Pharmaceutical Research (v.25, #8)

On The Rate and Extent of Drug Delivery to the Brain by Margareta Hammarlund-Udenaes; Markus Fridén; Stina Syvänen; Anubha Gupta (1737-1750).
To define and differentiate relevant aspects of blood–brain barrier transport and distribution in order to aid research methodology in brain drug delivery. Pharmacokinetic parameters relative to the rate and extent of brain drug delivery are described and illustrated with relevant data, with special emphasis on the unbound, pharmacologically active drug molecule. Drug delivery to the brain can be comprehensively described using three parameters: K p,uu (concentration ratio of unbound drug in brain to blood), CLin (permeability clearance into the brain), and V u,brain (intra-brain distribution). The permeability of the blood–brain barrier is less relevant to drug action within the CNS than the extent of drug delivery, as most drugs are administered on a continuous (repeated) basis. K p,uu can differ between CNS-active drugs by a factor of up to 150-fold. This range is much smaller than that for log BB ratios (K p), which can differ by up to at least 2,000-fold, or for BBB permeabilities, which span an even larger range (up to at least 20,000-fold difference). Methods that measure the three parameters K p,uu, CLin, and V u,brain can give clinically valuable estimates of brain drug delivery in early drug discovery programmes.
Keywords: blood–brain barrier; brain penetration; drug delivery; permeability clearance; unbound concentration

Discovery and Development of Toll-Like Receptor 4 (TLR4) Antagonists: A New Paradigm for Treating Sepsis and Other Diseases by Carlos G. Leon; Rita Tory; Jessica Jia; Olena Sivak; Kishor M. Wasan (1751-1761).
Sepsis remains the most common cause of death in intensive care units in the USA, with a current estimate of at least 750,000 cases per year, and 215,000 deaths annually. Despite extensive research still we do not quite understand the cellular and molecular mechanisms that are involved in triggering and propagation of septic injury. Endotoxin (lipopolysaccharide from Gram-negative bacteria, or LPS) has been implicated as a major cause of this syndrome. Inflammatory shock as a consequence of LPS release remains a serious clinical concern. In humans, inflammatory responses to LPS result in the release of cytokines and other cell mediators from monocytes and macrophages, which can cause fever, shock, organ failure and death. A number of different approaches have been investigated to try to treat and/or prevent the septic shock associated with infections caused by Gram-negative bacteria, including blockage of one or more of the cytokines induced by LPS. Recently several novel amphipathic compounds have been developed as direct LPS antagonists at the LPS receptor, TLR4. This review article will outline the current knowledge on the TLR4-LPS synthesis and discuss the signaling, in vitro pre-clinical and in vivo clinical evaluation of TLR4 antagonists and their potential use in sepsis and a variety of diseases such as atherosclerosis as well as hepatic and renal malfunction.
Keywords: drug discovery; LPS; sepsis; toll-like receptor antagonists

Investigation of Drug Delivery by Iontophoresis in a Surgical Wound Utilizing Microdialysis by Heidi J. Holovics; Carter R. Anderson; Barry S. Levine; Ho-Wah Hui; Craig E. Lunte (1762-1770).
This study investigated the penetration of lidocaine around and through a sutured incision following the application of iontophoretic and passive patches in the CD Hairless rat.Concentrations in localized areas (suture, dermis, subcutaneous, and vascular) were determined using microdialysis sampling followed by analysis using liquid chromatography with UV detection.Iontophoresis significantly enhanced the dermal penetration of lidocaine. In an intact skin model, dermal concentrations were 40 times greater following iontophoretic delivery compared to passive delivery. In a sutured incision model, iontophoresis enhanced localized concentrations in the dermis, suture, and subcutaneous regions by 6-, 15-, and 20-fold, respectively. Iontophoretic delivery to a region containing a sutured incision was focused to the incision resulting in a greater increase in the suture concentration and in the subcutaneous region directly below the incision.The four microdialysis probe design was successful in the determination of localized drug penetration in a sutured incision model. Iontophoresis enhanced skin penetration and allowed for site specific delivery when applied to a sutured incision.
Keywords: iontophoresis; lidocaine; liquid chromatography; microdialysis; percutaneous; skin; sutured incision; transdermal drug delivery

Probabilistic methods are insufficient for dealing with the vagueness inherent in human judgment of minimal data available during early drug development. We sought to use fuzzy set theory as a basis for quantifying and propagating vague judgment in a physiologically based pharmacokinetic (PBPK) model for diazepam disposition.First, using diazepam distribution data in rat tissues and fuzzy regression, we estimated fuzzy rat tissue-to-plasma partition coefficients (K p ’s). We scaled the coefficients prior to human PBPK modeling. Next, we constructed the fuzzy set of hepatic intrinsic clearance (CL int) by integrating CL int values measured in vitro from human hepatocytes. Finally, we used these parameters, and other physiological and biochemical information, to predict human diazepam disposition. We compared the simulated plasma kinetics with published concentration-time profiles.We successfully identified rat K p ’s by fuzzy regression. The predicted rat tissue concentration-time contours enveloped the animal tissue distribution data. For the human PBPK model, the mean in vivo plasma concentrations were contained in the simulated concentration-time envelopes.We present a novel computational approach for handling information paucity in PBPK models using fuzzy arithmetic. Our methodology can model the vagueness associated with human perception and interpretation of minimal drug discovery data.
Keywords: diazepam; fuzzy arithmetic; fuzzy regression; fuzzy sets; physiologically based pharmacokinetic model

Safe and Effective Permeation Enhancers for Oral Drug Delivery by Kathryn Whitehead; Natalie Karr; Samir Mitragotri (1782-1788).
The use of intestinal permeation enhancers to overcome the absorption challenges associated with oral drug delivery has been hampered by the notion that enhancer efficacy is directly linked to toxicity. This study attempts to gain insight into the principles governing the potency and toxicity behavior of enhancers.Fifty-one enhancers were selected from 11 chemical categories and their potency and toxicity were analyzed in Caco-2 monolayers at concentrations spanning three orders of magnitude.A small but significant fraction of the 153 enhancer formulations studied demonstrated unexpected but desired behavior, that is, substantial efficacy without marked toxicity. Our results revealed that both chemical category and concentration proved critical in determining the usefulness of many enhancers, and the concept of an enhancer’s ‘therapeutic window’ is discussed. Several of the most promising enhancers identified by the study were tested for their effect on the transport of the marker molecules mannitol and 70 kDa dextran across Caco-2 cells and were capable of increasing permeability more than 10-fold.The results presented here underscore the potential of chemical permeation enhancers while providing valuable direction as to what classes and concentrations of compounds are of interest when searching for safe and effective additions to oral formulations.
Keywords: Caco-2; oral delivery; permeation enhancers; potency; toxicity

To establish a semi-mechanistic pharmacokinetic/pharmacodynamic (pk/pd) model for racemic tramadol (T) integrating all the components with a significant contribution to T effects in rats, using cold allodynia in the Bennett model of neuropathic pain.Male Sprague-Dawley rats (n=53) were randomly allocated in six groups receiving saline, racemic T (5 mg/kg), RR-T (5 mg/kg), SS-T (5 mg/kg), RR-O-demethyltramadol [RR-M1 (1 mg/kg)] or SS-M1 (30 mg/kg) in two h intravenous infusion.The μ-opioid effects of RR-M1 (ERR-M1) were described with an effect compartment model. Contribution to analgesic response of RR-T resulted negligible. The monoamine re-uptake inhibition effects (E SS-M1,T) were modelled as an indirect response model incorporating a competitive interaction between SS-T and SS-M1. ERR-M1 and E SS-M1,T were finally considered as a two independent stimuli converging into a single and common antinoceptive stimulus. The estimates of the steady-state plasma concentrations eliciting half of maximum response for RR-M1, SS-T, and SS-M1 were 20.2, 230, and 869 ng/ml, respectively. RR-M1 is the main active component, but SS-T having a significant contribution.Cold allodynia in the Bennett model has proven an adequate experimental set up to develop complex pk/pd models in analgesia involving different mechanisms of action.
Keywords: neuropathic pain; racemic tramadol; rat model; semi-mechanistic pharmacokinetic/pharmacodynamic model

Synergistic Effect of Amlodipine and Atorvastatin in Reversing LDL-Induced Endothelial Dysfunction by R. Preston Mason; Ruslan Kubant; Gehan Heeba; Robert F. Jacob; Charles A. Day; Yehudi S. Medlin; Philipp Funovics; Tadeusz Malinski (1798-1806).
Statins and certain calcium channel blockers may improve nitric oxide (NO) release and endothelial function through various mechanisms, but their combined effects are not well understood.The separate versus combined effects of amlodipine (AML) and atorvastatin (AT) on NO and peroxynitrite (ONOO) were measured in human umbilical vein endothelial cells (HUVEC) in the presence and absence of low-density lipoprotein (LDL) using electrochemical nanosensors.The combination of AML (5 μmol/l) and AT (3-6 μmol/l) directly stimulated NO release that was about twofold greater than the sum of their separate effects (p < 0.05). This synergistic activity is attributed to enhanced endothelial NO synthase (eNOS) function and decreased cytotoxic ONOO. LDL (100 mg/dl) caused a dysfunction of HUVEC manifested by a 60% reduction in NO and an almost twofold increase in ONOO. Treatment with AML/AT partially reversed the effects of LDL on endothelial function, including a 90% increase in NO and 50% reduction in ONOO. Small-angle X-ray diffraction analysis indicates that AML and AT are lipophilic and share an overlapping molecular location in the cell membrane that could facilitate electron transfer for antioxidant mechanisms.These findings indicate a synergistic effect of AML and AT on an increase in NO concentration, reduction of nitroxidative stress. Also, AML/AT partially restored the NO level of LDL-induced dysfunctional endothelium. Their combined effects may be enhanced by antioxidant properties related to their intermolecular actions in the cell membrane and an increase in the expression and coupling of endothelial nitric oxide synthase.
Keywords: endothelium; LDL; nitric oxide; oxidative stress

Effect of Silymarin Supplement on the Pharmacokinetics of Rosuvastatin by Jian Wei Deng; Ji-Hong Shon; Ho-Jung Shin; Soo-Jin Park; Chang-Woo Yeo; Hong-Hao Zhou; Im-Sook Song; Jae-Gook Shin (1807-1814).
To evaluate the effect of silymarin on the pharmacokinetics of rosuvastatin in systems overexpressing OATP1B1 or BCRP transporters and in healthy subjects.The concentration-dependent transport of rosuvastatin and the inhibitory effect of silymarin were examined in vitro in OATP1B1-expressing oocytes and MDCKII-BCRP cells. For in vivo assessment, eight healthy male volunteers, divided into two groups, were randomly assigned to receive placebo or silymarin (140 mg) three times per day for 5 days. On day 4, all subjects received rosuvastatin (10 mg, 8 am) 1 h after the placebo or silymarin administration. A series of blood samples were collected for 72 h, and the plasma concentration of rosuvastatin was determined using LC-MS/MS.Based on the concentration dependency of rosuvastatin transport in the OATP1B1 and BCRP overexpression systems, rosuvastatin is a substrate for both transporters. Silymarin inhibited both OATP1B1- and BCRP-mediated rosuvastatin transport in vitro (K i 0.93 μM and 97 μM, respectively). However, no significant changes in AUC, half-life, V d/F, or Cl/F of rosuvastatin were observed in human subjects following pretreatment with silymarin.Silymarin does not appear to affect rosuvastatin pharmacokinetics in vivo, suggesting that silymarin, administered according to a recommended supplementation regimen, is not a potent modulator of OATP1B1 or BCRP in vivo.
Keywords: BCRP; OATP1B1; pharmacokinetics; rosuvastatin; silymarin

Role of Particle Size in Phagocytosis of Polymeric Microspheres by Julie A. Champion; Amanda Walker; Samir Mitragotri (1815-1821).
Polymeric microspheres are extensively researched for applications in drug and vaccine delivery. However, upon administration into the body, microspheres are primarily cleared via phagocytosis by macrophages. Although numerous studies have reported on the biochemical pathways of phagocytosis, relatively little is known about the dependence of phagocytosis on particle size. Here, we investigate the previously unexplained dependence of phagocytosis on particle size.Rat alveolar macrophages and IgG-opsonized and non-opsonized polystyrene microspheres were used as model macrophages and drug delivery particles. Phagocytosis, attachment and internalization were measured by flow cytometry and time-lapse video microscopy.Particles possessing diameters of 2–3 μm exhibited maximal phagocytosis and attachment. Rate of internalization, however, was not affected significantly by particle size. Maximal attachment of 2–3 μm microspheres is hypothesized to originate from the characteristic features of membrane ruffles in macrophages. Elimination of ruffles via osmotic swelling nearly eliminated the peculiar size-dependence of phagocytosis. A simple mathematical model is presented to describe the dependence of phagocytosis on particle size.The dependence of phagocytosis on particle size originated primarily from the attachment step. These results reveal the importance of controlling drug delivery particle size distribution and selecting the size appropriate for avoiding or encouraging phagocytosis.
Keywords: drug delivery; macrophage; microsphere; phagocytosis; size

Different Curcuminoids Inhibit T-Lymphocyte Proliferation Independently of Their Radical Scavenging Activities by Michael Deters; Heiko Knochenwefel; Daniel Lindhorst; Therese Koal; Hartmut H. Meyer; Wolfram Hänsel; Klaus Resch; Volkhard Kaever (1822-1827).
We investigated the inhibitory effects of curcumin, curcumin derivatives and degradation products on OKT3-induced human peripheral blood mononuclear cell (PBMC) proliferation and the role of their radical scavenging activity.OKT3-induced human PBMC proliferation was determined by measuring 3H-thymidine incorporation. Radical scavenging activity was evaluated by using an in vitro DPPH assay.OKT3-induced PBMC proliferation was inhibited by curcumin, isocurcumin, bisdesmethoxy-, diacetyl-, tetrahydro-, hexahydro-, and octahydrocurcumin as well as by vanillin, ferulic acid, and dihydroferulic acid with IC50-values of 2.8, 2.8, 6.4, 1.0, 25, 38, 82, 729, 457, and >1,000 μM, respectively. The investigated substances with the strongest effect on radical scavenging were tetrahydro-, hexahydro-, and octahydrocurcumin with IC50 values of 10.0, 11.7, and 12.3 μM, respectively. IC50-values of dihydroferulic acid, ferulic acid, and curcumin were 19.5, 37, and 40 μM. The substances with the lowest radical scavenging activities were vanillin, isocurcumin, diacetylcurcumin, and bisdesmethoxycurcumin with IC50 values higher than 100 μM each.Curcuminoid-induced inhibition of OKT3-induced PBMC proliferation depends on the number of carbon atoms and double bonds of the 1,6-heptadiene-3,5-dione structure as well as on the phenolic ring substitutes of the curcuminoids but is not correlated to their respective radical scavenging activity.
Keywords: antioxidants; curcumin; curcuminoids; immunosuppression; OKT3-induced human PBMC proliferation; radical scavengers

Interplay Between Intestinal pH, Transit Time and Feed Status on the In Vivo Performance of pH Responsive Ileo-Colonic Release Systems by Valentine C. Ibekwe; Hala M. Fadda; Emma L. McConnell; Mandeep K. Khela; David F. Evans; Abdul W. Basit (1828-1835).
Oral pH triggered drug delivery systems, for targeting to the lower gastrointestinal tract, show erratic behaviour in vivo. This study aimed to establish correlations between in situ gastrointestinal pH, transit time or feed status and the disintegration of pH-responsive dosage forms designed to dissolve above pH 7.Tablets (radiolabelled with Technetium 99m) coated with Eudragit S were administered to eight healthy subjects in a three-way crossover study after an overnight fast. Food was administered either 30 min after (pre-feed) or 4 h after (fasted) tablet ingestion. Concurrently, a Bravo® pH monitoring capsule (radiolabelled with Indium 111) was administered in a “freefall manner”. In a third arm of the study tablets were given immediately after breakfast (fed). Transit was followed by gamma scintigraphy.Gastrointestinal pH showed variability between and within individuals but no differences were seen between pre-feed and fasted states. Three tablets failed to disintegrate in pre-feed and fed regimens and one in the fasted state; this has been tentatively linked to ileocaecal pH and ileoceacal junction residence time. In vivo performance of “pH-responsive” dosage forms is complex and influenced by a multitude of factors other than just in situ pH.
Keywords: colonic delivery; enteric coating; inflammatory bowel disease; polymethacrylic acid methyl methacrylate ester copolymer; radiotelemetry

New Predictive Models for Blood–Brain Barrier Permeability of Drug-like Molecules by Sandhya Kortagere; Dmitriy Chekmarev; William J. Welsh; Sean Ekins (1836-1845).
The goals of the present study were to apply a generalized regression model and support vector machine (SVM) models with Shape Signatures descriptors, to the domain of blood–brain barrier (BBB) modeling.The Shape Signatures method is a novel computational tool that was used to generate molecular descriptors utilized with the SVM classification technique with various BBB datasets. For comparison purposes we have created a generalized linear regression model with eight MOE descriptors and these same descriptors were also used to create SVM models.The generalized regression model was tested on 100 molecules not in the model and resulted in a correlation r 2 = 0.65. SVM models with MOE descriptors were superior to regression models, while Shape Signatures SVM models were comparable or better than those with MOE descriptors. The best 2D shape signature models had 10-fold cross validation prediction accuracy between 80–83% and leave-20%-out testing prediction accuracy between 80–82% as well as correctly predicting 84% of BBB+ compounds (n = 95) in an external database of drugs.Our data indicate that Shape Signatures descriptors can be used with SVM and these models may have utility for predicting blood–brain barrier permeation in drug discovery.
Keywords: blood–brain barrier; principal component analysis; regression; shape signatures; support vector machine

Penetration Profile of Taurine in the Human Skin and Its Distribution in Skin Layers by D. L. P. da Silva; S. B. Thiago; F. A. Pessôa; Y. Mrestani; H. H. Rüttinger; J. Wohlrab; R. H. H. Neubert (1846-1850).
To measure in vitro release of taurine from a semisolid standard formulation (amphiphilic cream, DAC) containing 1% taurine, a multi-layer membrane system was used. The content and distribution of taurine in different healthy skin layers (stratum corneum, epidermis and dermis) before (native taurine) and after application of the DAC cream were determined using capillary electrophoresis.The release of taurine from the DAC cream was studied using a multilayer membrane system. Due to the high hydrophilic properties of taurine, the artificial model membranes consisted of collodion as matrix and glycerol as the acceptor phase. In order to determine whether taurine shows the potential for dermal penetration a Franz diffusion cell system was used. The distribution of taurine in the skin layers was determined before and after application of the DAC cream followed by the incubation in a Franz diffusion cell. The excised skin sample was cut in horizontal sections using a cryomicrotome. In order to detect taurine, fluorescamine was used as a derivatization agent.Experiments with a multilayer membrane system were performed to verify the release of taurine at different times (1, 2 and 5 h). Approximately 42.5% taurine was released from the semisolid standard formulation, accumulating in the first membrane (17.63%). The native taurine content was quantified in human isolated skin layer before and after the application of the semisolid standard formulation followed by incubation in a Franz-type diffusion cell for 1 and 5 h. No statistically significant difference (p < 0.05) of the taurine content in the skin layers existed between exposure times (1 and 5 h) studied. The highest taurine content was found in the epidermis both before (256.01 μg taurine/g skin layer) and after (555.5 μg taurine/g skin layer) the application of the DAC cream.The distribution profile of taurine in the skin layers was very similar for the times studied, which suggests that taurine is accumulated in specific cells of the skin. The study suggests that taurine is effectively released from the semisolid standard formulation and can be used for topical application in dermatopharmaceutics.
Keywords: capillary electrophoresis; Franz diffusion cell; multilayer membrane model; skin layers; taurine

Transport of the hepatobiliary scintigraphy agent Tc-99m mebrofenin (MEB) was characterized and simulation studies were conducted to examine the effects of altered hepatic transport on MEB pharmacokinetics in humans.MEB transport was investigated in Xenopus laevis oocytes expressing OATP1B1 or OATP1B3, and in membrane vesicles prepared from HEK293 cells transfected with MRP2 or MRP3. A pharmacokinetic model was developed based on blood, urine and bile concentration-time profiles obtained in healthy humans, and the effect of changes in hepatic uptake and/or excretion associated with disease states (hyperbilirubinemia and cholestasis) on MEB disposition was simulated.MEB (80 pM) transport by OATP1B1 and OATP1B3 was inhibited by rifampicin (50 μM) to 10% and 4% of control, respectively. MEB (0.4 nM) transport by MRP2 was inhibited to 12% of control by MK571 (50 μM); MRP3-mediated transport was inhibited to 5% of control by estradiol-17-beta-glucuronide (100 μM). A two-compartment model described MEB (2.5 mCi) systemic disposition in humans (systemic clearance = 16.2 ± 2.7 ml min−1 kg−1); biliary excretion was the predominant route of hepatic elimination (efflux rate constants ratio canalicular/sinusoidal = 3.4 ± 0.8). Based on simulations, altered hepatic transport markedly influenced MEB systemic and hepatic exposure.MEB may be a useful probe to assess how altered hepatic function at the transport level modulates hepatobiliary drug disposition.
Keywords: biliary excretion; hepatic transport; MRP2; OATPs; pharmacokinetic modeling and simulation; Tc-99m mebrofenin

Selective Contrast Enhancement of Individual Alzheimer’s Disease Amyloid Plaques Using a Polyamine and Gd-DOTA Conjugated Antibody Fragment Against Fibrillar Aβ42 for Magnetic Resonance Molecular Imaging by Muthu Ramakrishnan; Thomas M. Wengenack; Karunya K. Kandimalla; Geoffry L. Curran; Emily J. Gilles; Marina Ramirez-Alvarado; Joseph Lin; Michael Garwood; Clifford R. Jack Jr.; Joseph F. Poduslo (1861-1872).
The lack of an in vivo diagnostic test for AD has prompted the targeting of amyloid plaques with diagnostic imaging probes. We describe the development of a contrast agent (CA) for magnetic resonance microimaging that utilizes the F(ab′)2 fragment of a monoclonal antibody raised against fibrillar human Aβ42This fragment is polyamine modified to enhance its BBB permeability and its ability to bind to amyloid plaques. It is also conjugated with a chelator and gadolinium for subsequent imaging of individual amyloid plaquesPharmacokinetic studies demonstrated this 125I-CA has higher BBB permeability and lower accumulation in the liver and kidney than F(ab′)2 in WT mice. The CA retains its ability to bind Aβ40/42 monomers/fibrils and also binds to amyloid plaques in sections of AD mouse brain. Intravenous injection of 125I-CA into the AD mouse demonstrates targeting of amyloid plaques throughout the cortex/hippocampus as detected by emulsion autoradiography. Incubation of AD mouse brain slices in vitro with this CA resulted in selective enhancement on T 1-weighted spin-echo images, which co-register with individual plaques observed on spatially matched T 2-weighted spin-echo imageDevelopment of such a molecular probe is expected to open new avenues for the diagnosis of AD.
Keywords: Alzheimer’s disease; amyloid plaques; antibody fragments; contrast agent; magnetic resonance imaging

Photodynamic therapy (PDT), involving the combination of a photosensitizer and light, is being evaluated as a vascular disrupting therapy and drug delivery enhancement modality based on its effects on vascular perfusion and barrier function. Since tumor vasculature is the common route for the delivery of both blood and therapeutic agents, it is important to compare the effects of PDT on blood perfusion and substance transport.Tumor blood cell velocity and the extravasation of high molecular weight dextran molecules were continuously monitored by intravital fluorescence microscopy for up to 60 min after PDT using three doses of verteporfin in the MatLyLu prostate tumor model.PDT induced tumor perfusion disruption via thrombus formation. PDT using a higher dose of verteporfin was more effective in inhibiting blood perfusion while a lower dose verteporfin-PDT was more potent in enhancing dextran extravasation. The increase in dextran extravasation induced by PDT was dependent upon dextran molecular weight. A lower molecular weight dextran obtained a higher tumor accumulation after PDT than a higher molecular weight dextran.PDT with verteporfin had different effects on tumor vascular perfusion versus the extravasation of macromolecules. Optimal PDT conditions should be adjusted based on the therapeutic application.
Keywords: benzoporphyrin derivative (BPD); blood flow; drug delivery; photodynamic therapy (PDT); photosensitizer; vascular permeability; vascular targeting

Fragmentation of a Recombinant Monoclonal Antibody at Various pH by Georgeen Gaza-Bulseco; Hongcheng Liu (1881-1890).
To determine the relative importance of direct hydrolysis and β-elimination, two common mechanisms of antibody hinge region fragmentation, and the impact of the conserved N-linked oligosaccharides in affecting antibody fragmentation under various pH.A recombinant monoclonal antibody was incubated in buffers of various pH at 40°C for 5 weeks. The level of fragmentation was measured using size-exclusion-chromatography (SEC). The specific sites of fragmentation were determined by analyzing SEC fractions using liquid chromatography mass spectrometry (LC-MS).Direct hydrolysis was accelerated by acidic and basic pH, while β-elimination contributed to hinge region fragmentation at pH 7 and above. In addition, a shift of the major peptide bond hydrolysis sites in the hinge region towards the C-terminal direction with the decrease of sample pH from 9 to 5 was observed. At pH 4, the major cleavage site shifted outside the hinge region and was localized in the CH2 domain. Oligosaccharides did not affect hinge region fragmentation in the pH range of 5–9, however, at pH 4 oligosaccharides slowed down fragmentation in the CH2 domain.Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4.
Keywords: fragmentation; hinge region; mass spectrometry; recombinant monoclonal antibody

The Quantitative Prediction of CYP-mediated Drug Interaction by Physiologically Based Pharmacokinetic Modeling by Motohiro Kato; Yoshihisa Shitara; Hitoshi Sato; Kunihiro Yoshisue; Masaru Hirano; Toshihiko Ikeda; Yuichi Sugiyama (1891-1901).
The objective is to confirm if the prediction of the drug–drug interaction using a physiologically based pharmacokinetic (PBPK) model is more accurate. In vivo K i values were estimated using PBPK model to confirm whether in vitro K i values are suitable.The plasma concentration–time profiles for the substrate with coadministration of an inhibitor were collected from the literature and were fitted to the PBPK model to estimate the in vivo K i values. The AUC ratios predicted by the PBPK model using in vivo K i values were compared with those by the conventional method assuming constant inhibitor concentration.The in vivo K i values of 11 inhibitors were estimated. When the in vivo K i values became relatively lower, the in vitro K i values were overestimated. This discrepancy between in vitro and in vivo K i values became larger with an increase in lipophilicity. The prediction from the PBPK model involving the time profile of the inhibitor concentration was more accurate than the prediction by the conventional methods.A discrepancy between the in vivo and in vitro K i values was observed. The prediction using in vivo K i values and the PBPK model was more accurate than the conventional methods.
Keywords: CYP; drug interaction; enzyme inhibition; physiologically based pharmacokinetics

QSAR Modeling of the Blood–Brain Barrier Permeability for Diverse Organic Compounds by Liying Zhang; Hao Zhu; Tudor I. Oprea; Alexander Golbraikh; Alexander Tropsha (1902-1914).
Development of externally predictive Quantitative Structure–Activity Relationship (QSAR) models for Blood–Brain Barrier (BBB) permeability.Combinatorial QSAR analysis was carried out for a set of 159 compounds with known BBB permeability data. All six possible combinations of three collections of descriptors derived from two-dimensional representations of molecules as chemical graphs and two QSAR methodologies have been explored. Descriptors were calculated by MolconnZ, MOE, and Dragon software. QSAR methodologies included k-Nearest Neighbors and Support Vector Machine approaches. All models have been rigorously validated using both internal and external validation methods.The consensus prediction for the external evaluation set afforded high predictive power (R 2 = 0.80 for 10 compounds within the applicability domain after excluding one activity outlier). Classification accuracies for two additional external datasets, including 99 drugs and 267 organic compounds, classified as permeable (BBB+) or non-permeable (BBB−) were 82.5% and 59.0%, respectively. The use of a fairly conservative model applicability domain increased the prediction accuracy to 100% and 83%, respectively (while naturally reducing the dataset coverage to 60% and 43%, respectively). Important descriptors that affect BBB permeability are discussed.Models developed in these studies can be used to estimate the BBB permeability of drug candidates at early stages of drug development.
Keywords: combinatorial QSAR; k-nearest neighbors; model validation; predictors of BBB permeability; support vector machines

It has been reported that telithromycin is primarily metabolized via hepatic CYP3A1/2 in rats, the expression and/or mRNA level of hepatic CYP3A1/2 increase in rat model of diabetes mellitus induced by alloxan (DMIA) or streptozotocin (DMIS), and intestinal CYP3A1/2 enzyme activity decreases in rat model of DMIS. Thus, the pharmacokinetic changes of telithromycin in both models of diabetes mellitus compared with those in the control rats were evaluated.Telithromycin was administered (50 mg/kg) intravenously or orally to both rat models of diabetes and their respective control rats.After intravenous administration of telithromycin to both models of diabetes, the non-renal clearance (CLNR) was significantly faster (32.3 and 53.1% increase for rat models of DMIA and DMIS, respectively) and the AUC was significantly smaller (25.0 and 33.8% decrease, respectively) than those in their respective controls. However, after oral administration of telithromycin, the AUC was comparable to that in their respective controls.The faster CLNR after intravenous administration was due to increased hepatic CYP3A1/2 in both models of diabetes. The comparable AUC after oral administration was mainly due to decreased intestinal CYP3A1/2 activity. Alloxan and streptozotocin appear to influence some pharmacokinetics of telithromycin in a different fashion.
Keywords: diabetes mellitus; hepatic and intestinal CYP3A1/2; pharmacokinetics; rats; telithromycin

The aim of this work was to evaluate in vivo poly(lactide)-d-α-tocopheryl polyethylene glycol 1,000 succinate nanoparticles (PLA–TPGS NPs) for controlled and sustained small molecule drug chemotherapy.The drug-loaded PLA–TPGS NPs were prepared by the dialysis method. Particle size, surface morphology and surface chemistry, in vitro drug release and cellular uptake of NPs were characterized. In vitro and in vivo therapeutic effects of the nanoparticle formulation were evaluated in comparison with Taxol®.The PLA–TPGS NP formulation exhibited significant advantages in in vivo pharmacokinetics and xenograft tumor model versus the PLGA NP formulation and the pristine drug. Compared with Taxol®, the PLA–TPGS NP formulation achieved 27.4-fold longer half-life in circulation, 1.6-fold larger area-under-the-curve (AUC) with no portion located above the maximum tolerance concentration level. For the first time in the literature, one shot for 240 h chemotherapy was achieved in comparison with only 22 h chemotherapy for Taxol® at the same 10 mg/kg paclitaxel dose. Xenograft tumor model further confirmed the advantages of the NP formulation versus Taxol®.The PLA–TPGS NP formulation can realize a way of controlled and sustained drug release for more than 10 days, which relieves one of the two major concerns on cancer nanotechnology, i.e. feasibility.
Keywords: chemotherapeutic engineering; controlled release; nanomedicine; nanopharmaceutical engineering; paclitaxel

Using a combination of paclitaxel (PTX), and the apoptotic signaling molecule, C6-ceramide (CER), the enhancement in anti-proliferative effect of human aortic smooth muscle cells (SMC) was examined by administering in polymeric nanoparticles.PTX- and CER-loaded poly(ethylene oxide)-modified poly(epsilon caprolactone) (PEO-PCL) nanoparticles were formulated by solvent displacement and characterized. The uptake and intracellular localization of the nanoparticle in SMC was examined using Z-stack fluorescent confocal microscopy. Anti-proliferative and pro-apoptotic effects of SMC were determined upon administration of PTX and CER, either as single agent or in combination, in aqueous solution and in PEO-PCL nanoparticle formulations.High encapsulation efficiencies (i.e., >95%) of PTX and CER at 10% (w/w) loading were attained in the PEO-PCL nanoparticles of around 270 nm in diameter. Fluorescence confocal analysis showed that nanoparticle delivery did facilitate cellular uptake and internalization. Additionally, combination of PTX and CER delivery in PEO-PCL nanoparticles was significantly more effective in decreasing the proliferation of SMC, probably by enhancing the apoptotic response.The results of this study show that combination of PTX and CER when administered in PEO-PCL nanoparticles can significantly augment the anti-proliferative effect in SMC. This strategy may potentially be useful in the treatment of coronary restenosis.
Keywords: anti-proliferative effects; aortic smooth muscle cells; C6-ceramide; coronary restenosis; paclitaxel

Investigation into the Role of Tumor-Associated Macrophages in the Antitumor Activity of Doxil by Manuela Banciu; Raymond M. Schiffelers; Gert Storm (1948-1955).
Our recent studies show specific localization of long-circulating liposomes (LCL) within the endosomal/lysosomal compartment of tumor-associated macrophages (TAM). Based on this finding, the present study aims to investigate whether clinically applied LCL formulations such as Doxil (LCL-encapsulated doxorubicin), have alternative mechanisms of action additionally to direct drug-mediated cytotoxicity towards tumor cells.The antitumor activity of Doxil was evaluated in B16.F10 melanoma-bearing mice, in the presence and in the absence of TAM. To suppress TAM functions, liposomal clodronate (Lip-CLOD) was injected 24 h before the actual treatment. The effect of Doxil on the levels of angiogenic factors was determined using an angiogenic protein array. As positive control, the same experiments were conducted with LCL-encapsulated prednisolone phosphate (LCL-PLP), a tumor-targeted formulation with known strong anti-angiogenic/anti-inflammatory effects on TAM.Our results show that the antitumor efficacy of Doxil was only partially attributed to the inhibition of TAM-mediated angiogenesis whereas LCL-PLP inhibited tumor growth through strong suppressive effects on pro-angiogenic functions of TAM. As described previously, the main mechanism of Doxil might be a cytotoxic effect on tumor cells.Our findings suggest that the antitumor activity of Doxil does not depend mainly on the presence of functional TAM in tumors.
Keywords: angiogenic proteins; doxil; tumor-associated macrophages; tumor cells

Bioavailability and Bioequivalence: Focus on Physiological Factors and Variability by Vangelis Karalis; Panos Macheras; Achiel Van Peer; Vinod P. Shah (1956-1962).
This is a summary report of the EUFEPS & COST B25 conference on Bioavailability and Bioequivalence which focused on physiological factors and variability. This conference was held at The Royal Olympic Hotel in the centre of Athens (Greece) during the 1–2 of October in 2007. The issues discussed in the conference involved physiological factors affecting drug absorption, the role of pre-systemic effects on bioavailability (BA), the impact of variability in bioequivalence (BE) studies, and a final closing panel session on unresolved issues in BA/BE regulations. Several important aspects of drug absorption were highlighted. It was presented how the complexity of gastrointestinal (GI) physiology and the site dependent absorption can impact on drug BA. Similarly, the effects of food and formulation were also studied. The second session focused on integrating the complexities of GI into modeling the inter-individual variability of absorption and the prediction of first-pass metabolism from in-vitro data. The necessity to measure metabolites, the value of Biopharmaceutical Classification System (BCS), and the more recently proposed Biopharmaceutical Drug Disposition Classification System (BDDCS) were assessed as well. This session closed with presentations of pharmacokinetic software delegates. In the second day of the conference, the problem of high intra-subject variability in BE studies was analyzed. Study design considerations, the use of multiple-dose studies and the role of statistics in BE were also highlighted. Finally, the current thinking of regulatory authorities (EMEA and US-FDA) was presented. The conference closed with a last session on unresolved issues in the regulatory level.
Keywords: bioavailability and bioequivalence; highly variable drugs; impact of variability on BE studies; physiological factors affecting drug absorption

Multifunctional Nanoparticulate Polyelectrolyte Complexes by Sean M. Hartig; Rachel R. Greene; Jayasri DasGupta; Gianluca Carlesso; Mikhail M. Dikov; Ales Prokop; Jeffrey M. Davidson (1963-1963).

Flux Across Microneedle-treated Skin is Increased by Increasing Charge of Naltrexone and Naltrexol In Vitro by Stan L. Banks; Raghotham R. Pinninti; Harvinder S. Gill; Peter A. Crooks; Mark R. Prausnitz; Audra L. Stinchcomb (1964-1964).

AAPS Update (1965-1966).