Pharmaceutical Research (v.25, #3)

The Use of BDDCS in Classifying the Permeability of Marketed Drugs by Leslie Z. Benet; Gordon L. Amidon; Dirk M. Barends; Hans Lennernäs; James E. Polli; Vinod P. Shah; Salomon A. Stavchansky; Lawrence X. Yu (483-488).
We recommend that regulatory agencies add the extent of drug metabolism (i.e., ≥ 90% metabolized) as an alternate method in defining Class 1 marketed drugs suitable for a waiver of in vivo studies of bioequivalence. That is, ≥ 90% metabolized is an additional methodology that may be substituted for ≥ 90% absorbed. We propose that the following criteria be used to define ≥ 90% metabolized for marketed drugs: Following a single oral dose to humans, administered at the highest dose strength, mass balance of the Phase 1 oxidative and Phase 2 conjugative drug metabolites in the urine and feces, measured either as unlabeled, radioactive labeled or nonradioactive labeled substances, account for ≥ 90% of the drug dosed. This is the strictest definition for a waiver based on metabolism. For an orally administered drug to be ≥ 90% metabolized by Phase 1 oxidative and Phase 2 conjugative processes, it is obvious that the drug must be absorbed. This proposal, which strictly conforms to the present ≥ 90% criteria, is a suggested modification to facilitate a number of marketed drugs being appropriately assigned to Class 1.
Keywords: BCS; BDDCS; bioequivalence; elimination pathways

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure–function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. “Shielding” polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating ‘designer’ gene delivery vectors with enhanced properties.
Keywords: adeno-associated virus; directed evolution; gene delivery; neutralizing antibody; tropism

Inhibition of efflux pumps is an emerging approach in cancer therapy and drug delivery. Since it has been discovered that polymeric pharmaceutical excipients such as Tweens® or Pluronics® can inhibit efflux pumps, various other polymers have been investigated regarding their potential efflux pump inhibitory activity. Among them are polysaccharides, polyethylene glycols and derivatives, amphiphilic block copolymers, dendrimers and thiolated polymers. In the current review article, natural and synthetic polymers that are capable of inhibiting efflux pumps as well as their application in cancer therapy and drug delivery are discussed.
Keywords: drug delivery; efflux pump inhibitors; P-glycoprotein; P-gp inhibitors; polymeric inhibitors

To determine the elimination rates of subconjunctivally injected model drugs using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).Gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA) and gadolinium-albumin (Gd-albumin) were injected in rabbits. Experiments were performed in vivo and post mortem and injection volumes of 200 and 600 μl were administered. Signal intensity values from MR images were converted to concentration of contrast agent to determine the mass clearance rates from subconjunctival space.Injection volume did not have a significant effect on clearance rate for both Gd-DTPA and Gd-albumin. The clearance rate of Gd-DTPA in vivo was about nine times faster than that post mortem. The in vivo and post mortem clearance rates of Gd-albumin were not significantly different. The in vivo half-life of Gd-DTPA was about 22 min while that of Gd-albumin was about 5.3 h.DCE-MRI was used to quantitatively compare the subconjunctival clearance rates of Gd-DTPA and Gd-albumin. Dynamic clearance mechanisms present in vivo significantly reduced the subconjunctival concentration of Gd-DTPA but not Gd-albumin. Lymphatic clearance does not seem to be as significant as clearance by blood, as evidenced by data from Gd-albumin injections. Larger injection volumes may allow for longer retention times and prolonged release of drug.
Keywords: drug transport barrier; magnetic resonance imaging; ocular pharmacokinetics; subconjunctival injection; transscleral drug delivery

Calcipotriol Affects Keratinocyte Proliferation by Decreasing Expression of Early Growth Response-1 and Polo-like Kinase-2 by Jernej Kristl; Petra Slanc; Metka Krašna; Aleš Berlec; Matjaž Jeras; Borut Štrukelj (521-529).
Calcipotriol is a potent drug for topical treatment of psoriasis because it manages to inhibit keratinocyte proliferation. In the present study we investigated the effects of calcipotriol on gene expression in human keratinocytes in terms of mechanism of how calcipotriol decreases proliferation.Cell proliferation was analyzed by MTT assay. The differential display approach together with qPCR was used to assess the gene expression after treatment. In addition, Western immunoblotting revealed differences on the protein level. Finally, transfection of the KCs with specific small interfering RNA determined the genes necessary to inhibit proliferation.KCs proliferation was decreased in a concentration-dependent manner. Moreover, calcipotriol dowregulated the expression of two proliferation factors: early growth response-1 (EGR1) and polo-like kinase-2 (PLK2). The protein levels of EGR1 and PLK2 were also decreased. Specific siRNA against EGR1 and PLK2 in KCs resulted in marked reduction of EGR1 and PLK2 expression. In both cases, the reduction resolved in the decreased proliferation of KCs.This study provides a new insight into how calcipotriol affects proliferation of keratinocytes by decreasing the expression of EGR1 and PLK2. Furthermore, the results offer groundwork for developing novel compounds for the treatment of hiperproliferative skin disorders like psoriasis.
Keywords: calcipotriol; differential display; EGR1; PLK2; siRNA

To design and prepare cocrystals of indomethacin using crystal engineering approaches, with the ultimate objective of improving the physical properties of indomethacin, especially solubility and dissolution rate.Various cocrystal formers, including saccharin, were used in endeavours to obtain indomethacin cocrystals by slow evaporation from a series of solvents. The melting point of crystalline phases was determined. The potential cocrystalline phase was characterized by DSC, IR, Raman and PXRD techniques. The indomethacin–saccharin cocrystal (hereafter IND–SAC cocrystal) structure was determined from single crystal X-ray diffraction data. Pharmaceutically relevant properties such as the dissolution rate and dynamic vapour sorption (DVS) of the IND–SAC cocrystal were evaluated. Solid state and liquid-assisted (solvent-drop) cogrinding methods were also applied to indomethacin and saccharin.The IND–SAC cocrystals were obtained from ethyl acetate. Physical characterization showed that the IND–SAC cocrystal is unique vis-à-vis thermal, spectroscopic and X-ray diffraction properties. The cocrystals were obtained in a 1:1 ratio with a carboxylic acid and imide dimer synthons. The dissolution rate of IND–SAC cocrystal system was considerably faster than that of the stable indomethacin γ-form. DVS studies indicated that the cocrystals gained less than 0.05% in weight at 98%RH. IND–SAC cocrystal was also obtained by solid state and liquid-assisted cogrinding methods.The IND–SAC cocrystal was formed with a unique and interesting carboxylic acid and imide dimer synthons interconnected by weak N−H⋯O hydrogen bonds. The cocrystals were non-hygroscopic and were associated with a significantly faster dissolution rate than indomethacin (γ-form).
Keywords: crystal engineering; dissolution rate; indomethacin; pharmaceutical cocrystals; poorly soluble drugs

Toxicokinetic Study of Recombinant Human Heparin-Binding Epidermal Growth Factor-Like Growth Factor (rhHB-EGF) in Female Sprague Dawley Rats by Intira Coowanitwong; Susan K. Keay; Karthika Natarajan; Tushar S. Garimella; Clifford W. Mason; David Grkovic; Kenneth S. Bauer (542-550).
To determine the toxicity and pharmacokinetics of recombinant heparin-binding epidermal growth factor-like growth factor in female Sprague Dawley rats following intra-bladder and intravenous administration.rhHB-EGF was administered once daily for 6 or 27 days at doses of 3, 10, or 30 μg/kg. 125I-rhHB-EGF was administered on day 7 or 28 for pharmacokinetic analysis. Toxicity was assessed by general appearance and behavior, gross necropsy, blood chemistry and microscopic evaluation.Plasma AUCss of [125I] rhHB-EGF equivalents following IB administration for 7 days were 4.28 ± 2.29, 7.75 ± 2.70, and 7.11 ± 1.42 ng ml−1 h−1 at doses of 3, 10, and 30 μg/kg, respectively. Following IV administration, the AUCss on day 7 increased from 27.0 ± 2.66 to 124 ± 5.09 and 385.11 ± 7.57 ng ml−1 h−1 with increasing the dose from 3 to 10 and 30 μg/kg. Similar AUCss data was obtained after 28 day administration. No toxicity was evident upon gross examination. Histologic examination revealed subacute inflammation and lymphocytic infiltration of the urinary bladder in animals from all groups dosed by the IB route.Plasma and bladder concentrations of recombinant human [125I] rhHB-EGF equivalents were significantly lower following the IB route than following IV administration. Histologic tissue examination indicated no toxicity attributable to rhHB-EGF.
Keywords: HB-EGF; interstitial cystitis; intrabladder instillation; intravenous administration pharmacokinetics; NONMEM; toxicity; toxicokinetics

Delivery by Cationic Gelatin Nanoparticles Strongly Increases the Immunostimulatory Effects of CpG Oligonucleotides by Klaus Zwiorek; Carole Bourquin; Julia Battiany; Gerhard Winter; Stefan Endres; Gunther Hartmann; Conrad Coester (551-562).
Cationized gelatin nanoparticles (GNPs) were used as carrier to improve delivery of immunostimulatory CpG oligonucleotides (CpG ODN) both in vitro and in vivo.Uptake of CpG ODN-loaded cationized gelatin nanoparticles (CpG-GNPs) into murine myeloid dendritic cells (DCs) and their respective immunostimulatory activity was monitored. In vivo, induction of cytokine secretion by CpG-GNPs was measured. For experiments on primary human cells, prototypes of the three CpG ODN classes were adsorbed onto GNPs. Uptake and induction of proinflammatory cytokines were assessed in human plasmacytoid DCs and B cells, the only existing human target cells for CpG ODN.In the murine system, gelatin nanoparticle formulations enhanced the uptake and immunostimulatory activity of CpG ODN both in vitro and in vivo. Furthermore, delivery by cationized gelatin nanoparticles of CpG ODN of the classes B and C to primary human plasmacytoid DCs increased production of IFN-α, a key cytokine in the driving of both the innate and adaptive immune responses.GNPs can be used as a biodegradable and well tolerated carrier to deliver CpG ODN to their target cells and strongly increase activation of the immune system. This concept may be applied as novel adjuvant for antiviral and antitumoral vaccines.
Keywords: adjuvant; B cells; CpG oligonucleotide; dendritic cells; gelatin nanoparticles

The aim of this work was to compare the physicochemical characteristics of the phospholipids complex of puerarin (Pur) prepared by traditional methods (solvent evaporation, freeze-drying and micronization) and a supercritical fluid (SCF) technology. The physicochemical properties of the pure drug and the corresponding products prepared by two different SCF methods were also compared.Solid-state characterization of particles included differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), solubility, dissolution rate and scanning electron microscopy (SEM) examinations. Besides puerarin phospholipids complex (PPC) by four different methods, the solid-state properties of unprocessed, gas antisolvent (GAS) crystallized and solution enhanced dispersion by supercritical fluid (SEDS) precipitated puerarin samples were also compared. Crystallinity was assessed using DSC and XRPD. Drug-phospholipids interactions were characterized using Fourier transform infrared spectroscopy (FTIR). SEM was used to determine any morphological changes. Pharmaceutical performance was assessed in dissolution rate and solubility tests.The results of the physical characterization attested a substantial correspondence of the solid state of the drug before and after treatment with GAS technique, whereas a pronounced change in size and morphology of the drug crystals was noticed. The GAS-processed puerarin exhibited a better crystal shape confirmed by DSC, XRPD and IR. Polymorphic change of puerarin during SEDS coupled with the dramatic reduction of the dimensions determined a remarkable enhancement of its solubility and in vitro dissolution rate. Phospholipids complex prepared using supercritical fluid technology showed similar properties of physical state, thermal stability and molecular interaction with phospholipids (PC) to those of corresponding systems prepared by other three conventional methods namely solvent evaporation, freeze-drying and micronization as proved by XRPD, DSC, and FTIR. The best dissolution rate was obtained by SEDS-prepared complex, while the highest solubility was obtained for solvent evaporation method.Supercritical fluid technology for the preparation of puerarin and its phospholipids complex has been proven to have significant advantages over the solvent evaporation technique and other conventional methods.
Keywords: microparticles; phospholipids complex; physicochemical characterization; puerarin; supercritical fluids

Predominant Contribution of Rat Organic Anion Transporting Polypeptide-2 (Oatp2) to Hepatic Uptake of β-Lactam Antibiotics by Masanori Nakakariya; Taiki Shimada; Masanori Irokawa; Hiroyuki Koibuchi; Takashi Iwanaga; Hikaru Yabuuchi; Tomoji Maeda; Ikumi Tamai (578-585).
To identify the rat hepatic basolateral transporters involved in the hepatic uptake of β-lactam antibiotics using nafcillin as a model β-lactam antibiotic that undergoes extensive biliary excretion.Uptake by isolated rat hepatocytes and Xenopus laevis oocytes expressing organic anion transporting peptides (Oatp1, 2, and 4) and organic anion transporter (OAT2) was evaluated.Nafcillin uptake by isolated rat hepatocytes was saturable with the K m of 210 μM and was significantly inhibited by anionic compounds (estrone-3-sulfate and sulfobromophthalein), but not by cationic compounds (tetraethylammonium and 1-methyl-4-phenylpyridinium). In an in vitro uptake study by Xenopus oocytes expressing hepatic basolateral membrane transporters, nafcillin was transported by multiple Oatps with K m values of 4120 μM (Oatp1/Oatp1a1), 198 μM (Oatp2/Oatp1a4), and 1,570 μM (Oatp4/Oatp1b2), though it was not transported by hOAT2. Comparison of affinity and analysis by the relative activity factor method indicated that Oatp2 is the predominant contributor to the hepatic uptake of nafcillin. Cefadroxil, cefazolin, cefmetazole, cefoperazone, cefsulodin, and cephalexin, though not cefotaxime or ceftriaxone, were also substrates of Oatp2.These findings suggest that Oatp2 plays a key role in the hepatic uptake of nafcillin and most other β-lactam antibiotics in rats.
Keywords: biliary excretion; hepatic uptake; oatp; organic anion; transporter

The objective of this study was to develop poly(lactic-co-glycolic acid) (PLGA) injectable implants (i.e., millicylinders) with microencapsulated N-acetylcysteine (NAC) for site-specific controlled NAC release, for potential chemopreventive applications in persons with previously excised head and neck cancers.PLGA 50:50 (i.v. = 0.57 dl/g) implants with 1–10 wt% NAC free acid or 10 wt% NAC salts (NAC–Na+, NAC–Mg2+ and NAC–Ca2+) were prepared by solvent extrusion and/or fluid energy micronization (FEM) methods. X-ray diffraction (XRD), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) studies were performed to evaluate the physical mixing of NAC with PLGA. PLGA implant degradation was studied by kinetics of polymer molecular weight decline (gel permeation chromatography) and mass loss. Release studies were conducted in N2 purged PBS (pH 7.4) at 37°C in evacuated and sealed ampoules. NAC was quantified by HPLC at 210 nm.XRD, SEM and DSC studies indicated that NAC had dissolved in the polymer phase at 1–3.5% w/w loading, but became discretely suspended in the polymer at 6–10% w/w. Initial burst and long-term release rate increased with increased drug loading, and release was uncharacteristically rapid at higher loading (6–10% w/w). The cause of the rapid release was linked to extensive plasticization, matrix porosity and general acid catalysis of PLGA degradation caused by the NAC free acid. PLGA millicylinders loaded with 10% w/w NAC–Ca2+ and NAC–Mg2+salts exhibited reduced burst (34 vs 13–22% release within a day of incubation for NAC free acid vs NAC–Ca2+ and NAC–Mg2+salts, respectively) and slow and continuous complete release over 4 weeks without significant NAC-catalyzed degradation of PLGA. Release of NAC from NAC–Ca2+/PLGA implant was slower than that of NAC–Mg2+/PLGA consistent with the lower solubility of the former salt. NAC with its free thiol was rapidly converted to its cystine dimer in the presence of molecular oxygen. PLGA released samples in sealed and evacuated ampoules indicated >80% parent NAC remaining after the 1 month release analysis irrespective of initial NAC free acid and salt forms.By encapsulating the NAC–Mg2+ and NAC–Ca2+ salts in PLGA implants, the high initial burst, short release duration, and the general acid catalysis caused by the NAC free acid were each prevented and 1-month slow and continuous release was attained with minimal instability of the free thiol group.
Keywords: burst effect; controlled release; drug induced plasticization; drug induced PLGA degradation; head and neck cancer; N Acetylcysteine; NAC salts; PLGA degradation; PLGA implants

Mechanism of UVA-dependent DNA Damage Induced by An Antitumor Drug Dacarbazine in Relation to its Photogenotoxicity by Takuya Iwamoto; Yusuke Hiraku; Masahiro Okuda; Shosuke Kawanishi (598-604).
It has been reported that dacarbazine (DTIC) is photogenotoxic. The purpose of this study is to clarify the mechanism of photogenotoxicity induced by DTIC.We examined DNA damage induced by UVA-irradiated DTIC using 32P-5′-end-labeled DNA fragments obtained from human genes. Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in calf thymus DNA was measured by high performance liquid chromatograph with an electrochemical detector. Electron spin resonance (ESR) spin-trapping experiments were performed to detect radical species generated from UVA-irradiated DTIC.UVA-irradiated DTIC caused DNA damage at guanine residues, especially at the 5′-GGT-3′ sequence in the presence of Cu(II) and also induced 8-oxodG generation in calf thymus DNA. DTIC-induced photodamage to DNA fragments was partially inhibited by catalase, whereas 8-oxodG formation was significantly increased by catalase. NaN3, a carbene scavenger, inhibited DNA damage and 8-oxodG formation in a dose-dependent manner, suggesting that carbene intermediates are involved. The ESR spin-trapping experiments demonstrated the generation of aryl radicals in the process of photodegradation of DTIC.Photoactivated DTIC generates the carbene and aryl radicals, which may induce both DNA adduct and 8-oxodG formation, resulting in photogenotoxicity. This study could provide an insight into the safe usage of DTIC.
Keywords: aryl radical; carbene; dacarbazine; DNA damage; photogenotoxicity

To evaluate the effect of re-directing the uptake mechanism of polyplexes containing oligodeoxynucleotide (ODN) decoys to nuclear factor kappa B (NF-κB) from absorptive-mediated to receptor-mediated endocytosis.Complexes of ODNs and a co-polymer of biotin–polyethylenglycol and polyethylenimine (BPP) were targeted to brain-derived endothelial cells with a conjugate of antibody 8D3 and streptavidin (8D3SA). Size and stability of ODN/BPP complexes was measured by dynamic light scattering. Cellular uptake was studied by confocal microscopy. Cell viability and pharmacological effects were investigated on murine bEnd5 cells stimulated with tumor necrosis factor.ODN/BPP complexes showed sizes of 116 ± 2.3 nm, which increased by 40 nm when coupled to 8D3SA, and were stable in physiological fluids. Targeted complexes were internalized intact into endosomal compartments. Treatment conditions, which yielded significant inhibitory effects on mRNA expression of VCAM-1, ICAM-1, IκBα and iNOS by bEnd5 cells, did not affect viability. At 0.5 μM, decoy ODN significantly inhibited monocyte adhesion to bEnd5 monolayers when delivered as 8D3SA-targeted complex, while higher concentrations of untargeted complex were ineffective.The complex of NF-κB decoys and BPP, which can be targeted to transferrin receptors, is a promising drug candidate for neuroinflammatory diseases affecting the blood–brain barrier.
Keywords: blood–brain barrier; drug delivery; polyethylenimine; transferrin receptor; transcription factor decoy

Alternating Current (AC) Iontophoretic Transport across Human Epidermal Membrane: Effects of AC Frequency and Amplitude by Guang Yan; Qingfang Xu; Yuri G. Anissimov; Jinsong Hao; William I. Higuchi; S. Kevin Li (616-624).
As a continuing effort to understand the mechanisms of alternating current (AC) transdermal iontophoresis and the iontophoretic transport pathways in the stratum corneum (SC), the objectives of the present study were to determine the interplay of AC frequency, AC voltage, and iontophoretic transport of ionic and neutral permeants across human epidermal membrane (HEM) and use AC as a means to characterize the transport pathways.Constant AC voltage iontophoresis experiments were conducted with HEM in 0.10 M tetraethyl ammonium pivalate (TEAP). AC frequencies ranging from 0.0001 to 25 Hz and AC applied voltages of 0.5 and 2.5 V were investigated. Tetraethyl ammonium (TEA) and arabinose (ARA) were the ionic and neutral model permeants, respectively. In data analysis, the logarithm of the permeability coefficients of HEM for the model permeants was plotted against the logarithm of the HEM electrical resistance for each AC condition.As expected, linear correlations between the logarithms of permeability coefficients and the logarithms of resistances of HEM were observed, and the permeability data were first normalized and then compared at the same HEM electrical resistance using these correlations. Transport enhancement of the ionic permeant was significantly larger than that of the neutral permeant during AC iontophoresis. The fluxes of the ionic permeant during AC iontophoresis of 2.5 V in the frequency range from 5 to 1,000 Hz were relatively constant and were approximately 4 times over those of passive transport. When the AC frequency decreased from 5 to 0.001 Hz at 2.5 V, flux enhancement increased to around 50 times over passive transport.While the AC frequency for achieving the full effect of iontophoretic enhancement at low AC frequency was lower than anticipated, the frequency for approaching passive diffusion transport at high frequency was higher than expected from the HEM morphology. These observations are consistent with a transport model of multiple barriers in series and the previous hypothesis that the iontophoresis pathways across HEM under AC behave like a series of reservoirs interconnected by short pore pathways.
Keywords: AC; human skin; iontophoresis; transdermal; transport

The objective was to establish in vitro passive permeability (P e) vs in vivo fraction absorbed (f a)-relationships for each passage through the human intestine, liver, renal tubuli and brain, and develop a P e-based ADME/PK classification system (PCS). P e- and intestinal f a-data were taken from an available data set. Hepatic f a was calculated based on extraction ratios of the unbound fraction of drugs (with support from animal in vivo uptake data). Renal f a (reabsorption) was estimated using renal pharmacokinetic data, and brain f a was predicted using animal in vitro and in vivo brain P e-data. Hepatic and intestinal f a-data were used to predict bile excretion potential.Relationships were established, including predicted curves for bile excretion potential and minimum oral bioavailability, and a 4-Class PCS was developed: I (very high P e; elimination mainly by metabolism); II (high P e) and III (intermediate P e and incomplete f a); IV (low P e and f a). The system enables assessment of potential drug–drug transport interactions, and drug and metabolite organ trapping.The PCS and high quality P e-data (with and without active transport) are believed to be useful for predictions and understanding of ADME/PK, elimination routes, and potential interactions and organ trapping/toxicity in humans.
Keywords: absorption; classification system; drug–drug transport interactions; permeability; prediction

Involvement of Uric Acid Transporters in Alteration of Serum Uric Acid Level by Angiotensin II Receptor Blockers by Masanobu Sato; Takashi Iwanaga; Hideaki Mamada; Toshio Ogihara; Hikaru Yabuuchi; Tomoji Maeda; Ikumi Tamai (639-646).
To examine the mechanisms of the alteration of serum uric acid level by angiotensin II receptor blockers (ARBs), the effects of ARBs on renal uric acid transporters, including OAT1, OAT3, OAT4, and MRP4, were evaluated.Uptakes of uric acid by OAT1-expressing Flp293 cells, by Xenopus oocytes expressing OAT3 or OAT4, and by membrane vesicles from Sf9 cells expressing MRP4 were evaluated in the presence or absence of ARBs.All ARBs inhibited uptake of uric acid or estrone-3-sulfate by OAT1, OAT3 and OAT4 in concentration dependent manners. Among them, the IC50 values of valsartan, olmesartan and pratosartan for OAT3 were comparable to clinically observed unbound maximum plasma concentration of ARBs. Candesartan, losartan, and telmisartan inhibited ATP-dependent uptake of uric acid by MRP4 at 10 μM. The IC50 value of losartan for MRP4 was comparable to the estimated kidney tissue concentration of losartan. No ARBs showed trans-stimulatory effects on the uptake of estrone-3-sulfate by OAT4.Valsartan, olmesartan, and pratosartan could inhibit the OAT3-mediated uric acid secretion in clinical situations. Furthermore losartan could inhibit ATP-dependent uric acid secretion by MRP4. These effects may explain partially the alteration of serum uric acid level by ARBs.
Keywords: angiotensin II receptor blockers; kidney; MRP4; OAT; uric acid transporter

To compare the physical stability of amorphous molecular level solid dispersions of nifedipine and felodipine, in the presence of poly(vinylpyrrolidone) (PVP) and small amounts of moisture.Thin amorphous films of nifedipine and felodipine and amorphous molecular level solid dispersions with PVP were stored at various relative humidities (RH) and the nucleation rate was measured. The amount of water sorbed at each RH was measured using isothermal vapor sorption and glass transition temperatures (T g) were determined using differential scanning calorimetry. The solubility of each compound in methyl pyrrolidone was measured as a function of water content.Nifedipine crystallizes more easily than felodipine at any given polymer concentration and in the presence of moisture. The glass transition temperatures of each compound, alone and in the presence of PVP, are statistically equivalent at any given water content. The nifedipine systems are significantly more hygroscopic than the corresponding felodipine systems.Variations in the physical stability of the two compounds could not be explained by differences in T g. However, the relative physical stability is consistent with differences in the degree of supersaturation of each drug in the solid dispersion, treating the polymer and water as a co-solvent system for each drug compound.
Keywords: amorphous; crystallization; inhibition; nucleation; poly(vinylpyrrolidone); water

A Biodegradable pH-sensitive Micelle System for Targeting Acidic Solid Tumors by Vijay A. Sethuraman; Myung Cheon Lee; You Han Bae (657-666).
A new pH-sensitive micelle delivery system based on TAT cell penetrating peptide and biodegradable sulfonamide grafted disulfide polymer is presented. The system consists of two components: (1) A polymeric micelle made of Poly(l-lactic acid)-b-poly(ethylene glycol) (PLLA-b-PEG) conjugated to TAT (TAT-micelle), (2) A pH-sensitive diblock copolymer (poly(l-cystine bisamide-g-sulfadiazine))-b-PEG (PCBS-b-PEG). The anionic PCBS complexed with cationic TAT of TAT-micelles forms the final carrier. PCBS showed rapid degradation in the presence of cysteine. The TAT-micelles showed increase in particle size between pH 8.0 and 7.0 upon mixing with PCBS-b-PEG indicating complexation. As the pH was further decreased (pH 6.8 to 6.0) two populations were observed, one of normal TAT-micelles and the other of aggregated PCBS-b-PEG. Flow cytometry showed significantly higher uptake of TAT-micelles at pH 6.6 indicating deshielding compared to pH 7.4. The anticancer drug doxorubicin (DOX) was encapsulated into the TAT-micelles, and the in vitro cytotoxicity at different pHs was evaluated. The system was able to distinguish pHs 7.2 and 7.0 in terms of cytotoxicity.
Keywords: degradable pH sensitive polymer; disulfide polymer; pH-sensitive polymer; tumor pH

Implications on Emergence of Antimicrobial Resistance as a Critical Aspect in the Design of Oral Sustained Release Delivery Systems of Antimicrobials by Amnon Hoffman; Ehud Horwitz; Shmuel Hess; Ronit Cohen-Poradosu; Lilach Kleinberg; Anna Edelberg; Mervyn Shapiro (667-671).
To assess the effects of the unabsorbed fraction of an orally administered antimicrobial drug which enters the colon on the emergence of resistance among the natural microflora, a phenomenon largely overlooked so far despite its clinical importance, especially when sustained release formulations are used.Effects of an orally administered model β-lactam antibiotic (amoxicillin) on emergence of resistant bacteria were assessed using a microbiological assay for qualitative and quantitative determination of resistant bacteria in fecal samples of rats following gastric administration of the drug to rats for 4 consecutive days. Time- and site-controlled administration of a β-lactamase to the rat colon was assessed as a potential strategy for prevention the emergence of resistant bacteria following oral administration of incompletely absorbed antimicrobials.Emergence of resistant bacteria was demonstrated following oral administration of amoxicillin to rats, whereas de-activation of the β-lactam prior to entering the colon, by infusion of a β-lactamase into the lower ileum, was shown to prevent the emergence of resistant colonic bacteria.This study illustrates the need to consider the emergence of antimicrobial resistance as a goal equally important to microbiological and clinical cure, when designing oral sustained-release delivery systems of antimicrobial drugs.
Keywords: antimicrobial resistance; β-lactam antibiotic; colonic microflora; delayed action preparations; oral dosage form

To explore the relationship between the structure of block polypeptides and their self-assembly into hydrogels. To investigate structural parameters that influence hydrogel formation and physical properties.Three ABA triblock and two AB diblock coiled-coil containing polypeptides were designed and biologically synthesized. The triblock polypeptides had two terminal coiled-coil (A) domains and a central random coil (B) segment. The coiled-coil domains were different in their lengths, and tyrosine residues were incorporated at selected solvent-exposed positions in order to increase the overall hydrophobicity of the coiled-coil domains. The secondary structures of these polypeptides were characterized by circular dichroism and analytical ultracentrifugation. The formation of hydrogel structures was evaluated by microrheology and scanning electron microscopy.Hydrogels self-assembled from the triblock polypeptides, and had interconnected network microstructures. Hydrogel formation was reversible. Denaturation of coiled-coil domains by guanidine hydrochloride (GdnHCl) resulted in disassembly of the hydrogels. Removal of GdnHCl by dialysis caused coiled-coil refolding and hydrogel reassembly.Protein ABA triblock polypeptides composed of a central random block flanked by two coiled-coil forming sequences self-assembled into hydrogels. Hydrogel formation and physical properties may be manipulated by choosing the structure and changing the length of the coiled-coil blocks. These self-assembling systems have a potential as in-situ forming depots for protein delivery.
Keywords: coiled-coils; genetically engineered polymers; hydrogels; self-assembly

Intelligent Biosynthetic Nanobiomaterials (IBNs) for Hyperthermic Gene Delivery by Tze-Haw Howard Chen; Younsoo Bae; Darin Y. Furgeson (683-691).
Intelligent biosynthetic nanobiomaterials (IBNs) were constructed as recombinant diblock copolymers, notated as K8-ELP(1–60), containing a cationic oligolysine (VGK8G) and a thermosensitive elastin-like polypeptide (ELP) block with 60 repetitive pentapeptide units [(VPGXG)60; X is Val, Ala and Gly in a 5:2:3 ratio].K8-ELP(1–60) was synthesized by recursive directional ligation for DNA oligomerization. Purity and molecular weight of K8-ELP(1–60) were confirmed by SDS-PAGE and mass spectrometry. DNA polyplexes were prepared from K8-ELP(1–60) and pGL3-Control (pGL3–C) plasmid DNA (pDNA) and stability was evaluated by gel retardation, DLS, and DNA displacement with heparin. Thermal transition profiles were studied by measuring the turbidity change at 350 nm and the polyplexes were used to transfect MCF-7 cells with a concomitant cytotoxicity assay.SDS-PAGE and MALDI-TOF studies showed highly pure copolymers at the desired molecular weight. K8-ELP(1-60) condensed pDNA at a cation to anion (N/P) ratio above 0.25 with a tight distribution of particle size ranging from 115.5–32.4 nm with increasing N/P ratio. Thermal transition temperatures of K8-ELP(1-60)/pDNA and K8-ELP(1-60) alone were 44.9 and 71.5°C, respectively. K8-ELP(1-60)/pDNA complexes successfully transduced MCF-7 cells with qualitative expression of enhanced green fluorescent protein (EGFP) and minimal cytotoxicity compared to branched poly(ethyleneimine) controls.K8-ELP(1-60) was successfully designed and purified through recombinant means with efficient and stable condensation of pDNA at N/P ratios > 0.25 and polyplex particle size < 115 nm. MCF-7 cells successfully expressed EGFP with minimal cytotoxicity compared to positive controls; moreover, polyplexes retained sharp, thermotransitive kinetics within a narrow Tt range at clinically relevant hyperthermic temperatures, where the decrease of Tt was due to the increased hydrophobicity upon charge neutralization.
Keywords: nanobiomaterial; intelligent polymer; elastin-like polypeptide; gene delivery; gene vector; hyperthermia

In Vitro Chondrogenesis of Mesenchymal Stem Cells in Recombinant Silk-elastinlike Hydrogels by Mohamed Haider; Joseph Cappello; Hamidreza Ghandehari; Kam W. Leong (692-699).
In this study the chondrocytic differentiation and cartilage matrix accumulation of human mesenchymal stem cells (hMSCs) were investigated after encapsulation in a genetically engineered silk-elastinlike protein polymer SELP-47 K as an injectable matrix for delivery of cell-based therapeutics. hMSCs were encapsulated in SELP-47 K and cultured for 4 weeks in chondrogenic medium with or without transforming growth factor-β3 (TGF). Chondrogenic differentiation was evaluated by histological, RNA and biochemical analyses for the expression of cartilage extracellular matrix components.Histological and immunohistochemical staining revealed that the cells acquired a rounded morphology and were embedded in significant amounts of chondrogenic extracellular matrix. Reverse transcriptase (RT)-PCR showed an up-regulation in aggrecan, type II and type X collagen and SOX9 in presence of TGF-β3. By day 28, constructs cultured in the presence of TGF-β3 exhibited significant increase in sulfated glycosaminoglycan and total collagen content up to 65 and 300%, respectively.This study demonstrates that SELP-47 K hydrogel can be used as a scaffold for encapsulation and chondrogenesis of hMSCs. The ability to use recombinant techniques to precisely control SELP structure enables the investigation of injectable protein polymer scaffolds for soft-tissue engineering with varied physicochemical properties.
Keywords: chondrogenesis; genetically engineered polymers; hydrogels; silk-elastinelike polymers; tissue engineering

Evaluation of Conformation and Association Behavior of Multivalent Alanine-Rich Polypeptides by Robin S. Farmer; Ayben Top; Lindsey M. Argust; Shuang Liu; Kristi L. Kiick (700-708).
Helical alanine-rich polypeptides with functional groups displayed along the backbone can display desired molecules such as saccharides or therapeutic molecules at a prescribed spacing. Because these polypeptides have promise for application as biomaterials, the conformation and association of these molecules have been investigated under biologically relevant conditions.Three polypeptide sequences, 17-H-3, 17-H-6, and 35-H-6, have been produced through recombinant techniques. Circular dichroic (CD) spectroscopy was used to monitor the secondary structure of the polypeptides in PBS (phosphate buffered saline, pH 7.4). The aggregation behavior in PBS was monitored via analytical ultracentrifugation and non-denaturing polyacrylamide gel electrophoresis.The three polypeptides adopt a highly helical structure at low and ambient temperatures, and when heated, undergo a helix-to-coil transition, typical of other alanine-rich peptide sequences. The melting temperatures and van’t Hoff enthalpies, extracted from the CD data, suggest similar stability of the sequences. Although alanine-rich sequences can be prone to aggregation, there is no indication of aggregation for the three polypeptides at a range of concentrations relevant for possible biological applications.The helical polypeptides are monomeric under biologically relevant conditions enabling application of these polypeptides as useful scaffolds for ligand or drug display.
Keywords: aggregation; conformational behavior; multivalent scaffolds; polypeptides

A Protective Allergy Vaccine Based on CpG- and Protamine-Containing PLGA Microparticles by Julia M. Martínez Gómez; Stefan Fischer; Noèmi Csaba; Thomas M. Kündig; Hans P. Merkle; Bruno Gander; Pål Johansen (710-710).

AAPS Update (711-712).