European Journal of Pharmaceutics and Biopharmaceutics (v.65, #2)

APV diary (S1-S2).

High throughput screening of protein formulation stability: Practical considerations by Martinus A.H. Capelle; Robert Gurny; Tudor Arvinte (131-148).
The formulation of protein drugs is a difficult and time-consuming process, mainly due to the complexity of protein structure and the very specific physical and chemical properties involved. Understanding protein degradation pathways is essential for the success of a biopharmaceutical drug. The present review concerns the application of high throughput screening techniques in protein formulation development. A protein high throughput formulation (HTF) platform is based on the use of microplates. Basically, the HTF platform consists of two parts: (i) sample preparation and (ii) sample analysis. Sample preparation involves automated systems for dispensing the drug and the formulation ingredients in both liquid and powder form. The sample analysis involves specific methods developed for each protein to investigate physical and chemical properties of the formulations in microplates. Examples are presented of the use of protein intrinsic fluorescence for the analysis of protein aqueous properties (e.g., conformation and aggregation). Different techniques suitable for HTF analysis are discussed and some of the issues concerning implementation are presented with reference to the use of microplates.
Keywords: High throughput screening; Protein formulation; Spectroscopy; Physical and chemical characterization; Fluorescence; Aggregation; Stability;

Morphology of protein particles produced by spray freezing of concentrated solutions by Josh D. Engstrom; Dale T. Simpson; Edwina S. Lai; Robert O. Williams; Keith P. Johnston (149-162).
The mechanisms for the formation of high surface area lysozyme particles in spray freezing processes are described as a function of spray geometry and atomization, solute concentration and the calculated cooling rate. In the spray freeze-drying (SFD) process, droplets are atomized into a gas and then freeze upon contact with a liquid cryogen. In the spray freezing into liquid (SFL) process, a solution is sprayed directly into the liquid cryogen below the gas–liquid meniscus. A wide range of feed concentrations is examined for two cryogens, liquid nitrogen (LN2) and isopentane (i-C5). The particle morphologies are characterized by SEM micrographs and BET measurements of specific surface area. As a result of boiling of the cryogen (Leidenfrost effect), the cooling rate for SFL into LN2 is several orders of magnitude slower than for SFL into i-C5 and for SFD in the case of either LN2 or i-C5. For 50 mg/mL concentrated feed solutions, the slower cooling of SFL into LN2 leads to a surface area of 34 m2/g. For the other three cases with more rapid cooling rates, surface areas were greater than 100 m2/g. The ability to adjust the cooling rate to vary the final particle surface area is beneficial for designing particles for controlled release applications.
Keywords: Spray freezing into liquid (SFL); Spray freeze-drying (SFD); Liquid cryogen; Leidenfrost effect;

Stable high surface area lactate dehydrogenase particles produced by spray freezing into liquid nitrogen by Josh D. Engstrom; Dale T. Simpson; Carrie Cloonan; Edwina S. Lai; Robert O. Williams; G. Barrie Kitto; Keith P. Johnston (163-174).
Enzyme activities were determined for lactate dehydrogenase (LDH) powder produced by lyophilization, and two fast freezing processes, spray freeze-drying (SFD) and spray freezing into liquid (SFL) nitrogen. The 0.25 mg/mL LDH aqueous feed solutions included either 30 or 100 mg/mL trehalose. The SFL process produced powders with very high enzyme activities upon reconstitution, similar to lyophilization. However, the specific surface area of 13 m2/g for SFL was an order of magnitude larger than for lyophilization. In SFD activities were reduced in the spraying step by the long exposure to the gas–liquid interface for 0.1–1 s, versus only 2 ms in SFL. The ability to produce stable high surface area submicron particles of fragile proteins such as LDH by SFL is of practical interest in protein storage and in various applications in controlled release including encapsulation into bioerodible polymers. The SFL process has been scaled down for solution volumes <1 mL to facilitate studies of therapeutic proteins.
Keywords: Lactate dehydrogenase (LDH); Spray freezing into liquid (SFL); Spray freeze-drying (SFD); Gas–liquid interface; Atomization;

Development of a spray congealing process for the preparation of insulin-loaded lipid microparticles and characterization thereof by Angelika Maschke; Christian Becker; Daniela Eyrich; Josef Kiermaier; Torsten Blunk; Achim Göpferich (175-187).
A spray congealing process for the preparation of protein-loaded microparticles was developed. The influence of the process parameters atomization pressure and spraying temperature on particle size and process yield was investigated by experimental design. The employed spray congealing technique enabled the production of microparticles with yields ranging from 79% to 95% and median particle sizes (d(0.5)) from 182.2 to 315 μm. Insulin lipid microparticles could be prepared without any loss of insulin during the preparation process and the protein stability was not affected by the spray congealing process as investigated by HPLC-MS analysis. The stability of insulin encapsulated in lipid microparticles under release conditions over 28 days was assessed by investigating the residual insulin content. Starting after 3 days of release, a continuous increase of desamidoinsulin in the remaining particles of up to 7.5% after 28 days was observed. An additional degradation product was detected by HPLC and HPLC-MS analysis and identified as a covalent insulin dimer by MALDI-ToF. The microparticles did not show a burst release and testing the insulin lipid microparticles in a fibrin gel chondrocyte culture revealed that the released insulin was bioactive and had a significant effect on chondrocyte extracellular matrix production.
Keywords: Spray congealing; Lipid microparticles; Controlled release; Protein; Insulin; Experimental design;

Effect of surfactant on dissolution of spherical particles in micellar systems by Hussien Allaboun; Khouloud A. Alkhamis; Nawzat D. Al Jbour (188-197).
The influence of micelle-drug solubilization on the dissolution rate of monodisperse particles of benzocaine has been investigated. A model describing and predicting the initial dissolution rates of spherical particles was derived starting from the boundary layer theory. The dissolution rate of benzocaine spherical particles was determined in water and in solutions of sodium lauryl sulfate (SLS) under static conditions. The derived model was applied to the experimental data. The diffusion coefficients and the aqueous diffusion layer values were estimated from the experimental results and the aforementioned model. The diffusion coefficients and the boundary layer thickness values were also obtained experimentally from the rotating disk method and were used to predict the initial dissolution rates. Excellent correlations were obtained between the experimental and the calculated values at low micellar concentrations. However, obvious deviation was observed at high micellar concentrations. The results obtained from this study suggest that it is possible to predict the initial dissolution rates of monodisperse particles in micellar systems.
Keywords: Benzocaine; Micellar solubilization; Surfactants; Initial dissolution rates; Monodisperse; Particles;

Effect of poloxamers on nifedipine microparticles prepared by Hot Air Coating technique by Franco Pattarino; Lorella Giovannelli; Sonja Bellomi (198-203).
The Hot Air Coating (HAC) technique was used to prepare microparticles consisting of 30% nifedipine coated with different lipid mixtures. Cetearyl alcohol or cetearyl alcohol and 5% or 15% of a poloxamer (Pluronic F68 or Pluronic F127) were used as excipients. HAC products were analyzed in terms of morphology, flowability, thermal properties and nifedipine release behaviour, in order to elucidate the role played by the Pluronics on the physico-chemical and pharmaceutical characteristics of microparticles. HAC particles were spherical and their surface appeared scale-worked; thermal studies demonstrated the existence of relevant interactions among the system components and the dissolution experiments led to the hypothesis that the drug is released primarily by diffusion through the lipid coating: the poloxamer and its concentration have a significant influence on the pharmaceutical properties of the dosage form, as shown by the a parameter of Weibull model.
Keywords: Nifedipine; Hot Air Coating; Microparticles; Poloxamers; Drug release rate; Weibull model;

In this study, acrylamide (AAm) was grafted onto poly(vinyl alcohol) (PVA) with UV radiation at ambient temperature. The graft copolymer (PVA-g-PAAm) was characterized by using Fourier transform infrared spectroscopy (FTIR), elemental analysis and differential scanning calorimetry (DSC). Polymeric blend beads of PVA-g-PAAm and PVA with sodium alginate (NaAlg) were prepared by cross-linking with glutaraldehyde (GA) and used to deliver a model anti-inflammatory drug, diclofenac sodium (DS). Preparation condition of the beads was optimized by considering the percentage entrapment efficiency, particle size, swelling capacity of beads and their release data. Effects of variables such as PVA/NaAlg ratio, acrylamide content, exposure time to GA and drug/polymer ratio on the release of DS were discussed at three different pH values (1.2, 6.8, 7.4). It was observed that, DS release from the beads decreased with increasing PVA/NaAlg (m/m) ratio, drug/polymer ratio (d/p) and extent of cross-linking. However, DS release increased with increasing acrylamide content of the PVA-g-PAAm polymer. The highest DS release was obtained to be 92% for 1/1 PVA-g-PAAm/NaAlg ratio beads. It was also observed from release results that DS release from the beads through the external medium is much higher at high pH (6.8 and 7.4) than that at low pH (1.2). The drug release from the beads mostly followed Case II transport.
Keywords: Graft copolymer; Controlled release; Drug delivery systems; Diclofenac sodium; Hydrophilic polymers;

The present work explores, using response surface methodology, the main and interaction effects of some process variables on the preparation of a reversed chitosan–alginate polyelectrolyte complex (PEC) with entrapped α-amylase for stability improvement. A 33 full factorial design was used to investigate the effect of the chitosan and alginate concentrations and hardening time on the percent entrapment, time required for 50% (T 50) and 90% (T 90) enzyme release, and particle size. The beads were prepared by dropping chitosan containing α-amylase into a sodium alginate solution without any salt. The in vitro enzyme release profile of the beads was fitted to various release kinetics models to study the release mechanism. A topographical characterization was carried out using scanning electron microscopy (SEM), and the entrapment was confirmed using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zones III and IV. Beads prepared using 2.5% w/v chitosan and 3% w/v sodium alginate with a hardening time of 60 min had more than 90% entrapment and a T 90 value greater than 48 min. Moreover, the shelf-life of the enzyme-loaded beads was found to increase to 3.68 years, compared with 0.99 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare a reversed PEC of chitosan and alginate with good mechanical strength, provided both the reactants are in a completely ionized form at the time of the reaction. Proper selection of the reaction pH, polymer concentration and hence charge density, and hardening time is important and determines the characteristics of the PEC.
Keywords: Alginate; α-Amylase; Chitosan; In vitro release models; Polyelectrolyte complex (PEC); Stability;

Application of sucrose fatty acid esters in transdermal therapeutic systems by G. Csóka; S. Marton; R. Zelko; N. Otomo; I. Antal (233-237).
Transdermal therapeutic systems (TTSs) were studied applying different sucrose fatty acid esters (SEs) as drug delivery agents. Matrix and membrane controlled TTSs were prepared and compared. Membrane was made from a methacrylic polymer (Eudragit® NE) of pH independent permeability which can achieve diffusion controlled drug liberation. Model drug was a water soluble β-blocker, metoprolol, which has short biological half-life, so applying it in a TTS, the duration of its action could be prolonged. Sucrose fatty acid esters of different fatty acid chain lengths and consequently different hydrophilic–lipophilic balance (HLB) values were studied considering their effect on the metoprolol release from TTSs. Different mathematical models were applied for the evaluation of the release process. The results of the in vitro studies indicated that SEs of shorter fatty acid chain length and higher HLB value increased the amount of released drug about 10 times. SEs could be promising agents in transdermal therapeutic systems to control the drug release and cutaneous absorption.
Keywords: Sucrose fatty acid esters; Transdermal delivery; Metoprolol; Drug release; Mathematical models;

Diffusion of naltrexone across reconstituted human oral epithelium and histomorphological features by Libero Italo Giannola; Viviana De Caro; Giulia Giandalia; Maria Gabriella Siragusa; Giuseppina Campisi; Ada Maria Florena; Tomasz Ciach (238-246).
In transbuccal absorption a major limitation could be the low permeability of the mucosa which implies low drug bioavailability. The ability of naltrexone hydrochloride (NLX) to penetrate a resembling histologically human buccal mucosa was assessed and the occurrence of any histomorphological changes observed. We used reconstituted human oral (RHO) non-keratinised epithelium as mucosal section and a Transwell diffusion cells system as bicompartmental model. Buccal permeation was expressed in terms of drug flux (J s) and permeability coefficients (K p). Data were collected using both artificial and natural human saliva. The main finding was that RHO does not restrain NLX permeation. Drug transport across the epithelium was observed also in presence of various concentrations of penetration enhancers, without any significant differences. On the contrary, the flux throughout the mucosa was extensively affected by iontophoresis. Histologically, no sign of flogosis was observed in any specimen under experiment without iontophoresis, whereas cytoarchitectural changes, up to nuclear pycnosis or cellular swelling, were determined as a consequence of the application of electric fields.
Keywords: Buccal permeation; Transbuccal absorption; Naltrexone hydrochloride; Reconstituted human oral epithelium (RHO); Iontophoresis; Penetration enhancers;

Intranasal absorption of Δ9-tetrahydrocannabinol and WIN55,212-2 mesylate in rats by Satyanarayana Valiveti; Remigius U. Agu; Dana C. Hammell; Kalpana S. Paudel; D. Caroline Earles; Daniel P. Wermeling; Audra L. Stinchcomb (247-252).
The aim of this study was to examine the potential of the nasal route for systemic delivery of Δ9-tetrahydrocannabinol (Δ9-THC) and WIN55,212-2 mesylate. Anesthetized rats were surgically prepared to isolate the nasal cavity, into which Δ9-THC (10 mg/kg) or WIN55,212-2 (150 μg/kg) in propylene glycol alone or propylene glycol and ethanol (9:1) were administered. Rats were also administered Δ9-THC (1 mg/kg) and WIN55,212-2 (150 μg/kg) intravenously in order to determine absolute bioavailabilities of the nasal doses. Plasma Δ9-THC and WIN55,212-2 concentrations were determined by liquid chromatography/mass spectroscopy (LC/MS). The pharmacokinetics of the drugs after intranasal administration was best described by a one-compartment model with an absorption phase. WIN55,212-2 was absorbed more rapidly (T max  = 0.2–0.3 h) than Δ9-THC (T max  = 1.5–1.6 h) and to a higher extent than Δ9-THC. Addition of ethanol (10%) to the formulations had no significant effect on the C max after nasal administration (p  > 0.05). Furthermore, it had no significant effect on the absolute bioavailability (F abs): F abs  = 6.4 ± 2.4% and 9.1 ± 3.0% for Δ9-THC in propylene glycol, with and without ethanol, respectively. For WIN55,212-2, F abs  = 49.9 ± 6.9% (propylene glycol alone) and 56.6 ± 14.1% (propylene glycol with 10% ethanol). The results of the study showed that systemic delivery of Δ9-tetrahydrocannabinol and WIN55,212-2 could be achieved following nasal administration in rats.
Keywords: Δ9-Tetrahydrocannabinol; Nasal delivery; Pharmacokinetics; Ethanol; Propylene glycol; Cannabinoids;

The effect of suction during die fill on a rotary tablet press by S. Jackson; I.C. Sinka; A.C.F. Cocks (253-256).
Die fill on a rotary tablet press involves complex powder flow phenomena. Conventional techniques for measuring flowability do not normally provide information that is directly relevant to the design of powder feed systems or to the selection of press parameters for the die filling process. Sinka et al. [I.C. Sinka, L.C.R. Schneider, A.C.F. Cocks, Measurement of the flow properties of powders with special reference to die fill, in: International Journal of Pharmaceutics 280 (1–2) (2004) 27–38] used an experimental shoe–die system to characterise the flow behaviour of pharmaceutical powders. A rigorous data analysis procedure was developed by Schneider et al. [L.C.R. Schneider, I.C. Sinka, A.C.F. Cocks, Characterisation of the flow behaviour of pharmaceutical powders using a model die–shoe filling system, in: Powder Technology (in press)] to evaluate the experimental results, however, when scaling the results to a rotary tablet press, the die fill efficiency was underpredicted by a factor of approximately 2, because the experimental system did not capture major features of the rotary press flow process. The suction effect, whereby the lower punch is moved downwards while the top of the die is exposed to powder in the feed system, is a key element of the process. In this note we describe the development of a model shoe–die system that allows the effect of suction to be investigated. The results demonstrate the improvement offered by suction and illustrate how a fundamental understanding of die fill phenomena could assist the selection of process parameters to maximise the operational speed of a rotary press.
Keywords: Powder flow; Die fill; Suction fill; Tablet press;

by Alexander Nezlin (257-258).