European Journal of Pharmaceutics and Biopharmaceutics (v.61, #3)

APV Diary (S1-S2).

The bitter taste of drugs, food components, and any other substances which get in the mouth as dissolved in an aqueous solution, or in the saliva, can be strongly reduced or fully eliminated, if the bitter component forms an inclusion complex with an appropriate cyclodextrin (CD). The value of the complex association constant (determined by the structure of the bitter ‘guest’ molecule and the size and eventual substitution of the ‘host' CD molecule), the temperature and the host/guest ratio determine the extent of complexation of the guest molecule (percentage of complexation) at the equilibrium. The K ass for most drug/CD complexes at 36 °C buccal cavity temperature is between 102 and 104  mol−1. If the unit dose (of a sublingual or chewing tablet, chewing gum) with a bitter drug (molecular weight of about 150, forming a 1:1 complex with βCD) is approximately 10 mg then the βCD can be taken in a 5- or even 10-fold molar excess. Under such conditions more than 99% of the bitter drug is complexed, and because complexed molecules cannot react with the taste buds in the buccal cavity no bitter taste is perceived. Frequently, preparation of the drug/CD complex is not necessary, because the βCD is present in a large excess, dissolved very quickly in the saliva and results in a saturated CD solution. Therefore, the complexation of the bitter drug is completed very rapidly. Only dissolved substances have taste and only CD complexable drug molecules can become debittered by CDs. Bitter, astringent components of foods (e.g. soya), beverages (e.g. naringin in citrus fruit juice, or chlorogenic acid and polyphenols in coffee) cigarette smoke (nicotine) also can be complexed and their taste reduced or fully eliminated.
Keywords: Bitter taste; Debittering; Cyclodextrin; Inclusion complexation; Astringent taste; Taste of drugs; Palatability; Patient compliance;

Delivery of a lentiviral vector in a Pluronic F127 gel to cells of the central nervous system by Padraig M. Strappe; David W. Hampton; Begona Cachon-Gonzalez; James W. Fawcett; Andrew Lever (126-133).
Lentiviral vectors have been demonstrated as efficient tools for gene delivery to the CNS. We describe a novel approach for vector delivery using the thermoresponsive Gel, Pluronic F127 as a carrier. A HIV-1 lentiviral vector expressing GFP was contained in various concentrations of gel (15, 30 and 40%) and applied to cultures of 293T cells. FACS analysis of cells transduced with 8 ng of lentiviral vector revealed a similar transduction efficiency for each Gel concentration compared to vector added to cells without PF127. Primary Rat CNS mixed glial cultures were also transduced with lentiviral vector in 15% Pluronic F127 and results demonstrated a similar transduction efficiency of astrocytes compared to virus without gel and no evidence of cell toxicity or death. Stereotaxic delivery of viral vector in 15% PF127 to the rat brain resulted in transduction of cells, predominantly astrocytes close to the injection site. Pluronic F127 gel delivery of viral vectors to the CNS may provide a platform for localised release particularly in areas of brain or spinal cord injury.
Keywords: Lentiviral vector; Pluronic F127; Viral vector delivery; Central nervous system;

Development of an enteric-coated, layered multi-particulate formulation for ileal delivery of viable recombinant Lactococcus lactis by Nathalie Huyghebaert; An Vermeire; Pieter Rottiers; Erik Remaut; Jean Paul Remon (134-141).
Layering of recombinant hIL-10 producing Lactococcus lactis (L. lactis Thy12) on inert carriers is a promising technique for the preparation of a multi-particulate formulation of viable, hIL-10 producing L. lactis. To improve viability after layering and storage, L. lactis Thy12 was layered in different matrices (10% skim milk and/or 2.5, 5, 10% inulin). After layering, the highest viability was obtained in the 10% skim milk supplemented with 5% inulin matrix (8.7%). However, upon storage, 10% skim milk alone yielded the highest viability. Thereby, layered L. lactis Thy12 showed superior long term stability in comparison with freeze-dried L. lactis Thy12. The layering process was performed during 3 h without encountering technical problems, with good layer consistence and constant viability. Enteric properties were obtained with a 30% Eudragit® L30D-55 or 15% Eudragit® FS30D coating and maintained during an initial six months storage period (−20 °C/20% RH). After in vitro simulation of the gastric stage, only 5% of the bacteria remained viable in Eudragit® L30D-55 coated pellets, contrary to 85% in Eudragit® FS30D coated pellets, indicating its superior protective capacity against gastric fluid. After eight months storage (−20 °C), 80% of the initial L. lactis Thy12 remained viable in the Eudragit® FS30D coated pellets.
Keywords: Layering; Inulin; Eudragit; Viability; Recombinant Lactococcus lactis;

Pharmacokinetic comparison of two recombinant human granulocyte colony-stimulating factor after subcutaneous administration in rabbits by Jorge Ducongé; Leyanis Rodríguez-Vera; Carmen Valenzuela; Daniel Álvarez; Omar Ramírez; Kathya R. de la Luz-Hernández; Estela Y. Rabeza-Legón; Ángel Casacó; Eduardo Fernández-Sánchez (142-148).
A pharmacokinetic comparison between two formulations (LeukoCIM, CIMAB SA versus Neupogen®, Hoffman-La Roche, licensed by Amgen) of recombinant human granulocyte colony-stimulating factor (rhG-CSF) using non-compartmental analysis was performed in male F1 rabbits after a single subcutaneous 11.5 μg/kg dose to help decide whether to conduct further comparability tests. Unlike the absorption phase, a statistical difference was not detected between Neupogen® and LeukoCIM for clearance (18.69±11.83 versus 28.42±12.11 mL/h/kg, P=0.22). In addition, using a multivariate statistical analysis by independent samples test, a significant difference was not found between the two formulations (P=0.88). Finally, the results obtained in this study confirmed the pharmacokinetic comparability between both formulations, supporting the claim for further assessments following the current protocol on biogeneric equivalence.
Keywords: Pharmacokinetics; Recombinant human granulocyte colony-stimulating factor; Clearance;

The effect of powder blend and tablet structure on drug release mechanisms of hydrophobic starch acetate matrix tablets by B. van Veen; J. Pajander; K. Zuurman; R. Lappalainen; A. Poso; H.W. Frijlink; J. Ketolainen (149-157).
This study investigates the release mechanism of a hydrophilic drug (caffeine) from hydrophobic matrix tablets composed of starch acetate. Different particle size fractions of starch acetate were mixed with caffeine (22% V/V) to obtain various mixture organisations in the powder, as well as in the final tablet. The organisation of powder mixtures was calculated by the carrier payload of starch acetate particles, while the pore size distributions in tablets were measured by mercury intrusion porosimetry. A carrier payload below 1 indicated the existence of a free starch acetate particle surface, while numbers greater than 1 pointed to a complete occupation of the starch acetate particle surface area by caffeine particles. The carrier payload calculations gave a good prediction for the existence of a starch acetate matrix in the tablet structures. Caffeine matrices in tablets compressed from the mixtures could be detected by mercury intrusion porosimetry measurements. The existence of different matrices, as well as different pore networks, determined the physical changes of the tablets and the release mechanism of caffeine during dissolution tests. When a tablet contained only a caffeine matrix, rapid tablet disintegration and immediate release of the total amount of caffeine occurred. A single matrix of starch acetate resulted in tablets that remained intact, although cracks were formed. The co-existence of matrices of both materials created surface erosion of the tablet. The caffeine release profiles of tablets that remained intact or showed erosion were fitted by an equation containing both diffusional and relaxational factors to describe the effect of tablet porosity on drug release.
Keywords: Drug release; Powder blend; Particle size; Hydrophobic matrix tablet; Percolation;

The influence of measurement conditions on the Hammett acidity function of solid pharmaceutical excipients by Andrey V. Zinchuk; Bruno C. Hancock; Evgenyi Y. Shalaev; Renuka D. Reddy; Ramprakash Govindarajan; Elizabeth Novak (158-170).
In this work the Hammett acidity function has been measured to assess the relative acidity of excipients used in the preparation of pharmaceutical solid dosage forms. A systematic series of experiments is reported which illustrates how the selection of the measurement conditions can influence the results of such determinations. Although the technique is somewhat empirical and relies on several key assumptions it is shown that very consistent results can be achieved by carefully controlling the measurement conditions. It is also shown that by taking this approach laboratory-to-laboratory variation can be reduced to a negligible level and the influences of subtle changes in the acidity of pharmaceutical excipients due to intrinsic variations in their physical properties or due to different processing histories can be detected and quantified.
Keywords: Excipient; Hammett acidity function; Equivalent pH;

Baclofen-loaded microspheres in gel suspensions for intrathecal drug delivery: In vitro and in vivo evaluation by Frederic Lagarce; Nathalie Faisant; Jean-Claude Desfontis; Laurent Marescaux; Freddy Gautier; Joel Richard; Philippe Menei; Jean-Pierre Benoit (171-180).
Severe spasticity is a very disabling disorder treated by continuous baclofen intrathecal infusion which unfortunately remains an expensive and uncomfortable treatment. In order to address these issues, new sustained release formulations designed for intrathecal baclofen delivery were sought with the aim of minimising the burst effect of baclofen which can lead to toxicity.Baclofen was encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres which were then dispersed in chitosan thermosensitive gels, Pluronic® PF-127 gels, carboxymethylcellulose solutions or Ringer lactate solution. The release rate was assessed in vitro using continuous flow cells and in vivo after intrathecal injection in goats: baclofen was quantified in cerebrospinal fluid (CSF) and plasma, and the associated pharmacological effect was evaluated. The results showed that the burst effect was reduced by at least a factor of 2 in vitro, after microsphere dispersion in viscous media. In vivo, PF-127 gel was found to be the best vehicle to reduce the burst effect by a factor of 10 in CSF, and by a factor of 2 in plasma. The toxic effect of baclofen due to the burst effect was reduced by the dispersion in PF127 gels. Therapeutic levels of baclofen in CSF were maintained during at least 1 month.
Keywords: PLGA microsphere; Baclofen; Burst effect; Polymer gel; Intrathecal administration;

Final sterilisation of drug-loaded polymeric microspheres is problematic as dry heat or steam sterilisation are not applicable, and γ-irradiation may result in radiolytic scission of the polymer chains, and potentially damage the bioactive compound. Therefore, aseptic production is the method of choice to obtain a sterile product. A novel process for the production of microspheres is introduced based on the principle of double emulsion–solvent extraction. The process uses a flow-through ultrasonic cell for the preparation of the primary emulsion, in combination with a static micromixer for the production of the double emulsion. Because of its small scale, the equipment is readily accommodated in a laminar air-flow cabinet or an isolator. Thanks to the low technical complexity and easy handling of the process, only minimal manual interventions is required. Finally, the possibility for in-place cleaning and sterilisation makes the equipment and process well suited for aseptic microsphere preparation. Microspheres were prepared from poly(lactic-co-glycolic acid) (PLGA), and bovine serum albumin (BSA) served as model protein for microencapsulation. The BSA-in-PLGA (w/o) emulsions produced by the ultrasonic flow-through cell exhibited mean droplet sizes of <700 nm. Further processing into microspheres of 15–40 μm mean diameter resulted in approx. 70% BSA encapsulation efficiency. Batch-to-batch reproducibility was excellent. Microsphere batches produced under aseptic conditions to assure product sterility exhibited no microbial contamination when examined by a simplified sterility test. The presented technology offers great potential for aseptic microsphere production for batch-sizes suitable, e.g. for clinical investigations. Complete validation of product sterility would, however, demand more extended tests.
Keywords: PLGA microspheres; Solvent extraction; Ultrasonic emulsification; Flow-through sonication; Static micromixer; Aseptic processing;

Preparation of poly(N-isopropylacrylamide) copolymers and preliminary assessment of their acute and subacute toxicity in mice by Hugues Malonne; Frédéric Eeckman; David Fontaine; Anne Otto; Louis De Vos; André Moës; Jeanine Fontaine; Karim Amighi (188-194).
A subacute toxicity study was conducted to evaluate the oral toxicity profile of poly(N-isopropylacrylamide) (PNIPAAm) derivatives. These thermoresponsive polymers may have several potential pharmaceutical applications such as ingredient for oral solid dosage form. A preliminary acute oral toxicity study was performed with one of the polymer (PNIPAAm-co-NVA) at a unique dose of 4000 mg/kg body weight administered to six male and six female mice, to determine the dosage for further evaluation. No treatment-related effect was observed on behavior and health condition of the experimental animals during the 14 days observational period. The autopsy of the treated animals did not revealed any macroscopic changes in major organ aspects. Based on these preliminary results we selected a 2000 mg/kg body weight/day dose for the 28 days long subacute study. Three polymers were tested, namely PNIPAAm, PNIPAAm-co-NVA and PNIPAAm-co-AAc and compared to a saline control. No significant changes in clinical signs, body weight and food consumption, hematology, clinical chemistry or absolute organ weight were observed. Histological examination of excised major organs showed no marked differences between treated and control mice. In conclusion, PNIPAAm-co-NVA is well tolerated up to 4000 mg/kg body weight when administered orally. In addition, the subacute study indicated the absence of cumulative toxicity and a no-observed-adverse-effect level (NOAEL) of 2000 mg/kg was identified for PNIPAAm and its two copolymers. Further studies are mandatory.
Keywords: N-isopropylacrylamide; Acute toxicity; Subacute toxicity; Thermoresponsive polymer; PNIPAAm; PNIPAAm-co-NVA; PNIPAAm-co-AAc;

Chitosan and poly(methyl vinyl ether-co-maleic anhydride) microparticles as nasal sustained delivery systems by T. Cerchiara; B. Luppi; G. Chidichimo; F. Bigucci; V. Zecchi (195-200).
An original dosage form for nasal delivery based on the encapsulation of hydrophilic drug in chitosan-poly(methyl vinyl ether-co-maleic anhydride) (CH-PVM/MA) microparticles prepared by spray-drying technique was developed. Microparticles were characterized in terms of morphology, size, swelling properties, encapsulation efficiency and drug release. The physical state of the drug and the polymer was determined by scanning electron microscopy (SEM) and infrared spectroscopy (IR). Propranolol hydrochloride (PH) was a β-blocker, used for the treatment of hypertension and was chosen as a model of hydrophilic drug. SEM studies showed spherical particles with smooth surfaces for chitosan hydrochloride (CH-HCl), whereas rather gross surface defects resulted from the incorporation of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA). In vitro release studies revealed a sustained release of propranolol HCl from microparticles and in particular chitosan hydrochloride provided the lowest release of drug.
Keywords: Chitosan; Poly(methyl vinyl ether-co-maleic anhydride); Spray-drying; Propranolol hydrochloride; Mucoadhesion; Franz cells;

In the previous papers of our research group it was shown, that the Clausius-Mossotti-Debye equation for the quasi-static dielectric constant (ε r) can be extended to liquids if the parameter E i /E is introduced. Thus, it is possible to characterize polar liquids with the easily accessible parameter E i /E. This property is also reflected by the fact that the parameter E i /E can be directly related to the empirical E T(30) and the normalized E T N parameter to describe the polarity of liquids proposed by Reichardt (Chem. Rev. 94, 2319-2358) E i corresponds to the local mean field due to close molecule-molecule interactions after the application of an external electric field E. In a recent work of our research group it was also demonstrated that the modified Clausius-Mossotti-Debye equation and the study of the relaxation time can be related to percolation phenomena in binary solvent mixtures leading to a valuable insight of the structure of polar liquids and to a better understanding of binary systems. In the present paper it is demonstrated that percolation phenomena for binary DMSO/water mixtures, become visible due to changes of parameters describing the dielectric spectrums.The interpretation of the percolation effects leads to the following conclusion: DMSO/water mixtures seem to have in the whole range of miscibility the same microscopic structure like water with a coordination number z≈ between 4 and 6 which could be the reason for the high permeability of DMSO through biological membranes. (1) E T N = E T ( solvent ) − E T ( TMS ) E T ( water ) − E T ( TMS ) = E T ( solvent ) − 30.7 32.4
Keywords: Modification of the Clausius-Mossotti-Debye-Equation; Percolation phenomena; Binary polar solvent mixtures; Empirical solvent polarity parameter E T and E T N (minamalized); Dielectric spectroscopy;

Non-invasive pulmonary aerosol delivery in mice by the endotracheal route by Maytal Bivas-Benita; Raphaël Zwier; Hans E. Junginger; Gerrit Borchard (214-218).
In this report we present in detail a non-invasive pulmonary application method that can be a useful tool in studying drug and vaccine delivery to the lower airways. In this method the formulation is sprayed directly into the lungs of mice via the endotracheal route using a MicroSprayer™ aerolizer. Mean droplet size produced was 8 μm, appropriate for deposition in the large airways. Endotracheal application of suspension of fluorescent nanospheres, 200 nm in size, by this method resulted in nanoparticle deposition in the smaller airways (bronchi and bronchioles). Mice showed full recovery one day after administration of 50 μl of formulation. Furthermore, no mortality was observed as a result of the technique. We conclude that this endotracheal application is a useful tool for studying pulmonary drug delivery in mice. The technique is especially useful for the pulmonary application of vaccines, since it enables multiple administrations without a need for analgesics.
Keywords: Pulmonary delivery; Nanoparticles; Aerosol application; Mice; Endotracheal administration;