Pharmaceutical Research (v.26, #10)

Glial-derived neurotrophic factor (GDNF) is a potential therapy for stroke, Parkinson’s disease, or drug addiction. However, GDNF does not cross the blood-brain barrier (BBB). GDNF is re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR), which acts as a molecular Trojan horse to deliver the GDNF across the BBB. The pharmacokinetics (PK), toxicology, and safety pharmacology of the HIRMAb-GDNF fusion protein were investigated in Rhesus monkeys.The fusion protein was administered as an intravenous injection at doses up to 50 mg/kg over a 60 h period to 56 Rhesus monkeys. The plasma concentration of the HIRMAb-GDNF fusion protein was measured with a 2-site sandwich ELISA.No adverse events were observed in a 2-week terminal toxicology study, and no neuropathologic changes were observed. The PK analysis showed a linear relationship between plasma AUC and dose, a large systemic volume of distribution, as well as high clearance rates of 8–10 mL/kg/min.A no-observable-adverse-effect level is established in the Rhesus monkey for the acute administration of the HIRMAb-GDNF fusion protein. The fusion protein targeting the insulin receptor has a PK profile similar to a classical small molecule.
Keywords: blood-brain barrier; insulin receptor; GDNF; primate; Trojan horse

Arsonoliposomes for the Potential Treatment of Medulloblastoma by Marco E. Favretto; Showan Marouf; Panagiotis Ioannou; Sophia G. Antimisiaris; Terence L. Parker; Paraskevi Kallinteri (2237-2246).
To investigate the arsonoliposome effect on medulloblastoma cells (VC312Rs) related to uptake, endocytotic mechanism and cell viability.VC312R viability in presence of either arsonoliposomes or stealth liposomes was studied using MTT assay for 1–4 days. Fibroblasts (3T3) were used as control. Apoptosis was studied for 2 h, 5 h and 24 h. Bodipy-labelled arsonoliposome uptake (time- and dose-dependent) was estimated using FACS analysis. The endocytotic mechanism was investigated using inhibitors of clathrin- (chlorpromazine) and caveolae-mediated endocytosis (filipin).Arsonoliposomes affected significantly the VC312R viability compared to 3T3 cells and induced apoptosis to VC312Rs after 2 h of incubation. Apoptosis was not observed for 3T3 cells. Liposome uptake versus time showed a bimodal pattern. Clathrin-mediated endocytosis was the main endocytotic mechanism at low lipid concentrations and caveolae at higher ones; thus, dose-dependent uptake did not show a plateau at increased lipid concentrations.Arsonoliposomes showed “selective” toxicity towards medulloblastoma cells inducing apoptosis after 2 hs of incubation. Therefore, arsonoliposomes are promising anticancer vehicles for brain tumour treatment.
Keywords: apoptosis; arsonoliposomes; cellular uptake; liposomes; medulloblastoma

Application of Screening Methods, Shape Signatures and Engineered Biosensors in Early Drug Discovery Process by Izabela Hartman; Alison R. Gillies; Sonia Arora; Christina Andaya; Nitya Royapet; William J. Welsh; David W. Wood; Randy J. Zauhar (2247-2258).
In this study, two unreported estrogen antagonists were identified using a combination of computational screening and a simple bacterial estrogen sensor.Molecules here presented were initially part of a group obtained from a library of over a half million chemical compounds, using the Shape Signatures method. The structures within this group were then clustered and compared to known antagonists based on their physico-chemical parameters, and possible binding modes of the compounds to the Estrogen Receptor α (ERα) were analyzed. Finally, thirteen candidate compounds were purchased, and two of them were shown to behave as potential subtype-selective estrogen antagonists using a set of bacterial estrogen biosensors, which included sensors for ERα, ERβ, and a negative control thyroid hormone β biosensor. These activities were then analyzed using an ELISA assay against activated ERα in human MCF-7 cell extract.Two new estrogen receptor antagonists were detected using in silico Shape Signatures method with an engineered subtype-selective bacterial estrogen biosensor and commercially available ELISA assay. Additional thyroid biosensor control experiments confirmed no compounds interacted with human thyroid receptor β.This work demonstrates an effective combination of computational analysis and simple bacterial screens for rapid identification of potential hormone-like therapeutics.
Keywords: antagonist; drug discovery; estrogen receptor; estrogen receptor biosensor; shape signatures

Population Pharmacokinetic Model of the Pregabalin-Sildenafil Interaction in Rats: Application of Simulation to Preclinical PK-PD Study Design by Gregor Bender; James Gosset; Jeff Florian; Keith Tan; Mark Field; Scott Marshall; Joost DeJongh; Robert Bies; Meindert Danhof (2259-2269).
Preliminary evidence has suggested a synergistic interaction between pregabalin and sildenafil for the treatment of neuropathic pain. The focus of this study was to determine the influence of sildenafil on the pharmacokinetics (PK) of pregabalin with the objective of informing the design of a quantitative pharmacodynamic (PD) study.The pharmacokinetics were determined in rats following 2-hr intravenous infusions of pregabalin at doses of 4 mg/kg/hr and 10 mg/kg/hr with and without a sildenafil bolus (2.2 mg) and steady state infusion (12 mg/kg/hr for 6 h). This PK model was utilized in a preclinical trial simulation with the aim of selecting the optimal sampling strategy to characterize the PK-PD profile in a future study. Eight logistically feasible PK sampling strategies were simulated in NONMEM and examined through trial simulation techniques.A two-compartment population PK model best described pregabalin pharmacokinetics. Significant model covariates included either a binary effect of sildenafil administration (30.2% decrease in clearance) or a concentration-dependent effect due to sildenafil’s active metabolite.Analysis of simulations indicated that three post-PD samples had the best cost/benefit ratio by providing a significant increase in the precision (and minor improvement in bias) of both PK and PD parameters compared with no PK sampling.
Keywords: neuropathic pain; optimal sampling; synergistic interaction; trial simulation

Accelerated Blood Clearance Phenomenon Upon Repeated Injection of PEG-modified PLA-nanoparticles by Tsutomu Ishihara; Miho Takeda; Haruka Sakamoto; Ayumi Kimoto; Chisa Kobayashi; Naoko Takasaki; Kanae Yuki; Ken-ichiro Tanaka; Mitsuko Takenaga; Rie Igarashi; Taishi Maeda; Naoki Yamakawa; Yoshinari Okamoto; Masami Otsuka; Tatsuhiro Ishida; Hiroshi Kiwada; Yutaka Mizushima; Tohru Mizushima (2270-2279).
We recently developed prostaglandin E1 (PGE1)-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw = 17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats.The plasma levels of PGE1 and anti-PEG IgM antibody were determined by EIA and ELISA, respectively.Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw = 28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE1 than NP-L20.This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE1.
Keywords: ABC phenomenon; anti-PEG IgM antibody; biodegradable nanoparticles; encapsulation; prostaglandin E1

Present study was undertaken to gain insights into the mechanism of cell cycle arrest by ginseng saponin ginsenoside Rh2 (Rh2) using MCF-7 and MDA-MB-231 breast cancer cells.Cell viability and cell cycle distribution were determined by trypan blue dye exclusion assay and flow cytometry, respectively. Immunoblotting was performed to determine changes in protein levels. Knockdown of desired protein was achieved by transfection with small interfering RNA (siRNA).Rh2 treatment significantly inhibited viability of both cells in a concentration-dependent manner, which correlated with G0/G1 phase cell cycle arrest. Rh2-mediated cell cycle arrest was accompanied by down-regulation of cyclin-dependent kinases (Cdk) and cyclins leading to decreased interaction between cyclin D1 and Cdk4/Cdk6 and increased recruitment of p15 Ink4B and p27 Kip1 to cyclin D1/Cdk4 and cyclin D1/Cdk6 complexes. In addition, Rh2 treatment markedly reduced the levels of phosphorylated retinoblastoma protein (P-Rb) and decreased transcriptional activity of E2F1 in luciferase reporter assay. Rh2-induced cell cycle arrest was significantly attenuated by knockdown of p15 Ink4B and/or p27 Kip1 proteins.Rh2-mediated cell cycle arrest in human breast cancer cells is caused by p15 Ink4B and p27 Kip1 -dependent inhibition of kinase activities of G1-S specific Cdks/cyclin complexes.
Keywords: breast cancer; cell cycle arrest; ginsenoside Rh2; p15 and p27

Role of Convective Flow in Carmustine Delivery to a Brain Tumor by Davis Yohanes Arifin; Kam Yiu Timothy Lee; Chi-Hwa Wang; Kenneth A. Smith (2289-2302).
This paper presents a three-dimensional patient-specific simulation of carmustine delivery to brain tumor. The simulation investigates several crucial factors, particularly the role of convective flow, affecting drug delivery efficacy.The simulation utilizes a complete three-dimensional tissue geometry constructed from magnetic resonance images (MRI) of a brain tumor patient in whom commercially available Gliadel® wafers were implanted for sustained delivery of carmustine following excision of the tumor. This method permits an estimation of the convective flow field (in the irregularly shaped anatomical region) which can be used for prediction of drug penetration into the domain of interest, i.e. remnant tumor. A finite volume method is utilized to perform all simulations.Drug exposure exceeds its threshold therapeutic concentration (~15 μM) but for only a limited time (i.e. less than a week) and only in the immediately adjacent tissue (i.e. less than 2 mm). A quasi-steady transport process is established within 1 day following treatment, in which the drug is eliminated rapidly by transcapillary exchange, while its penetration into the tumor is mainly by diffusion. Convection appears to be crucial in influencing the drug distribution in the tumor: the remnant tumor near the ventricle is, by one to two orders of magnitude, less exposed to the drug than is the distal remnant tumor. Carmustine penetration from Gliadel® wafers implanted in brain is limited by rapid elimination via transcapillary exchange. Therefore, it could be useful to consider other therapeutic agents such as paclitaxel. In addition, local convective flow within the cavity appears to be a crucial factor in distributing the drug so that the tumor domain near the ventricle is prone to minimal drug exposure. Thus, complete removal of the tumor from this region is of particular concern.
Keywords: BCNU; computational fluid dynamics; convection; diffusion; Gliadel® wafer; glioblastoma

A Quantitative Kinetic Study of Polysorbate Autoxidation: The Role of Unsaturated Fatty Acid Ester Substituents by Jia Yao; Dushyanth K. Dokuru; Matthew Noestheden; SungAe S. Park; Bruce A. Kerwin; Janan Jona; Drazen Ostovic; Darren L. Reid (2303-2313).
To study the role of unsaturated fatty acid ester substituents in the autoxidation of polysorbate 80 using quantitative kinetics.Oxidation kinetics were monitored at 40°C in aqueous solution by tracking head space oxygen consumption using a fiber optic oxygen sensor with phase shift fluorescence detection. Radical chain initiation was controlled using an azo-initiator and assessed by Hammond’s inhibitor approach, allowing oxidizability constants (k p /(2k t )1/2) to be isolated. Reaction orders were determined using modified van’t Hoff plots and mixed polysorbate micelles.The oxidizability constant of polysorbate 80 ((1.07 ± 0.19) × 10−2 M−1/2 s−1/2) was found to be 2.65 times greater than polysorbate 20 ((0.404 ± 0.080) × 10−2 M−1/2 s−1/2). The additional reactivity of polysorbate 80 was isolated and was first-order in the unsaturated fatty acid ester substituents, indicating that the bulk of the autoxidative chain propagation is due to these groups. This data, and the observation of a half-order dependence on the azo-initiator, is consistent with the classical autoxidation rate law (-d[O2]/dt = k p [RH](Ri/2k t )1/2).Polysorbate 80 autoxidation follows the classical rate law and is largely dependent on the unsaturated fatty acid ester substituents. Clarification of the substituents’ roles will aid formulators in the selection of appropriate polysorbates to minimize oxidative liabilities.
Keywords: autoxidation; kinetics; micelle; oxidation; polysorbate

Permeation Enhancer-Containing Water-In-Oil Nanoemulsions as Carriers for Intravesical Cisplatin Delivery by Tsong-Long Hwang; Chia-Lang Fang; Chao-Huang Chen; Jia-You Fang (2314-2323).
In the present work, we developed water-in-oil (w/o) nanoemulsions for the intravesical administration of cisplatin.The nanoemulsions were made up of soybean oil as the oil phase and Span 80, Tween 80, or Brij 98 as the emulsifier system. α-Terpineol and oleic acid were incorporated as permeation enhancers. The physicochemical characteristics of droplet size, zeta potential, and viscosity were determined. Nanoemulsions were administered intravesically for 1 ~ 4 h to rats in vivo. Animals were subsequently sacrificed, and the bladders were harvested to examine drug accumulation and histology.Ranges of the mean size and zeta potential were 30 ~ 90 nm and −3.4 to −9.3 mV, respectively. The addition of enhancers further reduced the size of the nanoemulsions. The viscosity of all systems exhibited Newtonian behavior. The cisplatin-loaded nanoemulsions were active against bladder cancer cells. The nanoemulsions with Brij 98 exhibited the complete inhibition of cell proliferation. The encapsulation of cisplatin and carboplatin, another derivative of cisplatin, in nanoemulsions resulted in slower and more-sustained release. The amount of drug which permeated into bladder tissues significantly increased when using carriers containing Brij 98, with the α-terpineol-containing formulation showing the best result. The nanoemulsion with α-terpineol prolonged the duration of higher drug accumulation to 3 ~ 4 h. At the later stage of administration (3 ~ 4 h), this system increased the bladder wall deposition of cisplatin and carboplatin by 2.4 ~ 3.3-fold compared to the control solution. Histological examination of the urothelium showed near-normal morphology in rats instilled with these nanoemulsions. α-Terpineol possibly caused slight desquamation of umbrella cells.The nanoemulsions are feasible to load cisplatin for intravesical drug delivery.
Keywords: bladder; cisplatin; intravesical delivery; nanoemulsion; sustained release

In the present study, we have evaluated the pharmacokinetics and the in vivo prostate chemopreventive activity of broccoli sprouts.The in vivo pharmacokinetic profiles of sulforaphane (SFN) and SFN- glutathione (GSH) conjugate in rats after oral administration of 200 mg and 500 mg broccoli sprouts were analyzed. Next, 8-week old TRAMP mice were fed with dietary broccoli sprouts at two dosages (60 and 240 mg/mouse/day) for 16 weeks, and the mice were sacrificed to examine the pharmacodynamic response on prostate tumor and some biomarkers.SFN was readily released and conjugated with GSH in the rats after oral administration of broccoli sprouts. TRAMP mice fed with 240 mg broccoli sprouts/mouse/day exhibited a significant retardation of prostate tumor growth. Western blot analysis revealed that the expression levels of Nrf2, HO-1, cleaved-Caspase-3, cleaved-PARP and Bax proteins were increased, but that of Keap1 and Bcl-XL proteins were decreased. In addition, the phosphorylation and/or the expression level of Akt and its downstream kinase and target proteins, e.g. mTOR, 4E-BP1 and cyclin D1, were reduced.Our findings indicate that broccoli sprouts can serve as a good dietary source of SFN in vivo and that they have significant inhibitory effects on prostate tumorigenesis.
Keywords: broccoli sprout; Nrf2; prostate cancer; sulforaphane; TRAMP mice

The aim of this study is to develop novel polylactide/poly(ethylene glycol) (PLA/PEG) micelles as carrier of hydrophobic drug (paclitaxel) by direct dissolution method without using any organic solvents. The in vitro and in vivo release properties were studied in comparison with micelles prepared by dialysis.Drug encapsulation efficiency (EE) and loading content (LC) of the micelles were evaluated by high-performance liquid chromatography. Micelle diameters and structures were determined by dynamic light scattering and transmission electron microscopy. In vitro release was performed in phosphate-buffered saline (pH 7.4) at 37°C, and in vivo experiments were realized in lung cancer-bearing mice.Similar EE and LC values were obtained for micelles by direct dissolution method and those by dialysis. L- and D-PLA/PEG mixed micelles present higher drug encapsulation ability than separate micelles due to stereocomplexation. Micelle diameters are enlarged by drug-loading. Faster drug release was obtained for micelles by direct dissolution than those by dialysis. Compared with current clinical formulation and micelles by dialysis, paclitaxel-loaded micelles by direct dissolution showed the highest antitumor ability.The L- and D-PLA/PEG mixed micelles by direct dissolution method present many advantages such as easy formulation and absence of toxic organic solvents, which shows great potential as carrier of hydrophobic drugs.
Keywords: drug delivery; micelle; paclitaxel; polylactide; poly(ethylene glycol)

Characterization of a Microsphere Formulation Containing Glucose Oxidase and its In Vivo Efficacy in a Murine Solid Tumor Model by Qun Liu; Andrew Michael Rauth; Jiang Liu; Karlo Babakhanian; Xinyue Wang; Reina Bendayan; Xiao Yu Wu (2343-2357).
This work focused on the characterization and in vitro/in vivo evaluation of an alginate/chitosan microsphere (ACMS) formulation of glucose oxidase (GOX) for the locoregional delivery of reactive oxygen species for the treatment of solid tumors.The GOX distribution and ACMS composition were determined by confocal laser scanning microscopy and X-ray photoelectron spectroscopy. The mechanism of GOX loading and GOX-polymer interactions were examined with Fourier transform infrared spectroscopy and differential scanning calorimetry. In vitro cytotoxicity and in vivo efficacy of GOX-encapsulated ACMS (ACMS-GOX) were evaluated in EMT6 breast cancer cells and solid tumors.GOX was loaded into calcium alginate (CaAlg) gel beads via electrostatic interaction and the CaAlg-GOX-chitosan complexation likely stabilized GOX. Higher concentrations of GOX near the surface of ACMS were detected. GOX retained its integrity upon adsorption to CaAlg gel beads during the coating and after release from ACMS. ACMS-GOX exhibited cytotoxicity to the breast cancer cells in vitro and their efficacy increased with increasing incubation time. Intratumorally delivered ACMS-GOX significantly delayed tumor growth with much lower general toxicity than free GOX.The results suggest that the ACMS-GOX formulation has the potential for the intratumoral delivery of therapeutic proteins to treat solid tumors.
Keywords: antitumor activity; glucose oxidase; intratumoral delivery; microspheres; reactive oxygen species

Mutagenesis and Cysteine Scanning of Transmembrane Domain 10 of the Human Dipeptide Transporter by Liya Xu; Ian S. Haworth; Ashutosh A. Kulkarni; Michael B. Bolger; Daryl L. Davies (2358-2366).
The human dipeptide transporter (hPEPT1) facilitates transport of dipeptides and drugs from the intestine into the circulation. The role of transmembrane domain 10 (TMD10) of hPEPT1 in substrate translocation was investigated using cysteine-scanning mutagenesis with 2-Trimethylammonioethyl methanethiosulfonate (MTSET).Each amino acid in TMD10 was mutated individually to cysteine, and transport of [3H]Gly-Sar was evaluated with and without MTSET following transfection of each mutant in HEK293 cells. Similar localization and expression levels of wild type (WT) hPEPT1 and all mutants were confirmed by immunostaining and biotinylation followed by western blot analysis.E595C- and G594C-hPEPT1 showed negligible Gly-Sar uptake. E595D-hPEPT1 showed similar uptake to WT-hPEPT1, but E595K- and E595R-hPEPT1 did not transport Gly-Sar. Double mutations E595K/R282E and E595R/R282E did not restore uptake. G594A-hPEPT1 showed similar uptake to WT-hPEPT1, but G594V-hPEPT1 eliminated uptake. Y588C-hPEPT1 showed uptake of 20% that of WT-hPEPT1. MTSET modification supported a model of TMD10 with an amphipathic helix from I585 to V600 and increased solvent accessibility from T601 to F605.Our results suggest that G594 and E595 in TMD10 of hPEPT1 have key roles in substrate transport and that Y588 may have an important secondary mechanistic role.
Keywords: cysteine-scanning; human dipeptide transporter; protein structure-function; site-directed mutagenesis; transmembrane domain

AAPS Connection (2367-2368).