European Journal of Pharmaceutics and Biopharmaceutics (v.64, #3)

APV Dairy (S1-S2).

The objective of this study is to investigate the solubilization of poorly water-soluble anticancer drugs, octaethylporphine (OEP), meso-tetraphenyl porphine (mTPP) and camptothecin (CPT), in Pluronic and polyethylene glycol–distearoylphosphatidylethanolamine (PEG–DSPE) polymeric micelles. Three different Pluronic and PEG–DSPE polymers with various chain lengths were chosen and micelle formulations were prepared by using various drug:polymer ratios. Formulations were characterized by critical micellization concentration (CMC) values of copolymers, micelle particle size and distribution, zeta potential, loading efficiency and stability. Polymers formed very stable, low CMC micelles with smaller sizes than 100 nm. It was shown that drug loading efficiency highly depends on the polymer type, drug type and their ratios. The most efficient drug loading was obtained by loading mTPP in PEG2000–DSPE and Pluronic F127 micelles. This result is attributed to phenyl groups in mTPP might lead to attraction between alkyl groups in the polymer and increase drug incorporation. PEG–DSPE formulations had higher zeta potential values indicating that they would be more stable against aggregation than Pluronic micelles. From the drug assay aspect Pluronic micelles remained more stable in 3-month long stability test. These results showed that besides their solubilizing effects, polymeric micelles could be useful as novel drug carriers for hydrophobic drugs.
Keywords: Polymeric micelles; Anticancer drugs; Pluronics; Polyethylene glycol–distearoylphosphatidylethanolamine (PEG–DSPE); Critical micellization concentration (CMC);

Incorporation of camptothecin into N-phthaloyl chitosan-g-mPEG self-assembly micellar system by Praneet Opanasopit; Tanasait Ngawhirunpat; Amornrut Chaidedgumjorn; Theerasak Rojanarata; Auayporn Apirakaramwong; Sasiprapha Phongying; Chantiga Choochottiros; Suwabun Chirachanchai (269-276).
The capability of N-phthaloylchitosan-grafted poly (ethylene glycol) methyl ether (mPEG)(PLC-g-mPEG) to enhance the aqueous solubility and stability of the lactone form of camptothecin (CPT) was investigated. PLC-g-mPEG formed a core-shell micellar structure after dialysis of the polymer solutions in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) against water, with a critical micelle concentration (CMC) of 28 μg/ml. CPT was loaded into the inner core of the micelles by dialysis method. The results showed an increase in the CPT-loading amount with an increasing concentration of CPT. The stability of drug-loaded micelles was studied by gel-permeation chromatography (GPC), and their in vitro release behaviors were analyzed. Release of CPT from the micelles was sustained. When compared to the unprotected CPT, CPT-loaded PLC-g-mPEG micelles were able to prevent the hydrolysis of the lactone group of the drug. The kinetics of the CPT hydrolysis in human serum albumin (HSA) and fetal bovine serum (FBS) were pseudo-first order. The hydrolysis rate constants for CPT and CPT-loaded PLC-g-mPEG micelles in phosphate-buffered saline (PBS) pH 7.4, were 7.4 × 10−3  min−1 and 9.1 × 10−3  h−1, parallel to an increase in half-life of CPT from 94 min to 76.15 h, respectively.
Keywords: Polymeric micelles; Anticancer agent; Camptothecin; N-Phthaloylchitosan;

Pharmaceutical grade d,l-lactic acid, which is rather an economic source in comparison to lactide monomer, was utilized for synthesis of a series of copolymers with sebacic acid. Polymers were characterized by GPC, FTIR, NMR and DSC techniques, and formulated into blank and methotrexate (MTX) loaded microspheres by emulsion-solvent evaporation method. In vitro degradation of blank microspheres was studied by FTIR, GPC and SEM analysis. MTX loaded microspheres showed the encapsulation efficiency of 44–64% and were in the size range of 40–60 μm. These were used to study the release profile of the encapsulated drug. The release was found to be affected by the pH and buffer concentration of the release medium which was in turn revealed by solubility studies of MTX. The overall study demonstrates significance of drug as well as polymer properties on release. Biocompatibility of polymer was evaluated by injecting microspheres subcutaneously into Sprague–Dawley (SD) rat and no local histopathological abnormalities were found.
Keywords: PLA–PSA; Microspheres; Biodegradable polymers; Drug delivery; Biocompatibility; Drug release kinetics; Profile modeling;

Effect of drug type on the degradation rate of PLGA matrices by Steven J. Siegel; Jonathan B. Kahn; Kayla Metzger; Karen I. Winey; Kathryn Werner; Nily Dan (287-293).
We compare the rate of drug release through the degradation of 50:50 polylactic-co-glycolic acid polymer pellets, for six different drugs: Thiothixene, Haloperidol, Hydrochlorothiozide, Corticosterone, Ibuprofen, and Aspirin. Despite using the same polymer matrix and drug loading (20% by weight), we find that the rate of polymer degradation and the drug release profile differ significantly between the drugs. We conclude that the design of biodegradable polymeric drug carriers with high drug loadings must account for the effect of the drug on the polymer degradation and drug release rate.
Keywords: Controlled release; PLGA; Solubility; Diffusion-reaction; Kinetics;

There is an increasing interest in lipid based drug delivery systems due to factors such as better characterization of lipidic excipients and formulation versatility and the choice of different drug delivery systems. It is important to know the thermal characteristics, crystal habit, texture, and appearance of a new lipid matrix when determining its suitability for use in certain pharmaceutical application. It is line with this that this research was embarked upon to characterize mixtures of beeswax and theobroma oil with a view to applying their admixtures in drug delivery systems such as solid lipid nanoparticles and nanostructured lipid carriers. Admixtures of theobroma oil and beeswax were prepared to contain 25% w/w, 50% w/w, and 75% w/w of theobroma oil. The admixtures were analyzed by differential scanning calorimetry (DSC), small angle X-ray diffraction (SAXD), wide angle X-ray diffraction (WAXD), and isothermal heat conduction microcalorimetry (IMC). The melting behavior and microstructures of the lipid admixtures were monitored by polarized light microscopy (PLM). Transmission electron microscopy (TEM) was used to study the internal structures of the lipid bases. DSC traces indicated that the higher melting peaks were roughly constant for the different admixtures, but lower melting peaks significantly increased (p  < 0.05). The admixture containing 25% w/w of theobroma oil possessed highest crystallinity index of 95.6%. WAXD studies indicated different reflections for the different lipid matrices. However, new interferences were detected for all the lipid matrix admixtures between 2θ  = 22.0° and 2θ  = 25.0°. The lipid matrices containing 50% w/w and 25% w/w of theobroma oil showed absence of the weak reflection characteristic of pure theobroma oil, while there was disappearance of the strong intensity reflection of beeswax in all the lipid matrix admixtures at all stages of the study. PLM micrographs revealed differences with regard to the thermal and optical behaviors depending on the composition of the matrix. The lipid matrix consisting of 75% w/w of theobroma oil showed a spherulite texture after 4 weeks of isothermal storage. Crystallization exotherms of lipid matrices containing 50% w/w and 25% w/w of theobroma oil showed change in modification after 30 min with the latter having a greater time-dependent crystallization. Generally, low non-integral Avrami exponents and growth rate constants were obtained for all the lipid matrices, with the admixture containing 25% w/w theobroma oil having the lowest Avrami exponent and growth rate constant. Based on the results obtained, admixtures containing 50% w/w and 75% w/w of theobroma oil could be applied in the formulation of solid lipid nanoparticles and nanostructured lipid carriers as these lipid matrices possessed crystal characteristics that favour such drug delivery systems.
Keywords: Theobroma oil; Beeswax; Crystallinity; Crystallization kinetics; Differential scanning calorimetry; X-ray diffraction; Polarized light microscopy; Isothermal heat conduction microcalorimetry; Drug delivery;

Dynamic moisture sorption and desorption of standard and silicified microcrystalline cellulose by K. Kachrimanis; M.F. Noisternig; U.J. Griesser; S. Malamataris (307-315).
Moisture sorption and desorption isotherms of standard and silicified microcrystalline cellulose (MCC and SMCC) were determined using an automatic multi-sample gravimetric analyzer, and compared by fitting different kinetic models, including the excess surface work model (ESW), the BET and GAB model, Young and Nelson model and recently developed parallel exponential kinetics (PEK) model. It was found that silicification affects the moisture sorption and desorption properties of SMCC mainly at high relative humidity (above 50% and 70%, respectively). In general, the differences in the moisture sorption and desorption properties of MCC and SMCC can be elucidated by the different kinetic models. Particularly the PEK model shows that hysteresis is related primarily to a fast sorption process, which corresponds to bound water, and secondarily to a slow process, which corresponds to sorption of free water and that SMCC acquires more water than MCC at RH higher than 50% by the slow (secondary) sorption process. A possible mechanism for this process is presumably the hydrolysis of SiO2 particles and formation of silanol groups that act as a water reservoir, preventing the accumulation of more water in the polymer matrix and thus may be protecting the structure of SMCC from undergoing irreversible structural changes that would impair its performance as an excipient.
Keywords: Moisture sorption; Isotherms; Microcrystalline cellulose; Silicified microcrystalline cellulose; Parallel exponential kinetics;

The goal was to investigate the impact of NaCl on the physico-chemical properties of mannitol–sucrose formulations during freezing and drying, with special focus on mannitol hydrate formation. Differential scanning calorimetry (DSC) and low-temperature X-ray powder diffraction (LTXRD) were used to study the frozen solutions. After lyophilization the products were analyzed with DSC, temperature-modulated DSC (TMDSC), X-ray powder diffraction (XRD) and Karl-Fischer titration. DSC showed an inhibition of mannitol crystallization by sucrose and NaCl during freezing. The glass transitions of the maximally freeze-concentrated solutions (Tg′) were lowered by both mannitol and NaCl. By the application of an annealing step during lyophilization mannitol crystallinity could be increased. However, lyophilization with an annealing step promoted the formation of mannitol hydrate, which is known to undergo conversion into the anhydrous polymorphs of mannitol upon storage. LTXRD revealed that mannitol hydrate was formed at temperatures below −30 °C, but not at −27 °C. The tendency that mannitol hydrate is predominantly formed at lower temperature was confirmed by XRD of lyophilized products, produced at different annealing temperatures. For the development of lyophilization cycles the lowered Tg′, as well as the tendency to mannitol hydrate formation predominantly at lower temperature needs to be considered.
Keywords: Freezing; Lyophilization; Annealing; NaCl; Mannitol hydrate;

Visualizing the conversion of carbamazepine in aqueous suspension with and without the presence of excipients: A single crystal study using SEM and Raman microscopy by F. Tian; N. Sandler; K.C. Gordon; C.M. McGoverin; A. Reay; C.J. Strachan; D.J. Saville; T. Rades (326-335).
Visual observations of the hydration process of single carbamazepine (CBZ) crystals in water and in various excipient solutions [(1% w/v) – hydroxypropyl cellulose (HPC), poly(vinyl pyrrolidone) (PVP), sodium carboxymethylcellulose (CMC) at pH 7.5 and 3.0, and polyethylene glycol (PEG)] using scanning electron microscopy (SEM) are reported in this paper. Raman microscopy was used to confirm the chemical structures of the unconverted CBZ and the CBZ dihydrate (DH) needles. It was found that defect structures were a more important driving force than the nature of crystal faces for the initiation of the hydration, but face differences became obvious after 6 h immersion. The biggest crystal face grown from methanol, (100), was the slowest one to be covered with DH needles. A comparison of the molecular arrangements along the three crystal faces [(100), (010) and (001)] was carried out using crystal structure visualization software, and fewer polar groups exposed on the (100) face than on the (001) and (010) faces were found, explaining the comparatively weak interaction of the (100) face with water during hydration. Furthermore, investigation of the influence of excipients on the hydration of CBZ showed that both HPC and PVP strongly inhibited conversion, and no conversion of CBZ to DH was found after 18 h immersion in water. PEG and CMC (pH 7.5) were less potent inhibitors than HPC and PVP, and DH needles were observed on all the faces except the (100) face after 18 h immersion. No conversion was detected for the crystal immersed in CMC solution at pH 3.0. This is likely to be caused by the decreased polarity of CMC in water at pH 3.0 (pK a,cmc = 4.3), and thus a higher surface adsorption of CMC to the CBZ crystals in dispersion. The influence of excipients on the conversion of CBZ observed in this study agreed well with our previous quantitative studies using Raman spectroscopy. In this study, visual observation using electron microscopy has been demonstrated to be a unique and powerful tool to improve our understanding of polymorphic conversions of CBZ in aqueous suspension.
Keywords: Hydration; Carbamazepine; Single crystal; Dihydrate; Scanning electron microscopy; Raman microscopy; Excipient;

Estimation of the percolation thresholds in acyclovir hydrophilic matrix tablets by Inmaculada Fuertes; Antonia Miranda; Mónica Millán; Isidoro Caraballo (336-342).
The principles of percolation theory were applied to design controlled release matrix tablets containing acyclovir. This statistical theory studies disordered or chaotic systems where the components are randomly distributed in a lattice. The application of this theory to study the release and hydration rate of hydrophilic matrices allows to explain the changes in release and hydration kinetics of swellable matrix type controlled delivery systems. The objective of the present paper is to estimate the percolation threshold of HPMC K4M in matrices of acyclovir and to apply the obtained result to the design of hydrophilic matrices for the controlled delivery of this drug.Matrix tablets have been prepared using acyclovir as drug and HPMC K4M as matrix forming material, employing five different excipient/drug percentages. Dissolution studies were carried out using the paddle method. Water uptake measurements were performed using a modified Enslin apparatus. In order to estimate the percolation threshold, the behaviour of the kinetic parameters with respect to the excipient volumetric fraction at time zero plus initial porosity was studied. According to percolation theory, the critical points observed in dissolution and water uptake studies can be attributed to the excipient percolation threshold. This threshold was situated between between 20.76% and 26.41% v/v of excipient plus initial porosity.The knowledge of the percolation threshold of the components of the matrix formulations contributes to improve their design. First, reducing the time to market and second, increasing their robustness when they are prepared at Industrial scale, avoiding the formulation in the nearby of the percolation threshold.
Keywords: Percolation theory; Acyclovir; HPMC; Swelling matrix; Excipient percolation threshold; Controlled release;

The theoretically expected breaking strength of tablets from powder mixtures is often calculated by the weighted arithmetic mean from the breaking strength of the single components, which corresponds to a linear interpolation. The validity of this additivity of fracture strength shall be evaluated by the underlying model of parallel couplings. It assumes the components linked in parallel with respect to the direction of loading during diametrical strength testing. Parallel couplings were experimentally realised by the preparation of double layer tablets from crystalline and spray-dried lactose on the one hand and from maltitol and metamizol-sodium on the other. Constant total true volumes of the single substances and of layered powders in varying ratios of true volume were compressed on an eccentric tabletting machine to constant geometric mean punch force. Simulated crushing profiles of parallel couplings were derived from force–displacement profiles measured during diametrical compression of the one-component tablets. At given finely graded deformation levels, the forces exerted by the components during loading were added in the proportion of the true volume fractions of the components in the coupling. The results from the experiments and from the simulations are in good accordance. They demonstrate that a linear change of the crushing strength in dependence on the true volume fraction of the components can only be assumed if the single components deform to the same extent up to the point of fracture. This behaviour was approximately found with the parallel lactose system. In all other cases it must be expected that the crushing strength of parallel systems will be lowered beneath the weighted arithmetic mean values or even below the crushing strength of the single components. The latter was observed with the maltitol–metamizol combinations. Thus, if tablets from binary powder mixtures exhibit a crushing strength depression, this is not necessarily an indication of weak bonding between the components or of structural defects.
Keywords: Double layer tablets; Powder mixtures; Crushing strength; Additivity rule; Parallel couplings;

A new expert systems (SeDeM Diagram) for control batch powder formulation and preformulation drug products by Pilar Pérez; Josep M. Suñé-Negre; Montserrat Miñarro; Manel Roig; Roser Fuster; Encarna García-Montoya; Carmen Hernández; Ramón Ruhí; Josep R. Ticó (351-359).
The new SeDeM Method is proposed for testing the batch-to-batch reproducibility of the same active pharmaceutical ingredient (API) in powder form. The procedure describes the study of the galenic properties of substances in powder form in terms of the applicability of direct compression technology. Through experimental determination of the SeDeM Method parameters, and their subsequent mathematical treatment and graphical expression (SeDeM Diagram), three batches of the same API were analysed to determine whether it was suitable for direct compression. Batch-to-batch reproducibility of the results was verified. It was concluded that the SeDeM Method is suitable for testing batch-to-batch reproducibility of characteristics in powdered APIs substances. The results obtained confirm that the SeDeM Method is a useful, effective tool for drug-preformulation studies providing the pharmacotechnical data required when formulating a drug in tablet form. In addition, the results were effective for defining the most appropriate manufacturing technology.
Keywords: Expert system; Preformulation; Formulation; Bulk density (Da); Tapped density (Dc); Inter-particle porosity (Ie); Angle of repose (α); Flowability (t″); Particle size (%Pf); Direct compression (DC);

ucb-35440-3 is a new drug entity under investigation at UCB S.A. Due to its physicochemical characteristics, the drug, a poorly water-soluble weak base, shows poor solubility and dissolution characteristics. In rat, the low oral bioavailability (F  < 10%) is largely due to poor absorption. In order to enhance the solubility and dissolution characteristics, formulation of ucb-35440-3 as nanocrystals has been achieved in this study. Nanoparticles were prepared using high pressure homogenization and were characterized in terms of size and morphology. In vitro dissolution characteristics were investigated and compared to the un-milled drug in order to verify the theoretical hypothesis on the benefit of increased surface area. In vivo pharmacokinetic evaluation of ucb-35440-3 nanoparticles was also carried out on rats. Crystalline state evaluation before and following particle size reduction was conducted through polarized light microscopy and PXRD to denote any possible transformation to an amorphous state during the homogenization process. Drug chemical stability was also assessed following homogenization. The dissolution rate increased significantly at pH 3.0, 5.0 and 6.5 for ucb-35440-3 nanoparticles. However, the pharmacokinetic profile obtained yielded lower systemic exposure than the un-milled compound (in fed state), this although being thought to be the consequence of the drug and formulation characteristics.
Keywords: Nanoparticles; High pressure homogenization; Dissolution; Drug reprecipitation; In vivo evaluation;

Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions by Joni J. Palmgrén; Jukka Mönkkönen; Timo Korjamo; Anssi Hassinen; Seppo Auriola (369-378).
Loss of drug content during cell culture transport experiment can lead to misinterpretations in permeability analysis. This study analyses drug adsorption to various plastic containers and drug retention in cultured cells under in vitro conditions. The loss of various drugs to polystyrene tubes and well plates was compared to polypropylene and glass tubes both in deionised water and buffer solution. In cellular uptake experiments, administered drugs were obtained from cultured cells by liquid extraction. Samples were collected at various time points and drug concentrations were measured by a new HPLC–MS/MS method. Acidic drugs (hydrochlorothiazide, naproxen, probenicid, and indomethacin) showed little if any sorption to all tested materials in either water or buffer. In the case of basic drugs, substantial loss to polystyrene tubes and well plates was observed. After 4.5 h, the relative amount remaining in aqueous test solution stored in polystyrene tubes was 64.7 ± 6.8%, 38.4 ± 9.1%, 31.9 ± 6.7%, and 23.5 ± 6.1% for metoprolol, medetomidine, propranolol, and midazolam, respectively. Interestingly, there was no significant loss of drugs dissolved in buffer to any of the tested materials indicating that buffer reduced surficial interaction. The effect of drug concentration to sorption was also tested. Results indicated that the higher the concentration in the test solution the lower the proportional drug loss, suggesting that the polystyrene contained a limited amount of binding sites. Cellular uptake studies showed considerable retention of drugs in cultured cells. The amounts of absorbed drugs in cellular structures were 0.45%, 4.88%, 13.15%, 43.80%, 23.57% and 11.22% for atenolol, metoprolol, medetomidine, propranolol, midazolam, and diazepam, respectively. Overall, these findings will benefit development and validation of further in vitro drug permeation experiments.
Keywords: Drug loss; Plastic instruments; Cultured cells; HPLC–MS/MS; Drug analysis;

Evaluation of cocktail approach to standardise Caco-2 permeability experiments by Maija Koljonen; Kati S. Hakala; Tuula Ahtola-Sätilä; Leena Laitinen; Risto Kostiainen; Tapio Kotiaho; Ann Marie Kaukonen; Jouni Hirvonen (379-387).
The purpose of this study was to investigate the suitability and reliability of n-in-one approach using FDA suggested compounds for standardising Caco-2 permeability experiments. Special attention was paid to the evaluation of rank order correlation and mechanistic insights of compound permeability. Transport studies with antipyrine, metoprolol, ketoprofen, verapamil, hydrochlorothiazide, ranitidine, mannitol and fluorescein were performed in 12- and 24-well formats, as single compounds and in cocktails under iso-pH 7.4 and pH-gradient (pH 5.5 vs. 7.4) conditions. Compounds were quantified using n-in-one LC/MS/MS analysis. The cocktail-dosing proved to be a feasible method to determine the permeability of the Caco-2 cell line and to introduce external standards for permeability tests. Even though sink conditions were lost in cocktail experiments for highly permeable compounds, the rank order of compound permeability and the classification to low and high permeability compounds remained unchanged between single and cocktail studies and permeability values of 12- and 24-well formats were directly comparable. Under pH-gradient conditions the margin between high and low permeability compounds was narrower due to the lower permeability (higher fraction of ionisation) of basic molecules. Of the compounds studied, antipyrine, metoprolol, hydrochlorothiazide and mannitol are suitable for evaluation and standardisation purposes of passive permeability, while fluorescein would function as paracellular marker under iso-pH 7.4. As efflux activity may vary between cell batches, verapamil is a useful marker for P-glycoprotein.
Keywords: Caco-2 monolayers; n-in-one cocktail; Standardisation; Drug permeability; Transport mechanism;

Influence of scale-up on the abrasion of tablets in a pan coater by Ronny Mueller; Peter Kleinebudde (388-392).
The purpose of this study was to examine the influence of batch size during scale-up on the abrasion and edge splitting of flat faced lactose tablets. The weight loss of white tracer tablets in a batch of blue coated tablets was investigated in a laboratory scale pan coater and a pilot scale pan coater as a function of different pan speeds and mixing times. It was observed that increasing batch size resulted in a decreased weight loss due to less edge damaging. The higher number of tablet impacts at the pan wall in the laboratory scale compared to the pilot scale might be the reason for this phenomenon. The common assertion that an increase in batch size in scale-up leads to a higher abrasion or tablet damaging was not supported in the current study.
Keywords: Scale-up; Film coating; Abrasion; Pan speed; Bohle Film Coater;

Reciprocating dialysis tube method: Periodic tapping improved in vitro release/dissolution testing of suppositories by Soichi Itoh; Naomi Teraoka; Toshio Matsuda; Kousuke Okamoto; Tatsuya Takagi; Charles Oo; H. Danny Kao (393-398).
The reciprocating dialysis tube (RDT) method can be used for in vitro release/dissolution testing of suppositories and has been reported to show good in vitro and in vivo correlation. However, for suppositories with viscous excipients, the result remains variable and generally under-predicts in vivo absorption. The purpose of this study was to assess whether periodic tapping of the closure of the RDT could improve in vitro release testing of suppositories. Two commercially available acetaminophen suppositories (A and B) that showed characteristic release behavior under normal rectal temperatures (37 and 38 °C) were chosen as test suppositories. In the absence of tapping, suppository A showed different release profiles at 37 and 38 °C, but the difference disappeared with periodic tapping. This finding was consistent with minimum temperature effect in the rectal absorption of suppository A in rabbits. Suppository B showed distinct release profiles at 37 and 38 °C irrespective of tapping, and the rectal absorption of suppository B in rabbits was affected by temperature. The test variability (CV% and ranges of release values) was substantially reduced in the presence of tapping. In conclusion, the addition of periodic tapping to RDT method developed in this study could improve in vitro release testing of suppositories.
Keywords: In vitro and in vivo correlation; IVIVC; In vitro release; Dissolution test; Dialysis membrane; Suppository; Acetaminophen;

by Frank Schaubhut; Wolfgang Frieß (399).