharmaceutical screening method: After Flowing Through Immobilized Receptor (AFTIR; Dong et al., J Med Chem, 49: 1845-1854, 2006). This Echinacea component is the first small molecule that is able to specifically block “signal 2” of T-cell activation.In this study, we used the AFTIR method to further confirm that cynarin effectively blocked the binding between CD80 of B-cells and CD28 of T-cells, and provide details of its mechanism of action.The experimental results showed that cynarin blocked about 87% of the CD28-dependent “signal 2” pathway of T-cell activation under the condition of one to one ratio of T-cell and B-cell in vitro. Theoretical structure modeling showed that cynarin binds to the “G-pocket” of CD28 (Evans et al., Nat Immunol, 6:271-279, 2005), and thus interrupts the site of interaction between CD28 and CD80.These results confirm both that AFTIR is a promising method for screening selective active compounds from herbal medicine and that cynarin has great potential as an immuno-suppressive agent.
Keywords: blocking efficiency; cynarin; CD28; immuno-suppression; T-cell receptor

Intratracheal Versus Intravenous Liposomal Delivery of siRNA, Antisense Oligonucleotides and Anticancer Drug by Olga B. Garbuzenko; Maha Saad; Seema Betigeri; Min Zhang; Alexandre A. Vetcher; Viatcheslav A. Soldatenkov; David C. Reimer; Vitaly P. Pozharov; Tamara Minko (382-394).
To compare systemic intravenous and local intratracheal delivery of doxorubicin (DOX), antisense oligonucleotides (ASO) and small interfering RNA (siRNA).“Neutral” and cationic liposomes were used to deliver DOX, ASO, and siRNA. Liposomes were characterized by dynamic light scattering, zeta-potential, and atomic force microscopy. Cellular internalization of DOX, ASO and siRNA was studied by confocal microscopy on human lung carcinoma cells. In vivo experiments were carried out on nude mice with an orthotopic model of human lung cancer.Liposomes provided for an efficient intracellular delivery of DOX, ASO, and siRNA in vitro. Intratracheal delivery of both types of liposomes in vivo led to higher peak concentrations and much longer retention of liposomes, DOX, ASO and siRNA in the lungs when compared with systemic administration. It was found that local intratracheal treatment of lung cancer with liposomal DOX was more efficient when compared with free and liposomal DOX delivered intravenously.The present study outlined the clear advantages of local intratracheal delivery of liposomal drugs for the treatment of lung cancer when compared with systemic administration of the same drug.
Keywords: antisense oligonucleotides; imaging; liposomes; local lung delivery of siRNA; lung cancer; pulmonary delivery

Intrascleral Drug Delivery to the Eye Using Hollow Microneedles by Jason Jiang; Jason S. Moore; Henry F. Edelhauser; Mark R. Prausnitz (395-403).
This study tested the hypothesis that hollow microneedles can infuse solutions containing soluble molecules, nanoparticles, and microparticles into sclera in a minimally invasive manner.Individual hollow microneedles were inserted into, but not across, human cadaver sclera and aqueous solutions containing sulforhodamine or fluorescently tagged nanoparticles or microparticles were infused into sclera at constant pressure. The infused volume of fluid was measured and imaged histologically as a function of scleral thickness, infusion pressure, needle retraction depth and the presence of spreading enzymes (hyaluronidase and collagenase).Individual hollow microneedles were able to insert into sclera. Fluid infusion was extremely slow after microneedle insertion into the sclera without retraction, but partial retraction of the microneedle over a distance of 200–300 μm enabled infusion of 10–35 μl of fluid into the tissue. Scleral thickness and infusion pressure had insignificant effects on fluid delivery. Nanoparticle suspensions were also delivered into sclera, but microparticles were delivered only in the presence of hyaluronidase and collagenase spreading enzymes, which suggested the role of scleral glycosaminoglycans and collagen fibers as rate-limiting barriers.This study shows that hollow microneedles can infuse solutions into the sclera for minimally invasive delivery of soluble molecules, nanoparticles and microparticles.
Keywords: eye; hollow microneedle; MEMS; ocular drug delivery; sclera

Effect of Diabetes on Transscleral Delivery of Celecoxib by Narayan P. S. Cheruvu; Aniruddha C. Amrite; Uday B. Kompella (404-414).
To investigate the effects of diabetes on transscleral retinal delivery of celecoxib in albino and pigmented rats.Albino (Sprague Dawley—SD) and pigmented (Brown Norway—BN) rats were made diabetic by a single intraperitoneal injection of streptozotocin (60 mg/kg) following 24 h of fasting and diabetes was confirmed (blood glucose >250 mg/dL). Two months after diabetes induction, the integrity of blood-retinal-barrier in control versus diabetic rats from both strains was compared by using FITC-dextran leakage assay. Fifty microliter suspension of celecoxib (3 mg/rat) was injected periocularly in both the strains in one eye, 2 months following diabetes induction. The animals were euthanized at the end of 0.25, 0.5, 1, 2, 3, 4, 8, and 12 h post-dosing and celecoxib levels in ocular tissues and plasma were estimated using a HPLC assay.Diabetes (2-month duration) resulted in 2.4 and 3.5 fold higher blood-retinal barrier leakage in diabetic SD and BN rats, respectively, compared to controls. The area under tissue celecoxib concentration versus time curves (AUC) for sclera, cornea, and lens were not significantly different between control and diabetic animals. However, retinal and vitreal AUCs of celecoxib in treated eyes were approximately 1.5-fold and 2-fold higher in diabetic SD and BN rats, respectively, as compared to the controls.Transscleral retinal and vitreal delivery of celecoxib is significantly higher in diabetic animals of both strains. The increase in retinal delivery of celecoxib due to diabetes is higher in pigmented rats compared to albino rats. Higher delivery of celecoxib in diabetic animals compared to control animals can be attributed to the disruption of blood-retinal barrier due to diabetes.
Keywords: blood-retinal-barrier; diabetes; periocular injection; pigmentation; transscleral

Dexamethasone 21-Sulfate Improves the Therapeutic Properties of Dexamethasone Against Experimental Rat Colitis by Specifically Delivering the Steroid to the Large Intestine by Inho Kim; Hyesik Kong; Younghyun Lee; Sungchae Hong; Jungoh Han; Sunhwa Jung; Yunjin Jung; Young Mi Kim (415-421).
We investigated in vivo-colon targetability and therapeutic properties of DS against experimental rat colitis.The systemic absorption and colonic delivery of D after oral administration of DS was analyzed by examining the concentration of drugs in the GI tract, plasma, urine and feces. Therapeutic activity of DS was determined using a TNBS-induced rat colitis model. Adrenal suppression by DS administration was evaluated by monitoring the concentration of ACTH and corticosterone in the plasma.DS administered orally was delivered efficiently to the large intestine resulting in D accumulation at the target site. In addition, DS was not detectable in the plasma and was detected very low in the urine after DS administration. The fecal and urinary recovery of D (after DS administration) was much greater and less than that after D administration, suggesting that DS should exhibit enhanced therapeutic activity and reduced systemic side effects. Consistent with this notion, DS was more effective than D in healing rat colitis. Moreover, oral administration of either D or DS reduced the plasma corticosterone and ACTH levels from the normal levels, which is significantly greater for D.DS is a promising colon specific prodrug that improves therapeutic properties of D
Keywords: colon specific prodrug; dexamethasone; dexamethasone 21-sulfate; inflammatory bowel disease; systemic adverse effect

Enhancing Alendronate Release from a Novel PLGA/Hydroxyapatite Microspheric System for Bone Repairing Applications by Xuetao Shi; Yingjun Wang; Li Ren; Yihong Gong; Dong-An Wang (422-430).
The goal of this study was to exploit the multifunction of PLGA based microsphere as efficient alendronate delivery and also as potential injectable cell carrier for bone-repairing therapeutics.Novel poly (lactic-co-glycolic acid) (PLGA)-hybridizing -hydroxyapatite (HA) microspheres loaded with bisphosphonate-based osteoporosis preventing drugs, alendronate (AL), are prepared with solid/oil/water (s/o/w) or water/oil/water (w/o/w) technique. Macrophage resistance was evaluated by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DNA assay and Live/dead staining, and osteoblast proliferation and maturation was assessed by MTT assay, Alkaline phosphatase (ALP) activity assay and Real time-PCR.In such fabricated AL laden PLGA/HA microspheric composites (abbreviated “PLGA/HA-AL”), the introduction of HA component has been proven capable of largely enhancing drug encapsulation efficiency especially when the single emulsion protocol is adopted. The in-vitro drug (AL) releasing profile of PLGA/HA-AL system was plotted basing over 30 days’ data collection. It indicates a sustained releasing tendency despite a minimal burst at the very beginning. The in-vitro bone-repairing efficacy of PLGA/HA-AL system was first tested with macrophages that are identified as precursors of osteoclasts and potentially responsible for osteoporosis. The results indicated that the AL release significantly inhibited the growth of macrophages. Additionally, as a central executor for osteogenesis, osteoblasts were also treated with PLGA/HA-AL system in vitro. The outcomes confirmed that this controlled release system functions to improve osteoblast proliferation and also enables upregulation of a key osteogenic enzyme ALP.By pre-resisting osteoclastic commitment and promoting osteoblastic development in vitro, this newly designed PLGA/HA-AL controlled release system is promoting for bone-repairing therapeutics.
Keywords: alendronate; bone repair; drug delivery; hydroxyapatite; microspheres; PLGA

To introduce the design of the electrical Next Generation Impactor (eNGI), and validate its proposed function as a method of electrostatic characterization for pressurized metered dose inhaler (pMDI) formulations.Flixotide® (fluticasone propionate), Ventolin® (salbutamol sulphate), and QVAR® (beclomethasone dipropionate) were used as model pMDIs in this study. At an airflow rate of 30 l/min, five individual actuations of each pMDI were introduced into the electrical low-pressure impactor (ELPI), Next Generation Impactor (NGI), and the eNGI. Charge profiles for each actuation were measured by the ELPI and eNGI, while mass profiles were recorded by the all three impactors.The difference in estimated mass median aerodynamic diameters and geometric standard deviations for all pMDIs using the NGI and eNGI were not found to be statistically significant (p < 0.05). The mean charge profiles from the ELPI and eNGI overlap well between 0.54 and 6.61 μm (Flixotide® and Ventolin®), and between 0.615 and 11.72 μm (QVAR®), where the majority of the impacted doses were collected. Conclusion: For the analysis of pMDIs, the eNGI is comparable to the NGI in measuring particle size distribution, while still being comparable to the ELPI in measuring charge distribution.
Keywords: electrical next generation impactor (eNGI); electrostatics; ELPI

Nanometer- and Submicrometer-Sized Hollow Spheres of Chondroitin Sulfate as a Potential Formulation Strategy for Anti-inflammatory Encapsulation by Adriano V. Reis; Marcos R. Guilherme; Luiz H. C. Mattoso; Adley F. Rubira; Elias B. Tambourgi; Edvani C. Muniz (438-444).
The synthesis of nanometer and submicrometer hollow particles could be a motivating way to imprint new therapeutic properties into a chondroitin sulfate-based hydrogel formulation. The use of hollowed polymer structures as a formulation strategy is expected to have an impact in the effective therapy in the treatment of rheumatoid arthritis.Chemical modification of the chondroitin sulfate with glycidyl methacrylate (GMA) was performed in water under thermal and acid stimuli. The hydrogel spheres were formed upon cross-linking reaction of modified chondroitin sulfate (CSM) in a water-in-benzyl alcohol nano-droplet emulsion. 1H NMR and 13C NMR spectra showed that the carbon–carbon π-bonds coming from the GMA were incorporated onto backbones of CS. 13C-CP/MAS NMR spectra revealed that the formation of the CSM hydrogel spheres during the dispersion stage occurred by way of carbon–carbon π-bonds on the CSM structure. The spherical shapes of the particles with diameters in the range of 20 μm to 500 nm were very clearly verified by SEM images where the dark center and edge of the hollow spheres could be identified easily.Nanometer- and submicrometer-sized hydrogel spheres with hollow interior were produced from chondroitin sulfate by using a new strategy of hydrogel synthesis.
Keywords: chondroitin sulfate; free-radical polymerization; hollow spheres nanotechnology; hydrogel

Emerging Trends in Human ABC Transporters by Toshihisa Ishikawa (445-448).
obtained his Ph.D. degree from the Graduate School of Science, Hokkaido University, Japan in March 1982. In the same year, he received the scholarship of the Deutscher Akademischer Austauschdienst (DAAD) and moved to Germany. From 1982 till 1987, he was a postdoctoral fellow at the Institute of Physiological Chemistry (Prof. Helmut Sies), Medical School of University Düsseldorf, in Germany. In April 1987 he returned to Japan and was appointed Assistant Biochemist at the Department of Biochemistry, Medical School of Osaka University, Japan. In 1989, he went to Germany again to become the project leader at the Department of Tumor Biochemistry in German Cancer Research Institute (DKFZ), Heidelberg. In 1991, Dr. Ishikawa left Germany for U.S.A, as he was appointed Assistant Professor at the Department of Experimental Pediatrics, University of Texas M.D. Anderson Cancer Center in Huston, Texas, U.S.A. In Houston, he had an adjunct appointment as Assistant Professor at the Graduate School of Health Science Center, University of Texas. In 1993, he received the Achievement Award from the International Life Sciences Institute (Washington DC). In December 1995, he was appointed to Senior Scientist and Manager at the Department of Medicinal Biology in the Central Research of Pfizer, Inc. and thereafter he became the Director of the Department of Research Technology Development at the Japanese Headquarters of Pfizer, Inc. in Tokyo, 1999. Since June 1, 2000, Dr. Ishikawa is a Professor at the Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan. In 2003, he founded a bio-venture company named “MedicinalGenomix, Inc.”. He is also Professor (adjunct) at the Graduate School of Tokyo Medical and Dental University as well as Senior Guest Scientist (adjunct) at the Omics Research Center of RIKEN Yokohama Institute. Dr. Ishikawa was a member of the International Nomenclature Committee for Human ABC Transporter Genes. He directed the NEDO International Collaboration Research project entitled “International standardization of in-vitro functional assay methods for human drug transporters” in 2005–2008. He received the Research Award for his distinguished study on “Pharmacogenomics of human ABC transporters” as well as the Fellow Degree from the Japanese Society for Study of Xenobiotics (JSSX) in 2005 and 2008, respectively. In addition, Dr. Ishikawa is currently serving as Associate Editor for several journals including Journal of Experimental Therapeutics and Oncology and Pharmacogenomics. In 2004, Dr. Ishikawa organized the 24th International Symposium on “Pharmacogenomics in Cancer Chemotherapy: Recent Advances in ABC Transporters and Genome Analyses” in Sapporo, Japan. Most recently, he was one of the Steering Committee members to organize the FDA Critical Path Transporter Workshop held in Bethesda, MD on October 2–3, 2008. Thus, Dr. Ishikawa has become an internationally-recognized scientist in the research field on pharmacogenomics of human ABC transporter genes. In addition, he is also currently developing the world’s-fastest SNP detection system for clinical practice.

Cellular Phototoxicity Evoked Through the Inhibition of Human ABC Transporter ABCG2 by Cyclin-dependent Kinase Inhibitors In vitro by Ran An; Yuichiro Hagiya; Ai Tamura; Shanshan Li; Hikaru Saito; Daisuke Tokushima; Toshihisa Ishikawa (449-458).
The physiological importance of the human ATP-binding cassette (ABC) transporter ABCG2 has been recognized with regard to porphyrin-mediated photosensitivity. Functional impairment owing to inhibition of ABCG2 by drugs or its genetic polymorphisms may lead to the disruption of porphyrin homeostasis, which in turn causes cellular toxicity.We evaluated the impact on photosensitivity of the inhibition by cyclin-dependent kinase (CDK) inhibitors of ABCG2 function. For this purpose, we established new methods for photosensitivity assays by using Flp-In-293 cells and plasma membrane vesicles prepared from Sf9 insect cells. With the new methods, we subsequently tested CDK inhibitors, i.e., purvalanol A, WHI-P180, bohemine, roscovitine, and olomoucine.Among CDK inhibitors tested, purvalanol A was found to be the most potent inhibitor (IC50 = 3.5 μM) for ABCG2-mediated hematoporphyrin transport. At a concentration of 2.5 μM, it evoked the photosensitivity of ABCG2-expressing Flp-In-293 cells treated with pheophorbide a. WHI-P180 moderately inhibited ABCG2 function, exhibiting weak phototoxicity. In contrast, the phototoxicity of bohemine, roscovitine, and olomoucine were minimal in our assay system.It is suggested that the planar structure is an important factor for interactions with the active site of ABCG2. The present study provides a new approach to studying drug-induced phototoxicity in vitro.
Keywords: ABCG2 (BCRP/MXR); cyclin-dependent kinase inhibitor; pheophorbide a; phototoxicity; porphyrin

Metabolism and Renal Elimination of Gaboxadol in Humans: Role of UDP-Glucuronosyltransferases and Transporters by Xiao-Yan Chu; Yuexia Liang; Xiaoxin Cai; Karla Cuevas-Licea; Ronda K. Rippley; Kelem Kassahun; Magang Shou; Matthew P. Braun; George A. Doss; M. Reza Anari; Raymond Evers (459-468).
Gaboxadol, a selective extrasynaptic agonist of the delta-containing γ-aminobutyric acid type A (GABAA) receptor, is excreted in humans into the urine as parent drug and glucuronide conjugate. The goal of this study was to identify the UDP-Glucuronosyltransferase (UGT) enzymes and the transporters involved in the metabolism and active renal secretion of gaboxadol and its metabolite in humans.Methods. The structure of the glucuronide conjugate of gaboxadol in human urine was identified by LC/MS/MS. Human recombinant UGT isoforms were used to identify the enzymes responsible for the glucuronidation of gaboxadol. Transport of gaboxadol and its glucuronide was evaluated using cell lines and membrane vesicles expressing human organic anion transporters hOAT1 and hOAT3, organic cation transporter hOCT2, and the multidrug resistance proteins MRP2 and MRP4.Results. Our study indicated that the gaboxadol-O-glucuronide was the major metabolite excreted in human urine. UGT1A9, and to a lesser extent UGT1A6, UGT1A7 and UGT1A8, catalyzed the O-glucuronidation of gaboxadol in vitro. Gaboxadol was transported by hOAT1, but not by hOCT2, hOAT3, MRP2, and MRP4. Gaboxadol-O-glucuronide was transported by MRP4, but not MRP2.Conlusion. Gaboxadol could be taken up into the kidney by hOAT1 followed by glucuronidation and efflux of the conjugate into urine via MRP4.
Keywords: gaboxadol; glucuronidation; kidney; transporters

Major SNP (Q141K) Variant of Human ABC Transporter ABCG2 Undergoes Lysosomal and Proteasomal Degradations by Tomoka Furukawa; Kanako Wakabayashi; Ai Tamura; Hiroshi Nakagawa; Yoshihiro Morishima; Yoichi Osawa; Toshihisa Ishikawa (469-479).
Single nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCG2 gene have been suggested to be a significant factor in patients’ responses to medication and/or the risk of diseases. We aimed to evaluate the impact of the major non-synonymous SNP Q141K on lysosomal and proteasomal degradations.ABCG2 WT and the Q141K variant were expressed in Flp-In-293 cells by using the Flp recombinase system. Their expression levels and cellular localization was measured by immunoblotting and immunofluorescence microscopy, respectively.The protein level of the Q141K variant expressed in Flp-In-293 cells was about half that of ABCG2 WT, while their mRNA levels were equal. The protein expression level of the Q141K variant increased about two-fold when Flp-In-293 cells were treated with MG132. In contrast, the protein level of ABCG2 WT was little affected by the same treatment. After treatment with bafilomycin A1, the protein levels of ABCG2 WT and Q141K increased 5- and 2-fold in Flp-In-293 cells, respectively.The results strongly suggest that the major non-synonymous SNP Q141K affects the stability of the ABCG2 protein in the endoplasmic reticulum and enhances its susceptibility to ubiquitin-mediated proteasomal degradation.
Keywords: ABCG2; endoplasmic reticulum associated degradation (ERAD); proteasome; SNP; ubiquitin

Curcumin Inhibits the Activity of ABCG2/BCRP1, a Multidrug Resistance-Linked ABC Drug Transporter in Mice by Suneet Shukla; Hani Zaher; Anika Hartz; Björn Bauer; Joseph A. Ware; Suresh V. Ambudkar (480-487).
To evaluate the in vivo efficacy of curcumin as an inhibitor of the multidrug-resistance-linked ATP Binding Cassette (ABC) drug transporter, ABCG2.Photoaffinity labeling with [125I]-iodoarylazidoprazosin was used to characterize the interaction of sulfasalazine, a substrate of the mouse ABCG2, with human ABCG2. In addition, the inhibitory effect of curcumin on ABCG2 was evaluated in brain capillaries from rats. Furthermore, the effect of curcumin on absorption of orally administered sulfasalazine in wild-type and abcg2 −/− mice was also determined.Sulfasalazine interacted at the drug-substrate site(s) of human ABCG2. Curcumin inhibited ABCG2 activity at nanomolar concentrations at the rat blood-brain barrier in the ex vivo assay. Based on studies in wild type and abcg2 −/− mice, we observed that oral curcumin increased C max and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function.This study validates our previous in vitro results with human ABCG2 (Chearwae et al., Mol. Cancer Ther. 5:1995–2006, 2006) and provides the first in vivo evidence for the inhibition by curcumin of ABCG2-mediated efflux of sulfasalazine in mice. Based on these studies, we propose that non-toxic concentrations of curcumin may be used to enhance drug exposure when the rate-limiting step of drug absorption and/or tissue distribution is impacted by ABCG2.
Keywords: ABC transporter; ABCG2; curcumin; multidrug resistance; sulfasalazine

In Drug Delivery, Shape Does Matter by Samir Mitragotri (488-488).

AAPS Connection (489-490).