International Journal of Pharmaceutics (v.378, #1-2)

Permeation of astilbin and taxifolin in Caco-2 cell and their effects on the P-gp by Xiao-dan Wang; Min-xin Meng; Ling-bo Gao; Ting Liu; Qiang Xu; Su Zeng (1-8).
This study was designed to understand the transport profiles of astilbin and taxifolin in Caco-2 cell model and their effects on the function and expression of P-glycoprotein. The transport studies were examined using Caco-2 cells cultured on Transwell® inserts. Their effects on the function and expression of P-glycoprotein were detected using Western Blot and RT-PCR. The transport was concentration and temperature dependent. The apparent permeability (P app) of these two compounds in the secretory direction was larger than that in the absorptive direction in the concentration range of 10–1000 μM. Those compounds had no effects on the P-glycoprotein-mediated transport of Rhodamine 123. Caco-2 cells exposed to astilbin or taxifolin for 36 h exhibited higher P-glycoprotein activity through up-regulating P-glycoprotein expression at protein and mRNA levels. These results indicated that P-glycoprotein and Multidrug Resistance Protein 2 might play important roles in limiting the bioavailability of those compounds. Drugs which are the inhibitors of P-glycoprotein or Multidrug Resistance Protein 2 may increase the oral bioavailability of astilbin or taxifolin and the possibility of unwanted drug–food interactions. The increased expression of P-glycoprotein in Caco-2 cells may serve as an adaptation and defense mechanism in limiting the entry of xenobiotics into the body.
Keywords: Astilbin; Taxifolin; P-gp; MRP2; Caco-2 cell; Absorption;

We examined how melatonin absorption was affected by pharmaceutical excipients using the Ussing chamber technique with mounted rat gastrointestinal (GI) segments. Melatonin absorption occurs throughout the GI tract, with the greatest absorption being in the rectum and ileum and the least in the stomach. Melatonin can be classified as a low permeability drug. P-glycoprotein (P-gp) does not affect melatonin absorption but transported rhodamine 123, a well-known P-gp substrate. The possibility of saturating P-gp by melatonin was excluded. Sodium cholate (0.5%) increased melatonin absorption, but decreased absorption at higher concentrations (1.0% and 5.0%). Sodium oleate (0.5% and 1.0%) consistently decreased melatonin absorption. Pharmaceutical excipients increased the absorption of Lucifer yellow (100 μg/mL), a paracellular probe but decreased the absorption of melatonin above the critical micelle concentration (cmc), suggesting that melatonin was transported mainly by transcellular pathway. Sodium cholate and sodium oleate, when above the cmc, resulted in micellar complexes as revealed by 1H NMR spectra and particle size distribution. Histology tests showed mucosal damage of jejunum tissues in the presence of these excipients. The balance of tissue damage by the formation of micellar complexes could affect the melatonin absorption. This information on melatonin absorption behaviors and its modulation by pharmaceutical excipients can be used in further oral dosage formulations to affect circadian rhythm.
Keywords: Melatonin; Absorption mechanism; Effect of pharmaceutical excipients; P-glycoprotein efflux pump; Paracellular probe; Micellar complex; Histological damages;

Molecular states of prednisolone dispersed in folded sheet mesoporous silica (FSM-16) by Akinori Nishiwaki; Aya Watanabe; Kenjirou Higashi; Yuichi Tozuka; Kunikazu Moribe; Keiji Yamamoto (17-22).
Modes of molecular interaction between prednisolone and mesoporous materials have been investigated by the technique of solid-state NMR. Folded sheet mesoporous material (FSM-16) was used as host material and prednisolone was used as guest molecule. A suspension of FSM-16 in prednisolone dichloromethane solution was evaporated to prepare the evaporated samples. 13C NMR spectroscopy was used as well as powder X-ray diffractometry and differential scanning calorimetry. Crystalline behavior of prednisolone disappeared in the evaporated samples, indicating the monomolecular dispersion of prednisolone in FSM-16 matrices. NMR peak shifts and broadening could be attributed to the molecular interaction between the A ring of prednisolone and FSM-16. Thermal properties of prednisolone were investigated after heat treatment of the evaporated samples. The results indicated that the thermal stability of the dispersion made from FSM-16 of large pore size was superior to that from FSM-16 of small pore size. Hydrocortisone was used to compare the dispersion state with prednisolone. It was suggested that the double bond at the C-1 and C-2 positions of prednisolone might play an important role in the process of adsorption of prednisolone to FSM-16.
Keywords: Solid dispersion; Folded sheet mesoporous material; Molecular interaction; Solid-state NMR; Prednisolone;

An innovative floating gastro retentive dosage system: Formulation and in vitro evaluation by C. Sauzet; M. Claeys-Bruno; M. Nicolas; J. Kister; P. Piccerelle; P. Prinderre (23-29).
Over the years, different formulation technologies intended for gastro retentive dosage delivery were investigated and patented. The aim of this study was to develop an innovative floating gastro retentive dosage form (GRDF). The developed technology induces a low-density dosage form containing high active pharmaceutical ingredient (API) concentration by using a hydrophobic dusty powder excipient under specific conditions. The new dosage form was obtained by state of the art wet granulation manufacturing process. An experimental design using a discrete variable and four mixture variables was conducted in order to optimize API concentration and buoyancy of the new dosage form. An apparatus was developed to measure the apparent density of floating tablet. The GRDF was characterized for apparent density, buoyancy, porosity and dissolution using in vitro experimentations.
Keywords: Floating tablet; Gastro retentive dosage form; Experimental design; Hydrophobic dusty powder; Wet granulation;

Development of mucoadhesive microspheres of acyclovir with enhanced bioavailability by Yunying Tao; Yifan Lu; Yinjing Sun; Bing Gu; Weiyue Lu; Jun Pan (30-36).
Acyclovir-loaded mucoadhesive microspheres (ACV-ad-ms) using Ethylcellulose as matrix and Carbopol 974P NF as mucoadhesive polymer were prepared for the purpose of improving the oral bioavailability of acyclovir. The morphological properties of the microspheres were studied by optical microscopy and scanning electron microscopy (SEM). Drug loading and encapsulation efficiency was determined using HPLC method. In vitro and in vivo mucoadhesion of the microspheres was evaluated. Eggshell membrane was found to have a potential use for in vitro mucoadhesion measurement in place of stomach mucosa. In vitro drug release profiles and oral bioavailability of acyclovir in rats were also investigated. The release of the drug was influenced markedly by the medium pH and the proportion of Carbopol incorporated in the microspheres. The result of mucoadhesion study showed prolonged residence time of ACV-ad-ms in rats’ gastrointestinal tract. In pharmacokinetics study, relatively steady plasma drug concentrations were observed within 8 h after oral administration of ACV-ad-ms to rats. The AUC0−t and mean residence time (MRT) of ACV-ad-ms (6055.9 ng h/mL and 7.2 h) were significantly higher than that of ACV suspension (2335.6 ng h/mL and 3.7 h) (P  < 0.05), which indicated that the bioavailability of acyclovir was greatly improved due to the prolonged retention of ACV-ad-ms in gastrointestinal tract.
Keywords: Acyclovir; Mucoadhesive microspheres; Mucoadhesion; Bioavailability;

Audible acoustics in high-shear wet granulation: Application of frequency filtering by Erin M. Hansuld; Lauren Briens; Joe A.B. McCann; Amyn Sayani (37-44).
Previous work has shown analysis of audible acoustic emissions from high-shear wet granulation has potential as a technique for end-point detection. In this research, audible acoustic emissions (AEs) from three different formulations were studied to further develop this technique as a process analytical technology. Condenser microphones were attached to three different locations on a PMA-10 high-shear granulator (air exhaust, bowl and motor) to target different sound sources. Size, flowability and tablet break load data was collected to support formulator end-point ranges and interpretation of AE analysis. Each formulation had a unique total power spectral density (PSD) profile that was sensitive to granule formation and end-point. Analyzing total PSD in 10 Hz segments identified profiles with reduced run variability and distinct maxima and minima suitable for routine granulation monitoring and end-point control. A partial least squares discriminant analysis method was developed to automate selection of key 10 Hz frequency groups using variable importance to projection. The results support use of frequency refinement as a way forward in the development of acoustic emission analysis for granulation monitoring and end-point control.
Keywords: Acoustics; Process analytical technology; Fast Fourier transform; Partial least squares discriminant analysis; High-shear wet granulation; Granulation end-point;

Influence of heat treatment on spray-dried mixtures of Amioca® starch and Carbopol® 974P used as carriers for nasal drug delivery by D. Coucke; E. Pringels; P. Foreman; P. Adriaensens; R. Carleer; J.P. Remon; C. Vervaet (45-50).
A mucoadhesive spray-dried starch/poly(acrylic acid) powder underwent different heat treatments in order to induce cross-linking between the functional groups of starch (Amioca®) and poly(acrylic acid) (Carbopol® 974P). After heat treatment the water-absorbing capacity, viscosity and elasticity of the mucoadhesive powder increased. NMR analysis in combination with FT-IR indicated that heat treatment induced a low degree of cross-linking between the polymers. Nasal administration of Amioca®/Carbopol® 974P powders without heat treatment resulted in an absolute bioavailability in rabbits of 8.2 ± 3.0% for insulin. Due to the difference in water-absorbing capacity (which opened the tight junctions of the nasal mucosa), elasticity and plasticity (which reduced mucociliairy clearance and prolonged residence time) heat treatment at 120 °C improved the bioavailability: 26.4 ± 21.9, 36.5 ± 11.0 and 19.3 ± 17.3% after heat treatment during 30 min, 1 h and 4 h, respectively. Heat treatment at 60 °C was less efficient. This study demonstrated that the nasal insulin absorption improved via heat treatment of the Amioca®/Carbopol® 974P powder (prior to the addition of insulin). The bioavailability-enhancing effect of a 1 h heat treatment at 120 °C was confirmed using the same polymer matrix in combination with different drugs (salmon calcitonin, human growth hormone and metoprolol tartrate).
Keywords: Nasal delivery; Heat treatment; Amioca®/Carbopol® 974P powder formulation; Insulin; NMR spectroscopy;

The aims of the present study were to determine the antibacterial and antifungal activity as well as mutagenicity of Sechium edule fluid extract and to obtain a pharmaceutical formulation with them. The extract exhibited antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Candida spp. and Aspergillus spp. isolated from clinical samples from two hospitals of Tucuman, Argentina. Non-toxicity and mutagenicity on both Salmonella typhimurium TA98 and TA 100 strains until 100 μg/plate were observed. A hydrogel with carbopol acrylic acid polymer containing S. edule fluid extract as antibacterial, antimycotic and antioxidant agent was obtained. Microbiological, physical and functional stability of pharmaceutical formulation conserved at room temperature for 1 year were determined. Addition of antioxidant preservatives to store the pharmaceutical formulation was not necessary. The semisolid system showed antimicrobial activity against all Gram positive and Gram negative bacteria and fungi assayed. The minimal inhibitory concentration (MIC) values ranged from 20 to 800 μg/mL. Its activity was compared with a pharmaceutical formulation containing commercial antibiotic and antifungal. A pseudoplastic behavior and positive thixotropy were observed. Our current finding shows an antimicrobial activity of hydrogel containing S. edule extract on a large range of Gram negative and Gram positive multi-resistant bacteria and fungi. This topical formulation may be used as antimycotic and as antibacterial in cutaneous infections.
Keywords: Antimicrobial; Antioxidant; Hydrogel; Sechium edule; Topical formulation;

The aim of this study was to better understand the importance of coating and curing conditions of moisture-protective polymer coatings. Tablets containing freeze–dried garlic powder were coated with aqueous solutions/dispersions of hydroxypropyl methylcellulose (HPMC), poly(vinyl alcohol), ethyl cellulose and poly(methacrylate-methylmethacrylates). The water content of the tablets during coating and during storage at different temperatures and relative humidities (RH) was determined gravimetrically. In addition, changes in the allicin (active ingredient in garlic powder) content were monitored. During the coating process, the water uptake was below 2.7% and no drug degradation was detectable. Thermally induced drug degradation occurred only at temperatures above the coating temperatures. Different polymer coatings effectively decreased the rate, but not the extent of water uptake during open storage at room temperature and 75% RH. Tablets coated with poly(vinyl alcohol) and poly(methacrylate-methylmethacrylates) showed the lowest moisture uptake rates (0.49 and 0.57%/d, respectively). Curing at elevated temperature after coating did not improve the moisture-protective ability of the polymeric films, but reduced the water content of the tablets. Drug stability was significantly improved with tablets coated with poly(vinyl alcohol) and poly(methacrylate-methylmethacrylates).
Keywords: Moisture protection; Aqueous polymer coatings; Drug stability;

In vitro drug release studies of polymeric freeze-dried wafers and solvent-cast films using paracetamol as a model soluble drug by Joshua S. Boateng; Kerr H. Matthews; Anthony D. Auffret; Mike J. Humphrey; Howard N. Stevens; Gillian M. Eccleston (66-72).
Drug dissolution and release characteristics from freeze-dried wafers and solvent-cast films prepared from sodium carboxymethylcellulose (CMC) have been investigated to determine the mechanisms of drug release from the two systems. The formulations were prepared by freeze-drying (wafers) or drying in air (films), the hydrated gel of the polymer containing paracetamol as a model soluble drug. Scanning electron microscopy (SEM) was used to examine differences between the physical structure of the wafers and films. Dissolution studies were performed using an exchange cell and drug release was measured by UV spectroscopy at 242 nm. The effects of drug loading, polymer content and amount of glycerol (films) on the release characteristics of paracetamol were investigated. The release profiles of paracetamol from the wafers and films were also compared. A digital camera was used to observe the times to complete hydration and dissolution of the wafers containing different amounts of CMC and how that impacts on drug release rates. Both formulations showed sustained type drug release that was modelled by the Korsmeyer–Peppas equation. Changes in the concentration of drug and glycerol (films) did not significantly alter the rate of drug release while increasing polymer content significantly decreased the rate of drug release from both formulations. The results show that the rate of paracetamol release was faster from the wafers than the corresponding films due to differences in their physical structures. The wafers which formed a porous network, hydrated faster than the more dense and continuous, (non-porous) sheet-like structure of the films.
Keywords: Carboxymethylcellulose; Drug dissolution; Release mechanism; Films; Freeze-dried wafers; Paracetamol;

The purpose of this study was to investigate the olfactory transfer of a growth hormone releasing neuropeptide, hexarelin to the brain tissues by comparing brain uptake levels after intranasal administration with those after intravenous administration. The hexarelin nasal formulation was prepared using an aqueous cosolvent vehicle consisting of ethanol, propylene glycol, and n-tridecyl-β-d-maltoside as a permeation enhancer. Hexarelin was administered intravenously or intranasally to male rabbits at a dose of 1 mg/kg. Drug concentrations in the plasma, cerebrospinal fluid and six different regions of the brain, i.e., olfactory bulb (OB), olfactory tract (OT), anterior (CB1), middle (CB2), posterior (CB3) cerebrum, and cerebellum (CL) were analyzed by LC/MS method after solid phase extraction. The brain and cerebrospinal fluid levels achieved following intranasal administration were approximately 1.6 times greater than those attained after intravenous administration despite the intranasal plasma levels being significantly lower than the intravenous plasma levels. Intranasal administration resulted in significantly different spatial distribution patterns in various regions of brain with the rank order of COB  > COT  > CCB1, CB2, CB3  > CCL at 10, 20, and 40 min post-dosing, whereas intravenous administration yielded nearly similar distribution patterns in the brain. The intranasal administration into one nostril (left or right) exhibited markedly greater hexarelin concentrations in olfactory bulb and olfactory tract on the treated-side of brain tissues than those on the non-treated-side of the brain hemisphere. It was demonstrated that the hydrophilic neuropeptide hexarelin was transferred via olfactory pathway to the brain hemispheres and the drug transfer via this route significantly contributed to high brain concentrations after nasal administration to rabbits.
Keywords: Brain uptake; Hexarelin; Nasal drug delivery; Nose–brain transport; Olfactory pathway; Alkylglycoside;

The aim of this work was to clarify the role of Abcb1 and the possible involvement of Abcc2 and Abcg2 in liver, bile and brain disposition of amitriptyline (AMI).AMI was administrated to Abcb1a deficient mice (n  = 36): CF1 (−/−) and CF1 (+/+) mice received via intraperitoneal route (i.p.) 5 mg/kg AMI and CF1 (+/+) mice received i.p. 5 mg/kg AMI + 100 mg/kg quinidine (Abcb1 inhibitor). Then, Swiss mice (n  = 24) received i.p. 5 mg/kg AMI alone and in association with 200 mg/kg novobiocin (Abcg2 inhibitor), 20 mg/kg probenecid (Abcc2 inhibitor) and 100 mg/kg quinidine.Plasma concentrations of AMI were not influenced by novobiocin, probenecid and the lack of Abcb1, but were significantly increased by quinidine, resulting from the inhibition of hydroxylation mediated by CYP2D6. Brain distribution of AMI was not influenced by the lack of Abcb1 but was slightly significant with quinidine and not with novobiocin and probenecid. At the hepato-biliary interface, we showed the involvement of Abcb1, Abcc2 and Abcg2; indeed, AMI concentration was increased in liver and decreased in bile, where quinidine is the strongest inhibitor, followed by probenecid and novobiocin.These results show that in brain the effect of Abcb1, Abcc2 and Abcg2 should be negligible and that at the hepato-biliary level, Abcb1 plays a predominant role compared to Abcc2 and Abcg2.
Keywords: Amitriptyline; Biliary secretion; Brain distribution; Transporters inhibition;

Influence of dialkyne structure on the properties of new click-gels based on hyaluronic acid by Gabriella Testa; Chiara Di Meo; Stefania Nardecchia; Donatella Capitani; Luisa Mannina; Raffaele Lamanna; Andrea Barbetta; Mariella Dentini (86-92).
Hydrogels have been widely used in tissue engineering as a support for tissue formation and/or to deliver drug locally. A novel procedure for the in situ rapid chemical gelation of aqueous solutions of hyaluronan (HA) was employed. HA was functionalised with an arm bearing a terminal azido group (HAAA). When HAAA was mixed with a series of dialkyne reagents of different length, a 1,3-dipolar cycloaddition (“click-chemistry”) reaction took place in the presence of catalytic amount of Cu(I) resulting in fast gelation at room temperature. The resulting gels were characterised in terms of degree of cross-linking by 1H HR-MAS NMR. The kinetic of gelation and the determination of elastic moduli as well as the degree of swelling and the controlled release of a model drug, were studied as a function of chemical nature of the dialkyne group, catalyst concentration, HAAA concentration and temperature. All these variables allowed the swelling ratio and the extent of release of a drug, doxorubicin, entrapped within the gel, to be modulated. In all cases the kinetic of release reached the stationary state within 150 h. The height of the plateau was dependent on the overall (chemical and topological) degree of cross-linking.
Keywords: Hydrogels; Hyaluronan; Click-chemistry; Drug delivery systems;

Molecular charge mediated transport of a 13 kD protein across microporated skin by Sahitya Katikaneni; Advait Badkar; Sandeep Nema; Ajay K. Banga (93-100).
Transport of proteins across the skin is highly limited owing to their hydrophilic nature and large molecular size. This study was conducted to assess the skin transport abilities of a model protein across hairless rat skin during iontophoresis alone and in combination with microneedles as a function of molecular charge. The effect of microneedle pretreatment on electroosmotic flow was also investigated. Skin permeation experiments were carried out in vitro using daniplestim (DP) (MW, 12.76 kD; isoelectric point, 6.2) as a model protein molecule. The effect of molecular charge on protein transport was evaluated by performing studies in two different buffers – TRIS (pH 7.5) and acetate (pH 4.0). Iontophoretic transport mechanisms of DP varied with respect to molecular charge on the protein. The combination approach (iontophoresis and microneedles) gave much higher flux values compared to iontophoresis alone at both pH 4.0 and pH 7.5, however, the delivery in this case was also found to be charge dependent. The findings of this study indicate that electroosmosis persisted upon microporation, thus retaining skin's permselective properties. This enables us to explore the combination of microneedles and iontophoresis as a potential approach for delivery of proteins.
Keywords: Iontophoresis; Electroosmosis; Protein; Soluble microneedles; Molecular charge; Incorrect polarity;

Nortriptyline for smoking cessation: Release and human skin diffusion from patches by A. Melero; T.M. Garrigues; M. Alós; K.H. Kostka; C.M. Lehr; U.F. Schaefer (101-107).
The objective of this work was to develop a simple and inexpensive transdermal formulation containing Nortriptyline Hydrochloride (NTH) for smoking cessation support therapy. Hydroxypropyl-methyl-cellulose was chosen as polymer and a mixture of transdermal enhancers (selected from previous research) was incorporated. The formulations were characterised in terms of appearance, thickness, uniformity of NTH content, release and skin permeation. Release studies demonstrated controlled release for four formulations. Diffusion studies were performed through human heat separated epidermis (HHSE) using Franz Diffusion Cells (FDC). Patches provided different fluxes varying from 20.39 ± 7.09 μg/(cm2  h) to 256.19 ± 94.62 μg/(cm2  h). The penetration profiles of NTH within the stratum corneum (SC) and deeper skin layers (DSL) were established after three administration periods (3 h, 6 h, and 24 h). Skin changes induced by the application of the patches were observed by confocal laser scanning microscopy (CLSM). The highest flux obtained would provide the recommended doses for smoke cessation support therapy (25–75 mg per day) with a 2 cm × 2 cm patch or a 3.5 cm × 3.5 cm patch, respectively, without skin damage evidence.
Keywords: Nortriptyline Hydrochloride; Patch; Transdermal delivery; Release; Smoke cessation; Tape-stripping;

Collagen-based modified membranes for tissue engineering: Influence of type and molecular weight of GAGs on cell proliferation by Barbara Ruozi; Bruna Parma; Maria Antonietta Croce; Giovanni Tosi; Lucia Bondioli; Susanna Vismara; Flavio Forni; Maria Angela Vandelli (108-115).
This study aims to evaluate the effects of the two most widely used glycosaminoglycans (dermatan sulphate and heparin) on both the structural and biological properties of collagen-based modified membranes (COL/GAGs membranes) designed for tissue engineering. The molecular weight of dermatan sulphate and heparins was correlated with the membrane feasibility and the cell (fibroblasts and keratinocytes) ability to adhere and proliferate on the COL/GAG membranes.Microstructure and physico-chemical properties of COL/GAGs membranes were examined using scanning electron microscopy and differential scanning calorimetry; the free amino group content and the swelling properties were also detected. The morphology, proliferation and growth behaviour of keratinocytes and fibroblasts were investigated using microscopical approach and in vitro colorimetric assay. Both fibroblasts and keratinocytes are able to grow and proliferate on COL/dermatan sulphate membranes. Fibroblasts revealed significantly higher proliferation on the membranes prepared with heparin if compared to the proliferation on the membrane without heparin (COL membrane). Particularly, a combination of the membranes formulated adding high molecular weight dermatan sulphate and high molecular weight heparin could be suitable to be used as biomaterials for epidermal substitute.
Keywords: Collagen membranes; Glycosaminoglycans; Tissue engineering; Physico-chemical characterization; Cell adhesion; Cell proliferation;

Levitated single-droplet drying: Case study with itraconazole dried in binary organic solvent mixtures by Eva Wulsten; Filip Kiekens; Frederic van Dycke; Jody Voorspoels; Geoffrey Lee (116-121).
The objective of this case study of the single-droplet drying of two itraconazole/polymer formulations was to determine how the solvent system influences drying rate and dried particle morphology. A clear dual functionality of the two solutes could be identified. The polymeric component (PVP or HPMC) determined drying rate, whereas the drug determined end particle morphology. This could be related to solubilities of the two components in the binary solvent mixtures used. The formulation of a surface skin early on in drying only occurred with HPMC and strongly influenced drying rate but not dried particle morphology.
Keywords: Spray drying; Droplet drying; Particle morphology; Levitation;

This study discusses the effect of key factors like containers, buffers and the freeze (controlled vs. flash freezing) and thawing processes on the stability of a therapeutic protein fibroblast growth factor 20 (FGF-20). The freezing profiles monitored by 15 temperature probes located at different regions in a 2-L bottle during freezing can be grouped into three categories. A rapid drop in temperature was observed at the bottom followed by the top and middle center of the bottle. The freeze–thawing behavior in a 50 ml tube is considerably uniform, as expected. Among phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), citrate and histidine (each containing 0.5 M arginine-sulfate) buffer systems, a minimum pH change (0.4 pH unit vs. ∼1.7 pH unit) was observed for the phosphate buffer system. Thawing in a 50 ml tube at room temperature standing resulted in a significant phase separation in citrate, histidine and HEPES buffers; however, phase separation was least in the phosphate buffer system. These phase separations were found to be temperature dependent. No effect of Polysorbate 80 on freeze–thawing of FGF-20 was observed. Significant concentration gradients in major buffer components and protein concentration were observed during freeze–thawing in a 2-L bottle. The segregation patterns of the various components were similar with the top and bottom layers containing lowest and highest concentrations, respectively. In the formulation buffer no pH gradient was formed, and the precipitation of FGF-20 during thawing at the top layer was related to an insufficient amount of arginine-sulfate and the precipitation at the bottom layer was due to a salting out effect. The precipitate generated during thawing goes into solution easily upon mixing whole solution of the bottle and the various gradient formations do not cause any irreversible change in structure, stability and isoform distribution of FGF-20. Comparison of slow freezing and flash freezing data suggests that the gradients in excipient and protein concentrations are mainly formed during thawing.
Keywords: Fibroblast growth factor; Protein solubility; Protein stability; Folding/unfolding; Aggregation; Arginine; Salting out; Differential scanning calorimetry; Thermodynamics; Fluorescence; Circular dichroism;

Deep eutectic solvent (DES) is a new class of solvents typically formed by mixing choline chloride with hydrogen bond donors such as amines, acids, and alcohols. Most DES's are non-reactive with water, biodegradable, and have acceptable toxicity profiles. Urea–choline chloride and malonic acid–choline chloride eutectic systems were characterized using differential scanning calorimetry (DSC) and thermal microscopy. A potential new 2:1 urea–choline chloride cocrystal with a melting point of 25 °C was characterized at the eutectic composition. The formation of this cocrystal suggests that DES should not be universally explained by simple eutectic melting, and may be useful in guiding the search for new DES systems. The lack of nucleation of the malonic acid–choline chloride system prohibited the construction of a phase diagram for this system using DSC. We also investigated possible uses of DES in solubilizing poorly soluble compounds for enhanced bioavailability in early drug development such as toxicology studies. For five poorly soluble model compounds, solubility in DES is 5 to 22,000 folds more than that in water. Thus, DES can be a promising vehicle for increasing exposure of poorly soluble compounds in preclinical studies.
Keywords: Deep eutectic solvent (DES); Solubilization; Eutectic melting; Urea–choline chloride; Malonic acid–choline chloride;

The kinetic impact of intestinal glucuronidation metabolism on oral bioavailability (F) was assessed using reported human data of raloxifene, of which oral bioavailability was only 2%. Kinetic analysis showed that presystemic intestinal availability (Fpg) was 5.4%, whereas fraction absorbed (Ff) and hepatic availability (Fh) were 63% and 59.3%, respectively. Thus, Fpg was the lowest among factors, which affect oral bioavailability. In addition, Fpg was much lower than Fh, suggesting that intestinal glucuronidation metabolism has a greater impact on oral bioavailability than hepatic glucuronidation metabolism. It has been reported that UDP-glucuronosyltransferase (UGT) 1A1, UGT1A8, UGT1A9, and UGT1A10 are enzymes for raloxifene glucuronidation, and UGT1A8 and UGT1A10 are absent in the human liver, whereas UGT1A1, UGT1A8, UGT1A9, and UGT1A10 are present in the human intestine. Therefore, it is also suggested that intestinal glucuronidation catalyzed by UGTs, particularly UGT1A8 and UGT1A10, may play important roles in the first-pass metabolism, causing low oral bioavailability.
Keywords: Glucuronidation metabolism; Presystemic metabolism; Presystemic intestinal availability; Oral bioavailability;

The purpose of the present study was to investigate the applicability of the classical nucleation theory (CNT) for oral absorption simulation. The CNT was used to simulate the precipitation of low solubility basic compounds in the fasted state simulated intestinal fluid. The infusion–precipitation experiment data reported by Kostewicz et al. [Kostewicz, E.S., Wunderlich, M., Brauns, U., Becker, R., Bock, T., Dressman, J.B., 2004. Predicting the precipitation of poorly soluble weak bases upon entry in the small intestine. J. Pharm. Pharmacol. 56, 43–51] was used for validation. The surface tension of a drug and a pre-exponential factor were obtained by fitting to the experimental data. The CNT adequately simulated the precipitation characteristics of experimental data such as the increase of the precipitation rate and less sensitivity of maximum concentration by the increase of infusion rate.
Keywords: Oral absorption; Simulation; Precipitation; Classical nucleation theory; Basic compound; Bile micelles;

Pharmacokinetic study of a carbamazepine nanoemulsion in beagle dogs by Gislaine Kuminek; Jadel M. Kratz; Rodrigo Ribeiro; Regina G. Kelmann; Bibiana V. de Araújo; Helder F. Teixeira; Cláudia M.O. Simões; Letícia S. Koester (146-148).
This work describes the pharmacokinetics of a novel carbamazepine nanoemulsion. The plasma concentration profiles were determined in beagle dogs after i.v. bolus administration of a 5 mg/kg carbamazepine nanoemulsion and compared to the corresponding carbamazepine/hydroxypropyl-β-cyclodextrin complex solution. Both formulations showed similar pharmacokinetic profiles and could represent valuable formulations in case of emergencies, when a rapid action in the central nervous system is desirable.
Keywords: Carbamazepine; Nanoemulsion; Pharmacokinetics; Hydroxypropyl-β-cyclodextrin;

Stability of triptorelin in the presence of dermis and epidermis by Yogeshwar G. Bachhav; Yogeshvar N. Kalia (149-151).
An important issue with respect to the transdermal delivery of peptides is their stability during transit through the epidermis and dermis before entry into the systemic circulation. The objective of the present study was to evaluate the effect of epidermal and dermal tissue on the stability of the luteinizing hormone releasing hormone superagonist, triptorelin. The decapeptide was dissolved in PBS (pH 7.4) and placed in contact with (i) heat separated epidermis (HSE), (ii) dermatomed skin (0.75 mm; DS) and (iii) full thickness skin (FTS) and the extent of peptide biotransformation monitored as a function of time by HPLC. The results showed that triptorelin was metabolized when in contact with each of the skin tissues. However, there were marked differences with respect to the extent of peptide degradation. Triptorelin was least stable in the presence of FTS. After 3 h exposure to HSE, DS and FTS, the extent of triptorelin degradation was 15.0 ± 6.0%, 64.8 ± 9.9% and 100%, respectively. After 24 h, further triptorelin degradation had occurred in the samples in contact with HSE and DS—with 51.3 ± 6.0% and 87.8 ± 4.4%, respectively, of the peptide being degraded. The chromatograms revealed the presence of a degradation peak at a higher retention time than the parent molecule—most probably the free acid.
Keywords: Transdermal; Peptide; Stability; Triptorelin; Skin;

Microemulsion-based hydrogel formulation of penciclovir for topical delivery by Weiwei Zhu; Chenyu Guo; Aihua Yu; Yan Gao; Fengliang Cao; GuangXi Zhai (152-158).
The purpose of this study was to investigate microemulsion-based hydrogel (MBH) as a topical delivery system for penciclovir. Topical delivery of penciclovir in the forms of microemulsion, MBH and the commercial cream was evaluated in vitro and in vivo. The results of permeation test in vivo in mice showed that compared with the commercial cream, MBH and microemulsion could significantly increase the permeation of penciclovir into both epidermis and dermis. Stability test showed that MBH stored at 4 °C for 3 months had no significant change in physicochemical properties. Skin irritation test in rabbit demonstrated that single application or multiple applications of MBH did not cause any erythema or edema, slight skin irritation for microemulsion. Microstructure changes of skins after administration observed under light microscope and scanning electron microscope (SEM) might result from the interaction of the ingredients of microemulsion with skins, which was related with the permeation enhancement of penciclovir. It can be concluded that the MBH could be a promising vehicle for topical delivery of penciclovir.
Keywords: Penciclovir; Microemulsion; Hydrogel; Topical delivery; Microemulsion-based hydrogel;

Insulin has been encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres by solid-in-oil-in-oil (S/O/O) emulsion technique using DMF/corn oil as new solvent pairs. To get better encapsulation efficiency, insulin nanoparticles were prepared by the modified isoelectric point precipitation method so that it had good dispersion in the inner oil phase. The resulting microspheres had drug loading of 10% (w/w), while the encapsulation efficiency could be up to 90–100%. And the insulin release from the microspheres could last for 60 days. Microspheres encapsulated original insulin with the same method had lower encapsulation efficiency, and shorter release period. Laser scanning confocal microscopy indicated the insulin nanoparticle and original insulin had different distribution in microspheres. The results suggested that using insulin nanoparticle was better than original insulin for microsphere preparation by S/O/O method. Study about the secondary structure of insulin by Fourier transform infrared spectroscopy (FTIR) indicated high insulin structural integrity during the process. In vivo test showed insulin in microspheres retained its bioactivity. In addition, cytotoxicity evaluation by the MTT assay has proved that no extra toxicity was introduced into the microspheres during the emulsion process.
Keywords: Insulin nanoparticle; S/O/O emulsion; Microsphere; Protein delivery;

There are two generics of a parenteral lipid emulsion of prostaglandin E1 (PGE1) (Lipo-PGE1) in addition to two innovators. It was reported the change from innovator to generic in clinical practice caused the slowing of drip rate and formation of aggregates in the infusion line. Thus, we investigated the difference of pharmaceutical quality in these Lipo-PGE1 formulations. After mixing with some infusion solutions, the mean diameter and number of large particles were determined. Although the mean diameter did not change in any infusion solutions, the number of large particles (diameter >1.0 μm) dramatically increased in generics with Hartmann's solution pH 8 or Lactec® injection with 7% sodium bicarbonate. Next, we investigated the effect of these infusion solutions on the retention rate of PGE1 in lipid particles. The retention rate of PGE1 in these two infusion solutions decreased more quickly than that in normal saline. Nevertheless, there were no significant differences among the formulations tested. Our results suggest that there is no difference between innovators and generics except in mixing with these infusion solutions. Furthermore, that monitoring the number of large particles can be an effective means of evaluating pharmaceutical interactions and/or the stability of lipid emulsions.
Keywords: Emulsion; Prostaglandin E1; Lipid particle; Release; Generics;

Polyhydroxyethylaspartamide-based micelles for ocular drug delivery by C. Civiale; M. Licciardi; G. Cavallaro; G. Giammona; M.G. Mazzone (177-186).
In this paper three copolymers of polyhydroxyethylaspartamide (PHEA), bearing in the side chains polyethylene glycol (PEG) and/or hexadecylamine (C16) (PHEA-PEG, PHEA-PEG-C16 and PHEA-C16 respectively) have been studied as potential colloidal drug carriers for ocular drug delivery. The physical characterization of all three PHEA derivatives, using the Langmuir trough (LT) and micellar affinity capillary electrophoresis (MACE) techniques allowed to assume that whereas alone PHEA backbone is an inert polymer with respect to the interactions with lipid membranes and drug complexation, when PHEA chains are grafted with long alkyl chains like C16 or in combination C16 chains and hydrophilic chains like PEG, copolymers with lipid membrane interaction ability and drug complexation capability are obtained.In vitro permeability studies performed on primary cultured rabbit conjunctival and corneal epithelia cells, using PHEA-C16 and PHEA-PEG-C16 as micelle carriers for netilmicin sulphate, dexamethasone alcohol and dexamethasone phosphate, demonstrated that in all cases drug loaded PHEA-C16 and PHEA-PEG-C16 micelles provide a drug permeation across ocular epithelia greater than simple drug solutions or suspensions. In particular PHEA-PEG-C16 acts as the best permeability enhancer in our experimental model. In vivo bioavailability studies conducted with PHEA-PEG-C16 micelles loaded with dexamethasone alcohol, confirmed that this system also provides a drug bioavailability greater in comparison with that obtained with water suspension of the same drug after ocular administration to rabbits.
Keywords: Ocular drug delivery systems; Polymeric micelles; Alpha,beta-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA); Amphiphilic copolymers;

A simple chemometric approach to differentiate among the three crystalline polymorphs of the model drug Furosemide (FUR) in a pharmaceutical dosage form is presented. The proposed method is based on the principal component analysis with confidence regions (PCA-CR) comparison of the dissolution profiles of the test pharmaceutical formulation, and formulations containing the different polymorphs, employed as the corresponding references.For the elaboration of the references, FUR polymorphs I, II and III were prepared, characterized and compounded with the excipients found in the test commercial formulation. The dissolutions were carried out in a discriminating HCl–KCl dissolution medium (pH 2.2), and the corresponding profiles were constructed from the absorbances (274 nm) of the dissolution samples.PCA-CR was able to differentiate among the three crystalline polymorphs of FUR and to confirm the presence of polymorph I in the test sample, with 99% statistical confidence. The PCA-CR results were compared with those obtained by a bootstrap-mediated implementation of Moore and Flanner's difference factor (f 2). The same conclusion was reached employing an f 2-based comparison, despite its inability to differentiate between polymorphs II and III. Therefore, PCA-CR may be considered a complementary and useful tool for probing the polymorphic form present in a pharmaceutical formulation.
Keywords: PCA-CR; Crystal polymorphism; Chemometric method; Furosemide;

Chitosan-graft-polyethylenimine for Akt1 siRNA delivery to lung cancer cells by Dhananjay Jere; Hu-Lin Jiang; You-Kyoung Kim; Rohidas Arote; Yun-Jaie Choi; Cheol-Heui Yun; Myung-Haing Cho; Chong-Su Cho (194-200).
Efficient delivery of small interfering RNA (siRNA) remains a challenging task in RNA interference (RNAi) studies. In this study, we used chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer composed of chitosan and low molecular weight polyethylenimine (PEI) for the delivery of siRNA. The CHI-g-PEI carrier formed stable complexes with siRNA with compact spherical morphology. CHI-g-PEI delivered EGFP siRNA (siGFP) silenced EGFP expression nearly 2.5 folds higher than PEI25K at 50 pM siGFP concentration. Cell viability was found to be 2 folds high with CHI-g-PEI carrier than PEI25K. Also, our CHI-g-PEI carrier efficiently delivered Akt1 siRNA (siAkt) and thereby silenced onco-protein Akt1. Silencing of this crucial cell survival protein significantly reduced the lung cancer cell survival and proliferation. Additionally, Akt1 protein knock-down decreased A549 cell malignancy and metastasis. These findings suggest that the CHI-g-PEI carrier efficiently and safely delivered siRNA. Moreover, CHI-g-PEI mediated Akt1 siRNA delivery may immerge as a viable approach for lung cancer treatment.
Keywords: Polymeric carrier; Non-viral vector; SiRNA delivery; Akt; Chitosan; Polyethylenimine; Lung cancer;

Effect of acyl chain length on transfection efficiency and toxicity of polyethylenimine by Latha Aravindan; Katrina A. Bicknell; Gavin Brooks; Vitaliy V. Khutoryanskiy; Adrian C. Williams (201-210).
Polyethylenimine (PEI) is an efficient nonviral gene delivery vector because of its high buffering capacity and DNA condensation ability. In our study, the amino groups on the polymeric backbone were acylated using acetic or propionic anhydride to alter the protonation behaviour and the hydrophilic/hydrophobic balance of the polymer. The concentration of acylated primary amines was determined using trinitrobenzene sulphonic acid assay. Results showed that our modified polymers had lower buffering capacities in solutions compared to PEI. The polymers were complexed with plasmid encoding enhanced green fluorescent protein at three different ratios (1:1, 1:2 and 1:10 w/w DNA to polymer) to form polyplexes and their toxicities and transfection efficiencies were evaluated in HEK 293 cells. Acylation reduced the number of primary amines on the polymer and the surface charge, improving haemocompatibility and reducing cytotoxicity. The reduction in the concentration of amino groups helped to optimise DNA compaction and facilitated polyplex dissociation in the cell, which increased transfection efficiency of the modified polymers compared to the parent polymer. Polymers with buffering capacities greater than 50% and less than 80% relative to PEI, showed higher transfection efficiencies than PEI. The propionic anhydride modified polymers had appropriate interactions with DNA which provided both DNA compaction and polyplex dissociation. These systems interacted better with the cell membrane because of their slightly higher lipophilicity and formed polyplexes which were less cytotoxic than polyplexes of acetic anhydride modified polymers. Among the vectors tested, 1:0.3 mol/mol PEI:propionic anhydride in a 1:2 w/w DNA:polymer composition provided the best transfection system with improved transfection efficiency and reduced cytotoxicity.
Keywords: Polyethylenimine; Buffering capacity; Nonviral vectors; Haemolysis; Transfection; Biocompatibility;

Solubilisation of griseofulvin, quercetin and rutin in micellar formulations of triblock copolymers E62P39E62 and E137S18E137 by Maria Elenir N.P. Ribeiro; Ícaro G.P. Vieira; Igor Marques Cavalcante; Nágila M.P.S. Ricardo; David Attwood; Stephen G. Yeates; Colin Booth (211-214).
The solubilisation of two poorly soluble flavonoids, quercetin and rutin, in micellar solutions of mixtures of a block copolymer of ethylene oxide and styrene oxide (E137S18E137) with one of ethylene oxide and propylene oxide (E62P39E62) has been studied at 25 and 37 °C. Solubilisation capacities were higher than those for the model poorly water-soluble drug griseofulvin and comparable with published values for the solubilisation of rutin by β-cyclodextrin.
Keywords: Flavonoids; Rutin; Quercetin; Block copolymer micelles; Drug solubilisation;

Ultrasound-mediated oxygen delivery from chitosan nanobubbles by Roberta Cavalli; Agnese Bisazza; Alessandro Rolfo; Sonia Balbis; Daniele Madonnaripa; Isabella Caniggia; Caterina Guiot (215-217).
Ultrasound (US) energy combined with gas-filled microbubbles has been used for several years in medical imaging. This study investigated the ability of oxygen-loaded chitosan bubbles to exchange oxygen in the presence or in the absence of US. Oxygen delivery is enhanced by sonication and both frequency and time duration of US affected the exchange kinetics.
Keywords: Microbubbles; Oxygen; Ultrasound; Chitosan; Hypoxia;

Noticeboard (218).