PharmaGateway

Pharmaceutical Research: Effect of Treatment Regimen on the Immunogenicity of Human Interferon Beta in Immune Tolerant Mice

Kijanka; Jiskoot; Schellekens; Brinks — 2013-06-01

Type: Original Paper
Purpose

Interferon beta is commonly used as therapeutic in the first line of therapy for multiple sclerosis. However, depending on the product, it induces an antibody response in up to 60% of patients. This study evaluated the impact of therapy related factors like dose, route of administration and administration frequency on the immunogenicity of one of the originator interferon beta drugs (Betaferon®) in an immune tolerant transgenic mouse model.

Methods

Immune tolerant transgenic mice received injections with Betaferon® via different routes, doses and injection frequencies. Anti-drug antibody (ADA) production was measured by ELISA to assess immunogenicity.

Results

A single injection of Betaferon® was found to be sufficient for the induction of ADAs. The antibody titer was enhanced with increasing dose and treatment frequency. Among the tested administration routes, the intravenous route was the most immunogenic one, which is in contradiction with one of the dogma in immunogenicity research according to which subcutaneous administration is the most immunogenic route. Intramuscular, intraperitoneal and subcutaneous injections resulted in comparable immunogenicity.

Conclusion

This study shows that treatment related factors affect significantly immunogenicity of Betaseron® and therefore substantiate the need for further studies on these factors in patients.

Pharmaceutical Research: Co-transfection Gene Delivery of Dendritic Cells Induced Effective Lymph Node Targeting and Anti-tumor Vaccination

Chen; Ruan; Yao; Li; Hu; Tabata; Gao — 2013-06-01

Type: Original Paper
Purpose

Successful genetically engineered Dendritic Cell (DC) can enhance DC’s antigen presentation and lymph node migration. The present study aims to genetically engineer a DC using an efficient non-viral gene delivery vector to induce a highly efficient antigen presentation and lymph node targeting in vivo.

Methods

Spermine-dextran (SD), a cationic polysaccharide vector, was used to prepare a gene delivery system for DC engineering. Transfection efficiency, nuclear trafficking, and safety of the SD/DNA complex were evaluated. A vaccine prepared by engineering DC with SD/gp100, a plasmid encoding melanoma-associated antigen, was injected subcutaneously into mice to evaluate the tumor suppression. The migration of the engineered DCs was also evaluated in vitro and in vivo.

Results

SD/DNA complex has a better transfection behavior in vitro than commercially purchased reagents. The DC vaccine co-transfected with plasmid coding CCR7, a chemokine receptor essential for DC migration, and plasmid coding gp100 displayed superior tumor suppression than that with plasmid coding gp100 alone. Migration assay demonstrated that DC transfected with SD/CCR7 can promote DC migration capacity.

Conclusions

The study is the first to report the application of nonviral vector SD to co-transfect DC with gp100 and CCR7-coding plasmid to induce both the capacity of antigen presentation and lymph node targeting.

Pharmaceutical Research: Microfluidic Synthesis of PEG- and Folate-Conjugated Liposomes for One-Step Formation of Targeted Stealth Nanocarriers

Hood; Shao; Omiatek; Vreeland; DeVoe — 2013-06-01

Type: Original Paper
Purpose

A microfluidic hydrodynamic flow focusing technique enabling the formation of small and nearly monodisperse liposomes is investigated for continuous-flow synthesis of poly(ethylene glycol) (PEG)-modified and PEG-folate-functionalized liposomes for targeted drug delivery.

Methods

Controlled laminar flow in thermoplastic microfluidic devices facilitated liposome self-assembly from initial lipid compositions including lipid/cholesterol mixtures containing PEG-lipid and folate-PEG-lipid conjugates. Relationships among flow conditions, lipid composition, and liposome size were evaluated; their impact on PEG and folate incorporation were determined through a combination of UV–vis absorbance measurements and characterization of liposome zeta potential.

Results

PEG and folate were successfully incorporated into microfluidic-synthesized liposomes over the full range of liposome sizes studied. Efficiency of PEG-lipid incorporation was inversely correlated with liposome diameter. Folate-lipid was effectively integrated into liposomes at various flow conditions.

Conclusions

Liposomes incorporating relatively large PEG-modified and folate-PEG-modified lipids were successfully synthesized using the microfluidic flow focusing platform, providing a simple, low cost, rapid method for preparing functionalized liposomes. Relationships between preparation conditions and PEG or folate-PEG functionalization have been elucidated, providing insight into the process and defining paths for optimization of the microfluidic method toward the formation of functionalized liposomes for pharmaceutical applications.

Pharmaceutical Research: Erratum to: Design, Synthesis and Brain Uptake of LAT1-Targeted Amino Acid Prodrugs of Dopamine

2013-06-01

Type: Erratum
No abstract available.

Pharmaceutical Research: The Antitumor Effect of a New Docetaxel-Loaded Microbubble Combined with Low-Frequency Ultrasound In Vitro: Preparation and Parameter Analysis

2013-06-01

Type: Original Paper
Purpose

To develop a novel docetaxel (DOC)-loaded lipid microbubbles (MBs) for achieving target therapy and overcoming the poor water-solubility drawback of DOC.

Methods

A novel DOC-loaded microbubble (DOC + MB) was prepared by lyophilization and the physicochemical properties including ultrasound contrast imaging of the liver were measured. The anti-tumor effect of the DOC + MBs combined with low-frequency ultrasound (LFUS; 0.8Hz, 2.56 W/cm², 50% cycle duty) on the DLD-1 cancer cell line was examined using an MTT assay.

Results

The physicochemical properties of the two tested formats of DOC + MBs (1.0 mg and 1.6 mg) was shown: concentration, (6.74 ± 0.02) × 10⁸ bubbles/mL and (8.27 ± 0.15) × 10⁸ bubbles/mL; mean size, 3.296 ± 0.004 μm and 3.387 ± 0.005 μm; pH value, 6.67 ± 0.11 and 6.56 ± 0.05; release rate, 3.41% and 12.50%; Zeta potential, −37.95 ± 7.84 mV and −44.35 ± 8.70 mV; and encapsulation efficiency, 54.9 ± 6.21% and 46.3 ± 5.69%, respectively. Compared with SonoVue, the DOC + MBs similarly enhanced the echo signal of the liver imaging. The anti-tumor effect of the DOC + MBs/LFUS group was significantly better than that of DOC alone and that of the normal MBs/LFUS groups.

Conclusions

The self-made DOC + MBs have potential as a new ultrasound contrast agent and drug-loaded microbubble, and can obviously enhance the antitumor effect of DOC under LFUS exposure.

Pharmaceutical Research: Supersolubilization and Amorphization of a Model Basic Drug, Haloperidol, by Interaction with Weak Acids

Singh; Parikh; Sandhu; Shah; Malick; Singhal; Serajuddin — 2013-06-01

Type: Original Paper
Purpose

To present a novel approach of greatly enhancing aqueous solubility of a model weakly basic drug, haloperidol, by using weak acids that would not form salts with the drug and to attain physically stable form of amorphous drug by drying such aqueous solutions.

Method

Aqueous solubility of haloperidol in presence of increasing concentrations of four different weak organic acids (malic, tartaric, citric, fumaric) were determined. Several concentrated aqueous solutions with differing drug-to-acid molar ratios were dried in vacuum oven, and dried materials were characterized by DSC, powder XRD, dissolution testing, and stability study.

Result

Acids were selected such that they would not form salts with haloperidol. Haloperidol solubility increased greatly with increased concentrations of malic, tartaric and citric acids, reaching >300 mg/g of solution. In contrast to the haloperidol HCl aqueous solubility of 4 mg/g, this may be called supersolubilization. Fumaric acid did not cause such solubilization as it had low water solubility. Dried solids formed dispersions of amorphous haloperidol in acids that were either amorphous or partially crystalline. Amorphous haloperidol was physically stable and had better dissolution rate than HCl salt.

Conclusion

A novel method of drug solubilization in aqueous media by acid–base interaction is presented. Physically stable amorphous systems of drugs may also be prepared by using this organic solvent-free approach.

Pharmaceutical Research: Inhalable Powder Formulation of Pirfenidone with Reduced Phototoxic Risk for Treatment of Pulmonary Fibrosis

Onoue; Seto; Kato; Aoki; Kojo; Yamada — 2013-06-01

Type: Original Paper
Purpose

Orally-taken pirfenidone (PFD), an idiopathic pulmonary fibrosis drug, often causes severe phototoxicity. Present study aimed to develop a respirable powder formulation for PFD (PFD-RP) to minimize phototoxic risk.

Methods

Photochemical properties of PFD were examined using a reactive oxygen species (ROS) assay and photostability testing. PFD-RP was characterized with a focus on photostability, in vitro inhalation performance, and the efficacy in antigen-sensitized rats. Pharmacokinetic studies were conducted after oral and intratracheal administration of PFD formulations.

Results

Although PFD solution exhibited photodegradation under simulated sunlight (250 W/m²), both PFD powder and PFD-RP were photochemically stable. Laser diffraction and cascade impactor analyses on PFD-RP suggested its high dispersion and fine in vitro inhalation performance. Inhaled PFD-RP (300 μg-PFD/rat) could suppress antigen-evoked pulmonary inflammation in rats as evidenced by decreases in recruited inflammatory cells and neutrophilia-related biomarkers in the lung. Exposure of PFD to light-exposed tissues (skin and eye) after intratracheal administration of PFD-RP at a pharmacologically effective dose (300 μg-PFD/rat) was 90–130-fold less than that of the oral PFD dosage form at a phototoxic dose (160 mg/kg).

Conclusions

PFD-RP might be an attractive alternative to the current oral PFD therapy with a better safety margin.

Pharmaceutical Research: Effects of Iontophoresis, Hydration, and Permeation Enhancers on Human Nail Plate: Infrared and Impedance Spectroscopy Assessment

Benzeval; Bowen; Guy; Delgado-Charro — 2013-06-01

Type: Original Paper
Purpose

To investigate whether permeation enhancement techniques affect the nail plate.

Methods

Infrared and impedance spectroscopies examined the effects of hydration, iontophoresis and N-acetyl-L-cysteine on the human nail.

Results

While significant shifts to higher wavenumbers were observed for the symmetric and asymmetric -CH2 stretching vibrations these changes were essentially the same for the three treatments suggesting they were principally due to hydration alone. Spectral changes associated with amide bonds from nail protein were particularly evident post-treatment with N-acetyl-L-cysteine. The alternating current conductivity and permittivity of the nail, particularly at low frequencies, increased with hydration. Iontophoresis increased the low frequency ac conductivity of the nail but had less effect on the nail capacitance/permittivity. Further, the effects seemed to return gradually to baseline after termination of current passage. Treatment with N-acetyl-Lcysteine produced a greater perturbation, leading to increased low-frequency conductivity and a shift of the frequency-dependent conductivity region to a higher frequency.

Conclusions

Overall, the effects of iontophoresis on infrared and impedance spectroscopic profiles of the nail were attributable simply to increased hydration and similar to those observed after skin iontophoresis. In contrast, both spectroscopy techniques indicated that N-acetyl-L-cysteine disrupted nail structure in line with the enhancer’s known effect on keratin.

Pharmaceutical Research: Response to the Comment on the Article “Physiologically Based Modeling of Pravastatin Transporter-Mediated Hepatobiliary Disposition and Drug-Drug Interactions”

Varma; Lai; Feng; Litchfield; Goosen; Bergman — 2013-06-01

Type: Letter
No abstract available.

Pharmaceutical Research: Thrombus-Targeted Nanocarrier Attenuates Bleeding Complications Associated with Conventional Thrombolytic Therapy

Absar; Nahar; Kwon; Ahsan — 2013-06-01

Type: Original Paper
Purpose

To test the hypothesis that thrombus-specific tissue plasminogen activator (tPA)-loaded nanocarriers enhance thrombolytic efficacy and attenuate hemorrhagic complications.

Methods

A series of pegylated and non-pegylated tPA-loaded liposomes were prepared and their surfaces were decorated with the peptide sequence (CQQHHLGGAKQAGDV) of fibrinogen gamma-chain that binds with GPIIb/IIIa expressed on activated platelets. All formulations were characterized for physical properties, stability and in vitro release profile. The thrombolytic activities of tPA-loaded liposomes were tested by visual end-point detection, fibrin agar-plate and human blood clot-lysis assays. The thrombus-specificity of the peptide-modified-liposomes was evaluated by studying the binding of fluorescent peptide-liposomes with activated platelets. The pharmacokinetic profile and thrombolytic efficacy were evaluated in healthy rats and an inferior vena-cava rat model of thrombosis, respectively.

Results

Both pegylated and non-pegylated peptide-modified-liposomes showed favorable physical characteristics and colloidal stability. Formulations exhibited an initial burst release (40–50% in 30 min) followed by a continuous release of tPA (80–90% in 24 h) in vitro. Encapsulated tPA retained >90% fibrinolytic activity as compared to that of native tPA. Peptide-grafted-liposomes containing tPA demonstrated an affinity to bind with activated platelets. The half-life of tPA was extended from 7 to 103 and 141 min for non-pegylated and pegylated liposomes, respectively. Compared to native tPA, liposomal-tPA caused a 35% increase in clot-lysis, but produced a 4.3-fold less depletion of circulating fibrinogen.

Conclusions

tPA-loaded homing-peptide-grafted-liposomes demonstrate enhanced thrombolytic activity with reduced hemorrhagic risk.

Pharmaceutical Research: Aerodynamic Factors Responsible for the Deaggregation of Carrier-Free Drug Powders to Form Micrometer and Submicrometer Aerosols

Longest; Son; Holbrook; Hindle — 2013-06-01

Type: Original Paper
Purpose

To employ in vitro experiments combined with computational fluid dynamics (CFD) analysis to determine which aerodynamic factors were most responsible for deaggregating carrier-free powders to form micrometer and submicrometer aerosols from a capsule-based platform.

Methods

Eight airflow passages were evaluated for deaggregation of the aerosol including a standard constricted tube, impaction surface, 2D mesh, inward radial jets, and newly proposed 3D grids and rod arrays. CFD simulations were implemented to evaluate existing and new aerodynamic factors for deaggregation and in vitro experiments were used to evaluate performance of each inhaler.

Results

For the carrier-free formulation considered, turbulence was determined to be the primary deaggregation mechanism. A strong quantitative correlation was established between the mass median diameter (MMD) and newly proposed non-dimensional specific dissipation (NDSD) factor, which accounts for turbulent energy, inverse of the turbulent length scale, and exposure time. A 3D rod array design with unidirectional elements maximized NDSD and produced the best deaggregation with MMD<1 μm.

Conclusions

The new NDSD parameter can be used to develop highly effective dry powder inhalers like the 3D rod array that can efficiently produce submicrometer aerosols for next-generation respiratory drug delivery applications.

Pharmaceutical Research: Spray-Dried Chitosan Microparticles for Cellular Delivery of an Antigenic Protein: Physico-chemical Properties and Cellular Uptake by Dendritic Cells and Macrophages

Kusonwiriyawong; Lipipun; Vardhanabhuti; Zhang; Ritthidej — 2013-06-01

Type: Original Paper
Purpose

Spray-dried chitosan microparticles for cellular delivery of antigen to dendritic cells (DC) and macrophages (Mϕ) were investigated.

Methods

Chitosan microparticles were prepared by spray drying. For comparison, poly(lactic-co-glycolic acid) (PLGA) and poly(α-butyl cyanoacrylate) (BCA) micro-/nanoparticles were generated. Bovine serum albumin (BSA) was used as a model antigen. The particles were characterized in terms of size, morphology, surface charge, surface composition, protein content, entrapment efficiency, in vitro release, and protein integrity. Additionally, they were subject to cell viability and cellular uptake study with DC and Mϕ.

Results

Size of chitosan, PLGA, and BCA micro-/nanoparticles ranged between 3.11–7.18, 0.94–6.26, and 0.30–6.34 μm, respectively. Particle morphology and in vitro protein release varied, depending on polymer type, particle composition and preparation process parameters. Chitosan microparticles were cationic, while PLGA microparticles were neutral. BCA micro-/nanoparticles were either anionic or cationic, according to polymerization pH. Protein content and entrapment efficiency of chitosan and PLGA microparticles were relatively consistent. Only integrity and conformational structure of protein encapsulated in chitosan microparticles were completely retained. Chitosan and PLGA microparticles were non-toxic to DC and Mϕ, but the former were internalized more efficiently.

Conclusions

Spray-dried chitosan microparticles delivered the antigen efficiently to DC and Mϕ.

Pharmaceutical Research: Topically Applied Phospho-Sulindac Hydrogel is Efficacious and Safe in the Treatment of Experimental Arthritis in Rats

Mattheolabakis; Mackenzie; Huang; Ouyang; Cheng; Rigas — 2013-06-01

Type: Original Paper
Purpose

Formulate phospho-sulindac (P-S, OXT-328) in a Pluronic hydrogel to be used as a topical anti-inflammatory agent and study its efficacy, safety and pharmacokinetics in an arthritis model.

Methods

LEW/crlBR rats with Freund’s adjuvant-induced arthritis were treated with P-S formulated in Pluronic hydrogel (PSH). We determined the clinical manifestations of arthritis including the locomotor activity of the rats; evaluated joints for inflammation, bone resorption, cartilage damage, COX-2 expression and NF-κB activation; assayed plasma IL-6 and IL-10 levels; and studied the pharmacokinetics of P-S in rats after topical or oral administration.

Results

PSH applied at the onset of arthritis or when arthritis was fully developed, suppressed it by 56–82%, improved the locomotor activity of the rats 2.1–4.4 fold, suppressed synovial inflammation, bone resorption, cartilage damage, NF-κB activation and COX-2 expression but not plasma IL-6 and IL-10 levels. There were no side effects. PSH produced rapidly high local levels of P-S with <14% of P-S reaching the circulation, while orally administered P-S was rapidly metabolized generating much lower joint levels of P-S.

Conclusions

Topical application of PSH is efficacious and safe in the treatment of Freund’s adjuvant-induced arthritis; has a favorable pharmacokinetic profile; and likely acts by suppressing key pro-inflammatory signaling pathways.

Pharmaceutical Research: Comment on the Article “Physiologically Based Modeling of Pravastatin Transporter-Mediated Hepatobiliary Disposition and Drug-Drug Interactions”

Neuhoff; Tucker — 2013-06-01

Type: Letter
No abstract available.

Pharmaceutical Research: Formulating Weakly Basic HCl Salts: Relative Ability of Common Excipients to Induce Disproportionation and the Unique Deleterious Effects of Magnesium Stearate

John; Xu; Lupton; Harmon — 2013-06-01

Type: Original Paper
Purpose

Nine common excipients were examined to determine their ability to cause disproportionation of the HCl salt of a a weakly basic compound. The goal was to determine which excipients were problematic and correlate the results to known properties such as surface pH, slurry pH, or molecular structure. Such a correlation enables a general, simple excipient selection process.

Methods

Binary compacts and “pseudo formulations” are studied after stressing at 40°C/75%RH and 40°C/35% RH for up to 28 days. Near-Infrared (NIR) and X-Ray powder diffraction (XRPD) measurements monitored the conversion of the HCl salt to the free base.

Results

The excipients which induced measureable disproportionation were magnesium stearate, sodium croscarmellose, and sodium stearyl fumarate. Magnesium stearate induced the most extensive and rapid disproportionation at 40°C/75%RH and 40°C/35%RH. Samples containing magnesium stearate showed a unique and significant water uptake above 31%RH.

Conclusions

The problematic excipients are best explained by the proton accepting capacity of excipient carboxylate groups which have pK_a’s higher than the pH_max of the drug salt. Alternative lubricants and disintegrants are suggested and a simple excipient screening process is proposed. Magnesium stearate was the most deleterious excipient for HCl salts due to the formation of the deliquescent salt magnesium chloride.

Pharmaceutical Research: Enhanced Transfection by Antioxidative Polymeric Gene Carrier that Reduces Polyplex-Mediated Cellular Oxidative Stress

Lee; Kim; Lee; Kim; Jeong — 2013-06-01

Type: Original Paper
Purpose

To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection.

Methods

An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (H₂DCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level.

Results

The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential (ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system.

Conclusions

This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection.

Pharmaceutical Research: Design of Biorelevant Test Setups for the Prediction of Diclofenac In Vivo Features After Oral Administration

Guhmann; Thommes; Gerber; Pöllinger; Klein; Breitkreutz; Weitschies — 2013-06-01

Type: Original Paper
Purpose

Design of biorelevant test setups mimicking the physiological conditions experienced by drugs after oral administration along the passage through the mouth and the GI tract for the in vitro evaluation of diclofenac exhibiting multiple-peak phenomenon during absorption.

Methods

The biorelevant models simulated successively saliva (SSF, pH 6.2–6.75–7.4, 5 mL, 3 min), gastric (SGF-FaSSGF, pH 1.2–1.6, 50–250 mL, 30 min) and intestinal (FaSSIF, pH 6.8, 250 mL, 60 min) fluids. Applying these models, diclofenac free acid and its sodium/potassium salt were comparatively evaluated for dissolution and further characterized by HPLC, optical morphogranulometry, DSC and PXRD to elucidate peculiar behaviors.

Results

Diclofenac salts almost completely dissolved in SSF and showed a transitional dissolution pattern before complete precipitation in SGF/FaSSGF. This peculiar pattern correlated with simultaneous chemical modification and formation of agglomerates. With low dissolution in SSF and almost immediately complete precipitation, these behaviors were not observed with diclofenac free acid. Distinct diclofenac features were strongly determined by pH-modifications after oral administration.

Conclusions

The multiple-peak phenomenon observed after administrating a solution, suspension or dispersible formulation of diclofenac salts are likely caused by drug precipitation and agglomeration in the stomach leading to irregular gastric-emptying. Diclofenac free acid may provide more reliable in vivo features.

Pharmaceutical Research: A Physiologically Based Pharmacokinetic Model for Strontium Exposure in Rat

Pertinez; Chenel; Aarons — 2013-06-01

Type: Original Paper
Purpose

To develop a physiologically based pharmacokinetic (PBPK) model to describe the disposition of Strontium—a bone seeking agent approved in 2004 (as its Ranelate salt) for treatment of osteoporosis in post-menopausal women.

Methods

The model was developed using plasma and bone exposure data obtained from ovariectomised (OVX) female rats—a preclinical model for post-menopausal osteoporosis. The final PBPK model incorporated elements from literature models for bone seeking agents allowing for description of the heterogeneity of bone tissue and also for a physiological description of bone remodelling processes. The model was implemented in MATLAB in open and closed loop configurations, and fittings of the model to exposure data to estimate certain model parameters were carried out using nonlinear regression, treating data with a naïve-pooled approach.

Results

The PBPK model successfully described plasma and bone exposure of Strontium in OVX rats with parameter estimates and model behaviour in keeping with known aspects of the distribution and incorporation of Strontium into bone.

Conclusions

The model describes Strontium exposure in a physiologically rationalized manner and has the potential for future uses in modelling the PK-PD of Strontium, and/or other bone seeking agents, and for scaling to model human Strontium bone exposure.

Pharmaceutical Research: Optimization of the Fine Particle Fraction of a Lyophilized Lysozyme Formulation for Dry Powder Inhalation

Claus; Weiler; Schiewe; Friess — 2013-06-01

Type: Original Paper
Purpose

A new dry powder inhalation technology creates inhalable particles from a coherent lyophilized bulk at the time of inhalation. The aim of this study was to evaluate several approaches to improve the fine particle fraction (FPF) and to understand underlying mechanisms.

Methods

Lysozyme was chosen as model drug. Phenylalanine and valine were added, and the freezing process was varied. Lyophilisate characteristics as well as aerosolization behavior was analyzed.

Results

The addition of the crystalline amino acids rendered a dose independent three-fold increase of the FPF. This is possibly due to enhanced fracture properties of the lyophilisates upon impact of the air stream and reduced particle agglomeration/cohesion caused by a rougher surface. This positive effect was well preserved over 3 months of storage. The structure of the lyophilisate was influenced by the freezing process which in turn affected the aerosolization behavior. Liquid nitrogen and vacuum-induced freezing performed best, doubling the FPF. The special cake morphology with elongated channels enabled easy disintegration. The resulting large porous particles comprise a low density being advantageous for a high FPF.

Conclusion

The variation of the lyophilization process and formulation utilizing excipients enabled an optimization of the FPF of the novel lyophilisate based DPI system.

Pharmaceutical Research: Systems Pharmacology Modeling of Drug-Induced Modulation of Thyroid Hormones in Dogs and Translation to Human

Ekerot; Ferguson; Glämsta; Nilsson; Andersson; Rosqvist; Visser — 2013-06-01

Type: Original Paper
Purpose

To develop a systems pharmacology model based on hormone physiology and pharmacokinetic-pharmacodynamic concepts describing the impact of thyroperoxidase (TPO) inhibition on thyroid hormone homeostasis in the dog and to predict drug-induced changes in thyroid hormones in humans.

Methods

A population model was developed based on a simultaneous analysis of concentration-time data of T₄, T₃ and TSH in dogs following once daily oral dosing for up to 6-months of a myeloperoxidase inhibitor (MPO-IN1) with TPO inhibiting properties. The model consisted of linked turnover compartments for T₄, T₃ and TSH including a negative feedback from T₄ on TSH concentrations.

Results

The model could well describe the concentration-time profiles of thyroid hormones in dog. Successful model validation was performed by predicting the hormone concentrations during 1-month administration of MPO-IN2 based on its in vitro dog TPO inhibition potency. Using human thyroid hormone turnover rates and TPO inhibitory potency, the human T₄ and TSH concentrations upon MPO-IN1 treatment were predicted well_.

Conclusions

The model provides a scientific framework for the prediction of drug induced effects on plasma thyroid hormones concentrations in humans via TPO inhibition based on results obtained in in vitro and animal studies.

Pharmaceutical Research: Characterization of Spherulites as a Lipidic Carrier for Low and High Molecular Weight Agents

Zhang; Huang; Makhov; Gao; Zhang; Li — 2013-06-01

Type: Original Paper
Purpose

To develop spherulite formulations to achieve high entrapment efficiency for both small and macromolecules as well as cell-type specific delivery.

Methods

Spherulites of various compositions were prepared, and lipid-PEG was incorporated through post-insertion. Calcein and FITC-labeled albumin were employed as model drugs for small and macromolecules. The spherulites were characterized with respect to entrapment efficiency, size, structure, and release kinetics, and the morphology was examined via cryo-EM. Finally, SV119-decorated spherulites were examined for their selective uptake by cancer cells.

Results

The spherulites are 170 ~ 290 nm in size. A loading efficiency of 55 ~ 60% can be consistently achieved for both calcein and albumin under optimized conditions. Cryo-EM shows the onion-like morphology consistent with the structure of multilamellar liposomes. A t_1/2 of 39.3 h and 69.7 h in cargo release in serum was observed before and after PEG decoration, and incorporation of SV119 led to selective delivery of rhodamine-labeled spherulites to PC-3 tumor cells.

Conclusions

Our optimized formulations may represent a platform with simple preparation approach, relatively small particle size, high drug loading efficiency for both low and high molecular weight agents, and slow release kinetics for selective delivery of various types of therapeutics to target cells.

Pharmaceutical Research: AAPS Connection

2013-06-01

Type: News
No abstract available.

AAPS PharmSciTech: Plasticizer Effects on Physical–Mechanical Properties of Solvent Cast Soluplus® Films

Lim; Hoag — 2013-05-21

Type: Original Paper

Soluplus® is a novel amphiphilic polymer that has been shown to enhance the solubility and drug dissolution rate of poorly soluble drugs. However, there still is a lack of information regarding the physical mechanical properties of Soluplus® with addition of the plasticizers. This study characterized the mechanical properties of Soluplus® with four different plasticizers. The plasticizers selected were polyethylene glycol 6, triethyl citrate, propylene glycol, and glycerin; they were studied at three different levels (15%, 20%, and 25% w/w). The effects of these plasticizers on the glass transition temperature, tensile strength, percent elongation, and Young’s modulus of free films made from Soluplus® were measured and the toughness and ratio of tensile strength to Young’s modulus were calculated. These results showed these four plasticizers are capable to plasticizing Soluplus® as indicated by the glass transition temperature lowering, tensile strength, and Young’s modulus while increasing the percent elongation and film toughness. Among the plasticizers tested, polyethylene glycol 6 showed greatest changed in the mechanical properties studied.

Pharmaceutical Research: Improved Oral Bioavailability of BCS Class 2 Compounds by Self Nano-Emulsifying Drug Delivery Systems (SNEDDS): The Underlying Mechanisms for Amiodarone and Talinolol

Elgart; Cherniakov; Aldouby; Domb; Hoffman — 2013-05-18

Type: Original Paper
Purpose

Superior bioavailability of BCS Class 2 compounds incorporated into SNEDDS was previously reported. This study aims to elucidate the underlying mechanisms accountable for this phenomenon.

Methods

SNEDDS of amiodarone (AM) and talinolol were developed. Pharmacokinetic parameters were assessed in vivo. Effect on intestinal permeability, P-gp efflux and toxicity was evaluated in vitro (Caco-2) and ex vivo (Ussing). Solubilization was assessed in vitro (Dynamic Lipolysis Model). Effect on intraenterocyte metabolism was evaluated using CYP3A4 microsomes.

Results

Oral administration of AM-SNEDDS and talinolol-SNEDDS resulted in higher and less variable AUC and Cmax. In vitro, higher talinolol-SNEDDS Papp indicated Pgp inhibition. Lipolysis of AM-SNEDDS resulted in higher AM concentration in the fraction available for absorption. Incubation of AM-SNEDDS with CYP3A4 indicated CYP inhibition. SNEDDS didn’t alter mannitol Papp and TEER. SNEDDS effect was transient.

Conclusions

Multiple mechanisms are accountable for improved bioavailability and reduced variability of Class-2 compounds by SNEDDS: increased solubilization, reduced intraenterocyte metabolism and reduced P-gp efflux. SNEDDS effect is reversible and doesn’t cause intestinal tissue or cell damage. These comprehensive findings can be used for intelligent selection of drugs for which oral bioavailability will improve upon incorporation into SNEDDS, based on recognition of the drug’s absorption barriers and the ability of SNEDDS to overcome them.

Pharmaceutical Research: Preparation, Drug Release, and Cell Growth Inhibition of a Gelatin: Doxorubicin Conjugate

Wu; Cammarata; Park; Rhodes; Ofner — 2013-05-18

Type: Original Paper
Purpose

To demonstrate the feasibility of a novel macromolecular delivery system for doxorubicin (DOX) which combines pH dependent DOX release with a high molecular weight and biodegradable gelatin carrier.

Methods

DOX was conjugated to gelatin using an acid labile hydrazone bond and a glycylglycine linker. The gelatin-doxorubicin conjugate (G-DOX) was evaluated for hydrazide and DOX content by spectrophotometry, molecular weight by HPLC-SEC, in vitro DOX release at various pH, and cell growth inhibition using EL4 mouse lymphoma and PC3 human prostate cells.

Results

G-DOX hydrazide and DOX content was 47% and 5–7%, respectively of theoretical gelatin carboxylic acid sites. During preparation of G-DOX, the molecular weight decreased to 22 kDa. DOX release was 48% in pH 4.8 phosphate buffer, 22% at pH 6.5, but 10% at pH 7.4. The G-DOX IC50 values in EL4 and PC3 cells were 0.26 μM and 0.77 μM, respectively; the latter value 3 times greater than that of free DOX.

Conclusions

A 22 kDa macromolecular DOX conjugate containing 3.4–5.0% w/w DOX has been prepared. The pH dependent drug release in combination with a biodegradable gelatin carrier offer potential therapeutic advantages of enhanced tumor cell localization and reduced systemic toxicities of the drug.

Pharmaceutical Research: Co-delivery of Vascular Endothelial Growth Factor and Angiopoietin-1 Using Injectable Microsphere/Hydrogel Hybrid Systems for Therapeutic Angiogenesis

Shin; Lee; Ahn; Lee — 2013-05-18

Type: Original Paper
Purpose

We hypothesized that combined delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) using microsphere/hydrogel hybrid systems could enhance mature vessel formation compared with administration of each factor alone.

Methods

Hybrid delivery systems composed of alginate hydrogels and poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres containing angiogenic factors were prepared. The release behavior of angiogenic factors from hybrid systems was monitored in vitro. The hybrid systems were injected into an ischemic rodent model, and blood vessel formation at the ischemic site was evaluated.

Results

The sustained release over 4 weeks of both VEGF and Ang-1 from hybrid systems was achieved in vitro. Co-delivery of VEGF and Ang-1 was advantageous to retain muscle tissues and significantly induced vessel enlargement at the ischemic site, compared to mice treated with either VEGF or Ang-1 alone.

Conclusions

Sustained and combined delivery of VEGF and Ang-1 significantly enhances vessel enlargement at the ischemic site, compared with sustained delivery of either factor alone. Microsphere/hydrogel hybrid systems may be a promising vehicle for delivery of multiple drugs for many therapeutic applications.

Pharmaceutical Research: Bone Morphogenetic Protein-2 Release from Composite Hydrogels of Oligo(poly(ethylene glycol) fumarate) and Gelatin

Kinard; Chu; Tabata; Kasper; Mikos — 2013-05-18

Type: Original Paper
Purpose

Hydrogel composites of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (GMs) were investigated as carriers of bone morphogenetic protein-2 (BMP-2) for bone tissue engineering applications.

Methods

Hydrogel composites with different physical characteristics were prepared by changing the amount and type (acidic vs. basic) of gelatin incorporated in the OPF bulk phase. Composites with differing physical properties (degradation, swelling, and mechanical properties) and differing BMP-2 loading phase were investigated to determine the effect of these factors on BMP-2 release profiles over 28 days.

Results

Overall, higher gelatin amount increased the degradation and swelling of composites, and acidic GMs further increased the degradation and swelling and reduced the compressive modulus of the composites. The most significant factor affecting the release of BMP-2 from composites was the loading phase of the growth factor: GM loading reduced the burst release, increased BMP-2 release during the later phases of the experiment, and increased the cumulative release in faster degrading samples.

Conclusions

The results indicate that the physical properties and the BMP-2 release kinetics of hydrogel composites can be controlled by adjusting multiple parameters at the time of the hydrogel composite fabrication.

The AAPS Journal: Bioequivalence for Locally Acting Nasal Spray and Nasal Aerosol Products: Standard Development and Generic Approval

Li; Jin; Lee; Bai; Chowdhury; Caramenico; Conner — 2013-05-18

Type: Brief Communication

Demonstrating bioequivalence (BE) for nasal spray/aerosol products for local action has been very challenging because the relationship between the drug in systemic circulation and the drug reaching the nasal site of action has not been well established. Thus, the current BE standard for these drug/device combination products is based on a weight-of-evidence approach, which contains three major elements: equivalent in vitro performance, equivalent systemic exposure, and equivalent local delivery. In addition, formulation sameness and device similarity are evidences to support BE. This paper presents a comprehensive review of the scientific rationale of the current BE standard and their development history for nasal spray/aerosol products, as well as the Food and Drug Administration’s review and approval status of generic nasal sprays/aerosols with the application of these BE standard.

Pharmaceutical Research: Rapid Assessment of Homogeneity and Stability of Amorphous Solid Dispersions by Atomic Force Microscopy—From Bench to Batch

Lauer; Siam; Tardio; Page; Kindt; Grassmann — 2013-05-15

Type: Original Paper
Purpose

To verify the robustness and fundamental value of Atomic Force Microscopy (AFM) and AFM-based assays to rapidly examine the molecular homogeneity and physical stability of amorphous solid dispersions on Hot-Melt-Extrudates.

Methods

Amorphous solid dispersions were prepared with a Hot-Melt Extruder (HME) and profiled by Raman Microscopy and AFM following a sequential analytical routine (Multi-Scale-Imaging-of-Miscibiliy (MIMix)). Extrudates were analyzed before and after incubation at elevated temperature and humidity. The data were compared with published results as collected on miniaturized melt models. The value of molecular phase separation rates for long term stability prediction was assessed.

Results

Data recorded on the extrudates are consistent with those published, and they can be compared side by side. Such direct data comparisons allow the identification of possible sources of extrudate heterogeneities. The surface roughness analysis of fracture-exposed interfaces is a novel quantitative way to trace on the nanometer scale the efficiencies of differently conducted HME-processes. Molecular phase separation rates are shown to be relevant for long term stability predictions.

Conclusions

The AFM-based assessment of API:excipient combinations is a robust method to rapidly identify miscible and stable solid dispersions in a routine manner. It provides a novel analytical tool for the optimization of HME processes.

Pharmaceutical Research: The Immunogenicity of Polyethylene Glycol: Facts and Fiction

Schellekens; Hennink; Brinks — 2013-05-15

Type: Review Paper

An increasing number of pegylated therapeutic proteins and drug targeting compounds are being introduced in the clinic. Pegylation is intended to increase circulation time and to reduce an immunogenic response. Recently however a number of publications have appeared claiming that the polyethylene glycol (PEG) moiety of these products in itself may be immunogenic and that the induced anti-PEG antibodies are linked to enhanced blood clearance and reduced efficacy of the products. A critical review of the literature shows that most, if not all assays for anti-PEG antibodies are flawed and lack specificity. Also the biological effects induced by anti-PEG antibodies lack the characteristics of a bona fide antibody reaction. Standardization of the anti-PEG assays and the development of reference sera are urgently needed.

Pharmaceutical Research: Cellular Uptake Mechanism and Therapeutic Utility of a Novel Peptide in Targeted-Delivery of Proteins into Neuronal Cells

Fu; Zhao; Gao; Zhang — 2013-05-15

Type: Original Paper
Purpose

The peptide-based delivery system constitutes a potent approach to overcome the limitations of drug delivery in vitro and in vivo. We recently proposed a novel peptide RDP, which enables brain-targeting delivery of proteins into neuronal cells. Here we investigate the possible internalization mechanism of RDP, and identify the therapeutic effects of functional proteins when linked with RDP in brain disease.

Methods

The RDP fusion proteins are produced through recombinant DNA technology, and cell culture is used to investigate the uptake mechanism of RDP and its fusion protein. Experimental Parkinson’s disease (PD) model is prepared in mice by intra-striatal injection of 6-hydroxydopamine, and is tested by apomorphine- and amphetamine-induced rotation.

Results

The results suggest that the possible route for RDP cellular uptake might involve GABA receptor-dependent, clathrin-mediated endocytosis pathway. Additionally, the conjugate of RDP and glial cell-derived neurotrophic factor (GDNF) exhibits the neuroprotective effect in experimental PD animals, including reduction of apomorphine- and amphetamine-induced rotation following toxin administration.

Conclusions

RDP may become an effective tool for the targeted delivery of proteins into brain for disease treatment.

Pharmaceutical Research: The Co-Delivery of Oxaliplatin Abrogates the Immunogenic Response to PEGylated siRNA-Lipoplex

Alaaeldin; Abu Lila; Moriyoshi; Sarhan; Ishida; Khaled; Kiwada — 2013-05-15

Type: Original Paper
Purpose

In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex.

Methods

Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated.

Results

Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses.

Conclusions

Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.

Pharmaceutical Research: Improving Intracellular Doxorubicin Delivery Through Nanoliposomes Equipped with Selective Tumor Cell Membrane Permeabilizing Short-Chain Sphingolipids

2013-05-11

Type: Original Paper
Purpose

To improve nanoliposomal-doxorubicin (DoxNL) delivery in tumor cells using liposome membrane-incorporated short-chain sphingolipids (SCS) with selective membrane-permeabilizing properties. DoxNL bilayers contained synthetic short-chain derivatives of known membrane microdomain-forming sphingolipids; C₈-glucosylceramide (C₈-GluCer), C₈-galactosylceramide (C₈-GalCer) or C₈-lactosylceramide (C₈-LacCer).

Methods

DoxNL enriched with C₈-GluCer or C₈-GalCer were developed, optimized and characterized with regard to size, stability and drug retention. In vitro cytotoxic activity was studied in a panel of human tumor cell lines and normal cells. Intracellular Dox delivery was measured by flow cytometry and visualized by fluorescence microscopy. For a further understanding of the involved drug delivery mechanism confocal microscopy studies addressed the cellular fate of the nanoliposomes, the SCS and Dox in living cells.

Results

C₈-LacCer-DoxNL aggregated upon Dox loading. In tumor cell lines SCS-DoxNL with C₈-GluCer or C₈-GalCer demonstrated strongly increased Dox delivery and cytotoxicity compared to standard DoxNL. Surprisingly, this effect was much less pronounced in normal cells. Nanoliposomes were not internalized, SCS however transfered from the nanoliposomal bilayer to the cell membrane and preceded cellular uptake and subsequent nuclear localization of Dox.

Conclusion

C₈-GluCer or C₈-GalCer incorporated in DoxNL selectively improved intracellular drug delivery upon transfer to tumor cell membranes by local enhancement of cell membrane permeability.

AAPS PharmSciTech: Development and Evaluation of Sustained-Release Etoposide-Loaded Poly(ε-Caprolactone) Implants

2013-05-11

Type: Original Paper

Poly(ε-caprolactone) implants containing etoposide, an important chemotherapeutic agent and topoisomerase II inhibitor, were fabricated by a melt method and characterized in terms of content uniformity, morphology, drug physical state, and sterility. In vitro and in vivo drug release from the implants was also evaluated. The cytotoxic activity of implants against HeLa cells was studied. The short-term tolerance of the implants was investigated after subcutaneous implantation in mice. The original chemical structure of etoposide was preserved after incorporation into the polymeric matrix, in which the drug was dispersed uniformly. Etoposide was present in crystalline form in the polymeric implant. In vitro release study showed prolonged and controlled release of etoposide, which showed cytotoxicity activity against HeLa cells. After implantation, good correlation between in vitro and in vivo drug release was found. The implants demonstrated good short-term tolerance in mice. These results tend to show that etoposide-loaded implants could be potentially applied as a local etoposide delivery system.

Pharmaceutical Research: Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations, Part 3: Understanding Supersaturation Versus Precipitation Potential During the In Vitro Digestion of Type I, II, IIIA, IIIB and IV Lipid-Based Formulations

2013-05-10

Type: Original Paper
Purpose

Recent studies have shown that digestion of lipid-based formulations (LBFs) can stimulate both supersaturation and precipitation. The current study has evaluated the drug, formulation and dose-dependence of the supersaturation – precipitation balance for a range of LBFs.

Methods

Type I, II, IIIA/B LBFs containing medium-chain (MC) or long-chain (LC) lipids, and lipid-free Type IV LBF incorporating different doses of fenofibrate or tolfenamic acid were digested in vitro in a simulated intestinal medium. The degree of supersaturation was assessed through comparison of drug concentrations in aqueous digestion phases (AP_DIGEST) during LBF digestion and the equilibrium drug solubility in the same phases.

Results

Increasing fenofibrate or tolfenamic acid drug loads (i.e., dose) had negligible effects on LC LBF performance during digestion, but promoted drug crystallization (confirmed by XRPD) from MC and Type IV LBF. Drug crystallization was only evident in instances when the calculated maximum supersaturation ratio (SR^M) was >3. This threshold SR^M value was remarkably consistent across all LBF and was also consistent with previous studies with danazol.

Conclusions

The maximum supersaturation ratio (SR^M) provides an indication of the supersaturation ‘pressure’ exerted by formulation digestion and is strongly predictive of the likelihood of drug precipitation in vitro. This may also prove effective in discriminating the in vivo performance of LBFs.

Pharmaceutical Research: Effects of Cerium Oxide Nanoparticles on PC12 Neuronal-Like Cells: Proliferation, Differentiation, and Dopamine Secretion

2013-05-10

Type: Original Paper
Purpose

Oxidative stress has been found to play a key role in several diseases, that range from cancer to neurodegenerative disorders. Besides traditional anti-oxidant agents, in recent years much attention has been focused on nanotechnological solutions, including cerium oxide nanoparticles (nanoceria).

Methods

Thanks to its extraordinary catalytic properties, nanoceria mimics the activity of superoxide dismutase and of catalase, therefore acting as a reactive oxygen species (ROS) scavenger in many biological contexts. In this paper, we report on nanoceria interactions with PC12 cell line, that represents a valuable model for many features of central dopaminergic neurons.

Results

Nanoceria confirmed a strong anti-ROS action but, most interestingly, also showed beneficial effects on both cell differentiation and dopamine production.

Conclusions

Even if deeper examinations will be necessary in order to better clarify the mechanisms at the base of the documented effects, nanoceria demonstrated a significant potential as pharmacological agent in the treatment of neurological disorders.

Pharmaceutical Research: Novel Biotinylated Lipid Prodrugs of Acyclovir for the Treatment of Herpetic Keratitis (HK): Transporter Recognition, Tissue Stability and Antiviral Activity

Vadlapudi; Vadlapatla; Earla; Sirimulla; Bailey; Pal; Mitra — 2013-05-09

Type: Original Paper
Purpose

Biotinylated lipid prodrugs of acyclovir (ACV) were designed to target the sodium dependent multivitamin transporter (SMVT) on the cornea to facilitate enhanced cellular absorption of ACV.

Methods

All the prodrugs were screened for in vitro cellular uptake, interaction with SMVT, docking analysis, cytotoxicity, enzymatic stability and antiviral activity.

Results

Uptake of biotinylated lipid prodrugs of ACV (B-R-ACV and B-12HS-ACV) was significantly higher than biotinylated prodrug (B-ACV), lipid prodrugs (R-ACV and 12HS-ACV) and ACV in corneal cells. Transepithelial transport across rabbit corneas indicated the recognition of the prodrugs by SMVT. Average Vina scores obtained from docking studies further confirmed that biotinylated lipid prodrugs possess enhanced affinity towards SMVT. All the prodrugs studied did not cause any cytotoxicity and were found to be safe and non-toxic. B-R-ACV and B-12HS-ACV were found to be relatively more stable in ocular tissue homogenates and exhibited excellent antiviral activity.

Conclusions

Biotinylated lipid prodrugs demonstrated synergistic improvement in cellular uptake due to recognition of the prodrugs by SMVT on the cornea and lipid mediated transcellular diffusion. These biotinylated lipid prodrugs appear to be promising drug candidates for the treatment of herpetic keratitis (HK) and may lower ACV resistance in patients with poor clinical response.

Pharmaceutical Research: Erratum to: Protected Graft Copolymer Excipient Leads to a Higher Acute Maximum Tolerated Dose and Extends Residence Time of Vasoactive Intestinal Peptide Significantly Better than Sterically Stabilized Micelles

2013-05-09

Type: Erratum
No abstract available.

The AAPS Journal: Epigenetic Modifications of Nrf2 by 3,3′-diindolylmethane In Vitro in TRAMP C1 Cell Line and In Vivo TRAMP Prostate Tumors

Wu; Khor; Su; Saw; Shu; Cheung; Huang; Yu; Kong — 2013-05-09

Type: Original Paper

3,3′-diindolylmethane (DIM) is currently being investigated in many clinical trials including prostate, breast, and cervical cancers and has been shown to possess anticancer effects in several in vivo and in vitro models. Previously, DIM has been reported to possess cancer chemopreventive effects in prostate carcinogenesis in TRAMP mice; however, the in vivo mechanism is unclear. The present study aims to investigate the in vitro and in vivo epigenetics modulation of DIM in TRAMP-C1 cells and in TRAMP mouse model. In vitro study utilizing TRAMP-C1 cells showed that DIM suppressed DNMT expression and reversed CpG methylation status of Nrf2 resulting in enhanced expression of Nrf2 and Nrf2-target gene NQO1. In vivo study, TRAMP mice fed with DIM-supplemented diet showed much lower incidence of tumorigenesis and metastasis than the untreated control group similar to what was reported previously. DIM increased apoptosis, decreased cell proliferation and enhanced Nrf2 and Nrf2-target gene NQO1 expression in prostate tissues. Importantly, immunohistochemical analysis showed that DIM reduced the global CpG 5-methylcytosine methylation. Focusing on one of the early cancer chemopreventive target gene Nrf2, bisulfite genomic sequencing showed that DIM decreased the methylation status of the first five CpGs of the Nrf2 promoter region, corroborating with the results of in vitro TRAMP-C1 cells. In summary, our current study shows that DIM is a potent cancer chemopreventive agent for prostate cancer and epigenetic modifications of the CpG including Nrf2 could be a potential mechanism by which DIM exerts its chemopreventive effects.

The AAPS Journal: Impact of Anti-Drug Antibodies in Preclinical Pharmacokinetic Assessment

Thway; Magana; Bautista; Jawa; Gu; Ma — 2013-05-08

Type: Original Paper

The administration of human biotherapeutics is often associated with a higher incidence of immunogenicity in preclinical species. The presence of anti-drug antibodies (ADAs) in the test samples can affect the accurate measurement of therapeutic protein (TP) in bioanalytical methods designed to support pharmacokinetic (PK) and toxicokinetic (TK) assessments. The impact can vary depending on the bioanalytical method platform and study dosing design. The goal of this study is to evaluate the impact of ADA response on the bioanalytical methods in support of PK/TK and the associated study data interpretation. Sprague Dawley rats were administered with four weekly doses of 50 mg/kg TP, a humanized monoclonal antibody. The TP in serum samples was measured using three bioanalytical methods that quantified bound and/or unbound TP to ADA. The ADA response in the animals was classified into negative, low, medium, and high based on the magnitude of the response. The presence of ADA in samples led to discrepant TP measurements between the methods, especially at time points where the TP concentrations were low. This could be due to ADA interference to the accurate measurement of ADA-bound TP concentrations. The TP concentration at last time point (C_last) was reduced by 82.8%, 98.6%, and 99.8%, respectively, for samples containing low, medium, and high levels of ADA. The interfering effects of the ADA on bioanalytical methods and exposure were evident as early as 2 weeks post-dosing. This modeling approach can provide the better understanding of ADA impact on PK exposure in multiple doses.

AAPS PharmSciTech: Development and Characterization of Propranolol Selective Molecular Imprinted Polymer Composite Electrospun Nanofiber Membrane

2013-05-08

Type: Original Paper

Propranolol (PPL) imprinted microspheres (MIP) were successfully prepared via oil/water polymerization using a methyl methacrylate (MMA) monomer, PLL template, and divinylbenzene (DVB) cross-linker and favorably incorporated in a Eudragit-RS100 nanofiber membrane. A non-PPL imprinted polymer (NIP), without a template, was used as a control. The morphology and particle size of the beads were investigated using scanning electron microscopy. The results revealed that both MIP and NIP had a spherical shape with a micron size of approximately 50–100 μm depending on the amounts of DVB and PPL used. NIP2 (MMA/DVB, 75:2.5) and MIP8 (PPL/MMA/DVB, 0.8:75:2.5) were selected for reloading of PPL, and the result indicated that increasing the ratio of PPL to polymer beads resulted in increase PPL reloading (>80%). A total of 10–50% NIP2 or MIP8 was incorporated into a 40% (w/v) Eudragit-RS100 fiber membrane using an electrospinning technique. PPL could be bound to the 50% MIP8 composite fiber membrane with a higher extent and at a higher rate than the control (NIP2). Furthermore, the MIP8 composite fiber membrane showed higher selectivity to PPL than the other β-blockers (atenolol, metoprolol, and timolol). Thus, the MIP8 composite fiber membrane can be further developed for various applications in pharmaceutical and other affinity separation fields.

AAPS PharmSciTech: 25-Hydroxyvitamin D3-Loaded PLA Microspheres: In Vitro Characterization and Application in Diabetic Periodontitis Models

Li; Wang; Xiao; Bao; Li — 2013-05-08

Type: Original Paper

This study aimed at the preparation of a sustained-release 25-hydroxyvitamin D₃ (25OHD) treatment for diabetic periodontitis, a known complication of diabetes. 25OHD-loaded polylactic acid (PLA) microspheres were prepared using oil-in-water emulsion–solvent evaporation method. The prepared microspheres exhibited intact surfaces, with average sizes ranging from 42.3 to 119.4 μm. The encapsulation efficiency ranged from 79.2% (w/w) to 88.5% (w/w), and the drug content was between 15.8% (w/w) and 17.8% (w/w). Drug release from the produced microspheres followed a near-to-zero-order release pattern and lasted over 10 weeks. In an in vitro model of diabetic periodontitis, the abnormal morphological changes and the decrease in the cell viability of bone marrow stromal cells could be effectively attenuated after the 25OHD-loaded microsphere application. Additionally, in a rat model of diabetic periodontitis, alveolar bone loss was inhibited and osteoid formation in the periodontium was promoted upon 25OHD-loaded microsphere treatment. In conclusion, 25OHD-loaded PLA microspheres may provide an effective approach for the treatment of this disease.

Pharmaceutical Research: Oral Delivery of Low Molecular Weight Heparin by Polyaminomethacrylate Coacervates

Viehof; Lamprecht — 2013-05-07

Type: Original Paper
Purpose

Oral bioavailability of low molecular weight heparin (LMWH) can be achieved by several advanced drug delivery approaches. Here, a new preparation method for coacervates (CAs) using non-toxic polyethylene glycol derivates was developed.

Methods

LMWH were coacervated with polyaminomethacrylates (Eudragit® RL or RS) using polyethylene glycol (PEG) derivatives as non-toxic solvents. CAs were analyzed for their physicochemical properties and pharmacokinetic parameters were determined for different formulations in rabbits.

Results

CAs from both polymer types using various PEGs were of irregular shape and had particle sizes of around 40 μm, encapsulation efficiencies of >90%, and complete LMWH in vitro release was obtained within 2 h. In vivo, oral Absorption at doses of 300 IU/kg was rather low (F < 2.5%) while dose increase resulted in a maximum at 600 IU/kg (F_RL: 6.0 ± 1.2%; F_RS: 5.8 ± 2.5%) and 1,200 IU/kg did not result in higher bioavailability (F_RL: 4.6 ± 0.4%; F_RS: 4.1 ± 0.8%). CAs were applicable to various LMWH types where the oral availability decreased in the order fondaparinux>enoxaparin>nadroparin>certoparin depending mainly on the molecular weight.

Conclusions

CAs prepared by an organic solvent-free method allowed the oral delivery of LMWHs. The therapeutic efficiency and the simple and solvent-free manufacturing process underlines the high potential of this new preparation method.

Pharmaceutical Research: Preclinical Models Used for Immunogenicity Prediction of Therapeutic Proteins

Brinks; Weinbuch; Baker; Dean; Stas; Kostense; Rup; Jiskoot — 2013-05-07

Type: Review Paper

All therapeutic proteins are potentially immunogenic. Antibodies formed against these drugs can decrease efficacy, leading to drastically increased therapeutic costs and in rare cases to serious and sometimes life threatening side-effects. Many efforts are therefore undertaken to develop therapeutic proteins with minimal immunogenicity. For this, immunogenicity prediction of candidate drugs during early drug development is essential. Several in silico, in vitro and in vivo models are used to predict immunogenicity of drug leads, to modify potentially immunogenic properties and to continue development of drug candidates with expected low immunogenicity. Despite the extensive use of these predictive models, their actual predictive value varies. Important reasons for this uncertainty are the limited/insufficient knowledge on the immune mechanisms underlying immunogenicity of therapeutic proteins, the fact that different predictive models explore different components of the immune system and the lack of an integrated clinical validation. In this review, we discuss the predictive models in use, summarize aspects of immunogenicity that these models predict and explore the merits and the limitations of each of the models.

AAPS PharmSciTech: New Direct Compression Excipient from Tigernut Starch: Physicochemical and Functional Properties

Builders; Anwunobi; Mbah; Adikwu — 2013-05-07

Type: Original Paper

Tigernut starch has been isolated and modified by forced retrogradation of the acidic gel by freezing and thawing processes. Relevant physicochemical and functional properties of the new excipient (tigernut starch modified by acid gelation and accelerated (forced) retrogradation (ST_AM)) were evaluated as a direct compression excipient in relation to the native tigernut starch (ST_NA), intermediate product (tigernut starch modified by acid gelation (ST_A)), and microcrystalline cellulose (MCC). The particle morphology, swelling capacity, moisture sorption, differential scanning calorimeter (DSC) thermographs and X-ray powder diffraction (XRD) patterns, flow, dilution capacity, and tablet disintegration efficiency were evaluated. The particles of ST_NA were either round or oval in shape, ST_A were smooth with thick round edges and hollowed center while ST_AM were long, smooth, and irregularly shaped typically resembling MCC. The DSC thermographs of ST_NA and MCC showed two endothermic transitions as compared with ST_A and ST_AM which showed an endothermic and an exothermic. The moisture uptake, swelling, flow, and dilution capacity of ST_AM were higher than those of MCC, ST_A, and ST_NA. The XRD pattern and moisture sorption profile of ST_AM showed similarities and differences with ST_NA, ST_A, and MCC that relate the modification. Acetylsalicylic acid (ASA) tablets containing ST_AM disintegrated at 3 ± 0.5 min as compared with the tablets containing ST_NA, ST_A, and MCC which disintegrated at 8.5 ± 0.5, 10 ± 0.5, and 58 ± 0.8 min, respectively. The study shows the physicochemical properties of tigernut starch modified by forced retrogradation as well as its potential as an efficient direct compression excipient with enhanced flow and disintegration abilities for tablets production.

AAPS PharmSciTech: Formulation and In Vivo Evaluation of Orally Disintegrating Tablets of Clozapine/Hydroxypropyl-β-cyclodextrin Inclusion Complexes

Zeng; Wang; Zhang; Shi; Zong — 2013-05-07

Type: Original Paper

The aim of this study was to improve the solubility and oral bioavailability of clozapine (CLZ), a poorly water-soluble drug subjected to substantial first-pass metabolism, employing cyclodextrin complexation technique. The inclusion complexes were prepared by an evaporation method. Phase solubility studies, differential scanning calorimetry, X-ray powder diffraction, and Fourier transform infrared spectroscopy were used to evaluate the complexation of CLZ with hydroxypropyl-β-cyclodextrin (HP-β-CD) and the formation of true inclusion complexes. Characterization and dissolution studies were carried out to evaluate the orally disintegrating tablets (ODTs) containing CLZ/HP-β-CD complexes prepared by direct compression. Finally, the bioavailability studies of the prepared ODTs were performed by oral administration to rabbits. The ODTs showed a higher in vitro dissolution rate and bioavailability compared with the commercial tablets. It is evident from the results herein that the developed ODTs provide a promising drug delivery system in drug development, owing to their excellent performance of a rapid onset of action, improved bioavailability, and good patient compliance.

AAPS PharmSciTech: A Novel Multi-Unit Tablet for Treating Circadian Rhythm Diseases

Liu; Gong; Shi; Jiang; Zheng; Ge; Liu; Zhu — 2013-05-07

Type: Original Paper

This study aimed to develop and evaluate a novel multi-unit tablet that combined a pellet with a sustained-release coating and a tablet with a pulsatile coating for the treatment of circadian rhythm diseases. The model drug, isosorbide-5-mononitrate, was sprayed on microcrystalline cellulose (MCC)-based pellets and coated with Eudragit^® NE30D, which served as a sustained-release layer. The coated pellets were compressed with cushion agents (a mixture of MCC PH-200/ MCC KG-802/PC-10 at a ratio of 40:40:20) at a ratio of 4:6 using a single-punch tablet machine. An isolation layer of OpadryII, swellable layer of HPMC E5, and rupturable layer of Surelease^® were applied using a conventional pan-coating process. Central-composite design-response surface methodology was used to investigate the influence of these coatings on the square of the difference between release times over a 4 h time period. Drug release studies were carried out on formulated pellets and tablets to investigate the release behaviors, and scanning electron microscopy (SEM) was used to monitor the pellets and tablets and their cross-sectional morphology. The experimental results indicated that this system had a pulsatile dissolution profile that included a lag period of 4 h and a sustained-release time of 4 h. Compared to currently marketed preparations, this tablet may provide better treatment options for circadian rhythm diseases.

AAPS PharmSciTech: Batch-to-Batch Quality Consistency Evaluation of Botanical Drug Products Using Multivariate Statistical Analysis of the Chromatographic Fingerprint

Xiong; Yu; Qu — 2013-05-02

Type: Original Paper

Botanical drug products have batch-to-batch quality variability due to botanical raw materials and the current manufacturing process. The rational evaluation and control of product quality consistency are essential to ensure the efficacy and safety. Chromatographic fingerprinting is an important and widely used tool to characterize the chemical composition of botanical drug products. Multivariate statistical analysis has showed its efficacy and applicability in the quality evaluation of many kinds of industrial products. In this paper, the combined use of multivariate statistical analysis and chromatographic fingerprinting is presented here to evaluate batch-to-batch quality consistency of botanical drug products. A typical botanical drug product in China, Shenmai injection, was selected as the example to demonstrate the feasibility of this approach. The high-performance liquid chromatographic fingerprint data of historical batches were collected from a traditional Chinese medicine manufacturing factory. Characteristic peaks were weighted by their variability among production batches. A principal component analysis model was established after outliers were modified or removed. Multivariate (Hotelling T² and DModX) control charts were finally successfully applied to evaluate the quality consistency. The results suggest useful applications for a combination of multivariate statistical analysis with chromatographic fingerprinting in batch-to-batch quality consistency evaluation for the manufacture of botanical drug products.

Pharmaceutical Research: Chemical Drug Stability in Lipids, Modified Lipids, and Polyethylene Oxide-Containing Formulations

Stella — 2013-05-02

Type: Original Paper

To critique the stability complications seen in formulating poorly water-soluble, problematic drugs in lipids, modified lipids, and polyethylene oxide solvents and surfactants in hard and soft gelatin capsules as well as some parenterals, a literature search was performed and personal experiences, and those of colleagues, collated. The literature is replete with examples of molecules undergoing rapid oxidative degradation in the presence of polyethylene oxide based solvents and surfactants as well as in the presence of unsaturated lipids. More recently appreciated is instability caused by the reaction of amine and amide drugs, with formaldehyde, formic acid found in many of these solvents as impurities and other degradation byproducts of the solvents themselves. One would expect acylation and transacylation reactions to be more common than reported but the literature has some good examples. An added complexity is occasionally seen with the use of hard and soft gelatin capsules with these solvents. The chemical stability of drugs in liquid and semi-solid formulations in the presence of lipids, modified lipids, and polyoxyethylene oxide-based solvents and surfactants can be complex, further exacerbated by the use of gelatin capsules, and can lead to a plethora of degradation pathways often not seen when the same drugs are formulated in solid dosage forms.

Pharmaceutical Research: In Vitro Digestion of the Self-Emulsifying Lipid Excipient Labrasol® by Gastrointestinal Lipases and Influence of its Colloidal Structure on Lipolysis Rate

Fernandez; Jannin; Chevrier; Chavant; Demarne; Carrière — 2013-05-02

Type: Original Paper
Purpose

Labrasol^® is a self-emulsifying excipient used to improve the oral bioavailability of poorly water-soluble drugs. It is a mixture of acylglycerols and PEG esters, these compounds being substrates for digestive lipases. The characterization of Labrasol^® gastrointestinal lipolysis is essential for understanding its mode of action.

Methods

Labrasol^® lipolysis was investigated using either individual enzymes (gastric lipase, pancreatic lipase-related protein 2, pancreatic carboxyl ester hydrolase) or a combination of enzymes under in vitro conditions mimicking first the gastric phase of lipolysis and second the duodenal one. Specific methods for quantifying lipolysis products were established in order to determine which compounds in Labrasol^® were preferentially hydrolyzed.

Results

Gastric lipase showed a preference for di- and triacylglycerols and the main acylglycerols remaining after gastric lipolysis were monoacylglycerols. PEG-8 diesters were also hydrolyzed to a large extent by gastric lipase. Most of the compounds initially present in Labrasol^® were found to be totally hydrolyzed after the duodenal phase of lipolysis. The rate of Labrasol^® hydrolysis by individual lipases was found to vary significantly with the dilution of the excipient in water and the resulting colloidal structures (translucent dispersion; opaque emulsion; transparent microemulsion), each lipase displaying a distinct pattern depending on the particle size.

Conclusions

The lipases with distinct substrate specificities used in this study were found to be sensitive probes of phase transitions occurring upon Labrasol^® dilution. In addition to their use for developing in vitro digestion models, these enzymes are interesting tools for the characterization of self-emulsifying lipid-based formulations.

Pharmaceutical Research: Brushed Block Copolymer Micelles with pH-Sensitive Pendant Groups for Controlled Drug Delivery

Lee; Bae — 2013-05-02

Type: Original Paper
Purpose

To investigate the effects of small aliphatic pendent groups conjugated through an acid-sensitive linker to the core of brushed block copolymer micelles on particle properties.

Methods

The brushed block copolymers were synthesized by conjugating five types of 2-alkanone (2-butanone, 2-hexanone, 2-octanone, 2-decanone, and 2-dodecanone) through an acid-labile hydrazone linker to poly(ethylene glycol)-poly(aspartate hydrazide) block copolymers.

Results

Only block copolymers with 2-hexanone and 2-octanone (PEG-HEX and PEG-OCT) formed micelles with a clinically relevant size (< 50 nm in diameter), low critical micelle concentration (CMC, < 20 μM), and drug entrapment yields (approximately 5 wt.%). Both micelles degraded in aqueous solutions in a pH-dependent manner, while the degradation was accelerated in an acidic condition (pH 5.0) in comparison to pH 7.4. Despite these similar properties, PEG-OCT micelles controlled the entrapment and pH-dependent release of a hydrophobic drug most efficiently, without altering particle size, shape, and stability. The molecular weight of PEG (12 kDa vs 5 kDa) induced no change in pH-controlled drug release rates of PEG-OCT micelles.

Conclusion

Acid-labile small aliphatic pendant groups are useful to control the entrapment and release of a hydrophobic drug physically entrapped in the core of brushed block copolymer micelles.

Pharmaceutical Research: Distribution and Visualisation of Chlorhexidine Within the Skin Using ToF-SIMS: A Potential Platform for the Design of More Efficacious Skin Antiseptic Formulations

Judd; Scurr; Heylings; Wan; Moss — 2013-05-01

Type: Original Paper
Purpose

In order to increase the efficacy of a topically applied antimicrobial compound the permeation profile, localisation and mechanism of action within the skin must first be investigated.

Methods

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to visualise the distribution of a conventional antimicrobial compound, chlorhexidine digluconate, within porcine skin without the need for laborious preparation, radio-labels or fluorescent tags.

Results

High mass resolution and high spatial resolution mass spectra and chemical images were achieved when analysing chlorhexidine digluconate treated cryo-sectioned porcine skin sections by ToF-SIMS. The distribution of chlorhexidine digluconate was mapped throughout the skin sections and our studies indicate that the compound appears to be localised within the stratum corneum. In parallel, tape strips taken from chlorhexidine digluconate treated porcine skin were analysed by ToF-SIMS to support the distribution profile obtained from the skin sections.

Conclusions

ToF-SIMS can act as a powerful complementary technique to map the distribution of topically applied compounds within the skin.

Pharmaceutical Research: AAPS Connection

2013-05-01

Type: News
No abstract available.

Pharmaceutical Research: Emerging Topics in Structure-Based Virtual Screening

Rastelli — 2013-05-01

Type: Original Paper

Molecular dynamics simulations and the generation of ad hoc chemical libraries are playing an increasingly important and recognized role in structure-based virtual screening. These approaches are important for treating target flexibility and improving the drug discovery pipeline. In this article I will comment on these two topics and put them into perspective.

Pharmaceutical Research: Fluorogenic Tagging Methodology Applied to Characterize Oxidized Tyrosine and Phenylalanine in an Immunoglobulin Monoclonal Antibody

Zhou; Mozziconacci; Kerwin; Schöneich — 2013-05-01

Type: Original Paper
Purpose

Metal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences.

Methods

We developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl₂/L-ascorbic acid.

Results

Besides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1.

Conclusion

The fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.

Pharmaceutical Research: Synthetic FXR Agonist GW4064 Prevents Diet-Induced Hepatic Steatosis and Insulin Resistance

Ma; Huang; Yan; Gao; Liu — 2013-05-01

Type: Original Paper
Purpose

To examine the effect of farnesoid X receptor (FXR) activation by its synthetic agonist, 3-[2-[2-Chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) on diet-induced obesity and hepatic steatosis.

Methods

Fifteen week-old C57BL/6 mice fed with high-fat diet (HFD) or high-fat, high-cholesterol diet were treated by twice weekly injection of GW4064 (50 mg/kg) intraperitoneally or DMSO (carrier solution) for 6 weeks. Body weight, body composition and food intake were monitored weekly. Serum glucose and insulin levels and lipid content in the liver were measured at the end of study. Additionally, genes involved in lipogenesis, gluconeogenesis and inflammation were analyzed by real time PCR. CD36 protein level was detected by western blot.

Results

Activation of FXR by GW4064 suppressed weight gain in C57BL/6 mice fed with either HFD or high-fat and high-cholesterol diet. GW4064 treatment of mice significantly repressed diet-induced hepatic steatosis as evidenced by lower triglyceride and free fatty acid level in the liver. Analysis of genes involved in lipid metabolism showed GW4064 markedly reduced lipid transporter Cd36 gene expression without affecting expression of genes that are directly involved in lipogenesis. GW4064 treatment attenuated hepatic inflammation while having no effect on white adipose tissue. In addition, activation of FXR by GW4064 avoided diet-induced hyperinsulinemia and hyperglycemia through decreasing the transcript levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pase), two key enzymes in gluconeogenesis.

Conclusions

The results verify the important function of FXR in diet-induced obesity and suggest that FXR agonists are promising therapeutic agents for obesity-associated metabolic disorders.

Pharmaceutical Research: In Vitro and In Vivo Evaluation of Lipofufol, a New Triple Stealth Liposomal Formulation of Modulated 5-Fu: Impact on Efficacy and Toxicity

2013-05-01

Type: Original Paper
Purpose

Drug resistance and severe toxicities are limitations when handling 5-FU. We have developed a triple liposomal formulation of 5-FU combined to 2′-deoxyinosine and folinic acid to improve its efficacy-toxicity balance.

Methods

Stealth liposomes were obtained using the thin-film method. Antiproliferative activity was tested on human colorectal and breast cancer models using sensitive (HT29) and resistant (SW620, LS174t, MDA231) cell lines. In vivo, pharmacokinetics, biodistribution and safety studies were performed in rodents. Finally, efficacy was evaluated using two tumor-bearing mice models (LS174 and MDA231) with response and survival as main endpoints.

Results

LipoFufol is a 120-nm pegylated liposome, displaying 20–30% encapsulation rates. In vitro, antiproliferative activities were higher than 5-FU, and matched that of FolFox combination in colorectal models, but not in breast. Drug monitoring showed an optimized pharmacokinetics profile with reduced clearance and prolonged half-life. Liposome accumulation in tumors was shown by fluorescence-based biodistribution studies. Beside, milder neutropenia was observed when giving LipoFufol to animals with transient partial DPD-deficiency, as compared with standard 5-FU. In LS174t-bearing mice, higher response and 55% longer survival were achieved with Lipofufol, as compared with 5-FU.

Conclusion

The issues of drug-resistance and drug-related toxicity can be both addressed using a stealth liposomal formulation of modulated 5-FU.

Pharmaceutical Research: Bioresponsive Deciduous-Charge Amphiphiles for Liposomal Delivery of DNA and siRNA

Pierrat; Kereselidze; Wehrung; Zuber; Pons; Lebeau — 2013-05-01

Type: Original Paper
Purpose

Biolabile cationic lipids were developed for efficient intracellular delivery of DNA and siRNA.

Methods

The compounds have been designed starting from the membrane lipid DOPC in a way they may loose their cationic charge when exposed to an acidic and/or enzymatic stimulus, such as those met during the journey of a lipoplex in biological media.

Results

They demonstrated remarkable efficiency to deliver DNA in various cell lines (BHK-21, Calu-3, NCI-H292, and A549), with no significant cytotoxicity. Furthermore, two of the compounds (carbonate-based DOPC derivatives) revealed able to deliver small interfering RNA in U87Luc and A549Luc cancer cells and to mediate a selective 70–80% knockdown of the stably transfected luciferase gene.

Conclusions

The results show that the described bioresponsive cationic lipids have high DNA and siARN delivery activity which is encouraging in view of delivering a therapeutic nucleic acid to pulmonary tissues in vivo.

Pharmaceutical Research: In Vivo and Ex Vivo Inhibition of Spinal Nerve Ligation-Induced Ectopic Activity by Sodium Channel Blockers Correlate to In Vitro Inhibition of NaV1.7 and Clinical Efficacy: A Pharmacokinetic-Pharmacodynamic Translational Approach

Kalezic; Luo; Lund; Eriksson; Bueters; Visser — 2013-05-01

Type: Original Paper
Purpose

In vivo and ex vivo inhibition of ectopic activity of clinically used and newly developed sodium channel (NaV) blockers were quantified in the rat spinal nerve ligation (SNL) model using a pharmacokinetic-pharmacodynamic (PKPD) approach and correlated to in vitro NaV1.7 channel inhibition and clinical effective concentrations.

Methods

In vivo, drug exposure and inhibition of ectopic activity were assessed in anaesthetized SNL rats at two dose levels. Ex vivo, compounds were applied at increasing concentrations to dorsal root ganglias isolated from SNL rats. The inhibitory potency (IC₅₀) was estimated using PKPD analysis. In vitro IC₅₀ was estimated using an electrophysiology-based assay using recombinant rat and human NaV1.7 expressing HEK293 cells.

Results

In vivo and ex vivo inhibition of ectopic activity correlated well with the in vitro inhibition on the rat NaV1.7 channel. The estimated IC_50s for inhibition of ectopic activity in the SNL model occurred at similar unbound concentrations as clinical effective concentrations in humans.

Conclusions

Inhibition of ectopic activity in the SNL model could be useful in predicting clinical effective concentrations for novel sodium channel blockers. In addition, in vitro potency could be used for screening, characterization and selection of compounds, thereby reducing the need for in vivo testing.

Pharmaceutical Research: Importance of Peptide Transporter 2 on the Cerebrospinal Fluid Efflux Kinetics of Glycylsarcosine Characterized by Nonlinear Mixed Effects Modeling

Huh; Hynes; Smith; Feng — 2013-05-01

Type: Original Paper
Purpose

To develop a population pharmacokinetic model to quantitate the distribution kinetics of glycylsarcosine (GlySar), a substrate of peptide transporter 2 (PEPT2), in blood, CSF and kidney in wild-type and PEPT2 knockout mice.

Methods

A stepwise compartment modeling approach was performed to describe the concentration profiles of GlySar in blood, CSF, and kidney simultaneously using nonlinear mixed effects modeling (NONMEM). The final model was selected based on the likelihood ratio test and graphical goodness-of-fit.

Results

The profiles of GlySar in blood, CSF, and kidney were best described by a four-compartment model. The estimated systemic elimination clearance, volume of distribution in the central and peripheral compartments were 0.236 vs 0.449 ml/min, 3.79 vs 4.75 ml, and 5.75 vs 9.18 ml for wild-type versus knockout mice. Total CSF efflux clearance was 4.3 fold higher for wild-type compared to knockout mice. NONMEM parameter estimates indicated that 77% of CSF efflux clearance was mediated by PEPT2 and the remaining 23% was mediated by the diffusional and bulk clearances.

Conclusions

Due to the availability of PEPT2 knockout mice, we were able to quantitatively determine the significance of PEPT2 in the efflux kinetics of GlySar at the blood-cerebrospinal fluid barrier.

Pharmaceutical Research: Recombinant High Density Lipoprotein Nanoparticles for Target-Specific Delivery of siRNA

Rui; Tang; Li; Wei; Xu — 2013-05-01

Type: Original Paper
Purpose

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for treatments of numerous diseases. However, the progress towards broad application of siRNA requires the development of safe and effective vectors that target to specific cells. In this study, we developed a novel recombinant high density lipoprotein (rHDL) vector with high siRNA encapsulation efficiency.

Methods

They were prepared by condensing siRNA with various commercial cationic polymers and coating the polyplex with a layer of lipids and apolipoprotein AI (apo AI). The rHDL nanoparticles were used to transfect SMMC-7721 hepatoma cells with stable luciferase expression. The uptake and intracellular trafficing of siRNA were also investigated.

Results

Characterization studies revealed these rHDL nanoparticles had similar physical properties as natural HDLs. The various rHDL formulations had high silencing efficiency (more than 70% knockdown) in hepatocytes with minimum cytotoxicity. Moreover, the uptake of rHDL by SMMC-7721 was confirmed to be mediated through the natural HDL uptake pathway.

Conclusions

The work described here demonstrated the optimized rHDL nanoparticles may offer a promising tool for siRNA delivery to the liver.

Pharmaceutical Research: Ternary Complexes with Core-Shell Bilayer for Double Level Targeted Gene Delivery: In Vitro and In Vivo Evaluation

Fan; Yao; Du; Hou; Zhou; Lu; Meng; Zhang — 2013-05-01

Type: Original Paper
Purpose

Hyaluronic acid (HA)/polyethyleneimine-dexamethasone (PEI-Dex)/DNA ternary complexes with “core-shell” bilayer were developed for double level targeted gene delivery. A PEI₁₈₀₀-Dex, as a core, was applied to compact DNA into a nano-sized structure and facilitate the nuclear translocation of DNA after endocytosis into tumor cells, and a polyanion HA, as the outer corona, was employed to improve targeted tumor delivery and reduce cytotoxicity.

Methods

PEI-Dex was synthesized and characterized by ¹H NMR. The characterizations of ternary complexes were investigated. Their biological properties, including transfection efficiency, cytotoxicity, cellular uptake and in vivo efficacy were evaluated systemically.

Results

Ternary complexes with the size of about 160 nm exhibited the lowest cytotoxicity and the highest transfection efficiency in B16F10 cells among investigated complexes. The sub-cellular localization study confirmed that ternary complexes could facilitate more efficient cell uptake and nuclear transport of DNA than binary complexes. Moreover, Cy7-labeled ternary complexes obviously accumulated in the tumor after i.v. administration, indicating that ternary complexes could assist the DNA targeting to the tumor. In in vivo studies, HA/PEI₁₈₀₀-Dex/DNA ternary complexes showed confirmed anti-inflammation activity, and could significantly suppress tumor growth of tumor-bearing nude mice.

Conclusions

HA/PEI-Dex/DNA ternary complexes might be a promising targeted gene delivery system.

Pharmaceutical Research: Ternary Polymeric Nanoparticles for Oral siRNA Delivery

Zhang; He; Tang; Yin — 2013-05-01

Type: Original Paper
Purpose

Poor stability and inefficient absorption in the intestinal tract are major barriers confronting oral delivery of siRNA. We aimed to uncover if ternary polymeric nanoparticles (cationic polymer/siRNA/anionic component) can overcome these obstacles through changing the formulation-related parameters.

Methods

Ternary polymeric nanoparticles were prepared by ionic gelation of chitosan, N-trimethyl chitosan (TMC), or thiolated trimethyl chitosan (TTMC) with tripolyphosphate (TPP) or hyaluronic acid (HA), and siRNA was simultaneously encapsulated. Structural stabilities and siRNA protection of these nanoparticles were assessed in simulated intestinal milieu. Their transport across ex vivo rat ileum, macrophage uptake, in vitro gene silencing, and in vivo biodistribution after oral administration were investigated.

Results

Ternary polymeric nanoparticles formed by TTMC, siRNA, and TPP (TTMC/siRNA/TPP nanoparticles) showed suitable structural stability and siRNA protection in the intestinal tract, good permeability across ex vivo rat ileum, superior cellular uptake and gene silencing efficiency in Raw 264.7 cells, and high systemic biodistribution after oral administration.

Conclusions

TTMC/siRNA/TPP nanoparticles demonstrated efficient gene silencing in vitro and systemic biodistribution in vivo, therefore, they were expected to be potential vehicles for oral siRNA delivery.

Pharmaceutical Research: The Photolysis of Disulfide Bonds in IgG1 and IgG2 Leads to Selective Intramolecular Hydrogen Transfer Reactions of Cysteine Thiyl Radicals, Probed by Covalent H/D Exchange and RPLC-MS/MS analysis

Zhou; Mozziconacci; Kerwin; Schöneich — 2013-05-01

Type: Original Paper
Purpose

The evaluation of photo-instability of biotherapeutic products is mandated by regulatory agencies. Photo-irradiation can induce oxidative modifications in proteins, which may lead to undesired biological and therapeutic consequences. Among the modifications, epimerization of amino acid residues can occur upon photo-irradiation of IgGs.

Methods

We show here, that UV irradiation (λ = 253.7 nm) of IgG1 and IgG2 leads to the formation of intermediary carbon-centered radicals, validated by covalent incorporation of deuterium into the protein primary sequence.

Results

By MS/MS analysis we identified the sites of deuterium incorporation, such as the sequence QD [303:304, HC], present in the peptide of VVSVLTVVHQDWLNGK [294:309, HC] in both IgG1 and IgG2, and V [111, LC] and K [116, LC], present in the peptide VTVLGQPK [109:116, LC] in IgG2. Both peptides are in the proximity of intrachain disulfide bonds.

Conclusions

The exposure of IgG1 and IgG2 to UV-light (λ = 253.7 nm) generates specific carbon-centered radicals. The latter were evidenced by a covalent H-D exchange reaction that likely occurred through a hydrogen atom transfer reaction between cysteine thiyl radical and C-H bond.

Pharmaceutical Research: A New Approach to Dissolution Testing by UV Imaging and Finite Element Simulations

Boetker; Rantanen; Rades; Müllertz; Østergaard; Jensen — 2013-05-01

Type: Original Paper
Purpose

Most dissolution testing systems rely on analyzing samples taken remotely from the dissolving sample surface at different time points with poor time resolution and therefore provide relatively unresolved temporally and spatially information on the dissolution process. In this study, a flexible numerical model was combined with a novel UV imaging system, allowing monitoring of the dissolution process with sub second time resolution.

Methods

The dissolution process was monitored by both effluent collection and UV imaging of compacts of paracetamol. A finite element model (FEM) was used to characterize the UV imaging system.

Results

A finite element model of the UV imaging system was successfully built. The dissolution of paracetamol was studied by UV imaging and by analysis of the effluent. The dissolution rates obtained from the collected effluent were in good agreement with the numerical model. The numerical model allowed an assessment of the ability of the UV imager to measure dissolution—time profiles. The simulation was able to extend the experimental results to conditions not easily obtained experimentally.

Conclusions

Combining FEM,experimental dissolution data and UV imaging provided experimental validation of the FEM model as well as a detailed description of the dissolution process.

Pharmaceutical Research: Design and Characterization of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transporter Activity

2013-05-01

Type: Original Paper
Purpose

To synthesize a trifluorinated bile acid that can be used for ¹⁹F magnetic resonance imaging (MRI) of bile acid enterohepatic circulation, characterize its in vitro transporter affinity, stability, and ¹⁹F-MRI signal, and assess its ability to concentrate in the gallbladder of C57BL/6 mice.

Methods

Target compound CA-lys-TFA was synthesized and tested for affinity toward the apical sodium dependent bile acid transporter (hASBT) and the Na+/taurocholate cotransporting polypeptide (hNTCP). In a pilot study, fasted mice were gavaged with vehicle control, 150 mg/kg or 300 mg/kg CA-lys-TFA. CA-lys-TFA in gallbladder, liver and plasma at t = 5 h was quantified. Additionally, a 24-h time course (24 mice across eight time points) was studied using 50 mg/kg CA-lys-TFA.

Results

CA-lys-TFA was a potent substrate of hASBT (Kt = 39.4 μM, normalized Vmax = 0.853) and hNTCP (Kt = 8.99 μM, normalized Vmax = 0.281). ¹⁹F MRI phantom imaging showed linear signal-concentration dependence. In vivo studies showed that rapid accumulation of CA-lys-TFA in the gallbladder was maximal within 4–7 h.

Conclusions

These findings suggest that CA-lys-TFA, a fluorinated non-radioactive bile acid analogue, has potential for use in MRI to measure in vivo bile acid transport and diagnose bile acid malabsorption and other conditions associated with impaired bile acid transport.

Pharmaceutical Research: Engineered Andrographolide Nanoparticles Mitigate Paracetamol Hepatotoxicity in Mice

Roy; Das; Auddy; Saha; Mukherjee — 2013-05-01

Type: Original Paper
Purpose

Paracetamol (acetaminophen, APAP) overdose is often fatal due to progressive and irreversible hepatic necrosis. The aim of this work was to design Andrographolide (AG) loaded nanoparticles to prevent similar hepatic necrosis.

Methods

Functionalized AG-loaded PLGA nanoparticles carrying different densities of heparin were prepared following a facile emulsion solvent evaporation technique. Nanoparticle morphology, loading and release kinetics were studied. Hepatic localization of the nanoparticles was investigated in both normal and APAP damaged conditions using FITC fluorescent probe. Different serum parameters and liver histopathology were further examined as indicators of hepatic condition before and after treatment.

Result

A collection of heparin functionalized AG-loaded PLGA nanoparticles were designed. Low amount of heparin on the particle surface could rapidly localize the nanoparticles up to the liver. The new functionalized AG nanoparticles affect efficient hepatoprotection in experimental mouse APAP overdose conditions. AG nanoparticle hepatoprotection was due to the rapid regeneration of antioxidant capacity and hepatic GSH store.

Conclusions

Engineered nanoparticles loaded with AG provided a fast protection in APAP induced acute liver failure.

Pharmaceutical Research: Effects of Ionic Strength and Sugars on the Aggregation Propensity of Monoclonal Antibodies: Influence of Colloidal and Conformational Stabilities

Saito; Hasegawa; Kobayashi; Tomitsuka; Uchiyama; Fukui — 2013-05-01

Type: Original Paper
Purpose

To develop a general strategy for optimizing monoclonal antibody (MAb) formulations.

Methods

Colloidal stabilities of four representative MAbs solutions were assessed based on the second virial coefficient (B₂) at 20°C and 40°C, and net charges at different NaCl concentrations, and/or in the presence of sugars. Conformational stabilities were evaluated from the unfolding temperatures. The aggregation propensities were determined at 40°C and after freeze–thawing. The electrostatic potential of antibody surfaces was simulated for the development of rational formulations.

Results

Similar B₂ values were obtained at 20°C and 40°C, implying little dependence on temperature. B₂ correlated quantitatively with aggregation propensities at 40°C. The net charge partly correlated with colloidal stability. Salts stabilized or destabilized MAbs, depending on repulsive or attractive interactions. Sugars improved the aggregation propensity under freeze–thaw stress through improved conformational stability. Uneven and even distributions of potential surfaces were attributed to attractive and strong repulsive electrostatic interactions.

Conclusions

Assessment of colloidal stability at the lowest ionic strength is particularly effective for the development of formulations. If necessary, salts are added to enhance the colloidal stability. Sugars further improved aggregation propensities by enhancing conformational stability. These behaviors are rationally predictable according to the surface potentials of MAbs.

Pharmaceutical Research: Brain Extracellular γ-hydroxybutyrate Concentrations are Decreased by L-lactate in Rats: Role in the Treatment of Overdoses

Roiko; Vijay; Felmlee; Morris — 2013-05-01

Type: Original Paper
Purpose

L-lactate represents a potential treatment for GHB overdose by inhibiting GHB renal reabsorption mediated by monocarboxylate transporters. Our objective was to assess the dose-dependence of L-lactate treatment, with and without D-mannitol, on GHB toxicokinetics/toxicodynamics (TK/TD).

Methods

Rats were administered GHB 600 mg/kg i.v. with L-lactate (low and high doses), D-mannitol, or L-lactate (low dose) with D-mannitol. GHB-induced sleep time and GHB plasma, urine and brain extracellular fluid (ECF) concentrations (by LC/MS/MS) were determined. The effect of L-lactate and D-mannitol on the uptake and efflux of GHB was assessed in rat brain endothelial RBE4 cells.

Results

L-lactate treatment increased GHB renal clearance from 1.4 ± 0.1 ml/min/kg (control) to 2.4 ± 0.2 and 4.7 ± 0.5 ml/min/kg after low and high doses, respectively, and reduced brain ECF AUC values to 65 and 25% of control. Sleep time was decreased from 137 ± 12 min (control) to 91 ± 16 and 55 ± 5 min (low and high L-lactate, respectively). D-mannitol did not alter GHB TK/TD and did not alter L-lactate’s effects on GHB TK/TD. L-lactate, but not D-mannitol, inhibited GHB uptake, and increased GHB efflux from RBE4 cells.

Conclusions

L-lactate decreases plasma and brain ECF concentrations of GHB, decreasing sedative/hypnotic effects.

Pharmaceutical Research: Aggregation and Chemical Modification of Monoclonal Antibodies under Upstream Processing Conditions

Dengl; Wehmer; Hesse; Lipsmeier; Popp; Lang — 2013-05-01

Type: Original Paper
Purpose

To investigate antibody stability and formation of modified species under upstream processing conditions.

Methods

The stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC).

Results

Sensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations.

Conclusions

Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.

Pharmaceutical Research: Understanding the Effect of Environmental History on Bilayer Tablet Interfacial Shear Strength

Klinzing; Zavaliangos — 2013-05-01

Type: Original Paper
Purpose

To understand the effect of post production environmental conditions on the interfacial strength of bilayer tablets.

Methods

Bilayer tablets of microcrystalline cellulose/dicalcium phosphate were exposed to several humidity conditions higher/lower than production conditions and tested in shear to assess interfacial strength. Specific failure mechanisms were observed using x-ray microtomography and scanning electron microscopy.

Results

Transients in moisture diffusion of bilayer tablets with significant differential moisture absorption characteristics are responsible for the reduction of strength in both high and low moisture environments. X-ray microtomography and SEM experiments have shown that two different mechanisms of interfacial crack formation are present. For low moisture exposure, interfacial cracks close to the surface were produced, whereas at high moisture conditions, internal interfacial cracks were created. In both cases the fracture modes are consistent with the tensile stresses that develop locally due to the volumetric strains induced by moisture absorption.

Conclusions

The insight gained from this work will be useful for material selection and packaging of bilayer tablet systems. While additional work is needed to develop specific guidelines for the optimization of bilayer strength, the results presented here provide a rational basis upon which such work can be conducted.

Pharmaceutical Research: Preclinical Pharmacokinetic/Pharmacodynamic Models to Predict Schedule-Dependent Interaction Between Erlotinib and Gemcitabine

Li; Li; Cheng; Wang; Li; Zhou; Lu — 2013-05-01

Type: Original Paper
Purpose

To investigate the pharmacological effects of different erlotinib (ER) and gemcitabine (GM) combination schedules by in vitro and in vivo experiments and PK/PD models in non-small cell lung cancer cells.

Methods

H1299 cells were exposed to different ER combined with GM schedules. Cell growth inhibition was analyzed to evaluate these schedules. A preclinical in vivo study was then conducted to compare tumor suppression effects of different schedules in H1299 xenografts. PK/PD models were developed to quantify the anti-tumor interaction of ER and GM.

Results

Synergism was observed when ER preceded GM, but other sequences showed antagonism. The optimal in vitro schedule, or interval schedule, was applied to the animal study, which showed greater anti-tumor effect than simultaneous group. PK/PD models implied that interaction of the two drugs was additive in simultaneous treatment but synergistic in interval schedule. The simulation results showed that interval schedule can delay tumor growth for a longer time, and demonstrated more evident anti-tumor effect compared with simultaneous group if the treatment duration was longer.

Conclusions

Interval schedule of the two drugs can achieve synergistic anti-tumor effect, and is superior to simultaneous treatment.

Pharmaceutical Research: Rapid Vaccination Using an Acetalated Dextran Microparticulate Subunit Vaccine Confers Protection Against Triplicate Challenge by Bacillus Anthracis

2013-05-01

Type: Original Paper
Purpose

A rapid immune response is required to prevent death from Anthrax, caused by Bacillus anthracis.

Method

We formulated a vaccine carrier comprised of acetalated dextran microparticles encapsulating recombinant protective antigen (rPA) and resiquimod (a toll-like receptor 7/8 agonist).

Results

We were able to protect against triplicate lethal challenge by vaccinating twice (Days 0, 7) and then aggressively challenging on Days 14, 21, 28. A significantly higher level of antibodies was generated by day 14 with the encapsulated group compared to the conventional rPA and alum group. Antibodies produced by the co-encapsulated group were only weakly-neutralizing in toxin neutralization; however, survival was not dependent on toxin neutralization, as all vaccine formulations survived all challenges except control groups. Post-mortem culture swabs taken from the hearts of vaccinated groups that did not produce significant neutralizing titers failed to grow B. anthracis.

Conclusions

Results indicate that protective antibodies are not required for rapid protection; indeed, cytokine results indicate that T cell protection may play a role in protection from anthrax. We report the first instance of use of a particulate carrier to generate a rapid protective immunity against anthrax.

Pharmaceutical Research: Efficacy of Aerosolized Celecoxib Encapsulated Nanostructured Lipid Carrier in Non-small Cell Lung Cancer in Combination with Docetaxel

Patel; Chougule; I.; Patlolla; Wang; Singh — 2013-05-01

Type: Original Paper
Purpose

Evaluation of in-vivo anticancer activity of aerosolized Celecoxib encapsulated Nanolipidcarriers (Cxb-NLC) as a single therapeutic agent and combined with intravenously administered Docetaxel (Doc) against non-small cell lung cancer.

Methods

Cxb-NLC were prepared by high-pressure homogenization and were characterized for its physicochemical characteristics. Metastatic A549 tumor model in Nu/Nu mice was used to evaluate response of aerosolized Cxb-NLC & Doc. Isolated lung tumor samples were analyzed for: a) DNA fragmentation and cleaved caspase-3 by immunohistochemistry, b) apoptotic and angiogenic protein markers by western blot, c) global proteomic alterations by an isobaric labeling quantitative proteomic method and d) toxicity studies of NLC.

Results

The particle size of Cxb-NLC was 217 ± 20 nm, while entrapment efficiency was more than 90%. Cxb-NLC and Doc alone and in combination showed 25 ± 4%, 37 ± 5%, and 67 ± 4% reduction in tumor size respectively compared to control. Proteomic analysis with combination treatment further revealed significantly decreased expression of multiple pro-survival and pro-metastasis proteins as well as tumor invasion markers and the expression of S100 family proteins, such as S100A6 and S100P were decreased by 2.5 and 1.6 fold.

Conclusions

Combination therapy with Cxb-NLC and Doc showed significant reduction in tumor growth which was further confirmed by proteomic analysis.

AAPS PharmSciTech: Preparation and Evaluation of Solid Dispersions of A New Antitumor Compound Based on Early-Stage Preparation Discovery Concept

Hou; Ni; Cao; Lei; Cai; Zhang; Yu; Tan — 2013-04-30

Type: Original Paper

Ensuring sufficient drug solubility is a crucial problem in pharmaceutical-related research. For water-insoluble drugs, various formulation approaches are employed to enhance the solubility and bioavailability of lead compounds. The goal of this study was to enhance the dissolution and absorption of a new antitumor lead compound, T-OA. Early-stage preparation discovery concept was employed in this study. Based on this concept, a solid dispersion system was chosen as the method of improving drug solubility and bioavailability. Solid dispersions of T-OA in polyvinylpyrrolidone (PVP) K30 were prepared by the solvent evaporation method. Dissolution testing determined that the ideal drug-to-PVP ratio was 1:5. X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry were employed to confirm the formation of solid dispersions. Scanning electron microscopy demonstrated that T-OA was converted into an amorphous form. Both in vitro dissolution testing and the in vivo studies demonstrated that the solubility and bioavailability of T-OA were significantly improved when formulated in a solid dispersion with PVP. The dissolution rate of the T-OA/PVP solid dispersion was greatly enhanced relative to the pure drug, and the relative bioavailability of T-OA solid dispersions was found to be 392.0%, which is 4-fold higher than the pure drug.

AAPS PharmSciTech: Electrospun Chitosan Microspheres for Complete Encapsulation of Anionic Proteins: Controlling Particle Size and Encapsulation Efficiency

Choi; Kim; Kang; Jeong; Yoo — 2013-04-30

Type: Original Paper

Electrospinning was employed to fabricate chitosan microspheres by a single-step encapsulation of proteins without organic solvents. Chitosan in acetic acid was electrospun toward a grounded sodium carbonate solution at various electric potential and feeding rates. Electrospun microspheres became insoluble and solidified in the sodium carbonate solution by neutralization of chitosan acetate. When the freeze-dried microspheres were examined by scanning electron microscopy, the small particle size was obtained at higher voltages. This is explained by the chitosan droplet size at the electrospinning needle was clearly controllable by the electric potential. The recovery yield of chitosan microspheres was dependent on the concentration of chitosan solution due to the viscosity is the major factor affecting formation of chitosan droplet during curling of the electrospinning jets. For protein encapsulation, fluorescently labeled bovine serum albumin (BSA) was codissolved with chitosan in the solution and electrospun. At higher concentration of sodium carbonate solution and longer solidification time in the solution, the encapsulation efficiency of the protein was confirmed to be significantly high. The high encapsulation efficiency was achievable by instant solidification of microspheres and electrostatic interactions between chitosan and BSA. Release profiles of BSA from the microspheres showed that the protein release was faster in acidic solution due to dissolution of chitosan. Reversed-phase chromatography of the released fractions confirmed that exposure of BSA to acidic solution during the electrospinning did not result in structural changes of the encapsulated protein.

AAPS PharmSciTech: Binding Interactions of Naringenin and Naringin with Calf Thymus DNA and the Role of β-Cyclodextrin in the Binding

Yousuf; Enoch — 2013-04-27

Type: Original Paper

The interaction of naringenin (Nar) and its neohesperidoside, naringin (Narn), with calf thymus deoxyribonucleic acid (ctDNA) in the absence and the presence of β-cyclodextrin (β-CD) was investigated. The interaction of Nar and Narn with β-CD/ctDNA was analyzed by using absorption, fluorescence, and molecular modeling techniques. Docking studies showed the existence of hydrogen bonding, electrostatic and phobic interaction of Nar and Narn with β-CD/DNA. 1:2 stoichiometric inclusion complexes were observed for Nar and Narn with β-CD. With the addition of ctDNA, Nar and Narn resulted into the fluorescence quenching phenomenon in the aqueous solution and β-CD solution. The binding constant K_b and the number of binding sites were found to be different for Nar and Narn bindings with DNA in aqueous and β-CD solution. The difference is attributed to the structural difference between Nar and Narn with neohesperidoside moiety present in Narn.

AAPS PharmSciTech: Solid Lipid Nanoparticles as Effective Reservoir Systems for Long-Term Preservation of Multidose Formulations

Cerreto; Paolicelli; Cesa; Amara; D’Auria; Simonetti; Casadei — 2013-04-27

Type: Original Paper

Cosmetic multidose preparations, as well as pharmaceutical ones, are at risk of contamination by microorganisms, due to their high water content. Besides the risk of contamination during manufacturing, multidose cosmetic preparations may be contaminated by consumers during their use. In this paper, the results of the utilization of nanoparticles as reservoir systems of parabens, the most used class of preservatives, were reported. Two different systems, solid lipid nanoparticles (SLN) made of pure precirol and nanostructured lipid carriers (NLC) made of precirol and almond oil, containing three parabens as single molecules or as a mixture, were prepared and tested. All the systems were characterized for size, polydispersion index, zeta potential and encapsulation efficiency. Release experiments, carried out in steady state and sink conditions, allowed to evidence that both SLN and NLC were able to act as reservoir systems. The antimicrobial activity of the systems was tested against Candida albicans ATCC 10231 with repeat insult tests. The results of the release experiments and the antimicrobial tests showed very low water concentration of parabens still maintaining their antimicrobial activity.

AAPS PharmSciTech: Studies on Tolfenamic Acid–Chitosan Intermolecular Interactions: Effect of pH, Polymer Concentration and Molecular Weight

Ahmed; Sheraz; Rehman — 2013-04-27

Type: Original Paper

Solid-state properties of tolfenamic acid (TA) and its complexes with chitosan (CT) have been studied. Effect of medium pH, molecular weight of polymer and its different concentrations on these TA–CT complexes were studied in detail. Low and medium molecular weight CT have been used in different ratios at pH ranging from 4 to 6 and freeze-drying technique has been employed to modify the appearance of crystalline TA. Physical properties of the formed complexes have been studied by employing X-ray diffraction, differential scanning calorimetry and scanning electron microscopy; chemical structure has been studied using Fourier transform infrared spectroscopy. The results showed that both forms of the polymer exhibited complete conversion in 1:8 ratio at pH 4, 1:4 at pH 5 and 1:1 at pH 6 indicating a marked effect of pH on drug–polymer complexation. The percent crystallinity calculations indicated low molecular weight CT slightly more effective than the other form. No changes in the complexes have been observed during the 12 week storage under controlled conditions. Both forms of CT at different pH values indicated retardation of recrystallization in TA during cooling of the melt from 1:1 ratios exhibiting formation of strong intermolecular hydrogen bonding between the drug and the polymer.

Pharmaceutical Research: Nanoparticles in the Brain: A Potential Therapeutic System Targeted to an Early Defect Observed in Many Neurodegenerative Diseases

Gunawardena — 2013-04-27

Type: Review Paper

Currently, there are no effective treatments or cures for many neurodegenerative diseases affecting an aging baby-boomer generation. The ongoing problem with many of the current therapeutic treatments is that most are aimed at dissolving or dissociating aggregates and preventing cell death, common neuropathology often seen towards the end stage of disease. Often such treatments have secondary effects that are more devastating than the disease itself. Thus, effective therapeutics must be focused on directly targeting early events such that global deleterious effects of drugs are minimized while beneficial therapeutic effects are maximized. Recent work indicates that in many neurodegenerative diseases long distance axonal transport is perturbed, leading to axonal blockages. Axonal blockages are observed before pathological or behavioral phenotypes are seen indicating that this pathway is perturbed early in disease. Thus, developing novel therapeutic treatments to an early defect is critical in curing disease. Here I review neurodegenerative disease and current treatment strategies, and discuss a novel nanotechnology based approach that is aimed at targeting an early pathway, with the rationale that restoring an early problem will prevent deleterious downstream effects. To accomplish this, knowledge exchange between biologists, chemists, and engineers will be required to manufacture effective novel biomaterials for medical use.

Pharmaceutical Research: Imaging the Cytosolic Drug Delivery Mechanism of HDL-Like Nanoparticles

Lin; Chen; Ng; Cao; Zhang; Zheng — 2013-04-27

Type: Original Paper
Purpose

Molecular therapeutics often require an effective nanoparticle-based delivery strategy to transport them to cytosolic organelles to be functional. Recently, a cytosolic delivery strategy based on the scavenger receptor class B type I (SR-BI) mediated pathway has shown great potential for the effective delivery of theranostics agents into the cytoplasm of cells without detrimental endosomal entrapment. This study elucidates this unique delivery mechanism for improving cytosolic drug delivery.

Methods

Multifluorophore-labeled HDL-mimicking peptide phospholipid scaffold (HPPS) nanoparticles were developed. Fluorescence imaging was utilized to examine HPPS transporting payloads into cells step by step through sequential inhibition studies.

Results

HPPS specifically recognizes and binds to SR-BI, then interacts with SR-BI, which results in direct transport of payload molecules into the cell cytoplasm without entire particles internalization. The cytosolic transport of payloads occurred through a temperature- and energy-independent pathway, and was also different from actin- and clathrin-mediated endocytosis. Furthermore, this transport was significantly inhibited by disruption of lipid rafts using filipin or methyl-β-cyclodextrin.

Conclusions

The cytosolic delivery of payloads by HPPS via SR-BI targeting is predominately mediated through a lipid rafts/caveolae-like pathway. This cytosolic delivery strategy can be utilized for transporting molecular therapeutics that require their action sites to be within cytosolic organelles to enhance therapeutic effect.

Pharmaceutical Research: Poly(ethylene glycol)-poly(ε-caprolactone) Iodinated Nanocapsules as Contrast Agents for X-ray Imaging

Hallouard; Briançon; Anton; Li; Vandamme; Fessi — 2013-04-26

Type: Original Paper
Purpose

Synthesis and formulation of iodinated PCL-mPEG nanocapsules as new original blood pool contrast agents for computed tomography.

Methods

PCL-mPEG was synthesized and formulated following the emulsion–solvent diffusion process, in the form of iodinated nanocapsules. Physico-chemical characterization of such nano-materials was performed by DLS and transmission electron microscopy. A stability study of the nanocapsules suspension was followed-up to 3 month. Blood biocompatibility was performed. Finally, the nanocapsules suspension radiopacity was evaluated in vitro then in vivo in mice as micro-CT contrast agent.

Results

In this study, the iodine concentration in nanocapsules suspension was about 70 mgI/mL. Besides, these nanocarriers appeared non-toxic, and stable in suspension. In vivo, i.v. administration of 10 μL/g of mouse body weight of theses nano-particles induced a vascular contrast enhancement of 168 HU and a half-life in blood of 4.2 +/− 0.5 h. Elimination route of these particles appears mainly performed by the liver, without sequestration in spleen and lymph nodes confirming their stealth properties.

Conclusions

This study proposes the first example of iodinated biodegradable polymeric blood pool contrast agent, able to induce an exploitable contrast enhancement. The main advantage of polymeric system compared to lipid ones, lies in their stability and handling, e.g. towards drying for storage.

Pharmaceutical Research: In Vivo Investigation of Hybrid Paclitaxel Nanocrystals with Dual Fluorescent Probes for Cancer Theranostics

Hollis; Weiss; Evers; Gemeinhart; Li — 2013-04-26

Type: Original Paper
Purpose

To develop novel hybrid paclitaxel (PTX) nanocrystals, in which bioactivatable (MMPSense® 750 FAST) and near infrared (Flamma Fluor® FPR-648) fluorophores are physically incorporated, and to evaluate their anticancer efficacy and diagnostic properties in breast cancer xenograft murine model.

Methods

The pure and hybrid paclitaxel nanocrystals were prepared by an anti-solvent method, and their physical properties were characterized. The tumor volume change and body weight change were evaluated to assess the treatment efficacy and toxicity. Bioimaging of treated mice was obtained non-invasively in vivo.

Results

The released MMPSense molecules from the hybrid nanocrystals were activated by matrix metalloproteinases (MMPs) in vivo, similarly to the free MMPSense, demonstrating its ability to monitor cancer progression. Concurrently, the entrapped FPR-648 was imaged at a different wavelength. Furthermore, when administered at 20 mg/kg, the nanocrystal formulations exerted comparable efficacy as Taxol®, but with decreased toxicity.

Conclusions

Hybrid nanocrystals that physically integrated two fluorophores were successfully prepared from solution. Hybrid nanocrystals were shown not only exerting antitumor activity, but also demonstrating the potential of multi-modular bioimaging for diagnostics.

Pharmaceutical Research: HCV J6/JFH1 Tilts the Capability of Myeloid-Derived Dendritic Cells to Favor the Induction of Immunosuppression and Th17-Related Inflammatory Cytokines

2013-04-26

Type: Original Paper
Purpose

How HCV virus affects the function of dendritic cells (DCs) and their ability to induce CD4+ T cell response remains not fully understood. This study was done to elucidate the impact of HCV on the function of DCs and on DC’s capability to induce CD4+ T-cell response.

Methods

Monocyte-derived DCs (MoDCs) were treated with cell-culture HCV (HCVcc). The effects of HCVcc on DC maturation, CD40L-induced DC maturation, and cytokine production and the capacity of DCs to induce Th cytokine production of allogeneic CD4+ T cells were evaluated.

Results

HCVcc exposure increased expression of both IL-6 and IL-10 by MoDCs. HCV-exposed MoDCs also selectively facilitated allogeneic CD4+ T cells to further produce Th17-related cytokines interleukin 1 (IL-1), IL-6, and IL-17A. Pretreatment of IL-17A inhibited HCV production in Huh7.5 cells, suggesting that induction of Th17 cells may be beneficial to host anti-HCV immunity. Paradoxically, induction of IL-10 expression and the failure of HCV-exposed MoDCs to facilitate other Th cell development may hinder the anti-viral immunity.

Conclusions

This study highlights both the therapeutic potential of IL-17A in treating HCV infection and the cautious consideration of HCV-induced immunosuppression in DC-based therapy.

The AAPS Journal: Pre-Existing Biotherapeutic-Reactive Antibodies: Survey Results Within the American Association of Pharmaceutical Scientists

Xue; Fiscella; Rajadhyaksha; Goyal; Holland; Gorovits; Morimoto — 2013-04-26

Type: Brief Communication

The immunogenicity profile of a biotherapeutic is determined by a multitude of product and patient-related risk factors that can influence the observed incidence and clinical consequences of immunogenicity. Pre-existing antibodies, i.e., biotherapeutic-reactive antibodies present in samples from treatment-naïve subjects, have been commonly observed during immunogenicity assessments; however their relevance in terms of the safety and efficacy of a biotherapeutic is poorly understood. An American Association of Pharmaceutical Scientists-sponsored survey was conducted to gather information about the prevalence, nature, and consequences of pre-existing antibodies in clinical and nonclinical studies. The survey results indicate that pre-existing antibodies against a variety of biotherapeutics (e.g., mAbs, fusion proteins) are frequently encountered, especially in the context of autoimmune diseases, but that the methods and approaches used to detect, characterize, and report these antibodies vary. In most cases, pre-existing antibodies did not appear to have clinical consequences; however, a few of the respondents reported having observed an effect on pharmacokinetic, pharmacodynamic, safety, and/or efficacy parameters. The findings from this survey are an important first step in evaluating the potential risks associated with the presence of pre-existing antibodies and highlight the importance of standardizing the approaches for detection and characterization of these antibodies. Cross-industry sharing of case studies and relevant data collection will help better inform biotherapeutic risk/benefit profiles and provide deeper understanding of the biological consequences of pre-existing antibodies.

AAPS PharmSciTech: Highly Sensitive Spectrofluorimetric Method for Determination of Certain Aminoglycosides in Pharmaceutical Formulations and Human Plasma

Omar; Nagy; Hammad; Aly — 2013-04-26

Type: Original Paper

A simple, reliable, highly sensitive and selective spectrofluorimetric method has been developed for determination of certain aminoglycosides namely amikacin sulfate, tobramycin, neomycin sulfate, gentamicin sulfate, kanamycin sulfate and streptomycin sulfate. The method is based on the formation of a charge transfer complexes between these drugs and safranin in buffer solution of pH 8. The formed complexes were quantitatively extracted with chloroform under the optimized experimental conditions. These complexes showed an excitation maxima at 519–524 nm and emission maxima at 545–570 nm. The calibration plots were constructed over the range of 4–60 pg mL⁻¹ for amikacin, 4–50 pg mL⁻¹ for gentamicin, neomycin and kanamycin, 4–40 pg mL⁻¹ for streptomycin and 5–50 pg mL⁻¹ for tobramycin. The proposed method was successfully applied to the analysis of the cited drugs in dosage forms. The proposed method was validated according to ICH and USP guidelines with respect to specificity, linearity, accuracy, precision and robustness. The high sensitivity of the proposed method allowed determination of amikacin and gentamicin in spiked and real human plasma.

Pharmaceutical Research: Preparation, Characterization, and Release of Amoxicillin from Electrospun Fibrous Wound Dressing Patches

Sofokleous; Stride; Edirisinghe — 2013-04-25

Type: Original Paper
Purpose

To produce electrospun polymeric fibrous wound dressing patches that can release the antibiotic drug amoxicillin in a controlled manner.

Methods

Poly(D,L-lactide-co-glycolide) acid (PLGA) fibrous dressings with entrapped amoxicillin were produced by electrospinning. The morphology and successful entrapment of amoxicillin in the PLGA fibrous dressings were validated by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) spectroscopy. The rate of drug release from the dressing patches was measured in various media for a period of 21 days using UV spectroscopy.

Results

PLGA fibres entrapping amoxicillin were collected for 300 s and then cut to form square patches with an average weight of 55 mg. Each dressing patch contained ~2 mg of amoxicillin. The mean fibre diameter was 2.2 ± 0.4 μm. The drug release from the PLGA dressings was found to be different for each medium during the 21-day release period with the highest and lowest concentration of drug released observed when the dressings were immersed in simulated body fluid (SBF) and phosphate buffered saline (PBS), respectively.

Conclusions

The release profiles obtained in this study and the well-established biocompatibility of PLGA indicate that the fibre-based patches with entrapped amoxicillin fabricated in this work are very well suited for applications in wound healing and infection control.

Pharmaceutical Research: Trastuzumab Labeled to High Specific Activity with 111In by Conjugation to G4 PAMAM Dendrimers Derivatized with Multiple DTPA Chelators Exhibits Increased Cytotoxic Potency on HER2-Positive Breast Cancer Cells

Chan; Cai; Reilly — 2013-04-25

Type: Original Paper
Purpose

To conjugate trastuzumab with/without NLS peptides to G4 PAMAM dendrimers derivatized with DTPA and determine the specific radioactivity (SA) for ¹¹¹In labeling, HER2 immunoreactivity and cytotoxicity on breast cancer (BC) cells.

Methods

G4 dendrimers were reacted with DTPA then conjugated through a thiol to maleimide-derivatized trastuzumab. The SA achievable was determined by incubating 2 to 20 μg with 60 MBq of ¹¹¹In. HER2 immunoreactivity, internalization and nuclear importation were measured. The effect of ¹¹¹In-DTPA-G4-trastuzumab (5.9 MBq/μg) on the clonogenic survival (CS) of SK-Br-3 or MDA-MB-231 cells with high or low HER2 density, respectively was compared to ¹¹¹In-DTPA-NLS-trastuzumab (0.5 MBq/μg). DNA double-strand breaks (DSBs) were measured.

Results

DTPA-G4-trastuzumab was labeled with ¹¹¹In to a SA (23.6 MBq/μg) which was 100-fold higher than ¹¹¹In-DTPA-NLS-trastuzumab. ¹¹¹In-DTPA-G4-trastuzumab and ¹¹¹In-DTPA-G4-NLS-trastuzumab retained HER2 immunoreactivity and were internalized and imported into the nucleus of BC cells. G4-radioimmunoconjugates were 2–4 fold and 9-fold more cytotoxic to SK-Br-3 and MDA-MB-231 cells, respectively than ¹¹¹In-DTPA-NLS-trastuzumab which was associated with an increase in DNA DSBs.

Conclusions

Conjugation of trastuzumab to G4 PAMAM dendrimers modified with 30 DTPA permitted high SA ¹¹¹In labeling which increased their cytotoxic potency for BC cells with high or low HER2 density.

Pharmaceutical Research: The Use of Borneol as an Enhancer for Targeting Aprotinin-Conjugated PEG-PLGA Nanoparticles to the Brain

Zhang; Han; Qin; Lu; Wang — 2013-04-25

Type: Original Paper
Purpose

To evaluate the effect of borneol on the brain targeting efficiency of aprotinin-conjugated poly (ethyleneglycol)–poly (L-lactic-co-glycolic acid) nanoparticles (Apr-NP) and the activity of huperzine A (Hup A) loaded nanoparticles to AD rats .

Method

Apr-NP was prepared by emulsion and solvent evaporation method. The uptake of Apr-NP alone or combined with borneol by brain capillary endothelial cells (BCECs) was evaluated by incorporating coumarin-6 as a tracer. In vivo imaging and the distribution of Hup A in the brain were measured to investigate the brain delivery of Apr-NP in rats, with or without the oral administration of borneol. Morris water maze was used to evaluate the memory improvement effect of Hup A loaded nanoparticles (Apr-NP-Hup).

Results

Co-incubation with borneol could increase the uptake of nanoparticles by BCECs. Nanoparticles delivered into the rat brain were enhanced significantly by the co-administration of borneol. The pharmacological effects of Hup A loaded nanoparticles on improving the memory impairment of AD rats were greatly improved when combined with borneol.

Conclusions

Borneol is a promising enhancer for brain-targeting delivery systems. When co-administered with aprotinin-modified nanoparticles, borneol could improve the brain targeting efficiency of nanoparticles significantly.

The AAPS Journal: Non-Arrhenius Protein Aggregation

Wang; Roberts — 2013-04-25

Type: Original Paper

Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

AAPS PharmSciTech: Inulin-Based Tablet in Capsule Device for Variable Multipulse Delivery of Aceclofenac: Optimization and In Vivo Roentgenography

Sharma; Pathak — 2013-04-25

Type: Original Paper

The aim of the study was to develop single-unit tablet in capsule system of aceclofenac for the treatment of late night pain and morning stiffness associated with rheumatoid arthritis. The system was conceptualized as a three-component design (1) a hard gelatin enteric-coated capsule (for carrying two pulses), (2) first-pulse granules (for rapid release in intestine), and (2) second-pulse matrix tablet (for slow release in colon). An appropriate integration of pH-sensitive (Eudragit S100) and bacteria-responsive (inulin) functions, on the basis of 3² factorial design, led to formulation of TICS 1–9 that were screened for in vitro release. TICS 2 with biphasic drug release of 98.64% from first-pulse granules in simulated intestinal fluid (12 h) and 97.82% from second-pulse matrix tablet in simulated colonic fluid (24 h) was the optimized formulation that exhibited Fickian diffusion of drug (n = 0.363). In vivo fluoroscopy in rats traced the intact tablet to colon in 7.5 h that got eroded at the tenth hour. This demonstrated the colon-specific delivery of the matrix tablet affirming the potential of the system to obviate the need for two-time administration of drug at odd hours. The experimental design was validated by extra design check point, and diffuse reflectance spectroscopy and DSC revealed absence of chemical interaction between the formulation excipients.

AAPS PharmSciTech: Dissolution Studies of Poorly Soluble Drug Nanosuspensions in Non-sink Conditions

2013-04-25

Type: Original Paper

Sink conditions used in dissolution tests lead to rapid dissolution rates for nanosuspensions, causing difficulties in discriminating dissolution profiles between different formulations. Here, non-sink conditions were studied for the dissolution testing of poorly water-soluble drug nanosuspensions. A mathematical model for polydispersed particles was established to clarify dissolution mechanisms. The dissolution of nanosuspensions with either a monomodal or bimodal size distribution was simulated. In the experimental part, three different particle sizes of indomethacin nanosuspensions were prepared by the wet milling technique. The effects of the dissolution medium pH and agitation speed on dissolution rate were investigated. The dissolution profiles in sink and non-sink conditions were obtained by changing the ratio of sample amount to the saturation solubility. The results of the simulations and experiments indicated that when the sample amount was increased to the saturation solubility of drug, the slowest dissolution rate and the best discriminating dissolution profiles were obtained. Using sink conditions or too high amount of the sample will increase the dissolution rate and weaken the discrimination between dissolution profiles. Furthermore, the low solubility by choosing a proper pH of the dissolution medium was helpful in getting discriminating dissolution profiles, whereas the agitation speed appeared to have little influence on the dissolution profiles. This discriminatory method is simple to perform and can be potentially used in any nanoproduct development and quality control studies.

AAPS PharmSciTech: Positively Charged Polymeric Nanoparticle Reservoirs of Terbinafine Hydrochloride: Preclinical Implications for Controlled Drug Delivery in the Aqueous Humor of Rabbits

Tayel; El-Nabarawi; Tadros; Abd-Elsalam — 2013-04-25

Type: Original Paper

Frequent instillation of terbinafine hydrochloride (T HCl) eye drops (0.25%, w/v) is necessary to maintain effective aqueous humor concentrations for treatment of fungal keratitis. The current approach aimed at developing potential positively charged controlled-release polymeric nanoparticles (NPs) of T HCl. The estimation of the drug pharmacokinetics in the aqueous humor following ocular instillation of the best-achieved NPs in rabbits was another goal. Eighteen drug-loaded (0.50%, w/v) formulae were fabricated by the nanopreciptation method using Eudragit® RS100 and chitosan (0.25%, 0.5%, and 1%, w/v). Soybean lecithin (1%, w/v) and Pluronic® F68 (0.5%, 1%, and 1.5%, w/v) were incorporated in the alcoholic and aqueous phases, respectively. The NPs were evaluated for particle size, zeta potential, entrapment efficiency percentage (EE%), morphological examination, drug release in simulated tear fluid (pH 7.4), Fourier-transform IR (FT-IR), X-ray diffraction (XRD), physical stability (2 months, 4°C and 25°C), and drug pharmacokinetics in the rabbit aqueous humor relative to an oily drug solution. Spherical, discrete NPs were successfully developed with mean particle size and zeta potential ranging from 73.29 to 320.15 nm and +20.51 to +40.32 mV, respectively. Higher EE% were achieved with Eudragit® RS100-based NPs. The duration of drug release was extended to more than 8 h. FT-IR and XRD revealed compatibility between inactive formulation ingredients and T HCl and permanence of the latter’s crystallinity, respectively. The NPs were physically stable, for at least 2 months, when refrigerated. F5-NP suspension significantly (P < 0.05) increased drug mean residence time and improved its ocular bioavailability; 1.657-fold.

Pharmaceutical Research: Polydopamine-Based Surface Modification for the Development of Peritumorally Activatable Nanoparticles

Gullotti; Park; Yeo — 2013-04-23

Type: Original Paper
Purpose

To create poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), where a drug-encapsulating NP core is covered with polyethylene glycol (PEG) in a normal condition but exposes a cell-interactive TAT-modified surface in an environment rich in matrix metalloproteinases (MMPs).

Methods

PLGA NPs were modified with TAT peptide (PLGA-pDA-TAT NPs) or dual-modified with TAT peptide and a conjugate of PEG and MMP-substrate peptide (peritumorally activatable NPs, PANPs) via dopamine polymerization. Cellular uptake of fluorescently labeled NPs was observed with or without a pre-treatment of MMP-2 by confocal microscopy and flow cytometry. NPs loaded with paclitaxel (PTX) were tested against SKOV-3 ovarian cancer cells to evaluate the contribution of surface modification to cellular delivery of PTX.

Results

While the size and morphology did not significantly change due to the modification, NPs modified with dopamine polymerization were recognized by their dark color. TAT-containing NPs (PLGA-pDA-TAT NPs and PANPs) showed changes in surface charge, indicative of effective conjugation of TAT peptide on the surface. PLGA-pDA-TAT NPs and MMP-2-pre-treated PANPs showed relatively good cellular uptake compared to PLGA NPs, MMP-2-non-treated PANPs, and NPs with non-cleavable PEG. After 3 h treatment with cells, PTX loaded in cell-interactive NPs showed greater toxicity than non-interactive ones as the former could enter cells during the incubation period. However, due to the initial burst drug release, the difference was not as clear as microscopic observation.

Conclusions

PEGylated polymeric NPs that could expose cell-interactive surface in response to MMP-2 were successfully created by dual modification of PLGA NPs using dopamine polymerization.

Pharmaceutical Research: Fatty Acid-Based Strategy for Efficient Brain Targeted Gene Delivery

Shen; Yu; Meng; Li; Lv; Lu — 2013-04-23

Type: Original Paper
Purpose

To investigate a fatty acid-based strategy for efficient brain targeted gene delivery and to understand mechanism(s) of this small molecule-mediated brain gene delivery strategy.

Methods

A series of fatty acids (FAs) were conjugated with polyethylenimine (PEI_25k). A near-infrared fluorescence probe, IR820, was used to study in vivo and ex vivo brain targeting ability of these fatty acid-PEI conjugates (FA-PEIs). Brain uptake of FA-PEI_25k/rhodamine-6-isothiocyanate (RITC)-labeled DNA nanoparticles was investigated via a fluorescence imaging method. Moreover, pEGFP was used as a model gene to study in vitro and in vivo transfection effect of the ideal FA-PEI_25k conjugate.

Results

FA modification did not have interference with the complexation between DNA and the PEI_25k. The FA-PEI_25k conjugates showed excellent brain targeting ability compared with unmodified PEI_25k. Among these FA-PEI_25k conjugates studied, myristic acid (MC)-PEI_25k showed sustained brain distribution profile and higher brain DNA uptake. Furthermore, MC-PEI_25k/pEGFP nanoparticles was able to achieve efficient in vitro and in vivo gene transfection. GFP expression was observed at different brain regions in vivo.

Conclusions

These results demonstrated that the small molecule fatty acid, particularly myristic acid-based brain gene delivery strategy, is promising to mediate efficient gene transfection in the brain.

AAPS PharmSciTech: Simultaneous Determination of Polyethylene Glycol-Conjugated Liposome Components by Using Reversed-Phase High-Performance Liquid Chromatography with UV and Evaporative Light Scattering Detection

Shibata; Yomota; Okuda — 2013-04-23

Type: Original Paper

Liposomes incorporating polyethylene glycol (PEG)-conjugated lipids (PEGylated liposomes) have attracted attention as drug delivery carriers because they show good in vivo stability. The lipid component of PEGylated liposomal formulations needs to be quantified for quality control. In this study, a simple reversed-phase high-performance liquid chromatography (HPLC) method with an evaporative light-scattering detector (ELSD) was established for simultaneous determination of hydrogenated soy phosphatidylcholine, cholesterol, PEG-conjugated lipid, and hydrolysis products of phospholipid in PEGylated liposomal formulations. These lipids were separated using a C18 column with a gradient mobile phase consisting of ammonium acetate buffer and ammonium acetate in methanol at a flow rate of 1.0 ml/min. This method provided sufficient repeatability, linearity, and recovery rate for all lipids. However, the linearity and recovery rates of cholesterol achieved using a ultraviolet (UV) detector were better than those achieved using an ELSD. This validated method can be applied to assess the composition change during the preparation process of liposomes and to quantify lipid components and hydrolysis products contained in a commercially available liposomal formulation DOXIL®. Taken together, this reversed-phase HPLC-UV/ELSD method may be useful for the rapid or routine analysis of liposomal lipid components in process development and quality control.

Alert: 2013 AAPS National Biotechnology Conference

2012-10-25

Type: Alert

Alert: 2013 AAPS Annual Meeting and Exposition

2009-05-26

Type: Alert

Alert: 2011 Impact Factors Released for the Official AAPS Journals

2008-10-03

Type: Alert