Pharmaceutical Research (v.26, #12)

Physiologically-Based PK/PD Modelling of Therapeutic Macromolecules by Peter Thygesen; Panos Macheras; Achiel Van Peer (2543-2550).
Therapeutic proteins are a diverse class of drugs consisting of naturally occurring or modified proteins, and due to their size and physico-chemical properties, they can pose challenges for the pharmacokinetic and pharmacodynamic studies. Physiologically-based pharmacokinetics (PBPK) modelling has been effective for early in silico prediction of pharmacokinetic properties of new drugs. The aim of the present workshop was to discuss the feasibility of PBPK modelling of macromolecules. The classical PBPK approach was discussed with a presentation of the successful example of PBPK modelling of cyclosporine A. PBPK model was performed with transport of the cyclosporine across cell membranes, affinity to plasma proteins and active membrane transporters included to describe drug transport between physiological compartments. For macromolecules, complex PBPK modelling or permeability-limited and/or target-mediated distribution was discussed. It was generally agreed that PBPK modelling was feasible and desirable. The role of the lymphatic system should be considered when absorption after extravascular administration is modelled. Target-mediated drug disposition was regarded as an important feature for generation of PK models. Complex PK-models may not be necessary when a limited number of organs are affected. More mechanistic PK/PD models will be relevant when adverse events/toxicity are included in the PK/PD modelling.
Keywords: convective distribution; cyclosporin A; erythropoietin; interspecies scaling; macromolecules; monoclonal antibodies; natural cell lifespan concept; neonatal Fc receptors; non-linear pharmacokinetics; permeability-limited distribution; physiologically-based pharmacokinetic modelling; PK/PD modelling; target-mediated drug disposition

An Approach to the Validation of Flow Cytometry Methods by Jo Cunliffe; Nicola Derbyshire; Sue Keeler; Ruth Coldwell (2551-2557).
This publication outlines an approach for the validation of flow cytometry methods used in the analysis of a wide range of biomarkers. It is written as a guidance document for method validation in a GLP environment, and from the viewpoint of the pharmaceutical industry, but its relevance is wide-ranging. The approach to method validation described is intended as a starting point for further discussion, as well as providing reference material to colleagues developing fit-for-purpose flow cytometry methods. Pre-validation steps are discussed as prerequisite assessments to determine method and reagent suitability, and to minimise variables during the full validation process. The guide to method validation takes account of the many flow cytometry assay types in use, and provides guidance on the types of assessments necessary to produce a fit-for-purpose method suitable for use in a regulatory environment.
Keywords: biomarker; flow cytometry; GLP; method validation; regulatory

Silibinin Suppresses Spontaneous Tumorigenesis in APC min/+ Mouse Model by Modulating Beta-Catenin Pathway by Subapriya Rajamanickam; Manjinder Kaur; Balaiya Velmurugan; Rana P. Singh; Rajesh Agarwal (2558-2567).
Here we assessed whether silibinin, a nontoxic chemopreventive agent, inhibits spontaneous intestinal tumorigenesis in APC min/+ mouse model, a genetically predisposed animal model of human familial adenomatous polyposis (FAP).Six-week-old APC min/+ mice were divided into four groups and orally gavaged with 0.2 ml vehicle, or 250, 500 and 750 mg silibinin/kg body weight in 0.2 ml vehicle for five days/week. After 6 weeks, polyp burden was analyzed and tissues examined for molecular alterations.Silibinin treatments decreased total number of intestinal polyps by 34% (P < 0.01), 42% (P < 0.01) and 55% (P < 0.001), respectively. Immunohistochemical analysis showed that silibinin dose-dependently decreases (P < 0.001) proliferation and induces (P < 0.001) apoptosis only in intestinal polyps without any considerable effects on normal crypt-villi in APC min/+ or wild-type mice. Further analysis of polyps showed that silibinin decreases β-catenin, cyclin D1, c-Myc and phospho-glycogen synthase kinase-3β expression. Silibinin treatment also decreased phospho-Akt, cyclooxygenase-2, inducible nitric oxide synthase, nitrotyrosine and nitrite levels in polyps, the well-known mediators of intestinal/colon carcinogenesis.Together, these results establish silibinin efficacy in a well-established genetic model of FAP, APC min/+ mouse, and suggest that this natural agent modulates various molecular pathways including β-catenin in its overall chemopreventive efficacy against intestinal carcinogenesis.
Keywords: beta-catenin; chemoprevention; colon cancer; COX-2; silibinin

Poly(ethyleneglycol) 500 Dimethylether as Novel Solvent for Injectable In Situ Forming Depots by Karin Schoenhammer; Holger Petersen; Frank Guethlein; Achim Goepferich (2568-2577).
Poly(D,L-lactide-co-glycolide) (PLGA) solutions in poly(ethyleneglycol)600 (PEG600), N-methyl-2-pyrrolidone (NMP) and poly(ethyleneglycol)500dimethylether (PEG500DME) as a novel solvent, were investigated as suitable for use in injectable in situ forming depots (ISFD).The hemolytic potential of the solvents was investigated. Viscosimetry was used to determine rheological properties of solvents and PLGA solutions. DSC was used to evaluate the stability of the PLGA solutions through investigation of the melting behavior of semicrystalline PEGs which depended on tempering and glass transition temperature of the PLGA. Phase separation was studied to determine ternary phase diagrams. In vitro release kinetics of the solvents and the surrogate methylene blue were investigated.Significantly less hemolysis was observed for PEG500DME compared to PEG600 and NMP. Newtonian fluid properties were found for all polymer solutions. A melting point depression of the solvents was detected in presence of PLGA. The duration of tempering of the polymer solutions showed no impact on their melting behavior. The initial in vitro release of methylene blue was according to the solvent diffusion kinetics.Low hemolytic potential, suitable viscosity for injection, stability of PLGA solutions in PEG500DME and the correlation between phase separation and in vitro release confirmed the potential of PEG500DME as a promising solvent for ISFD.
Keywords: degradation; in situ forming depot; poly(D,L-lactide-co-glycolide); stability

To study the feasibility of Leucine-Aspartic Acid-Valine (LDV) as targeting ligand and drug carrier for targeted delivery to integrin α4β1 over-expressing cancer cells.Poly(L,D,V) was randomly copolymerized using N-carboxyanhydrides of leucine, β-benzyl-aspartic acid, and valine. Oligo(LDV), consisting of 2-6 LDV units, were synthesized by solid phase protein synthesis (SPPS) method. Binding of Leu-Asp-Val, Val-Asp-Leu, and Leu-Asn-Val, and internalization of FITC labeled LDV by wild-type and integrin α4 knock-down A375 cells were studied. Cytotoxicity of poly(L,D,V)-Dox, oligo(LDV)-Dox, and doxorubicin (Dox) was also determined on wild-type, integrin α4 knock-down A375 cells, and normal human epithelial keratinocytes (NHEK).LDV was essential for the specific binding and internalization by cells expressing integrin α4β1. Cytotoxicity of poly(L,D,V)-Dox and oligo(LDV)-Dox was integrin α4-dependent, while free Dox did not show this differential effect. No observable cytotoxicity trend was found when increasing LDV repeating unit. Poly(L,D,V) was relatively more effective than oligo(LDV) for the delivery of Dox to A375.LDV containing moieties bind specifically to integrin α4β1 expressing cancer cells. The binding, internalization, and cytotoxicity depend on the level of integrin α4β1 expression. Poly(L,D,V) and oligo(LDV) were both effective in the in vitro targeted delivery of Dox to integrin α4β1 over-expressing A375 cells.
Keywords: binding specificity; cellular uptake; LDV; oligo(LDV); targeted delivery

The objective of present work was to develop a mannose-anchored, engineered nanoparticulate system for efficient delivery of amphotericin B to macrophages. Furthermore, the effect of spacer on macrophage targeting was also evaluated.PLGA was conjugated to mannose via direct coupling (M-PLGA) and via PEG spacer (M-PEG-PLGA), and engineered PLGA nanoparticles (M-PNPs and M-PEG-PNPs) were prepared from respective conjugates. These prepared engineered PNPs were characterized for size, polydispersity index (PDI), surface charge, and drug entrapment efficiency (% DEE). Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were employed to study the shape and surface morphology of engineered PNPs. Macrophage targeting was evaluated via cellular uptake, ex vivo antileishmanial activity and in vivo biodisposition pattern of engineered PNPs in macrophage-rich organs.The developed engineered PNPs were found to be of nanometric size (<200 nm) and to have low PDI (<0.162) and good entrapment efficiency (%DEE >53.0%). AFM and TEM revealed that both M-PNPs and M-PEG-PNPs had smooth surface and spherical topography. Engineered PNPs with spacer showed enhanced uptake, potential antileishmanial activity and higher disposition in macrophage-rich organs, suggesting improved macrophage targeting.The results suggest that engineering of nanoparticles could lead to development of efficient carrier for macrophage targeting.
Keywords: Amphotericin B; J774A.1 Cell; macrophages; nanoparticles; spacer

The objective of this study was to investigate the effects of polymer type and storage relative humidity (RH) on the crystallization kinetics of felodipine from amorphous solid dispersions.Crystallization of the model drug felodipine from amorphous solid dispersion samples containing poly(vinyl pyrrolidone) (PVP) and hypromellose acetate succinate (HPMCAS) were evaluated. Samples at three different drug–polymer weight ratios (10, 25, and 50 wt. % polymer) were prepared and stored at six different RHs (0%, 32%, 52% or 66%, 75%, 86%, and 93%). Periodically, the fraction of the drug that had crystallized from the samples was quantified using powder X-ray diffractometry (PXRD).Felodipine crystallization rates from PVP-containing dispersions were found to be very sensitive to changes in storage RH, while crystallization rates from HPMCAS-containing dispersions were not. PVP and HPMCAS were similar in terms of their ability to inhibit crystallization at low RH, but when the storage RH was increased to 75% or above, felodipine crystallization from PVP-containing solid dispersions proceeded much faster. It is hypothesized that this trend was caused by moisture-induced drug–polymer immiscibility in PVP-felodipine system. For PVP-containing solid dispersion samples stored at 75% RH and above, crystallization of the model drug felodipine seemed to approach a kinetic plateau, whereby a fraction of the drug still remained amorphous even after storage for 500 days or more.The physical stability of solid dispersions as a function of RH is highly dependent on the polymer used to form the solid dispersion, with PVP-containing dispersions being much less physically stable at high RH than HPMCAS-containing dispersions.
Keywords: crystallization; felodipine; hygroscopicity; powder X-ray diffractometry; relative humidity

Long- and Short-Range Electrostatic Interactions Affect the Rheology of Highly Concentrated Antibody Solutions by Ravi Chari; Kavita Jerath; Advait V. Badkar; Devendra S. Kalonia (2607-2618).
To explain the differences in protein-protein interactions (PPI) of concentrated versus dilute formulations of a model antibody.High frequency rheological measurements from pH 3.0 to 12.0 quantitated viscoelasticity and PPI at high concentrations. Dynamic light scattering (DLS) characterized PPI in dilute solutions.For concentrated solutions at low ionic strength, the storage modulus, a viscosity component and a measure of PPI, is highest at the isoelectric point (pH 9.0) and lowest at pH 5.4. This profile flattens at higher ionic strength but not completely, indicating PPI consist of long-range electrostatics and other short-range attractions. At low concentrations, PPI are near zero at pI but become repulsive as the pH is shifted. Higher salt concentrations completely flatten this profile to zero, indicating that these PPI are mainly electrostatic.This discrepancy occurs because long-range interactions are significant at low concentrations, whereas both long- and short-range interactions are significant at higher concentrations. Computer modeling was used to calculate antibody properties responsible for long- and short-range interactions, i.e. net charge and dipole moment. Charge-charge interactions are repulsive while dipole-dipole interactions are attractive. Their net effect correlated with the storage modulus profile. However, only charge-charge repulsions correlated with PPI determined by DLS.
Keywords: computer modeling; dipole; highly concentrated protein solutions; protein-protein interactions; viscosity

“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide by Abdulgader Baoum; Sheng-Xue Xie; Amir Fakhari; Cory Berkland (2619-2629).
Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks.Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure.TAT/pDNA complexes were condensed into small, 50–100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these “soft” crosslinks could be competitively disrupted to facilitate DNA release.Small and stable TAT-Ca/pDNA complexes were obtained via “soft” calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.
Keywords: A549 cells; gene delivery; plasmid DNA; polyethylenimine; TAT

Reverse Iontophoresis of Amino Acids: Identification and Separation of Stratum Corneum and Subdermal Sources In Vitro by Camille C. Bouissou; Jean-Philippe Sylvestre; Richard H. Guy; M. Begoña Delgado-Charro (2630-2638).
To differentiate the stratum corneum (SC) and subdermal sources of amino acids (AAs) extracted by reverse iontophoresis.13 zwitterionic AAs were quantified in this in vitro study. Repetitive tape-stripping permitted the distribution of the analytes to be determined in the SC. Iontophoresis experiments were performed in which the subdermal chamber contained either phosphate-buffered saline (PBS) only, or a mixture of the 13 AAs in PBS.AAs were homogeneously distributed across the SC and broadly divided into three groups (high, medium, low) in terms of total amount present. As expected, extraction to the cathode for the essentially neutral analytes involved was more efficient. Initial samples obtained during the first hour of iontophoresis primarily extracted AAs from the SC. The fluxes observed in the latter half of the 6-h experiment, on the other hand, correlated well with the corresponding subdermal concentrations.A relatively short extraction period (~1 h) by reverse iontophoresis can be used to evaluate the content of AAs in the SC. Once this ‘reservoir’ has been depleted, reverse iontophoresis can then monitor the subdermal concentrations of the AAs. The latter appears most useful for compounds which are present at lower levels in the SC.
Keywords: amino acids; non-invasive monitoring; reverse iontophoresis; skin reservoir; stratum corneum; tape-stripping

The capability of the electrostatic next generation impactor (eNGI) has been investigated as a tool capable of measuring the electrostatic charge of single (Flixotide™; containing fluticasone propionate (FP)) and combination (Seretide™; FP and salmeterol xinafoate (SX)) pressurised metered dose inhalers (pMDIs) at different flow rates.Aerosol mass distributions were investigated at 30, 60 and 90 l.min−1 and simultaneous charge measurements recorded.Analysis of the mass distribution data indicated a flow dependent relationship, where the aerosol performance (aerodynamic diameter <5 μm) of FP significantly increased between 30 l.min−1 and 60 l.min−1 for both formulations. No significant increase in SX was observed for Seretide with increased flow rate. Analysis of the charge distribution indicated both formulations to primarily charge negatively with a concurrent increase in charge with increased flow rate. Interestingly, the charge-tomass ratio remained relatively constant between 30 l.min−1 and 60 l.min−1 and increased at 90 l.min−1, indicating that charging was majorly influenced at the highest flow rate.This study has shown how the eNGI could be used as a simple Pharmacopeia based methodology for the evaluation of mass and charge profiles of single and combination pMDIs at a series of flow rates.
Keywords: electrostatic NGI; electrostatics; eNGI pMDI; flow rate; inhalation

We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate.AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with 99mTc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of 99mTc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration.HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with 99mTc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected 99mTc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater 99mTc-HYNIC-cys-AnxA5 accumulation. 99mTc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.
Keywords: annexinA5; apoptosis; cell death; molecular imaging; SPECT/CT

Engineering of Crystalline Combination Inhalation Particles of a Long-Acting β2-agonist and a Corticosteroid by Chonladda Pitchayajittipong; Jagdeep Shur; Robert Price (2657-2666).
Engineering of inhalation particles incorporating, in each individual particle, a combination of a long-acting β-agonist and a glucocorticosteroid in a pre-determined and constant ratio for delivery via a dry powder inhaler (DPI).Individual crystalline particles containing both the glucocorticosteroid fluticasone propionate (FP) and long-acting β-agonist salmeterol (SX) were prepared, in a ratio of 10:1, using the solution atomization and crystallization by sonication (SAX) process. Combination drug particles were characterized by particle size, morphology, crystallinity and aerosolisation efficiency using inertial impaction.Combination drug particles were spherical and crystalline, with a median diameter of 4.68 ± 0.01 μm. Aerosolisation of formulations containing combination drug particles resulted in greater uniformity in delivery ratios of both actives across all stages of the impactor before and after storage.Actives in a pre-determined dose ratio can be crystallised in a single particle using the SAX process.
Keywords: combination products; dry powder inhaler; glucocorticosteroid; long-acting β-agonist; SAX

Antiangiogenic Activity of Orally Absorbable Heparin Derivative in Different Types of Cancer Cells by Dong Yun Lee; Sung Won Lee; Sang Kyoon Kim; Myungjin Lee; Hyo Won Chang; Hyun Tae Moon; Youngro Byun; Sang Yoon Kim (2667-2676).
Orally absorbable anticancer medications have great advantages for conventional cancer therapies to patients. Here we evaluated the potent anticancer effect of orally absorbable LHD, a chemical conjugate of low-molecular-weight heparin and deoxycholic acid, on tumor graft growth models.We characterized the angiogenic factors, such as VEGF, heparanase, and MMPs, of murine squamous cell carcinoma (SCC7), melanoma (B16F10) or lung carcinoma (LLC1). Two weeks after oral administration of LHD into these cancer-cell-bearing mice, we evaluated the antiangiogenic activity of LHD.Although all cancer cells expressed the angiogenic factors, SCC7 cells had much higher angiogenic potential and grew rapidly after implantation into mice. When orally administered, LHD delayed tumor graft growth regardless of cancer types. Particularly, LHD powerfully diminished the SCC7-derived tumor growth. Also, the expression of angiogenic factors in all kinds of tumor tissues was decreased, thereby attenuating the neovascularization in tumor tissue.Our study shows that LHD has potent anticancer and antiangiogenic effect on at least three kinds of tumor cells. LHD can be specifically used for preventing neovascularization in tumor tissue because it has therapeutical potential as an antiangiogenic drug and can be orally absorbed.
Keywords: angiogenesis; anticancer effect; heparin derivative; oral absorption

AAPS Connection (2677-2678).