European Journal of Pharmaceutics and Biopharmaceutics (v.62, #1)

APV Diary (S1).

by Robert Gurny; Kim Briggs (1).

For many new chemical entities (NCE) of very low solubility oral bioavailability enhancement by micronisation is not sufficient, the next step taken was nanonisation. The production of drug nanocrystals by bottom up techniques (precipitation) is briefly described, main focus is given on particle diminution by high pressure homogenisation. Homogenisation can be performed in water (DissoCubes) or alternatively in non-aqueous media or water-reduced media (Nanopure). There is also a combination process of precipitation followed by a second high energy step, e.g. homogenisation (NANOEDGE). The result is a suspension of drug nanocrystals in a liquid, the so-called nanosuspension. Presented are the physical background of the diminution process, effects of production parameters (power density, number of homogenisation cycles) on crystal size, clinical batch production and scaling up of the production. As an important point the transfer of the liquid nanosuspensions to patient convenient oral dosage forms such as tablets and capsules is described.
Keywords: Drug nanocrystals; DissoCubes; Nanopure; Poorly soluble drugs; Bioavailability; High pressure homogenisation;

Chitosan microparticles containing plasmid DNA as potential oral gene delivery system by Ülker Guliyeva; Filiz Öner; Şule Özsoy; Rıfkı Haziroğlu (17-25).
The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. Chitosan has been shown to effectively bind DNA in saline or acetic acid solution and protect DNA from nuclease degradation. In this study, pDNA (plasmid DNA) was encapsulated in chitosan microparticles. Chitosan–DNA microparticles were prepared using a complex coacervation process and stability of plasmid DNA was investigated in this complex. The chitosan–DNA microparticles could protect the encapsulated plasmid DNA from nuclease degradation. Release of pDNA from microparticles was studied in simulated gastric, simulated intestinal medium and acidic PBS (phosphate buffer saline) (pH 4.5) buffer at 37 °C, and released pDNA was assayed spectrophotometrically. In vitro release of pDNA from chitosan microparticles was dependent on pH, as the pH of the release medium increased release profile decreased. In in vivo-animal studies blue color was observed with X-gal (4-chloro-5-bromo-3-indolyl-β-galactosidase) staining of histological stomach and small intestine sections after oral administration of pDNA–chitosan microparticles as an indicator of exogeneous gene expression.
Keywords: Chitosan; Microparticles; Gene therapy; Oral gene delivery; Plasmid DNA; Beta-galactosidase expression;

Preparation of Tc-99m-macroaggregated albumin from recombinant human albumin for lung perfusion imaging by A.P. Hunt; M. Frier; R.A. Johnson; S. Berezenko; A.C. Perkins (26-31).
Human serum albumin (HSA) extracted from pooled blood taken from human donors is used in the production of 99mTc-labelled macroaggregated albumin (MAA) for lung perfusion imaging. However, concerns for the safety of blood-derived products due to potential contamination by infective agents (e.g. new variant CJD), make alternative production methods necessary. Recombinant DNA technology is a promising method of albumin production avoiding problems associated with human-derived HSA. This paper presents results comparing MAA prepared from recombinant human albumin (rHA, Recombumin®) (rMAA) with in-house produced HSA MAA (hMAA) and commercially available MAA (cMAA). 99mTc-MAA was prepared using previously published production methods by heating a mixture of albumin and stannous chloride in acetate buffer (pH 5.4) at 70 °C for 20 min. Parameters investigated include aggregate size, radiolabelling efficiency, radiochemical and aggregate stability at 4 °C and in vitro (in whole human blood) at 37 °C and biodistribution studies. Results showed that rMAA could be produced with similar morphology, labelling efficiency and stability to hMAA and cMAA. Our findings confirm that rHA shows significant potential as a direct replacement for HSA in commercially available MAA.
Keywords: Serum albumin; Macroaggregates; Recombinant albumin; Lung perfusion; Radiopharmaceutical; Lung imaging; Diagnosis of pulmonary embolism;

Synthesis of a novel galactosylated lipid and its application to the hepatocyte-selective targeting of liposomal doxorubicin by Shao-ning Wang; Yi-hui Deng; Hui Xu; Hong-bing Wu; Ying-kun Qiu; Da-wei Chen (32-38).
This paper described the synthesis of a novel galactosylated lipid with mono-galactoside moiety, (5-Cholesten-3β-yl) 4-oxo-4-[2-(lactobionyl amido) ethylamido] butanoate (CHS-ED-LA), and the targetability of doxorubicin (DOX), a model drug, in liposomes containing 10% mol/mol CHS-ED-LA (galactosylated liposomes, GalL) to the liver was studied. The weighted-average overall drug targeting efficiency (Te *) was used to evaluate the liver targetability of GalL DOX. The results showed that GalL DOX gave a relatively high (Te *)liver value of 64.6%, while DOX in conventional liposome (CL DOX) only gave a (Te *)liver value of 21.8%. In the liver, the GalL DOX was mainly taken up by parenchymal cells (88% of the total hepatic uptake). Moreover, preinjection of asialofetuin significantly inhibited the liver uptake of GalL DOX (from 70 to 12% of the total injected dose). It was suggested that liposomes containing such novel galactosylated lipid, CHS-ED-LA, had a great potential as drug delivery carriers for hepatocyte-selective targeting.
Keywords: Galactosylated lipid; Hepatocyte-selective targeting; Parenchymal cells; Liposomes; Doxorubicin;

Development of a fluorescence-based assay for screening of modulators of human Organic Anion Transporter 1B3 (OATP1B3) by C. Baldes; P. Koenig; D. Neumann; H.-P. Lenhof; O. Kohlbacher; C.-M. Lehr (39-43).
The organic anion transporting protein 1B3 (OATP1B3), formerly termed OATP8, is responsible for uptake and subsequent elimination of multiple amphipathic drugs by the liver. In silico methods for the prediction of transport rates for drugs and drug-like molecules might provide an important tool in drug development. Most prediction methods however require a large training set of in vitro experimental data in order to yield reliable results. To obtain these data, we have developed a fluorescence-based assay that allows screening a relatively high number of substances for their transporter affinity.HEK293 cells overexpressing OATP1B3 (HEK-OATP8) [Y. Cui, J. Konig, D. Keppler, Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2, Mol. Pharmacol. 60 (2001) 934–943.] were tested for transport of Fluo-3. Fluo-3 uptake could be seen in a concentration-dependent manner. Uptake can be inhibited completely by the addition of the known OATP1B3-inhibitor rifampicin proving that Fluo-3 is transported by OATP1B3. To verify the suitability of the system to identify modulators of OATP1B3, we tested known substrates for competitively inhibiting the Fluo-3 transport by giving them simultaneously with a 2 μM Fluo-3-solution to the cells. The transport of Fluo-3 was decreased by all test substrates in a concentration dependent manner.
Keywords: active transport; bioavailability; hepatic uptake; OATP; human; substrate specifity;

Pharmacokinetics of intravenously administered stealth liposomal doxorubicin modulated with verapamil in rats by Jian-Cheng Wang; Xiao-Yan Liu; Wan-Liang Lu; Alex Chang; Qiang Zhang; Boon-Cher Goh; How-Sung Lee (44-51).
Treatment of cancer through co-administration of anticancer drugs and multidrug resistance (MDR) modulators as a strategy to overcome drug resistance has been extensively explored. However, success has been limited by pharmacokinetic interactions because of non-specific blockade of P-glycoprotein (P-gp) in normal tissues or inability to reach relevant concentrations clinically. We hypothesized that stealth liposomal co-encapsulation of doxorubicin (DOX) with a P-glycoprotein inhibitor, verapamil (DARSLs), may overcome these limitations. Using intravenous (i.v.) administrations, the effects of verapamil (VER) either free (FV) or liposome co-encapsulated with DOX (DARSLs) on the pharmacokinetics and tissue distribution characteristics of DOX either as free (FD) or liposome-encapsulated (LD) were evaluated in normal rats. FV increased (P<0.05) the plasma AUC of free DOX (FD). Preparations containing LD had significant prolonged systemic exposure and slow tissue distribution of DOX. LDFV (liposomal DOX with free verapamil) and DARSLs shared similar DOX pharmacokinetics but the latter showed slower DOX distribution in most tissues studied and slower (P<0.05) DOX biliary transport. The addition of VER into LD in these two preparations significantly increased the AUC (P<0.01) and reduced the clearance (P<0.01) of DOX when compared to LD. Specifically, DARSLs reduced initial DOX distribution to the heart (P<0.05) corresponding to initial alleviation (P<0.05) of bradycardia when compared to other DOX with VER preparations. In conclusion, liposomal co-encapsulation of DOX with VER has promise of significant therapeutic advantages, and should be explored further in therapeutic studies with animal tumor xenograft models.
Keywords: Doxorubicin; Verapamil; Stealth liposomes; Pharmacokinetics; Biodistribution;

Enhancement of intestinal absorption of poorly absorbed hydrophilic compounds by simultaneous use of mucolytic agent and non-ionic surfactant by Shinya Takatsuka; Takeo Kitazawa; Takahiro Morita; Yuji Horikiri; Hiroyuki Yoshino (52-58).
The effect of co-administration of a mucolytic agent with a penetration enhancer was assessed on the intestinal absorption of poorly absorbed hydrophilic compounds. Fluorescein isothiocyanate-labeled dextran with average molecular weight of ca. 4.4 kDa (FD-4) was used as a model compound, and N-acetylcysteine (NAC) was used as a mucolytic agent. Sodium caprate (C10), tartaric acid (TA), sodium taurodeoxycholate (TDC), sodium dodecyl sulfate (SDS), p-t-octyl phenol polyoxyethylene-9.5 (Triton X®−100, TX-100) were selected as penetration enhancers with different mechanisms of action. Various dosing solutions containing a penetration enhancer in the absence or in the presence of NAC were directly administered into the exposed rat jejunum, and the bioavailability of FD-4 up to 2 h was determined. The extent of improvement by co-administration was highly dependent on the penetration enhancer species applied. The observed enhancement was thought to result from the mucolytic activity of NAC, which can reduce the mucus viscosity and facilitate the penetration of FD-4 to mucosal membrane. Among the combinations tested, the simultaneous administration of NAC and TX-100 provided the highest enhancement (22.5-fold) of intestinal FD-4 absorption compared to the control. Although the detailed mechanism for the observed drastic improvement is unclear, one possible reason was thought to be due to the improved diffusivity of TX-100 micellar system in the mucus layer. All these results suggest that the combination of a mucolytic agent and a non-ionic surfactant may have potential as an enhancing system for peroral delivery of poorly absorbed hydrophilic compounds like protein and peptide drugs.
Keywords: Mucolytic agent; Penetration enhancer; Intestinal absorption; Poorly absorbed hydrophilic compounds; Non-ionic surfactant;

Ophthalmic delivery systems based on drug–polymer–polymer ionic ternary interaction: In vitro and in vivo characterization by Giuseppina Sandri; Maria Cristina Bonferoni; Patrizia Chetoni; Silvia Rossi; Franca Ferrari; Celestino Ronchi; Carla Caramella (59-69).
Aim of the work was to develop mucoadhesive eyedrops containing tetrahydrozoline hydrochloride (TZ), a decongestant drug, and based on a ternary interaction drug–polymer–polymer. The anionic polymers assessed were the anionic hyaluronic acid (HA) and polyacrylic acid (PAA), the cationic chitosan (HCS) and the polyelectrolyte gelatin (G). Formulations based on the ternary systems TZ/G/HA, TZ/HCS/HA, TZ/G/PAA and TZ/HCS/PAA at the stoichiometry ratios between cationic and anionic polymers and containing a 10 and 20 fold excess of the anionic polymers were prepared. The formulations were characterized for in vitro mucoadhesive and release properties. The ex vivo/in vivo residence properties were assessed for the formulations that combined the better in vitro mucoadhesive and release properties. The physical stability of the formulations selected was determined following steam sterilization and storage at 25 and 40 °C.The synergistic effect of G with HA and PAA improves the mucoadhesion of the formulations while the interaction of HCS with HA and PAA is likely to produce higher neutralization of the anionic polymer charge and minor chain flexibility resulting in a limited mucoadhesion improvement. Both G and HCS participate to control drug release.The selected formulations demonstrate to possess consistency (viscosity) sensitive to the ions of the medium, and probably for this reason the ex vivo/in vivo residence properties could not directly correlated to mucoadhesion and to drug release control properties. However, the formulations are able to maintain levels of TZ detectable until 20 min after the instillation in rabbits, while TZ was not detectable since 3 min after instillation of the drug solution.The physical stability, following steam sterilization and storage, the low viscosity combined with good residence time in conjunctival sac make the TZ/G/20HA the more promising formulation.
Keywords: Tetrahydrozoline hydrochloride; Hyaluronic acid; Polyacrylic acid; Chitosan; Gelatin; Mucoadhesion properties; Drug release properties, Ex vivo/in vivo residence properties; Physical stability; Steam sterilization;

A new commercially available extended release matrix material, Kollidon® SR, composed of polyvinylacetate (PVA) and polyvinylpyrrolidone (PVP), was evaluated with respect to its ability to modulate the in vitro release of the weakly basic drug ZK 811 752. The effect of different formulation and process parameters on the release kinetics of ZK 811 752 from PVA/PVP based matrix tablets was investigated as a function of the (i) nature of excipient added to the drug-polymer mixtures, (ii) method of manufacturing (direct compression versus wet granulation), and (iii) effect of a post-compression curing step. ZK 811 752 containing extended release matrix tablets were successfully prepared by using Kollidon® SR. The drug release from the matrix tablets increased by the addition of excipients such as maize starch, lactose and calcium phosphate. Addition of the highly swellable maize starch and the water-soluble lactose accelerated the drug release in a more pronounced manner compared to the water-insoluble calcium phosphate. Compound release from matrix tablets prepared by wet granulation was faster compared to the drug release from tablets prepared by direct compression. Post compression curing did not influence the drug release rate from drug-lactose-Kollidon® SR formulations. Stability studies demonstrated no degradation of the drug substance and reproducible drug release patterns for matrix tablets stored at 25 °C/60% RH and 30 °C/70% RH for up to 6 months.
Keywords: Weakly basic drug; Controlled release; Polyvinylacetate/polyvinylpyrrolidone; Matrix tablets; Fumaric acid;

Study of formulation variables influencing the drug release rate from matrix tablets by experimental design by Sandra Furlanetto; Marzia Cirri; Francesca Maestrelli; Giovanna Corti; Paola Mura (77-84).
Experimental design was utilized to simultaneously investigate the effect of varying the type of diluent (insoluble Calcium phosphate or water-soluble arabic gum) and the diluent/matrix ratio on the drug release behaviour from both lipophilic (glyceryl behenate, Compritol®) or hydrophilic (hydroxypropylmethylcellulose) matrix tablets. Ketoprofen, theophylline and sodium sulphadiazine were selected as model drugs on the basis of their respectively very low, medium and high water-solubility, in order to evaluate the influence of this parameter as well. The selected response variables were the dissolution efficiency (i.e. the area under the dissolution curve) after one and six hours and the time necessary to dissolve 10% drug. Tablets obtained by direct compression of drug-diluent-matrix ternary mixtures prepared according to the experimental plan provided for by an asymmetric screening matrix, were tested for drug release properties using a USP paddle apparatus. Graphic analysis of the effects allowed identification, for each examined drug, of the formulation factors active on the selected responses and determination of the proper level of the variables to be selected for the response improvement. The different results obtained with the three examined drugs pointed out the role of the drug solubility in determining the influence of formulation parameters on drug release rate from matrix tablets.
Keywords: Experimental design; Asymmetric screening matrix; Matrix tablets; Drug release rate; Formulation optimisation;

Each year millions of people are overexposed to the sun resulting in photodamage of the skin. Secondary photoprotection is the application of medicinal agents to the body after sun exposure to reduce this damage. The objective of this study was to determine the affects of hydroxypropyl-β-cyclodextrin (HPCD) complexation on the secondary photoprotective properties of topically applied ibuprofen. Complexation of ibuprofen by HPCD was demonstrated by differential scanning calorimetry, while solubilities were determined using HPLC. A linear (r 2>0.999) relationship was found between ibuprofen solubility and HPCD concentration. For subsequent experiments, the concentration of ibuprofen was held constant at the solubility in 10% HPCD (10.6 mg/ml), while the HPCD concentration varied from 0 to 20% (w/w). In vitro transdermal permeation experiments demonstrated a parabolic relationship between transdermal kinetic parameters and HPCD concentration, with maximum values for both flux and skin accumulation occurring with the 10% HPCD formulation. In vivo experiments were performed by exposing hairless mice to UV radiation and applying ibuprofen–HPCD formulations topically at various times following UV exposure. Edema and epidermal lipid damage data demonstrated that application of ibuprofen–HPCD formulations within 1 h of UV exposure provided significant photoprotection.
Keywords: Cyclodextrin; Photoprotection; Ultraviolet radiation; Transdermal; Skin accumulation; Ibuprofen; Complexation;

Rheological characterisation of dextran–concanavalin A mixtures as a basis for a self-regulated drug delivery device by M. Joan Taylor; Sangeeta Tanna; Tarsem S. Sahota; Barbara Voermans (94-100).
The rheological characterisation of glucose sensitive mixtures containing dextran and concanavalin A (con A) with and without glucose, was undertaken using oscillatory rheometry at 20 and 37 °C so that comparative data could be gathered in the linear viscoelastic (LVE) range. Measurements for a series of mixtures showed that complex viscosity is a function not only of the con A concentration but of the content and molecular weight of the dextran used. The extent of liquefaction on addition of glucose also depended on these factors. The tan delta profiles confirmed the change from semi-solid towards fluid behaviour. This occurs when glucose effects dismantling of the three-dimensional structure of the dextran–con A system by competitive binding to the glucose receptors in the protein. For the mixtures studied, the changes occurred between contents of 0 and 1% (w/w) glucose at 20 and 37 °C and form a useful basis for the formulation of a self-regulating delivery device for the control of hyper-and hypoglycaemia in diabetes.
Keywords: Insulin delivery; Formulation; Concanavalin A; Viscosity; Glucose; Diabetes; Self-regulating; Dextran;

Microwave vacuum drying is getting more and more popular thanks to its known advantageous and unique features, but its non-uniform electric field can cause nonhomogeneous temperature distribution in the workload. The origin and effect of a generated hot-spot is influenced by the electromagnetic and thermodynamic features of the microwave system and the workload. In the case of single/one pot technology, the geometry and the construction of the microwave cavity is primarily designed for high-shear granulation. As for the workload, its composition has first-order effect on the electric field pattern. The aim of our study is to present a rational decision procedure based on basic practical experiments and the elaborated ‘3D layered thermography’ technique to make it possible to choose the most suitable diluent to formulate a heat sensitive active pharmaceutical ingredient given its stability due to temperature distribution within the workload. Comparing two commonly used diluents, namely microcrystalline cellulose and corn starch, it was found that in the case of different actives with different acceptable temperature limits different diluents are recommended. Drying of a composition consists of an active ingredient characterized by a temperature limit of 70 °C. Using corn starch is safer when the workload is less endangered than when using microcrystalline cellulose. Above this temperature limit microcrystalline cellulose becomes beneficial.
Keywords: High shear granulation; Microwave vacuum drying; Thermography; Temperature distribution; Hot-spot; Heat sensitive;

In the field of the temperature sensitive drug delivery systems, we studied on the surface modification of liposomes by using poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and polyethyleneglycol (PEG) to increase the release of doxorubicin (DOX) from liposomes and prolong the stability of liposomes in the presence of serum. The release of DOX from the PNIPAM-AAM/PEG modified liposomes is enhanced around the transition temperature of the polymer. In addition, the stability of the PNIPAM-AAM/PEG modified liposomes in serum shows a high level comparing with polymer unmodified liposomes. These results suggest that the modification on the surface of liposomes with both PNIPAM-AAM and PEG enhances the drug release from liposomes and reduces the protein adsorption in serum.
Keywords: Protein adsorption; Temperature-sensitive liposome; Poly(N-isopropylacrylamide); Surface modification;

by Richard Süverkrüp (117).