International Journal of Pharmaceutics (v.356, #1-2)

Electrically controlled release of sulfosalicylic acid from crosslinked poly(vinyl alcohol) hydrogel by Kanokporn Juntanon; Sumonman Niamlang; Ratana Rujiravanit; Anuvat Sirivat (1-11).
Electrically controlled drug delivery using poly(vinyl alcohol) (PVA) hydrogels as the matrix/carriers for a model drug was investigated. The drug-loaded PVA hydrogels were prepared by solution-casting using sulfosalicylic acid as the model drug and glutaraldehyde as the crosslinking agent. The average molecular weight between crosslinks, the crosslinking density, and the mesh size of the PVA hydrogels were determined from the equilibrium swelling theory as developed by Peppas and Merril, and the latter data were compared with those obtained from scanning electron microscopy. The release mechanisms and the diffusion coefficients of the hydrogels were studied using modified Franz-Diffusion cells in an acetate buffer with pH 5.5 and temperature 37 °C during a period of 48 h, in order to determine the effects of crosslinking ratio, electric field strength, and electrode polarity. The amounts of drug released were analyzed by UV–vis spectrophotometry. The amounts of drug released vary linearly with square root of time. The diffusion coefficients of drug-loaded PVA hydrogels decrease with increasing crosslink ratio. Moreover, the diffusion coefficients of the charged drug in the PVA hydrogels depend critically on the electric field strength between 0 and 5 V as well as on the electrode polarity. Thus, the release rate of sulfosalicylic acid can be altered and controlled precisely through electric field stimulation.
Keywords: Poly(vinyl alcohol) hydrogels; Crosslink; Diffusion coefficient; Electrical control drug release;

Application and limitation of inhibitors in drug–transporter interactions studies by Qing Wang; Robert Strab; Paula Kardos; Chrissa Ferguson; Jibin Li; Albert Owen; Ismael J. Hidalgo (12-18).
The objective of the present study was to investigate the reliability of transporter inhibitors in the elucidation of drug–transporter interactions when multiple transporters are present in a test system. The bidirectional permeabilities of digoxin, estrone-3-sulfate (E3S), and sulfasalazine, substrates of P-gp, BCRP/MRP2 and unspecified efflux transporters, respectively, were examined in Caco-2 and MDR-MDCK cells in the absence and presence of transporter inhibitors: CsA (P-gp), FTC (BCRP) and MK571 (MRP). Digoxin showed significant efflux ratios (ER) in both Caco-2 (ER = 17) and MDR-MDCK (ER = 120), whereas E3S and sulfasalazine only showed significant efflux in Caco-2 (ER = 15 and 88, respectively) but not in MDR-MDCK cells (ER = 1.1 and 1.3, respectively). CsA at 10 μM showed complete inhibition of digoxin efflux, partial inhibition of E3S efflux and no effect on sulfasalazine efflux. FTC and MK571 had different inhibitory effects on the efflux of these compounds. The present study shows evidence of the functional expression of multiple efflux transporter systems in Caco-2 cells. Although the use of Caco-2 cells and selected inhibitors of efflux transporters can provide useful mechanistic information on drug–drug interactions involving efflux transporters, the potential cross-reaction of inhibitors with multiple transporters makes it difficult to discern the role of individual transporters in drug transport or drug–drug interactions.
Keywords: Caco-2; Transporter; Inhibition; Interaction;

Rheological and functional characterization of new antiinflammatory delivery systems designed for buccal administration by Luana Perioli; Cinzia Pagano; Stefania Mazzitelli; Carlo Rossi; Claudio Nastruzzi (19-28).
The aim of the present paper was to investigate the influence of different formulation parameters on the rheological and functional properties of emulgels (gelified emulsions), intended for the buccal administration of the antiinflammatory drug flurbiprofen. The influence of formulation parameters, such as (a) the amount of gelling polymeric emulsifier (Pemulen® 1621 TR-1) used, (b) the oil to water ratio present in the O/W emulgel and finally (c) the pH of the formulation, was studied by a experimental design (DoE) approach. Formulations were analyzed in term of size and morphology of the internal semi-solid oil droplets as well as in term of rheological properties in the presence or in the absence of flurbiprofen by “shear stress vs. shear rate tests” and “frequency sweep tests”. Emulgels were also characterized in vitro both by bioadhesion tests and release studies. In particular release studies demonstrated that flurbiprofen is released by the emulgels in a controlled manner, the drug release efficacy within the first 100 min was comprised between 50 and 80% of the total amount of the drug. Finally, in vivo tests on healthy volunteers have demonstrated that emulgels were able to remain on buccal mucosa for an average period of 1 h, moreover emulgels did not have bad taste and volunteers referred that were agreeable and pleasant.
Keywords: Buccal delivery; Mucoadhesion; Emulgels; Flurbiprofen; Rheology;

Synthesis and evaluation of a novel ligand for folate-mediated targeting liposomes by Guangya Xiang; Jun Wu; Yanhui Lu; Zhilan Liu; Robert J. Lee (29-36).
Folate receptors (FRs) have been identified as cellular surface markers for cancer and leukemia. Liposomes containing lipophilic derivatives of folate have been shown to effectively target FR-expressing cells. Here, we report the synthesis of a novel lipophilic folate derivative, folate-polyethylene glycol-cholesterol hemisuccinate (F-PEG-CHEMS), and its evaluation as a targeting ligand for liposomal doxorubicin (L-DOX) in FR-expressing cells. Liposomes containing F-PEG-CHEMS, with a mean diameter of 120 ± 20 nm, were synthesized by polycarbonate membrane extrusion and were shown to have excellent colloidal stability. The liposomes were taken up selectively by KB cells, which overexpress FR-α. Compared to folate-PEG-cholesterol (F-PEG-Chol), which contains a carbamate linkage, F-PEG-CHEMS better retained its FR-targeting activity during prolonged storage. In addition, F-PEG-CHEMS containing liposomes loaded with DOX (F-L-DOX) showed greater cytotoxicity (IC50  = 10.0 μM) than non-targeted control L-DOX (IC50  = 57.5 μM) in KB cells. In ICR mice, both targeted and non-targeted liposomes exhibited long circulation properties, although F-L-DOX (t 1/2  = 12.34 h) showed more rapid plasma clearance than L-DOX (t 1/2  = 17.10 h). These results suggest that F-PEG-CHEMS is effective as a novel ligand for the synthesis of FR-targeted liposomes.
Keywords: Liposomes; Targeted drug delivery; Folate receptor; Nanotechnology; Doxorubicin; Cancer;

We have previously shown that the stability and permeability of peptides can be greatly improved by conjugation with lipoamino acids such as 2-aminododecanoic acid (C12Laa). However, the increase in lipophilicity which this conjugation provides can also cause a significant decrease in the compound's water solubility. In this study, we coupled C12Laa to the N-terminus of endomorphin1 (Endo-1, Tyr-Pro-Trp-Phe-NH2), and addressed its solubility issue by formulating C12Laa-Endo-1 into phosphatidylcholine liposomes. The aqueous solubility of the lipidic analogue was greatly improved, facilitating the accurate analysis of the compound in in vitro assays. The metabolic stability and in vitro endothelial permeability of the C12Laa-Endo-1 liposomal formulation was assessed using Caco-2 cells, and compared with the formulation of the parent peptide Endo-1. The liposome-encapsulated C12Laa-Endo exhibited significant increases in both stability and permeability. These results suggest that the combination of chemical modification and liposome formulation has great potentials in improving the bioavailability of neuroactive peptides.
Keywords: Liposomes; Lipopeptide; Drug delivery; Endomorphin; Opioid peptide delivery;

Some considerations about the hydrophilic–lipophilic balance system by Ricardo C. Pasquali; Melina P. Taurozzi; Carlos Bregni (44-51).
The methods and results obtained by Griffin et al. in the determination of the hydrophilic–lipophilic balance (HLB) values of non-ionic surfactants and of required HLB values of oil mixtures are reviewed in the present work. HLB values published by Griffin were compared with those obtained by calculations from theoretic chemical formulas. Griffin HLB values of polyoxyethylene alkyl ethers, polyoxyethylene monoesters and propylene glycol monoesters coincide with those obtained from such theoretical chemical formulations. These results demonstrate that, for these surfactants, Griffin did not experimentally obtain their HLB values, but instead calculated them from theoretic formulae. For the calculation of the HLB values of glycerol monostearate, sorbitan fatty acid esters and polyoxyethylene sorbitan fatty acid esters, Griffin's assumptions were possibly based upon the mean saponification values of the ester and the acid of the fatty acid. It is concluded that the HLB values of non-ionic surfactants were not rigorously defined. Moreover, Griffin could not demonstrate the validity of the assumption that individual required HLB values can be added up to obtain the overall required HLB value of an oil mixture. The HLB and required HLB values published by Griffin should only be taken as approximate guidelines.
Keywords: HLB; HLB system; Non-ionic surfactants; Required HLB;

High-amylose sodium carboxymethyl starch (HASCA), produced by spray-drying (SD), was previously shown to have interesting properties as a promising pharmaceutical sustained drug-release tablet excipient for direct compression, including ease of manufacture and high crushing strength.This study describes the effects of some important formulation parameters, such as compression force (CF), tablet weight (TW), drug-loading and electrolyte particle size, on acetaminophen-release performances from sustained drug-release matrix tablets based on HASCA. An interesting linear relationship between TW and release time was observed for a typical formulation of the system consisting of 40% (w/w) acetaminophen as model drug and 27.5% NaCl as model electrolyte dry-mixed with HASCA. Application of the Peppas and Sahlin model gave a better understanding of the mechanisms involved in drug-release from the HASCA matrix system, which is mainly controlled by surface gel layer formation. Indeed, augmenting TW increased the contribution of the diffusion mechanism. CFs ranging from 1 to 2.5 tonnes/cm2 had no significant influence on the release properties of tablets weighing 400 or 600 mg. NaCl particle size did not affect the acetaminophen-release profile.Finally, these results prove that the new SD process developed for HASCA manufacture is suitable for obtaining similar-quality HASCA in terms of release and compression performances.
Keywords: Drug delivery; Sustained release; Excipient; Polymer; Tablet; Matrix; Starch; Amylose; In vitro;

The activity of phenylalanine ammonia lyase (PAL) encapsulated in cellulose nitrate microcapsules is only 23% of the activity of PAL in Tris buffer. This lower activity is partially due to its incomplete encapsulation. The objective of the study was to maximize the encapsulation efficiency (EE) of PAL by optimizing different formulation parameters. PAL was purified using size exclusion chromatography and then radioiodinated using the Iodo-gen reaction. Use of 125I-PAL showed that PAL had an EE of 45% in cellulose nitrate microcapsules. Formulation parameters including concentration of polymer in solution, stirring speed and ratio of aqueous phase volume to organic phase volume were individually optimized to maximize the EE of PAL. The reformulated microcapsules showed an EE of PAL of 80%. The dramatic increase in EE was reflected in a marked (119%) increase in the activity of encapsulated PAL compared to its activity in the original microcapsules. Eighty two percent of the encapsulated PAL was physically present in the aqueous core while 18% was entrapped in the microcapsule membrane. Distribution of PAL activity in the microcapsule was in concordance with its physical distribution.
Keywords: Microcapsules; Phenylalanine ammonia lyase; Phenylketonuria; Encapsulation efficiency; Catalytic activity; Protein formulation;

Cationic lipid-based gene delivery systems have shown promise in transfecting cells in vitro and in vivo. However, liposome/DNA complexes tend to form aggregates after preparation. Lyophilization of these systems, therefore, has become of increasing interest. In this study, we investigated the feasibility of preserving complexes as a dried preparation using a modified dehydration rehydration vesicle (DRV) method as a convenient and reliable procedure. We also studied storage stability of a lyophilized novel cationic gene delivery system incorporating sucrose, isomaltose and isomaltotriose. Liposomes were composed of 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and l-dioleoylphosphatidylethanolamine (DOPE), plus sucrose, isomaltose or isomaltotriose. Lyophilized liposome/DNA complexes were stored at −20, 25, 40 and 50 °C and their stability was followed for 50 days. Liposome/DNA complexes with sucrose could be stored even at 50 °C without large loss of transfection efficiency. The transfection efficiency of formulations stored at various temperatures indicated that the stabilizing effect of sugars on plasmid DNA was higher in the following order: isomaltotriose < isomaltose < sucrose, which was inverse to the order of their glass transition temperature (T g) values. It was concluded that we could prepare novel lyophilized liposome/DNA complexes with high transfection efficiency and stability, which might be concerned that sucrose stabilized plasmid DNA in liposomes by directly interacting with plasmid DNA rather than by vitrifying to a high T g solid.
Keywords: Cationic liposome; Sucrose; Dehydration rehydration vesicle; Transfection; Storage stability; Lyophilization;

Swelling, melting point reduction and solubility of PEG 1500 in supercritical CO2 by Irene Pasquali; Lidia Comi; Francesca Pucciarelli; Ruggero Bettini (76-81).
The knowledge of the solubility of PEG 1500 as well as the swelling and melting point variation in supercritical CO2 in a relatively high-pressure range is a necessary prerequisite to set-up pharmaceutical processes dealing with the polymer in the molten state.Experiments carried out in a pressurized view cell indicated that the PEG 1500 progressively decreases its melting point and increases its volume as a consequence of the absorption of the CO2. The melting point depression was pronounced (from 46 to 28 °C) up to 8.7 MPa. Thereafter a constant value was attained. Analogously, under CO2 the polymer increased its volume (about 34%) until 10 MPa; after this pressure, the polymer volume no longer increased.PEG 1500 showed solubility in SC-CO2 at 35 and 55 °C in the 10–40 MPa range in the order of 10−6  mole fraction. An empirical model based on solubility parameters was used to fit the experimental data and to predict the maximum concentration achievable by the polymer in the dense gas, as well as to quantify the polymer concentration at low pressures where the experimental determination may be extremely difficult.
Keywords: Supercritical CO2; PEG 1500; Solubility; Melting point depression; Polymer swelling; Heuristic model;

The aim of this study was to evaluate the transdermal permeation enhancement of N-trimethyl chitosan (TMC) with different degrees of quaternization (DQ). TMCs with DQ of 40 and 60% (TMC40 and TMC60) were synthesized and characterized by 1H NMR. Testosterone (TS) used as an effective drug, four different gels were prepared without enhancer, with 5% TMC40, 5% TMC60 or 2% Azone, respectively as enhancer. The effect of TMC60 on the stratum corneum was studied by Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) combined with the technique of deconvolution. The results showed that TMC60 could significantly affect the secondary structure of keratin in stratum corneum. In vitro permeation studies were carried out using Franz-diffusion cells and in vivo studies were performed in rabbits. Both in vitro and in vivo permeation studies suggested the transdermal permeation enhancement of TMCs. Compared to the TS gel without enhancer, TS gels with enhancers all showed significant enhancing effect on transdermal permeation of TS (P  < 0.05). Meanwhile, compared to 2% Azone, 5% TMC60 had a stronger enhancement (P  < 0.05) while 5% TMC40 had a similar effect (P  > 0.05). The results suggested that the enhancement of TMCs increased with the increase of DQ.
Keywords: N-Trimethyl chitosan; Transdermal permeation; Enhancement; Testosterone; Gels;

Gastroretentive drug delivery system of DA-6034, a new flavonoid derivative, for the treatment of gastritis by Sun Woo Jang; Jung Woo Lee; Sung Hyun Park; Jeong Hoon Kim; Moohi Yoo; Dong Hee Na; Kang Choon Lee (88-94).
A gastroretentive drug delivery system of DA-6034, a new synthetic flavonoid derivative, for the treatment of gastritis was developed by using effervescent floating matrix system (EFMS). The therapeutic limitations of DA-6034 caused by its low solubility in acidic conditions were overcome by using the EFMS, which was designed to cause tablets to float in gastric fluid and release the drug continuously. The release of DA-6034 from tablets in acidic media was significantly improved by using EFMS, which is attributed to the effect of the solubilizers and the alkalizing agent such as sodium bicarbonate used as gas generating agent. DA-6034 EFMS tablets showed enhanced gastroprotective effects in gastric ulcer-induced beagle dogs, indicating the therapeutic potential of EFMS tablets for the treatment of gastritis.
Keywords: DA-6034; Flavonoid derivative; Gastroretentive drug delivery; Effervescent floating matrix system; Gastroprotective effect;

In situ gelling xyloglucan/pectin formulations for oral sustained drug delivery by Kunihiko Itoh; Masayuki Yahaba; Akie Takahashi; Reina Tsuruya; Shozo Miyazaki; Masatake Dairaku; Mitsuo Togashi; Ryozo Mikami; David Attwood (95-101).
This study has examined the gelation and release characteristics of mixtures of xyloglucan, which has thermally reversible gelation characteristics, and pectin, the gelation of which is ion responsive, with the aim of formulating an in situ gelling vehicle suitable for oral sustained drug delivery. An investigation of the effect of the inclusion of pectin (0.75% (w/w)) on the rheological properties of gels formed from solutions of xyloglucan (1.5 and 2.0% (w/w)) showed a significantly greater gel strength when pectin was present in the formulation. The in vitro release of paracetamol from gels containing 1.5% (w/w) xyloglucan, and 1.5 or 2.0% (w/w) xyloglucan/0.75% (w/w) pectin was diffusion-controlled. Measurement of plasma levels of paracetamol after oral administration to rats of a solution containing 1.5% (w/w) xyloglucan and 0.75% (w/w) pectin showed that a more sustained release and higher drug bioavailability was achieved from the gels formed by the in situ gelation of this formulation compared to that of a 1.5% (w/w) xyloglucan solution; 0.75% (w/w) solutions of pectin did not form gels under these conditions. Visual observation of the contents of the rat stomach at intervals after oral administration showed that the inclusion of pectin in the xyloglucan solutions was effective in reducing gel erosion, so sustaining drug release.
Keywords: Xyloglucan; Pectin; In situ gelation; Sustained release; Oral drug delivery; Paracetamol;

Applicability of DPI formulations for novel neurokinin receptor antagonist by M. Kumon; Y. Yabe; Y. Kasuya; M. Suzuki; A. Kusai; E. Yonemochi; K. Terada (102-109).
A novel triple neurokinin receptor antagonist (TNRA) could have pharmaceutical efficacy for asthma and/or chronic obstructive pulmonary disease. TNRA is potentially developed as inhalation medicine. The aim of this investigation was to evaluate the applicability of dry powder inhaler (DPI) formulation for TNRA. DPI formulation containing lactose was used for this feasibility study. Mechanofusion process for surface modification was applied on lactose particles to prepare four different DPI formulations. The mixture of TNRA and lactose was administered to rats intratracheally using an insufflator. The deposition pattern and blood concentration profile of TNRA were evaluated. Although there was no significant difference in deposition on deep lungs between the four formulations, DPI formulations containing mechanofusion-processed lactose showed longer T max and t 1/2 and higher AUC0–∞ and MRT compared to that containing intact lactose. On the other hand, the contact angle measurement showed that the mechanofusion process decreased the polar part of the surface energy of the lactose. Therefore, the prolongation of the wetting of the formulated powder mixture seemed to delay the dissolution of TNRA deposited in respiratory tract. It was concluded that DPI formulation containing mechanofusion-processed lactose could be suitable for inhalation of TNRA.
Keywords: Triple neurokinin receptor antagonist; Dry powder inhaler; Mechanofusion; Intratracheal administration; Wettability;

Lyophilised wafers are being developed as topical drug delivery systems for the treatment of chronic wounds. This study describes the formulation of xanthan wafers containing a selective, insoluble MMP-3 inhibitor (UK-370,106) and a non-ionic surfactant, designed to release accurate doses of UK-370,106 directly to a suppurating wound bed. Stability of UK-370,106 in the wafer compared to a non-lyophilised gel suspension was investigated using a combination of light scattering, thermal and microscopic techniques. Particle size distributions in UK-370,106-loaded wafers were constant throughout an accelerated stability study (12 weeks, 40 °C) while the mean particle size in a non-lyophilised suspension increased by 15 μm in the same period. Thermal analysis of UK-370,106-loaded wafers highlighted an unexpected interaction between the drug and the surfactant that was further investigated using simple mixtures of each component. It was concluded that an in situ solvate of UK-370,106 and the non-ionic surfactant can form and that this may have implications towards the stability of UK-370,106 during the formulation process. Further concerns regarding high water contents (14%) in the wafer and its effect on product stability were unfounded and it was concluded that these novel delivery systems provided a viable alternative to gel suspensions.
Keywords: Lyophilised wafer; Wound healing; UK-370,106; Surfactant; Stability; Thermal analysis;

We have prepared poly(ethylene oxide) (PEO) tablets which have three-layered structure by direct compression. Carbopol (CP) was coated on both sides of the central PEO matrix which contains solid-dispersed nifedipine (NP) in PEG4000. For comparison, physical mixture of PEO with poly(ethylene glycol 4000) (PEG4000) solid dispersion was also prepared. The differential scanning calorimetry (DSC) thermogram and X-ray diffraction (XRD) pattern obtained after 4 weeks of storage indicated that the crystallinity of PEG4000 in solid dispersion only slightly increased upon aging during this storage period. The formation of crystalline domain of NP, PEO or sodium dodecyl sulfate (SDS) was not observed. CP layers decreased the surface area exposed to dissolution medium, and after swelling, they also covered the exposed side area of the tablet. It seems that swelling and morphological change of CP layers minimize the erosional release for rapidly erodible PEO200K (Mw 200,000) and change the NP release to a diffusion-controlled process. For PEO900K (Mw 900,000), initial release rate was slower than that of PEO200K, possibly due to the slower swelling and erosional release from the side of the tablet. Diffusional release seemed to be the dominating mechanism for the release of NP from PEO7000K (Mw 7,000,000) tablet. Physical mixture of PEO and CP delayed the release of NP remarkably. The increase in pH, ionic strength and buffer concentration of the dissolution medium decreased the release rate. The data obtained for capped and blended tablets were fitted using the power law equation to understand the release mechanism. These results provided some useful information on parameters which can be modulated in the design of a controlled release dosage form for NP.
Keywords: Nifedipine; Carbopol; Three-layered tablet; Controlled release; Swelling;

N-Epoxymethyl-1,8-naphthalimide (ENA) is a novel antiproliferative drug candidate with potent anticancer and antifungal activity. It has an aqueous solubility of 0.0116 mg/mL and also exhibits hydrolytic instability with a first-order hydrolysis rate of 0.051 h−1. The present preformulation study aimed to characterize the physicochemical properties of ENA and develop an early injectable solution formulation for preclinical studies. To minimize hydrolysis, ENA is proposed to be formulated as either lyophilized powders or nonaqueous solutions followed by solubilization/reconstitution prior to administration. ENA solubilization was investigated in both aqueous media (by cosolvency, micellization and complexation) and nonaqueous solutions (mixture of Cremophor EL and ethanol). It is found that none of the solubilization techniques in aqueous media could increase ENA solubility to a desired level of several hundreds μg/mL at pharmaceutically acceptable excipient concentrations (≤10%). In contrast, a combination of 70% Cremophor EL and 30% ethanol (v/v) proved effective in solubilizing ENA at 4 mg/mL, which exhibited good physical and chemical stability on storage at both 4 °C and room temperature over 4 months. No precipitation was observed upon 5–20 times dilution by the saline; in addition, less than 5% of ENA was hydrolyzed in 4 h for the saline-diluted aqueous solutions. This nonaqueous ENA formulation is thus proposed for further preclinical studies, which can be reconstituted, prior to administration, by the 5–20 times infusion fluids (saline, 5% dextrose, etc.) to the desired drug dosing concentration at the acceptable excipient level. The approach used in this work could serve as a useful reference in formulating nonpolar drugs with hydrolytic instability.
Keywords: N-Epoxymethyl-1,8-naphthalimide (ENA); Solubilization; Early formulation; Preformulation; Hydrolytic instability;

Breath-synchronized plume-control inhaler for pulmonary delivery of fluticasone propionate by Stephen B. Shrewsbury; Thomas A. Armer; Stephen P. Newman; Gary Pitcairn (137-143).
A novel breath-synchronized, plume-control inhaler (Tempo™ inhaler) was developed to overcome limitations of a pressurized metered-dose inhaler. This report compared the Tempo inhaler and a commercial inhaler for fine particle distribution and lung deposition of fluticasone propionate. In vitro fine particle distribution was determined using the Andersen Cascade Impactor at inspiration rates of 28.3 and 45 L/min. In vivo lung deposition was assessed in a randomized, two-arm, crossover study of 99mTc-radiolabeled fluticasone propionate in 12 healthy adult subjects, analyzed by gamma scintigraphy. In vitro: fine particle fractions at 28.3 and 45 L/min were 88.6 ± 3.6% and 89.2 ± 3.0% (Tempo inhaler) versus 40.4 ± 4.7% and 43.1 ± 4.4% (commercial inhaler). In vivo: lung deposition was 41.5 ± 9.8% (Tempo inhaler) versus 13.8 ± 7.4% (commercial inhaler) and oropharyngeal deposition was 18.3 ± 7.7% (Tempo inhaler) versus 76.8 ± 7.1% (commercial inhaler). Variability of lung deposition was reduced from 55% (commercial inhaler) to 24% (Tempo inhaler) of the delivered dose. The Tempo inhaler produced significantly higher fine particle fraction values, reduced oropharyngeal deposition by 75%, and increased whole, central, intermediate, and peripheral lung delivery by more than 200%. Thus, the Tempo inhaler enhances efficient drug delivery to the lungs.
Keywords: Aerosol drug delivery; Fluticasone propionate; Breath-synchronized plume-control inhaler; Lung deposition;

Topical photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is an alternative therapy for many non-melanoma skin cancers. The major limitation of this therapy, however, is the low permeability of ALA through the stratum corneum (SC) of the skin. The objective of the present work was to characterize ethosomes containing ALA and to enhance the skin production of protoporphyrin IX (PpIX), compared to traditional liposomes. Results showed that the average particle sizes of the ethosomes were less than those of liposomes. Moreover, the entrapment efficiency of ALA in the ethosome formulations was 8–66% depending on the surfactant added. The particle size of the ethosomes was still approximately <200 nm after 32 days of storage. An in vivo animal study observed the presence of PpIX in the skin by confocal laser scanning microscopy (CLSM). The results indicated that the penetration ability of ethosomes was greater than that of liposomes. The enhancements of all the formulations were ranging from 11- to 15-fold in contrast to that of control (ALA in an aqueous solution) in terms of PpIX intensity. In addition, colorimetry detected no erythema in the irradiated skin. The results demonstrated that the enhancement ratio of ethosome formulations did not significantly differ between the non-irradiated and irradiated groups except for PE/CH/SS, which may have been due to a photobleaching effect of the PDT-irradiation process.
Keywords: 5-Aminolevulinic acid; Liposomes; Ethosomes; Penetration; Photodynamic therapy; Confocal laser scanning microscopy;

This research was conducted to investigate the physico-mechanical characteristics of the EC-based coating membranes plasticized with two informal ingredients of vitamin resources, cholecalciferol and α-tocopherol, with respect to the commercial plasticizer DBS. Proceeding the experiment, free thin polymer sheetings of the sample formulations, incorporating incremental weight percents of the individual plasticizers were prepared employing a revised casting method of delayed solvent evaporation whereby similar flat specimens of standard dimensions were subjected to tensile loadings and extensions. The data were analyzed through the known equations of membrane theory in spherical subjects considering the complete symmetry of assumingly spherical pellets and/or granules. The relative tensile parameters of the experimental and commercial plasticizers in the resilient region were also estimated to fairly decide on a moderate explanation of a strong, hard, and tough structure among the specimens. The results implied the great compatibility of the oily soluble vitamins in EC networks projecting higher factors of safety and greater ultimate strength, toughness, and young coefficient of the formulations compared to the specimens plasticized with the commercial DBS within a concentration range of 40–50% (w/w) of the polymer solids. α-Tocopherol represented supremacy over colecalciferol to result in relatively a 2-fold (and practically a 4-fold with respect to DBS) greater increase in the modulus of resilience. The vitamin compounds and in essential α-tocopherol, in consequence, can properly be applied at concentrations of 40–50% (w/w) as efficient plasticizers to provide a greater protection of the structure against sudden fractures of dynamic and continuously increasing environmental and biological stresses.
Keywords: Ethyl cellulose; Vitamin D3; Vitamin E; Free film; Plasticizer; Membrane theory;

2-(1-Propene)oxyethyl phthalimide (3) was synthesized from reaction of 1-(2-chloroethoxy)propene (2) with potassium-t-butoxide and polymerized by cationic polymerization method using BF3·OEt2 as an initiator at −78 °C to obtain polymer 4. Then, polymer 4 was hydrazinolized by hydrazine and gave polymer 5 containing pendent amine groups. Three non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen, ketoprofen and naproxen were covalently linked to polymer 5 through amide groups by reacting chloroacylated drugs 6a–6c with amine groups of polymer 5 to obtain three polymeric prodrugs 7a–7c. All the synthesized compounds were characterized by FT-IR, 1H and 13C NMR spectroscopy techniques. The polymer–drug conjugates were hydrolyzed in cellophane member dialysis bags containing aqueous buffered solutions (pH 1, 7.4 and 10) at 37 °C and the hydrolysis solutions were detected by UV spectrophotometer at selected intervals. The results showed that the drugs could be released by hydrolysis of the amide bonds. The release profiles indicated that the hydrolytic behavior of polymeric prodrugs strongly depends on the pH of the hydrolysis solution. The results suggested that the vinyl ether type polymers could be the useful carriers for release of profens in controlled release systems.
Keywords: Ibuprofen; Ketoprofen; Naproxen; Vinyl ether polymers; Polymeric prodrugs; Controlled release;

Preparation and characterization of mucinated agarose: A mucin–agarose physical crosslink by Philip F. Builders; Olobayo O. Kunle; Michael U. Adikwu (174-180).
Efficient, biocompatible and biodegradable new polymer materials are continually being sought to meet the challenging needs of drug delivery. Mucinated agarose, a physical crosslink of mucin and agarose, which are both biodegradable natural polymers, has been successfully prepared by a temperature controlled coarcervation technique of aqueous dispersions of equal concentrations of both polymers. Some functional and physicochemical characteristics of the new polymer such as swelling, moisture uptake, mucoadhesive as well as the thermal properties were determined and compared to those of agarose and mucin alone. Turbidimetric interaction between the aqueous dispersions of mucin and agarose was used to determine the concentration ratio of optimum interaction between the two polymers. A concentration ratio mix of four parts mucin and six parts agarose was obtained as the concentration ratio of optimum interaction. A 1:1 dispersion mix was, however, used for the crosslinking process. The mucinated agarose showed characteristic swelling, mucoadhesiveness, moisture uptake and DSC thermal properties that were different from those of mucin and agarose alone. The results indicated that there was formation of a crosslink between mucin and agarose.
Keywords: Mucin; Agarose; Mucinated agarose; Temperature controlled coarcervation; Physical crosslink;

The effects of two vasoactive agents, phenylephrine and tolazoline, were determined on the dermatopharmacokinetics and systemic disposition of model compounds, salicylate (SA) and FITC-dextran 4 kDa (FD-4), following their intracutaneous (i.c.) injection. The determined blood flow in skin was lowered and increased by i.c. injection of phenylephrine and tolazoline, respectively. Dermatopharmacokinetics and the systemic disposition of SA and FD-4 with and without vasoactive agents were analyzed using a compartment model. As a result, the rate constant, k sc, from skin to systemic circulation of SA after i.c. injection with phenylephrine was almost zero, and the rate constant, k sm, from skin to muscle increased about 2.4-fold compared with the control group (without vasoactive agents). In contrast, the rate constants, k sc and k sm, after i.c. injection of SA with tolazoline were increased about 1.9- and 2.5-fold, respectively, compared with the control. In FD-4 disposition, k sc and k sm decreased to about 0.3-fold and increased to about 4.0-fold compared with the control after i.c. injection with phenylephrine. The k sc and k sm of FD-4 increased with tolazoline about 2.2- and 4.3-fold compared with the control, respectively. These data suggest that these vasoactive agents can be used to modify the dermatopharmacokinetics of topically or intracutaneously applied drugs.
Keywords: Vasoactive agent; Intracutaneous injection; Salicylate; FITC-dextran; Dermatopharmacokinetics;

Comparison of single and multiple dose pharmacokinetics between patches and conventional tablets of Huperzine A (Hup-A) was performed in beagle dogs to evaluate the patches’ controlled drug release characteristics in vivo, a newly developed transdermal system for treatment of Alzheimer disease. Results showed that transdermal administration of Hup-A prolonged T max value (24 h vs. 3 h, P  < 0.01), lowered C max value (3.4 ± 0.2 ng mL−1 vs. 9.8 ± 1.0 ng mL−1, P  < 0.01), and produced a relatively constant serum concentration within 84 h after a single transdermal dose of 4 mg/20 cm2 Hup-A patches. Following application of the patches, Hup-A serum concentrations increased for approximately 12–24 h, reaching an average C max of 3.4 ± 0.2 ng mL−1. Thereafter, a serum concentration of at least 2.1 ng mL−1 was maintained for up to 84 h. The serum concentration was maintained within the range of 2.4–4.3 ng mL−1 during 2-week wearing period after multiple dosing, and the degree of fluctuation at the steady state of td and po administration was significantly different (0.51 vs. 1.99, P  < 0.01). This study indicates that Hup-A patches exhibited good controlled-release properties in vivo, maintained a relatively constant serum concentration within 3.5 d after wearing, and are suitable for twice-weekly application.
Keywords: Huperzine A; Dogs; Pharmacokinetics; Transdermal administration;

Crosslinking of drug–alginate granules by D. Mukhopadhyay; D. Saville; I.G. Tucker (193-199).
Paracetamol–alginate (Keltone HVCR) (1:1) granules were prepared by a wet granulation process followed by crosslinking of dried granules in calcium chloride solution. The effect of shear (planetary (low), Brabender (high)), binder quantity (1:2, 1:1 water:powder) and drug particle size (PS 98, 275 μm) were studied using a factorial design. Supporting studies were carried out varying binder quantity and alginate grade (viscosity). In the pre-treated granules, drug entrapment was mainly influenced by drug PS, where the smaller particles showed better embedding. After crosslinking, drug particle size continued to be the most important factor influencing drug recovery. All factors influenced early stage release where high shear, high binder, small drug PS granules showed least release and the low shear, low binder and large drug PS granules showed greatest release. Some significant two-factor interactions were found. Granule consolidation (shown by SEM) and particle embedding increased with binder quantity and reduced as the alginate viscosity was increased. Crosslinking, as shown by Na and Ca contents was >90%. The impact of granule consolidation on drug entrapment and recovery was relatively small (<10%) when compared to its effect on early stage drug release (>60%) which may be important if these granule systems are to be used for taste improvement.
Keywords: Shear rate; Binder quantity; Drug particle size; Alginate viscosity; Granule crosslinking; Granule properties;

Perindopril Erbumine (PER) is one of the newly used angiotensin-converting enzyme inhibitors (ACE inhibitors) and is used for the treatment of patients with hypertension and symptomatic heart failure. It has two main degradation pathways, i.e. the degradation by hydrolysis and the degradation by cyclization. An isothermal heat conduction microcalorimetry (MC) and high pressure liquid chromatography (HPLC) were used for the characterization of aqueous solutions of PER and its stability properties. The rates of heat evolved during degradation of perindopril were measured by MC as a function of temperature and pH and from these data rate constant and change in enthalpy of the reactions were determined. With the HPLC method the concentration of perindopril and its degradation products were measured as a function of time in aqueous solutions of different pH that were stored at different temperatures. We demonstrated that reactions of degradation of perindopril at observed conditions follow the first order kinetics. The Arrhenius equation for each pH was determined. At pH 6.8 only one degradation pathway is present, i.e. the degradation by hydrolysis. Degradation constants for this pathway calculated from MC data are in good agreement with those obtained from HPLC. MC as a non-specific technique was shown to be useful in studies of PER when one reaction was present in the sample and also when more chemical and physical processes were simultaneously running.
Keywords: Isothermal microcalorimetry; Chromatography; Perindopril; Solution stability; Degradation kinetic;

In situ fiber optic method for long-term in vitro release testing of microspheres by Jennifer M. Voisine; Banu S. Zolnik; Diane J. Burgess (206-211).
The objective of this study was to develop an in vitro release method for relatively unstable drugs in long-term modified release (MR) formulations, such as microspheres. Drug stability in the release medium can complicate in vitro release testing of such delivery systems. To overcome this problem, a method has been developed where the model drug, cefazolin, and its degradation products are monitored simultaneously, using UV fiber optic probes, to account for cumulative drug release from poly(lactic-co-glycolic) acid (PLGA) microspheres. United States Pharmacopeia (USP) Apparatus 2 and 4 were used to evaluate cefazolin release throughout the 30-day study period. Cefazolin exhibits an isosbestic point (wavelength where the drug and the degradation products have the same absorbance). Cumulative drug release was compared at the isosbestic (288 nm) point and at the UV max (270 nm). Monitoring at the isosbestic point allowed determination of total drug release with approximately 100% release by day 25. Whereas, at the UV max approximately 61% release was detected by day 25 as a result of drug degradation. Problems were encountered using USP Apparatus 2 with the in situ UV fiber optic probes as a result of microsphere accumulation at and interference with the probe detection window.
Keywords: Cefazolin; Isosbestic point; Modified release; PLGA microspheres; Fiber optic probes;

Carboxylated high amylose starch as pharmaceutical excipients by Louis Philippe Massicotte; Wilms Emmanuel Baille; Mircea Alexandru Mateescu (212-223).
Carboxymethyl high amylose starch (CM-HAS) and succinate high amylose starch (S-HAS) were proposed as pharmaceutical excipients for oral drug delivery, providing a significant gastroprotection to dosage forms of pancreatic enzymes (alpha-amylase, lipase and trypsin) compared to unprotected enzymes. In acidic medium, carboxylic groups are protonated (at least in tablet surface) ensuring local buffering properties and giving a compact shape of the tablets. The enzymes were formulated individually or in association as three enzymes formulation. After the first hour of incubation (over a 2 h experiment) in simulated gastric fluid (SGF), the three pancreatic enzymes retained an overall (average of the three enzymes) activity of 72% when formulated as tablets with CM-HAS excipient and 77% when formulated with S-HAS excipient. Furthermore, after incubation in SGF, the delivery of 75% of the total remaining enzymatic activity in the simulated intestinal fluid (SIF) taken 180 and 170 min for CM-HAS and S-HAS, respectively. Both formulations with carboxylated starch as excipient have a high loading capacity (up to 70–80% enzymes), which is of interest for pancreatic enzymes replacement therapy of pancreatitis. An advantage of these formulations is that gastroprotection is afforded by the carboxylated matrices (carboxylic groups), without enteric coating.
Keywords: Carboxymethyl high amylose starch; Succinate high amylose starch; Alpha-amylase; Lipase; Trypsin; Oral drug delivery;

Validation of a continuous granulation process using a twin-screw extruder by B. Van Melkebeke; C. Vervaet; J.P. Remon (224-230).
Using twin-screw granulation as particle size enlargement technique, the effect of modifying the screw configuration (number of mixing zones, configuration of kneading block) on granule quality, tablet properties and mixing efficiency was investigated. The amount of oversized agglomerates and yield was significantly influenced by the presence of an extra conveying element at the screw end. Changing the staggering angle of the kneading block significantly affected yield and granule friability. The 90° configuration resulted in a lower yield and granule friability. Disintegration time was the only tablet property significantly influenced by the screw configuration as disintegration was significantly faster when an extra conveying element was placed at the screw end. The influence of tracer addition method (wet vs. dry) on mixing efficiency inside the extruder barrel was investigated by means of different tracers: riboflavin (0.05%) suspended in the granulation liquid and hydrochlorothiazide (2.5%) added separately as powder. Mixing efficiency in function of time and granule size (above and below 1400 μm) was tested using riboflavine sodium phosphate (0.05%) dissolved in the granulation liquid. Since a good mixing efficiency was obtained independent of tracer addition method, tracer solubility, granulation time and granule size, continuous granulation using a twin-screw extruder was identified as a robust process.
Keywords: Continuous process; Wet granulation; Twin-screw extruder; Screw configuration; Mixing efficiency;

Pharmacokinetics and pharmacodynamics of exenatide following alternate routes of administration by Bronislava R. Gedulin; Pamela A. Smith; Carolyn M. Jodka; Kim Chen; Sunil Bhavsar; Loretta L. Nielsen; David G. Parkes; Andrew A. Young (231-238).
Exenatide is a 39-amino acid peptide incretin mimetic approved for adjunctive treatment of type 2 diabetes. It shares several glucoregulatory activities with the mammalian hormone, glucagon-like peptide-1 (GLP-1). In clinical use, subcutaneous exenatide injections demonstrate glucoregulatory and weight loss effects with sustained plasma concentrations in the 50–100 pM range. We investigated the pharmacokinetics of exenatide in normoglycemic rats and biological activity in diabetic db/db mice after delivery to various epithelial surfaces of the intestinal and respiratory tracts. In rats, elimination kinetics were similar for all routes of administration (median k e 0.017 min−1). Bioavailability (versus intravenous administration) and C max per unit dose differed markedly. For gastrointestinal administration, sublingual administration invoked the highest bioavailability (0.37%); in db/db mice, potentially therapeutic concentrations were obtainable. In contrast, intraduodenal bioavailability was low (0.0053%). In regard to respiratory surfaces, bioavailability of intratracheal exenatide was up to 13.6%, and for nasal administration, 1.68%. Both routes of administration produced therapeutic plasma concentrations and glucose-lowering in db/db mice. At high doses, aerosolized exenatide also achieved effective concentrations and glucose-lowering. In summary, the intestinal tract seems to have limited potential as a route of exenatide administration, with sublingual being most promising. In contrast, the respiratory tract appears to be more viable, comparing favorably with the clinically approved subcutaneous route. Despite little optimization of the delivery formulation, exenatide bioavailability compared favorable to that of several commercially available bioactive peptides.
Keywords: Exenatide; Exendin-4; Delivery routes; Type 2 diabetes;

Inhaled nanoparticles—A current review by Wei Yang; Jay I. Peters; Robert O. Williams (239-247).
The field of nanotechnology may hold the promise of significant improvements in the health and well being of patients, as well as in manufacturing technologies. The knowledge of this impact of nanomaterials on public health is limited so far. This paper briefly reviews the unique size-controlled properties of nanomaterials, their disposition in the body after inhalation, and the factors influencing the fate of inhaled nanomaterials. The physiology of the lung makes it an ideal target organ for non-invasive local and systemic drug delivery, especially for protein and poorly water-soluble drugs that have low oral bioavailability via oral administration. The potential application of pulmonary drug delivery of nanoparticles to the lungs, specifically in context of published results reported on nanomaterials in environmental epidemiology and toxicology is reviewed in this paper.
Keywords: Nanotechnology; Nanoparticle; Nanotoxicology; Pulmonary delivery; Poorly water-soluble drug; Protein/peptide drug;

The micellization of a model cationic drug, diminazene diaceturate (DIM) and a series of new diblock copolymers, carboxymethyldextran-poly(ethylene glycols) (CMD-PEG), were evaluated as a function of the ionic charge density or degree of substitution (DS) of the carboxymethyldextran block and the molar ratio, [+]/[−], of positive charges provided by the drug to negative charges provided by CMD-PEG. Micelles ([+]/[−] = 2) incorporated up to 64% (w/w) DIM and ranged in hydrodynamic radius (R H) from 36 to 50 nm, depending on the molecular weight and DS of CMD-PEG. The critical association concentration (CAC) was on the order of 15–50 mg/L for CMD-PEG of DS > 60%, and ca. 100 mg/L for CMD-PEG of DS ∼ 30%. The micelles were stable upon storage in solution for up to 2 months and after freeze-drying in the presence of trehalose. They remained intact within the 4 < pH < 11 range and for solutions of pH 5.3, they resisted increases in salinity up to ∼0.4 M NaCl in the case of CMD-PEG of high DS. However, micelles of DIM and a CMD-PEG of low DS (30%) disintegrated in solutions containing more than 0.1 M NaCl, setting a minimum value to the DS of copolymers useful in in vivo applications. Sustained in vitro DIM release was observed for micelles of CMD-PEG of high DS ([+]/[−] = 2).
Keywords: Polyion complex micelles; Dextran; Electrostatic interactions; Polyelectrolytes; Diminazene diaceturate; Hydrophilic drug;

Nanoparticles of quaternized chitosan derivatives as a carrier for colon delivery of insulin: Ex vivo and in vivo studies by Akbar Bayat; Farid A. Dorkoosh; Ahmad Reza Dehpour; Leila Moezi; Bagher Larijani; Hans E. Junginger; Morteza Rafiee-Tehrani (259-266).
The aim of the present study was to develop insulin nanoparticulate systems by using chitosan (CS), triethylchitosan (TEC) and dimethyl-ethylchitosan (DMEC, a new quaternized derivative of chitosan) for colon delivery.The nanoparticles were prepared by the polyelectrolyte complexation (PEC) method. Particle size distribution, zeta potential and polydispersity index of the nanoparticles were determined using dynamic light scattering technique. Transmission electron microscopy (TEM) was also used to observe the morphology of the nanoparticles. It was found that the nanoparticles carried positive charges and showed a size distribution in the range of 170–270 nm with spherical morphology and smooth surface structure. The amount of insulin loaded into the nanoparticles was determined by measuring the association efficiency and also the content of insulin in the nanoparticles. Insulin loading was found to be more than 80% for all of the nanoparticles. In vitro release studies showed a small burst effect at the beginning and then a sustained release characteristic for 5 h. Ex vivo investigations revealed better insulin transport across the colon membrane of rats for nanoparticles made with quaternized derivatives than those made of chitosan. In vivo studies in rats have showed enhanced colon absorption of insulin by using these nanoparticles compared to free insulin in diabetic rats. The insulin absorption from the rat's colon was evaluated by its hypoglycemic effect.
Keywords: Chitosan; Triethyl chitosan; Dimethylethyl chitosan; Insulin; Nanoparticles; Ex vivo and in vivo studies;

Histological examination of PLGA nanospheres for intratracheal drug administration by Kaori Hara; Hiroyuki Tsujimoto; Yusuke Tsukada; C.C. Huang; Yoshiaki Kawashima; Masahiro Tsutsumi (267-273).
Polylactide–glycolide (PLGA) nanospheres were reported as useful pulmonary drug delivery carriers for improving the pharmacological effect of drug. This paper describes the pathological and histological examinations of tissues after intratracheal instillation of drug encapsulated PLGA nanospheres. After intratracheally introducing FITC encapsulated PLGA nanospheres (dispersed in the 0.5 ml saline followed by mixing with an equal volume of air) to a rat, FITC was found existing in the rat's lungs, liver, kidney, brain, spleen and pancreas as demonstrated by immuno-histo-chemical staining with the dye.In this study, FITC stayed in alveoli at least for 1.5 h after the intratracheal administration of the PLGA nanospheres, but the FITC almost disappeared 24 h later.In addition, it was found that the PLGA nanospheres were absorbed in the blood immediately (within 0.25 h after the intratracheal administration) through the type 1 alveolar epithelium cell. Furthermore, the PLGA nanospheres were found resistant to uptake by macrophages such as alveolus macrophages and kupffer cells.The results showed that the possibility to induce tissue damage caused by the excessive immune response from the deposition of PLGA nanospheres was very low, because the nanospheres were not treated as foreign substances.
Keywords: Drug delivery system; PLGA nanosphere; Intratracheal instillation; Immuno-histo-chemical staining;

In order to prolong the in vivo residence of glycyrrhetinic acid, a liposomes with surface modified by methoxy poly(ethyleneglycol) (mPEG)-PLA was prepared for the first time. The liposomes (C-LP) and long-circulating liposomes (LC-LP) were prepared by film-dispersion method using soybean phospholipid/cholesterol mixture (1:0.5 mol/mol), containing 5% (w/w) sodium deoxycholate, and 2% (w/w) mPEG-PLA (only for LC-LP). The diameter of LC-LP was 243.3 ± 17.1 nm. The pharmacokinetics behaviors of the conventional injection and liposomes (C-LP and LC-LP) were compared after a single intravenous injection to rats. Pharmacokinetic parameters were calculated based on a two-compartment open model analysis. LC-CP had a 1.7-fold longer residence time (MRT), a 2.75-fold larger AUC and a 0.4-fold lower clearance compared with conventional injection, respectively. These results combined suggested that the LC-CP had a well-improved residence in rats.
Keywords: Glycyrrhetinic acid; mPEG-PLA; Long-circulating; Physicochemical characteristics; Pharmacokinetics;

Optimization of tocol emulsions for the intravenous delivery of clarithromycin by Jie Li; Shufang Nie; Xingang Yang; Changguang Wang; Shuxia Cui; Weisan Pan (282-290).
In the present study, novel less-painful tocol emulsions for the intravenous delivery of clarithromycin were prepared and optimized. The therapeutically effective concentration of clarithromycin, 5 mg/ml, was achieved using tocopherol succinate (TS) combined with oleic acid as lipophilic counterions. The possibility of employing the microdialysis technique to investigate the distribution of the drug in emulsions was explored. A three-level three-factorial Box–Behnken experimental design was utilized to conduct the experiments. The effects of selected variables, tocopherol succinate/oleic acid relation, poloxamer 188 content and 0.1 M NaOH amount, on three considered responses were investigated. The particle size, ζ potential and the oil phase distribution of clarithromycin for the optimized formulation were observed to be 138.5 nm, −32.16 mV and 97.28%, respectively. The emulsions prepared with the optimized formula demonstrated good physical stability during storage at 4 °C and room temperature. The histopathological examination for rabbit ear vein irritation test indicated that the irritation of clarithromycin could be eliminated by formulating the drug in a tocol emulsion.
Keywords: Clarithromycin; Tocol emulsion; Tocopherol succinate; Microdialysis; Box–Behnken design; Vein irritation;

Stability of luciferase plasmid entrapped in cationic bilayer vesicles by A. Manosroi; K. Thathang; R.G. Werner; R. Schubert; J. Manosroi (291-299).
Characteristics and physical stability of luciferase plasmid (pLuc) entrapped in cationic bilayer vesicles prepared from various molar ratios of amphiphiles (DPPC, Tween61 or Span60), cholesterol (Chol) and cationic charge lipid (DDAB) were investigated. The cationic liposomes were composed of DPPC/Chol/DDAB in the molar ratio of 7:2:1. The cationic (Tween61 or Span60) niosomes were composed of Tween61/Chol/DDAB or Span60/Chol/DDAB in the molar ratio of 1:1:0.05. The maximum loading of pLuc was 15.29, 22.70, and 18.92 μg/mg of the total lipids or surfactants of liposomes, Tween61 and Span60 niosomes, respectively. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM). The particle sizes of the vesicles in nanosize range (160–850 nm) were determined by Photon Correlation Spectroscopy (PCS). Gel electrophoresis and gel documentation were modified to determine the entrapment efficiency of pLuc in cationic bilayer vesicles. The cationic bilayer vesicles gave the pLuc entrapment efficiency of 100%. The pLuc entrapped in cationic liposomes exhibited higher stability than pLuc in solution and pLuc entrapped in cationic Tween61 or Span60 niosomes, when stored at 4, 30 and 50 °C for 8 weeks. After 8 weeks at 4 °C, pLuc contents remained in cationic liposomes was 2 and 3 times higher than cationic Span60 and Tween61 niosomes, respectively. After 3 weeks, 50 and 2% of pLuc was remained in cationic liposomes at 30 and 50 °C respectively, whereas all pLuc in cationic Span 60 and Tween61 niosomes were degraded within 2 and 1 week, respectively. At 30 and 50 °C, pLuc in an aqueous solution or in bilayer vesicular formulations were not in a stable supercoil form. This study has indicated that the stability of pLuc can be enhanced by entrapping in cationic liposomes more than in niosomes. Higher temperature with increase storage time can affect the stability of pLuc even entrapped in bilayer vesicles.
Keywords: Bilayer vesicles; Liposomes; Niosomes; Luciferase plasmid; Stability; Entrapment efficiency;

A liposomal formulation study of 2,7-dichlorodihydrofluorescein for detection of reactive oxygen species by Yasuyuki Sadzuka; Kouhei Nakagawa; Hisashi Yoshioka; Takashi Sonobe (300-305).
It is known that reactive oxygen species (ROS) are connected with various diseases, and many studies have examined redox conditions in the body. However, there is a problem with stability of ROS and selectivity of detection probe. In this study, we aimed to stabilize the detection probe, and have tried to encapsulate the probe in polyethyleneglycol (PEG)-modified liposomes. Dichlorodihydrofluorescein (DCDHF) is oxidized to dichlorofluorescein (DCF), a highly fluorescent product, by ROS. We tried liposomalization of DCDHF probes by the Bangham method. However, it was found that DCDHF was oxidized during preparation and converted to DCF. In contrast, when we performed to encapsulate DCDHF in the liposome after the preparation of empty liposome, the stabilized DCDHF encapsulated liposome was successfully proposed by the addition of DCDHF solution by freeze-drying and re-hydration. Furthermore, the encapsulated efficacy of PEG-modified liposomes was higher than unmodified liposomes. This DCDHF liposome was examined for reactivity with hydroxyl radical and peroxynitrite as highly ROS. It was confirmed that DCDHF liposome had a protective effect on the hydroxyl radical, though an effect of the liposomalization of DCDHF was not shown on reactivity of the peroxynitrite. Therefore, it is likely that the liposomalization of DCDHF has selectivity for certain radical species. It is hoped that these results can be applied to novel and simple diagnostics for redox detection of conditions in the body.
Keywords: Liposome; Reactive oxygen species (ROS); Peroxynitrite (ONOO); Hydroxyl radical (•OH); Dichlorodihydrofluorescein (DCDHF);

Various biomacromolecules (BMs) such as proteins, DNA, and carbohydrates are extremely difficult to be dissolved in a single organic solvent phase for sustained release or targeted delivery formulation. In this study, three different BMs could be solubilized in selected organic solvents by forming poly(ethylene glycol) (PEG)-assisted nano-complexes while maintaining their structural integrity. Dynamic light scattering (DLS) and atomic force microscopy (AFM) analysis revealed that proteins, DNA, and carbohydrate polymers could be nano-complexed with PEG in various organic solvents. The diameter of nano-complexes decreased roughly from ∼600 nm to ∼100 nm with increasing weight ratio of PEG/BM. The present solubilization technique could be potentially applied for sustained release formulations of various therapeutic biological drugs.
Keywords: Biomacromolecules; Bovine serum albumin; DNA; Hyaluronic acid; Poly(ethylene glycol); Solubilization in organic solvents;

Synthesis and evaluation of functional hyperbranched polyether polyols as prospected gene carriers by Leto-Aikaterini Tziveleka; Anna-Maria G. Psarra; Dimitris Tsiourvas; Constantinos M. Paleos (314-324).
Hyperbranched polyether polyols have been partially functionalized with quaternary or tertiary ammonium groups. Five derivatives have been prepared bearing 4, 8 and 12 quaternary or 4 and 21 tertiary ammonium groups. The resulting dendritic polymers interact with plasmid DNA affording the corresponding polyplexes. The complexes were physicochemically characterized while their transfection ability was assessed by gel retardation assay, ethidium bromide exclusion assay and cell culture transfection. All the investigated polymers were shown to have marginal to low cytotoxicity in mammalian cells. Transfection efficiency comparable to that of polyethylenimine was exhibited by selected quaternized polymers. However, the introduction of tertiary amino groups on polyglycerol did not improve the transfection of the ineffective parent polymer, despite the fact that the derivatives obtained exhibited additional buffering capacity (sponge effect). The observed transfection efficiency for the quaternized polymers has been attributed to the destabilization of the lysosomal membrane originating from the interaction between the cationic polymers and the anionic moieties located at the membrane. These results are encouraging for the prospective application of these polyols as gene delivery vectors.
Keywords: Gene delivery; Hyperbranched polyether polyols; Polyplex; Quaternary ammonium group; Tertiary ammonium group; Transfection;

Radiation sterilisation of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles by O. Maksimenko; E. Pavlov; E. Toushov; A. Molin; Y. Stukalov; T. Prudskova; V. Feldman; J. Kreuter; S. Gelperina (325-332).
Doxorubicin-loaded poly(butyl cyanoacrylate) (PBCA) nanoparticles were prepared by anionic polymerisation under non-aseptic conditions. The feasibility of sterilisation of this formulation using either γ-irradiation or electron beam irradiation was investigated. The irradiation doses ranged from 10 to 35 kGy. Bacillus pumilus was used as the official test microorganism. The bioburden of the untreated formulation was found to be 100 CFU/g. Microbiological monitoring revealed that at this level of the bioburden the irradiation dose of 15 kGy was sufficient for sterilisation of the nanoparticles. The formulation showed excellent stability with both types of irradiation in the investigated dose range. The irradiation did not influence the physicochemical parameters of the drug-loaded and empty nanoparticles, such as the mean particle size, polydispersity, and aggregation stability. The molecular weights of the PBCA polymer as well as the polydispersity indices (M w/M n) remained nearly unchanged. The drug substance was stable to radiolysis. Additionally, the presence of irradiation-induced radicals was evaluated by ESR spectroscopy after storage of the particles at ambient temperature. The paramagnetic species found in the formulation were mainly produced by irradiation of mannitol and dextran used as excipients.
Keywords: Irradiation; Sterilisation; Nanoparticles; Poly(butyl cyanoacrylate); Doxorubicin;

Solid lipid nanoparticles for pulmonary delivery of insulin by Jie Liu; Tao Gong; Hualin Fu; Changguang Wang; Xiuli Wang; Qian Chen; Qin Zhang; Qin He; Zhirong Zhang (333-344).
Growing attention has been given to the potential of pulmonary route as an alternative for non-invasive systemic delivery of therapeutic agents. In this study, novel nebulizer-compatible solid lipid nanoparticles (SLNs) for pulmonary drug delivery of insulin were developed by reverse micelle-double emulsion method. The influences of the amount of sodium cholate (SC) and soybean phosphatidylcholine (SPC) on the deposition properties of the nanoparticles were investigated. Under optimal conditions, the entrapment delivery (ED), respirable fraction (RF) and nebulization efficiency (NE) of SLNs could reach 96.53, 82.11 and 63.28%, respectively, and Ins–SLNs remained stable during nebulization. Fasting plasma glucose level was reduced to 39.41% and insulin level was increased to approximately 170 μIU/ml 4 h after pulmonary administration of 20 IU/kg Ins–SLNs. A pharmacological bioavailability of 24.33% and a relative bioavailability of 22.33% were obtained using subcutaneous injection as a reference. Incorporating fluorescent-labelled insulin into SLNs, we found that the SLNs were effectively and homogeneously distributed in the lung alveoli. These findings suggested that SLNs could be used as a potential carrier for pulmonary delivery of insulin by improving both in vitro and in vivo stability as well as prolonging hypoglycemic effect, which inevitably resulted in enhanced bioavailability.
Keywords: Solid lipid nanoparticles; Insulin; Nebulization; Hypoglycemic effect; Bioavailability;

The purpose of this study was to determine optimal lipid concentration range for lyophilization of sterically stabilized phospholipid nanomicelles (SSM) and the freeze drying feasibility of self-associated therapeutic peptide–SSM assemblies. SSM at 5–20 mM 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–N-methoxy-poly(ethylene glycol 2000) (DSPE–PEG2000) were analyzed for particle size and viscosity before and after freeze drying which showed no significant changes (p  > 0.05). However, a steep increase in viscosity was seen for SSM above 15 mM phospholipid implying micelle–micelle interaction. Greater shrinkage of lyophilized cakes was observed below 10 mM phospholipid while they were more fibrous above 15 mM. Therefore, 10–15 mM DSPE–PEG2000 was chosen as the optimal phospholipid concentration for lyophilized SSM. When vasoactive intestinal peptide (VIP), glucagon-like peptide 1 (GLP-1) or gastric inhibitory peptide (GIP) (each, 67 μM) was added to SSM (10 mM), formulations showed no significant change in particle size, peptide fluorescence and peptide α-helicity before and after lyophilization. In conclusion, we found that peptide drug–SSM interactions are conserved during lyophilization.
Keywords: Nanomedicine; Nanobiotechnology; Drug delivery; Nanocarrier; DSPE–PEG2000; VIP; GLP-1; GIP;

Effect of pluronic F68 block copolymer on P-glycoprotein transport and CYP3A4 metabolism by Jiangeng Huang; Luqin Si; Lingli Jiang; Zhaoze Fan; Jun Qiu; Gao Li (351-353).
The aim of this work was to investigate the effects of pluronic F68 block copolymer on the P-gp-mediated transport of celiprolol (CEL) and CYP3A4-mediated formation of midazolam (MDZ) metabolite 1′-hydroxymidazolam. Over a range from 0.03 to 0.3%, pluronic F68 increased apical-to-basolateral permeability (AP-BL) and decreased basolateral-to-apical permeability (BL-AP) of the P-gp substrate CEL in Caco-2 cell monolayer with the efflux ratio values of 2.8 ± 0.3 (0.03%), 2.6 ± 0.3 (0.1%), 2.3 ± 0.2 (0.3%), respectively. CEL transport across the intestinal mucosa in the everted gut sac model was also enhanced by the P-gp inhibitor verapamil and the pharmaceutical excipient pluronic F68. Furthermore, CYP3A4-catalyzed formation of 1′-hydroxymidazolam was inhibited by pluronic F68 with IC50 and K i values of 0.11 and 0.16 mg/ml, respectively. The results indicate that pluronic F68 is a potent in vitro inhibitor of both P-gp and CYP3A4, suggesting a potential for pluronic F68 to modify the pharmacokinetics of orally administered drugs that are P-gp and/or CYP3A4 substrates in vivo.
Keywords: P-glycoprotein; Pluronic F68 block copolymer; CYP3A4; Caco-2; Everted gut sac;

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