European Journal of Pharmaceutics and Biopharmaceutics (v.68, #2)
Quantum dots – Nano-sized probes for the exploration of cellular and intracellular targeting
by W.A. Hild; M. Breunig; A. Goepferich (pp. 153-168).
Nanoparticles emerged as promising tool in drug targeting, since, after appropriate modification, they are able to deliver their payload to specific sites, like tissues, cells, or even certain cellular organelles. In this context, the delivery of nanoparticles from the circulation into the target cells represents a crucial step. Here, model drug delivery systems such as quantum dots are ideal candidates to elucidate this process in more detail, since they provide outstanding features like a small and uniform size, unique optical properties for most sensitive detection and modifiable surfaces. Recent progress in the surface chemistry of quantum dots expanded their use in biological applications, reduced their cytotoxicity and rendered quantum dots a powerful tool for the investigation of distinct cellular processes, like uptake, receptor trafficking and intracellular delivery. In this review, we will not only describe the ideal attributes of QDs for biological applications and imaging but also their distinct specific and non-specific pathways into the cells as well as their intracellular fate.
Keywords: Abbreviations; 5HT; 5-hydroxytryptamine; AFM; atomic force microscopy; AMPA; α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid; ANG II; angiotensin II; AT; angiotensin receptor; CCV; clathrin-coated vesicle; CHO; Chinese hamster ovary; CLSM; confocal laser scanning microscopy; CME; clathrin-mediated endocytosis; CPP; cell penetrating peptide; Da; dalton; DHLA; dihydrolipoic acid; DLS; dynamic light scattering; EDC; 1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; EPR; enhanced permeability and retention; FR; folate receptor; GPCR; G-protein coupled receptor; HEK; human embryonic kidney; Her2; human epidermal growth factor receptor 2; HIV; human immunodeficiency virus; HIV TAT; human immunodeficiency virus transactivator protein; IL-2; interleukin-2; LDL; low-density lipoprotein; MLS; mitochondrial localization sequence; MMP; matrix metalloprotease; MRI; magnetic resonance imaging; NGF; nerve growth factor; NHS; N; -hydroxysuccinimide; NIR; near infrared; NLS; nuclear localization sequence; PEG; polyethylene glycol; PET; positron emission tomography; PSMA; prostate specific membrane antigen; PTD; protein transduction domain; QD; quantum dot; RES; reticulo endothelial system; ROS; reactive oxygen species; SERT; serotonin transporter protein; SNAC; serotonin-labeled CdSe nanocrystals; SPECT; single photon emission computerized tomography; SPION; superparamagnetic iron oxide nanoparticle; sulfo-SMCC; sulfosuccinimidyl-4-(; N; -maleimidomethyl)-cyclohexane-1-carboxylate; SV40; simian virus 40; TEM; transmission electron microscopy; Tf; transferrin; TfR; transferrin receptor; TOP; trioctylphosphine; TOPO; trioctylphosphine oxide; TRK; receptor tyrosine kinaseDrug targeting; Quantum dot; Nanoparticle; Cellular uptake; Endocytosis; Cytotoxicity
Development of HSA-free formulations for a hydrophobic cytokine with improved stability
by Andrea Hawe; Wolfgang Friess (pp. 169-182).
The goal was to characterize a hydrophobic cytokine with respect to oxidation and aggregation, as well as its adsorption to the container at different pH and ionic strength conditions. The tendency of the cytokine to adsorb on surfaces and its low solubility at physiological pH were the main challenges during the development of HSA-free formulations for the cytokine. When the formulation pH exceeded 5.5 precipitation led to significantly higher turbidity. This turbidity increase and elevated aggregation as determined by HP-SEC and DLS was more pronounced at higher glycine and NaCl concentrations. With rising pH protein adsorption was more distinct compared to pH 3.0. However, protein adsorption could be minimized by polysorbate 20 or the use of glass type I+. FTIR revealed a reduced thermal stability at higher pH values indicated by a declining denaturation temperature. Five liquid formulations in the pH range 3.5–4.5 and five lyophilized formulations at pH 4.0–5.0 were stored for 6 months and the stability was evaluated with respect to aggregation and chemical modification. Liquid formulations at pH 3.5–4.0 and lyophilized formulations at pH 4.0–5.0 were most stable during 6 months at 2–8°C.
Keywords: Hydrophobic cytokine; HSA-free formulation; Protein adsorption; Stability
Stabilization of IgG by supercritical fluid drying: Optimization of formulation and process parameters
by Nataša Jovanović; Andréanne Bouchard; Gerard W. Hofland; Geert-Jan Witkamp; Daan J.A. Crommelin; Wim Jiskoot (pp. 183-190).
The aim of this study was to stabilize human serum immunoglobulin G (IgG) by a supercritical fluid (SCF) drying process. Solutions containing IgG (20mg/ml) and trehalose or hydroxypropyl-β-cyclodextrin in a 1:4 (w/w) ratio were sprayed into a SCF phase consisting of CO2 and ethanol at 100bar and 37°C. Initially, a set of drying conditions previously developed to successfully stabilize lysozyme and myogobin formulations was used [N. Jovanović, A. Bouchard, G.W. Hofland, G.J. Witkamp, D.J.A. Crommelin, W. Jiskoot, Eur. J. Pharm. Sci. 27 (2006) 336–345]. Dried formulations were analyzed by Karl Fisher titration, scanning electron microscopy, X-ray powder diffraction, and modulated DSC. Protein structure in the solid-state was studied by FTIR and after reconstitution by UV/Vis, circular dichroism and fluorescence spectroscopy, GPC and SDS–PAGE. When IgG was dried under the above-mentioned conditions, substantial amounts of insoluble aggregates were formed. Addition of buffer helped to reduce the fraction of insoluble material but not of soluble aggregates. Full stabilization could be achieved by adjusting the process conditions: drying without ethanol while keeping the other conditions the same, or drying with ethanol at a temperature below the critical point (20°C). In conclusion, it is possible to stabilize human IgG by SCF drying provided that the formulation and process conditions are tailored to meet the specific requirements of the protein.
Keywords: Hydroxypropyl-β-cyclodextrin; Human serum immunoglobulin G; Protein stability; Supercritical fluid drying; Trehalose
Preparation and physicochemical characterization of supercritically dried insulin-loaded microparticles for pulmonary delivery
by Maryam Amidi; Hubert C. Pellikaan; Anne H. de Boer; Daan J.A. Crommelin; Wim E. Hennink; Wim Jiskoot (pp. 191-200).
In the search for non-invasive delivery options for the increasing number of therapeutic proteins, pulmonary administration is an attractive route. Supercritical fluid (SCF) drying processes offer the possibility to produce dry protein formulations suitable for inhalation. In this study, insulin-loaded microparticles suitable for pulmonary administration were prepared and characterized. N-Trimethyl chitosan (TMC), a polymeric mucoadhesive absorption enhancer and dextran, a non-permeation enhancer, were used as carriers for insulin. The particles were prepared by spraying an acidic water/DMSO solution of insulin and polymer into supercritical carbon dioxide. The mean size of the particles was 6–10μm (laser diffraction analysis) and their volume median aerodynamic diameter ca. 4μm (time-of-flight analysis). The particles had a water content of ca. 4% (w/w) (Karl–Fischer), and neither collapsed nor aggregated after preparation and storage. In the freshly prepared dried insulin powders, no insulin degradation products were detected by HPLC and GPC. Moreover, the secondary and tertiary structures of insulin as determined by circular dichroism and fluorescence spectroscopy were preserved in all formulations. After one-year storage at 4°C, the particle characteristics were maintained and the insulin structure was largely preserved in the TMC powders. In conclusion, SCF drying is a promising, protein-friendly technique for the preparation of inhalable insulin-loaded particles.
Keywords: N; -Trimethyl chitosan microparticles; Supercritical carbon dioxide; Pulmonary delivery; Insulin; Physicochemical characterization
The addition of calcium ions to starch/Carbopol® mixtures enhances the nasal bioavailability of insulin
by E. Pringels; C. Vervaet; R. Verbeeck; P. Foreman; J.P. Remon (pp. 201-206).
To evaluate the influence of calcium poly(acrylates) on the nasal absorption of insulin in rabbits, starch/poly(acrylic acid) (ratio 25/75) (SD 25/75) was neutralised with NaOH and/or Ca(OH)2. After neutralisation, a mixture of sodium and/or calcium carboxylate was formed depending on the Ca(OH)2 concentration in the formulation. IR spectroscopy confirmed that most of the calcium molecules in the formulation interacted with acid groups of the acrylic acid polymer. Addition of Ca(OH)2 to aqueous dispersions containing starch/poly(acrylic acid) yielded powders with an enhanced absorption of insulin after nasal delivery to rabbits in comparison with the equivalent powder without Ca(OH)2. A mixture of SD 25/75 and Ca(OH)2 at a ratio of 90/10 neutralised to pH 7.4 with NaOH induced the highest absorption of insulin, obtaining a bioavailability of ±29% (vs. 19% for an equivalent formulation without Ca(OH)2). This increase in nasal delivery was possibly due to a higher elasticity after dispersing this formulation in nasal fluid and to a higher water absorbing capacity. Furthermore, after nasal delivery of (SD 25/75)/Ca(OH)2 90/10, a decrease in tmax was observed, possibly due to a progressive dissociation of Ca2+-ions after hydration of the powder resulting in the closing of the tight junctions.
Keywords: Powder formulation; Divalent cations; Carboxylate; Nasal bioavailability; Insulin
Lipid nanoparticles for alkyl lysophospholipid edelfosine encapsulation: Development and in vitro characterization
by Ander Estella-Hermoso de Mendoza; Marta Rayo; Faustino Mollinedo; María J. Blanco-Prieto (pp. 207-213).
The ether lipid 1- O-octadecyl-2- O-methyl- rac-glycero-3-phosphocholine, edelfosine (ET-18-OCH3) is the prototype molecule of a promising class of antitumor drugs named alkyl-lysophospholipid analogues (ALPs) or antitumor ether lipids. This drug presents a very important drawback as can be the dose dependent haemolysis when administered intravenously. Lipid nanoparticles have been lately proposed for different drug encapsulation as an alternative to other controlled release delivery systems, such as liposomes or polymeric nanoparticles. The aim of this study was to develop a lipid nanoparticulate system that would decrease systemic toxicity as well as improve the therapeutic potential of the drug. Lipids employed were Compritol® 888 ATO and stearic acid. The nanoparticles were characterized by photon correlation spectroscopy for size and size distribution, and atomic force microscopy (AFM) was used for the determination of morphological properties. By both differential scanning calorimetry (DSC) and X-ray diffractometry, crystalline behaviour of lipids and drug was assessed. The drug encapsulation efficiency and the drug release kinetics under in vitro conditions were measured by HPLC-MS. It was concluded that Compritol® presents advantages as a matrix material for the manufacture of the nanoparticles and for the controlled release of edelfosine.
Keywords: Lipid nanoparticles; Edelfosine; Drug delivery; Atomic force microscopy; Differential scanning calorimetry; X-ray diffractometry
Effects of process and formulation parameters on characteristics and internal morphology of poly(d,l-lactide-co-glycolide) microspheres formed by the solvent evaporation method
by Shirui Mao; Yi Shi; Luk Li; Jing Xu; Andreas Schaper; Thomas Kissel (pp. 214-223).
Taking ABT627 as a hydrophobic model drug, poly-(lactic-co-glycolic acid) (PLGA) microspheres were prepared by an emulsion solvent evaporation method. Various process parameters, such as continuous phase/dispersed phase (CP/DP) ratio, polymer concentration, initial drug loading, polyvinyl alcohol concentration and pH, on the characteristics of microspheres and in vitro drug release pattern of ABT627 were investigated. Internal morphology of the microspheres was observed with scanning electron microscopy by stereological method. CP/DP is a critical factor in preparing microspheres and drug loading increased significantly with increasing CP/DP ratios accompanied by a remarkably decreased burst release. At CP/DP ratio 20, microspheres with a core-shell structure were formed and the internal porosity of the microspheres decreased with increasing CP/DP ratio. Increase in PLGA concentration led to increased particle sizes and decreased drug release rates. ABT627 release rate increased considerably with increasing PVA concentrations in the continuous phase from 0.1% to 0.5%. The maximum solubility of ABT627 in PLGA was approximately 30%, under which ABT627 was dispersed in PLGA matrix in a molecular state. Increase in initial drug loading had no significant influence on particle size, drug encapsulation efficiency, burst release and internal morphology. However, drug release rate decreased at higher drug loading. Independent of process parameters, ABT627 was slowly released from the PLGA microspheres over 30 days, by a combination of diffusion and polymer degradation. During the first 13 days, ABT627 was mainly released by the mechanism of diffusion demonstrated by the unchanged internal morphology. In contrast, a core-shell structure of the microspheres was observed after being incubated in the release medium for 17 days, independent of drug loading, implying that the ABT627/PLGA microspheres degraded by autocatalytic effect, starting from inside of the matrix. In conclusion, hydrophobic drug release from the PLGA microspheres is mainly dependent on the internal morphology and drug distribution state in the microspheres.
Keywords: PLGA; Microspheres; Internal morphology
Chitosan-based spray-dried respirable powders for sustained delivery of terbutaline sulfate
by Tristan P. Learoyd; Jane L. Burrows; Eddie French; Peter C. Seville (pp. 224-234).
In this study, we describe the preparation of highly dispersible dry powders for pulmonary drug delivery that display sustained drug release characteristics. Powders were prepared by spray-drying 30% v/v aqueous ethanol formulations containing terbutaline sulfate as a model drug, chitosan as a drug release modifier and leucine as an aerosolisation enhancer. The influence of chitosan molecular weight on the drug release profile was investigated by using low, medium and high molecular weight chitosan or combinations thereof. Following spray-drying, resultant powders were characterised using scanning electron microscopy, laser diffraction, tapped density analysis, differential scanning calorimetry and thermogravitational analysis. The in vitro aerosolisation performance and drug release profile were investigated using Multi-Stage Liquid Impinger analysis and modified USP II dissolution apparatus, respectively. The powders generated were of a suitable aerodynamic size for inhalation, had low moisture content and were amorphous in nature. The powders were highly dispersible, with emitted doses of over 90% and fine particle fractions of up to 82% of the total loaded dose, and mass median aerodynamic diameters of less than 2.5μm. A sustained drug release profile was observed during dissolution testing; increasing the molecular weight of the chitosan in the formulation increased the duration of drug release.
Keywords: Spray-drying; Leucine; Modified release; Inhalation; Aerosolisation; Chitosan
Chitosan microspheres for intrapulmonary administration of moxifloxacin: Interaction with biomembrane models and in vitro permeation studies
by Cinzia A. Ventura; Silvana Tommasini; Emanuela Crupi; Ignazio Giannone; Venera Cardile; Teresa Musumeci; Giovanni Puglisi (pp. 235-244).
Chitosan microspheres loaded moxifloxacin were prepared to obtain sustained release of the drug after intrapulmonary administration. The microspheres were produced by the spray-drying method using glutaraldehyde as the crosslinking agent. The particles were spherical with a smooth but distorted surface morphology and were of small size, ranging from 2.5 to 6.0μm, thus suitable for inhalation. In vitro release studies showed a significant burst effect for all crosslinked systems, followed by a prolonged moxifloxacin release, particularly in the presence of the highest glutaraldehyde concentration. Lipid vesicles made of dipalmitoylphosphatidylcholine (DPPC) were used as an in vitro biomembrane model to evaluate the influence of chitosan microspheres on the interaction of moxifloxacin with biological membranes. Differential scanning calorimetry was used as a simple and non-invasive technique of analysis. Moxifloxacin freely permeates through DPPC liposomes, interacting with the hydrophobic zone of the bilayers (lowering of the Δ H value and loss of the cooperativity of the main transition peak). Uncrosslinked microspheres rapidly swelled and dissolved releasing free chitosan that was able to interact with liposomes (increase of Δ H value), probably altering the biomembrane permeability to the drug. Crosslinked microspheres did not show this property. Pulmonary absorption of moxifloxacin-loaded chitosan microspheres was evaluated compared to the free drug. A monolayer of Calu-3 human bronchial epithelial cells mounted on Franz diffusion cells was used as an in vitro bronchial epithelium model. Microspheres retard the absorption of moxifloxacin and within 6h the cumulative amount of permeated drug was about 18%, 11% and 7% (w/w) for free moxifloxacin, moxifloxacin-loaded crosslinked and moxifloxacin-loaded uncrosslinked microspheres, respectively.
Keywords: Chitosan microparticles; Moxifloxacin; Biomembrane interaction; In vitro; permeation; Calu-3 cell monolayer
Spray-dried microspheres based on methylpyrrolidinone chitosan as new carrier for nasal administration of metoclopramide
by Elisabetta Gavini; Giovanna Rassu; Corrado Muzzarelli; Massimo Cossu; Paolo Giunchedi (pp. 245-252).
The work purpose was to study the application of 5-methylpyrrolidinone chitosan (MPC) for preparing mucoadhesive microparticles for the nasal administration of drugs.Microspheres were produced by the spray-drying technique using MPC; metoclopramide hydrochloride (MC) was chosen as model drug. Chitosan microparticles were prepared as a comparison.The microparticles obtained were characterised (encapsulation efficiency, morphology, size and drug release behaviour). In-vitro mucoadhesive tests, swelling tests and ex-vivo studies using sheep nasal mucosa were performed. The hydrogel formation from microspheres was studied in different media and at different pHs.Microspheres are able to control the in-vitro MC release. MPC microparticles show good in-vitro mucoadhesive properties and ex-vivo controlled permeation profiles. The hydrogel formation is dependent mainly on the medium used: ionically crosslinked hydrogel was hypothesized.These in-vitro and ex-vivo preliminary results show that spray-dried microspheres based on MPC could be a suitable nasal delivery system for the administration of metoclopramide.
Keywords: Microspheres; Nasal delivery; Spray-drying; Mucoadhesion; Nasal sheep mucosa
Development of time-, pH-, and enzyme-controlled colonic drug delivery using spray-dried chitosan acetate and hydroxypropyl methylcellulose
by Jurairat Nunthanid; Kampanart Huanbutta; Manee Luangtana-anan; Pornsak Sriamornsak; Sontaya Limmatvapirat; Satit Puttipipatkhachorn (pp. 253-259).
A colonic drug delivery with a new concept based on a combination of time-, pH-, and enzyme-controlled system was developed. Spray-dried chitosan acetate (CSA) prepared from low molecular weight chitosan was characterized. A combination of CSA and hydroxypropyl methylcellulose (HPMC) was used as new compression-coats for 5-aminosalicylic acid (5-ASA) tablets. Factors affecting in-vitro drug release, i.e. % weight ratio of coating polymers, enzyme activity, pH of media, and excipients in core tablets, were evaluated. The tablets compression-coated with HPMC:CSA at 60:40 and 50:50% weight ratio providing lag times about 5–6h were able to pass through the stomach (stage I, 0.1N HCl) and small intestine (stage II, pH 6.8, Tris–HCl). The delayed release was time- and pH-controlled owing to the swelling with gradual dissolving of CSA and HPMC in 0.1N HCl and the less solubility of CSA at higher pH. After reaching the colon (stage III, pH 5.0, acetate buffer), the dissolution of CSA at low pH triggered the drug release over 90% within 14h. Furthermore, the degradation of CSA by β-glucosidase in the colonic fluid enhanced the drug release while adding the disintegrant or the osmotic agent in the core tablets would affect the drug release.
Keywords: Chitosan acetate; Colonic drug delivery; Compression-coated tablets; Spray drying; Characterization
Novel preparation of enteric-coated chitosan-prednisolone conjugate microspheres and in vitro evaluation of their potential as a colonic delivery system
by Tomoko Oosegi; Hiraku Onishi; Yoshiharu Machida (pp. 260-266).
After chitosan-succinyl-prednisolone conjugate (Ch-SP) was synthesized, conjugate microspheres (Ch-SP-MS), Eudragit L100-coated Ch-SP-MS and Eudragit S100-coated Ch-SP-MS, were prepared under novel preparative conditions. Namely, sonication was utilized to prepare finer Ch-SP-MS, and the addition ratio of Eudragit was reduced to yield Eudragit-coated Ch-SP-MS with higher drug content. Ch-SP-MS and Eudragit-coated Ch-SP-MS had mean sizes of 1.3μm and approximately 30μm, respectively, and showed prednisolone (PD) contents of 4.6% (w/w) and approximately 3% (w/w), respectively. Morphological changes of all the types of microparticles in different pH media were observed by scanning electron microscopy and confocal laser scanning microscopy. Both methods gave similar results. Both types of Eudragit-coated Ch-SP-MS protected Ch-SP-MS from morphological change at pH 1.2, and regenerated Ch-SP-MS fast at pH 6.8 and 7.4. For all types of microparticles, release of PD was suppressed at pH 1.2, but caused gradually at pH 6.8. These particle characteristics and in vitro behaviors demonstrated that the present Eudragit-coated Ch-SP-MS were considered potentially suitable for in vivo or practical application as a specific delivery system of PD to IBD sites.
Keywords: Chitosan-prednisolone conjugate microspheres; Eudragit-coated microparticles; Particle characteristics; Morphological change; Drug release; Specific delivery
Inulin–iron complexes: A potential treatment of iron deficiency anaemia
by Giovanna Pitarresi; Giuseppe Tripodo; Gennara Cavallaro; Fabio Salvatore Palumbo; Gaetano Giammona (pp. 267-276).
The aim of this work was that to synthesize macromolecular derivatives based on inulin able to complex iron and useful in the treatment of iron deficiency anaemia. Carboxylated or thiolated/carboxylated inulin derivatives were obtained by single or double step reactions, respectively. The first one was obtained by reaction of inulin (INU) with succinic anhydride (SA) alone obtaining INU–SA derivative; the second one was obtained by the reaction of INU with succinic anhydride and subsequent reaction of INU–SA with cysteine; both derivatives were treated with ferric chloride in order to obtain the INU–SA–FeIII and INU–SA–Cys–FeIII complexes. Both complexes showed an excellent biodegradability in the presence of inulinase and pronounced mucoadhesion properties; in particular, thiolated derivative INU–SA–Cys showed greater mucoadhesive properties than polyacrylic acid chosen, as a positive reference polymer, and a good iron release profile in condition mimicking the intestinal tract. These results suggest the potential employment of such systems in the oral treatment of iron deficiency anaemia or as supplement of iron in foods.
Keywords: Inulin; Iron complexes; Thiolated polymers; Mucoadhesion; Colon release
Effect of maltodextrin and superdisintegrant in directly compressible powder mixtures prepared via co-spray drying
by Y. Gonnissen; J.P. Remon; C. Vervaet (pp. 277-282).
The effect of maltodextrins and superdisintegrants on the tablet properties was evaluated in directly compressible powders coprocessed via spray drying. Powder mixtures containing acetaminophen, mannitol, erythritol and different maltodextrin types were prepared via co-spray drying and physically mixed with crospovidone (6% w/w, Kollidon® CL) in order to evaluate the influence of maltodextrin grade (amylose/amylopectin ratio) on powder hygroscopicity, flowability, density and compactability. In addition, different superdisintegrant types and grades (6% w/w) were co-spray dried to evaluate their effect on tablet disintegration time. Tablet disintegration was affected by the amylose/amylopectin ratio of the maltodextrins. Tablets containing Glucidex® 2 (1–5% amylose) had a longer disintegration time compared to Glucidex® 9 (20% amylose) (11.8min versus 5.7min) and Unipure DC (50–70% amylose) (1min). The disintegration time of tablets containing a coprocessed superdisintegrant was long due to loss of superdisintegrant during processing (preferential depositing on the spray dryer wall) and was in the following order: Kollidon® CL® XL®® CL-M® XL-10=Ac-Di-Sol®. A combination of acetaminophen, mannitol, erythritol, Glucidex® 9 and Kollidon® CL was selected for further formulation and process optimisation of co-spray dried powders intended for direct compression.
Keywords: Co-spray drying; Continuous processing; Maltodextrin; Superdisintegrant; Acetaminophen; Carbohydrates; Compression
Dissolution enhancement of fenofibrate by micronization, cogrinding and spray-drying: Comparison with commercial preparations
by Markus Vogt; Klaus Kunath; Jennifer B. Dressman (pp. 283-288).
Several techniques were compared for improving the dissolution of fenofibrate, a poorly soluble drug. Particle size reduction was realized by jet milling (micronization; cogrinding with lactose, polyvinylpyrrolidone or sodium lauryl sulphate) and by media milling using a bead mill (nanosizing) with subsequent spray-drying. Solid state characterization by X-ray diffraction and Differential Scanning Calorimetry verified the maintenance of the crystalline state of the drug after dry milling and its conversion to the amorphous state during spray-drying. Micronization of fenofibrate enhanced its dissolution rate in biorelevant media (8.2% in 30min) compared to crude material (1.3% in 30min). Coground mixtures of the drug increased the dissolution rate further (up to 20% in 30min). Supersaturated solutions were generated by nanosizing combined with spray-drying, this process converted fenofibrate to the amorphous state. Fenofibrate drug products commercially available on the German and French markets dissolved similarly to crude or micronized fenofibrate, but significantly slower than the coground or spray-dried fenofibrate mixtures. The results suggest that cogrinding and spray-drying are powerful techniques for the preparation of rapidly dissolving formulations of fenofibrate, and could potentially lead to improvements in the bioavailability of oral fenofibrate products.
Keywords: Biorelevant media; Cogrinding; Dissolution rate enhancement; Fenofibrate; Jet milling; Micronization; Particle size reduction; Spray-drying
Swellable elementary osmotic pump (SEOP): An effective device for delivery of poorly water-soluble drugs
by Javad Shokri; Parinaz Ahmadi; Parisa Rashidi; Mahbobeh Shahsavari; Ali Rajabi-Siahboomi; Ali Nokhodchi (pp. 289-297).
A new type of elementary osmotic pump (EOP) tablet for efficient delivery of poorly water-soluble/practically insoluble drugs has been designed. Drug release from the system, called swellable elementary osmotic pump (SEOP), is through a delivery orifice in the form of a very fine dispersion ready for dissolution and absorption. SEOP tablets were prepared by compressing the mixture of micronized drug and excipients into convex tablets. Factors affecting the release of drug from the SEOP tablets containing a poorly water-soluble drug, indomethacin, have been explored. The release behaviour of indomethacin from different formulations of this dosage form was studied at pH 6.8 for a period of 24h. The formulations were compared based on four comparative parameters, namely, D24h (total release after 24h), tL (lag time), RSQzero ( R square of zero order equation) and D%zero (percentage deviation from zero order kinetics). The drug release profile from osmotic devices showed that the type of polymer in the core formulation can markedly affect the drug release. The results showed that concentration of wetting agent in the core formulation was a very important parameter in D24h and release pattern of indomethacin from SEOP system. Increasing the amount of wetting agent to an optimum level (60mg) significantly increased D24h and improved zero order release pattern of indomethacin. Increasing concentration of caster oil (hydrophobic) in the semipermeable membrane of the device or hydrophilic plasticizer (glycerin) in coating formulation markedly increased tL and decreased D24h. The results also demonstrated that aperture size is a critical parameter and should be optimized for each SEOP system. Optimum aperture diameter for the formulations studied here was determined to be 650μm for zero order release pattern. tL and D%zero were dramatically decreased whereas D24h and RSQzero increased with increasing the aperture size to optimum level. This study also revealed that optimization of semipermeable membrane thickness is very important for approaching zero order kinetics.
Keywords: Controlled drug delivery; Indomethacin; SEOP; Zero order release; Lag time
Solubility-modulated monolithic osmotic pump tablet for atenolol delivery
by Longxiao Liu; Xiaocui Wang (pp. 298-302).
A method for the preparation of monolithic osmotic pump tablet was obtained by modulating atenolol solubility with acid. Tartaric acid was used as solubility promoter, sodium chloride as osmotic agent and polyvinyl pyrrolidone as retardant agent. Ethyl cellulose was employed as semipermeable membrane containing polyethylene glycol 400 as plasticizer. The formulation of atenolol monolithic osmotic pump tablet was optimized by orthogonal design and evaluated by similarity factor ( f2). The optimal monolithic osmotic pump tablet was found to be able to deliver atenolol at the rate of approximate zero-order up to 24h, independent of release media and agitation rate. The approach of solubility-modulated by acid-alkali reaction might be used for the preparation of osmotic pump tablet of other poorly water-soluble drugs with alkaline or acid groups.
Keywords: Atenolol; Monolithic osmotic pump tablet; Solubility-modulated; Orthogonal design
Sustained-release matrix tablets of metformin hydrochloride in combination with triacetyl-β-cyclodextrin
by Giovanna Corti; Marzia Cirri; Francesca Maestrelli; Natascia Mennini; Paola Mura (pp. 303-309).
The low bioavailability and short half-life of metformin hydrochloride (MH) make the development of sustained-release forms desirable. However, drug absorption is limited to the upper gastrointestinal (GI) tract, thus requiring suitable delivery systems providing complete release during stomach-to-jejunum transit. This study was undertaken to develop a MH sustained-release formulation in compliance with these requirements. The strategy proposed is based on direct-compressed matrix tablets consisting of a combination of MH with the hydrophobic triacetyl-β-cyclodextrin (TAβCD), dispersed in a polymeric material. Different polymers were tested as excipients, i.e. hydroxypropylmethylcellulose, xanthan gum, chitosan, ethylcellulose, Eudragit®L100-55, and Precirol®. Compatibility among the formulation components was assessed by DSC analysis. All the tablets were examined for drug release pattern in simulated gastric and jejunal fluids used in sequence to mimic the GI transit. Release studies demonstrated that blends of a hydrophobic swelling polymer (hydroxypropylmethylcellulose or chitosan) with a pH-dependent one (Eudragit®L100-55) were more useful than single polymers in controlling drug release. Moreover, the main role played by the MH–TAβCD system preparation method (i.e. grinding or spray-drying) in determining the behaviour of the final formulation was evidenced. In fact, for a given matrix-tablet composition, different sustained-release effects were obtained by varying the relative amounts of MH–TAβCD as ground or spray-dried product. In particular, the 1:1 (w/w) blend of such systems, dispersed in a Eudragit–chitosan polymeric matrix, fully achieved the prefixed goal, giving about 30% released drug after 2h at gastric pH, and overcoming 90% released drug within the subsequent 3h in jejunal fluid.
Keywords: Metformin hydrochloride; Triacetyl-β-cyclodextrin; Sustained release; Matrix tablets
New levodopa sustained-release floating minitablets coated with insoluble acrylic polymer
by J. Goole; Ph. Deleuze; F. Vanderbist; K. Amighi (pp. 310-318).
The aim of this study was to develop a new coated multiple-unit sustained-release floating system that is able to float over an extended period of time. Levodopa was used as a model drug. The system consisted of a 3mm drug-containing gas-generating core, prepared by melt granulation and subsequent compression, and coated with a flexible polymeric membrane. Eudragit® RL30D and ATEC were used as a film former and a plasticizer, respectively. The coating level was fixed at 20% (w/w). The floating lag time decreased as the proportion of effervescent agents increased. The optimized coated floating minitablets could float within 20min and remained buoyant for more than 13h. In addition, a sustained release of levodopa for more than 20h was observed.
Keywords: Multiple-unit system; Minitablets; Floating; Sustained release; Coating; Eudragit; ®; RL30D; Levodopa
A sugar cane native dextran as an innovative functional excipient for the development of pharmaceutical tablets
by Eddy Castellanos Gil; Antonio Iraizoz Colarte; Abdelsam El Ghzaoui; Denis Durand; Jean Louis Delarbre; Bernard Bataille (pp. 319-329).
We reported the physical chemical characterization of a new series of native dextran (B110-1-2). The chemical structure of the polymer was characterized by IR,1H and13C NMR spectroscopy and compared with that of a commercial native dextran B512-F obtained from Sigma Company. Molecular weights of the product and different commercial dextran fractions of Leuconostoc mesenteroides from 43000 to 170000 average molecular weight ( Mw) were established by the analysis of intrinsic viscosity in aqueous solutions and compared with those obtained by gel permeation chromatography (GPC). The critical overlap concentration around 9g/L was obtained. No interactions of powder mixtures with different commercial excipients (lactose, cetyl alcohol, HPMC) and drugs (propranolol hydrochloride, acetyl salicyclic acid, isosorbide dinitrate, lobenzarit disodium, and nifedipine) were demonstrated by differential scanning calorimetry (DSC) analysis. Tablets obtained by direct compression showed good physical–mechanical and technological properties. Dextran B110-1-2 has similar physical chemical properties as commercial Sigma B512-F. Water uptake, erosion and dissolution profile studies for dextran tablets established that glucose polymer with molecular weight Mw⩾2×106 is suitable for the development of controlled release solid dosage forms (soluble drugs). Fraction of dextran ( Mw 40000–170000) could be more useful for immediate release tablets.
Keywords: Native dextran; Molecular weight; Intrinsic viscosity; Tablets; Dissolution profiles
Dissolution improvement of four poorly water soluble drugs by cogrinding with commonly used excipients
by Markus Vogt; Klaus Kunath; Jennifer B. Dressman (pp. 330-337).
The rate of the dissolution of four poorly soluble drugs (EMD 57033, albendazole, danazol and felodipine) was improved by cogrinding them with various excipients (lactose monohydrate, corn starch, polyvinylpyrrolidone, hydroxypropylmethyl cellulose and sodium lauryl sulphate) using a jet-milling technique. Solid state characterization studies by X-ray diffraction and differential scanning calorimetry verified the maintenance of the crystalline state of the active substances after milling. In vitro dissolution of the coground mixtures in biorelevant media was much faster than from micronised drug in the corresponding physical mixtures for all four compounds. Supersaturated solutions were generated in some cases (EMD 50733 and felodipine), but this phenomenon appeared to be drug- and excipient-specific. Cogrinding with lactose monohydrate resulted in fast dissolution with unstable supersaturation for EMD 57033. Cogrinding the same drug with PVP or HPMC produced a more sustained supersaturation. SLS accelerated the dissolution of EMD 50733 but inhibited supersaturation. The results suggest that the cogrinding with selected excipients is a powerful tool to accelerate the dissolution of poorly soluble drugs without converting the drug to the amorphous form or changing the particle size.
Keywords: Albendazole; Cogrinding; Danazol; Dissolution rate enhancement; Felodipine; Jet-milling; Particle size reduction; Poorly soluble drugs; Solubility; Supersaturation
Cogrinding enhances the oral bioavailability of EMD 57033, a poorly water soluble drug, in dogs
by Markus Vogt; Maria Vertzoni; Klaus Kunath; Christos Reppas; Jennifer B. Dressman (pp. 338-345).
The oral bioavailability of EMD 57033, a calcium sensitizing agent with poor solubility, was compared in dogs using four solid dosage form formulation approaches: a physical blend of the drug with excipients, micronization of the drug, preparation of coground mixtures and spray-drying of the drug from a nanocrystalline suspension. The formulations contained generally accepted excipients such as lactose, hydroxypropylmethyl cellulose and sodium lauryl sulphate in usual quantities. Drug micronization and cogrinding was realized by a jet-milling technique. Nanoparticles were created by media milling using a bead mill. All formulations were administered orally as dry powders in hard gelatine capsules. While micronization increased the absolute bioavailability of the solid drug significantly compared to crude material (from nondetectable to 20%), cogrinding with specific excipients was able to almost double this improvement (up to 39%). With an absolute bioavailability of 26%, spray-dried nanoparticular EMD 57033 failed to show the superior bioavailability that had been anticipated from in vitro data. The control solution prepared with cyclodextrin was shown to have an absolute bioavailability of 57% (vs. i.v. infusion). It was concluded that cogrinding can be a useful tool to improve the bioavailability of poorly soluble drugs from a solid dosage form format.
Keywords: Bioavailability; Cogrinding; In vitro; –; in vivo; correlation; Jet-milling; Dissolution rate enhancement; Micronization; Particle size reduction; Poorly soluble drugs; Spray-drying
Formation of physically stable amorphous phase of ibuprofen by solid state milling with kaolin
by Subrata Mallick; Satyanarayan Pattnaik; Kalpana Swain; Pintu K. De; Arindam Saha; Gaurisankar Ghoshal; Arijit Mondal (pp. 346-351).
Ibuprofen was milled in the solid state with kaolin (hydrated aluminium silicate) in different ratio to examine the extent of transformation from crystalline to amorphous state. The physical stability of the resultant drug was also investigated. X-ray powder diffractometry (XRD) and birefringence by Scanning Electron Microscopy (SEM) studies indicated almost complete amorphization of the drug on ball milling with kaolin at 1:2 ratio. Fourier transform infrared spectroscopy (FTIR) data showed a reduction in the absorbance of the free and the hydrogen-bonded acid carbonyl peak of carboxylic acid group accompanied by a corresponding increase in the absorbance of the carboxylate peak, indicating an acid–base reaction between the carboxylic acid containing ibuprofen and kaolin on milling. The extent of amorphization and reduction in the carbonyl peak and increase in carboxylate peak was a function of kaolin concentration in the milled powder. On storage of milled powder (at 40°C and 75% RH for 10 weeks), XRD and birefringence of SEM study showed the absence of reversion to the crystalline state and FTIR data revealed continued reduction of carbonyl peak, whereas, ibuprofen converted from its crystalline acid form to amorphous salt form on milling with kaolin. Kaolin-bound state of ibuprofen was physically stable during storage. In-vitro dissolution studies revealed that percent release of ibuprofen from the kaolin co-milled powder is in the order: 1:2>1:1>1:0.5>1:0.1>milled alone ibuprofen>crystalline ibuprofen.
Keywords: Amorphization; Solid state milling; XRD; SEM; FTIR; Kaolin; Ibuprofen; In-vitro dissolution
In-silico model of skin penetration based on experimentally determined input parameters. Part I: Experimental determination of partition and diffusion coefficients
by Steffi Hansen; Andreas Henning; Arne Naegel; Michael Heisig; Gabriel Wittum; Dirk Neumann; Karl-Heinz Kostka; Jarmila Zbytovska; Claus-Michael Lehr; Ulrich F. Schaefer (pp. 352-367).
Mathematical modeling of skin transport is considered a valuable alternative of in-vitro and in-vivo investigations especially considering ethical and economical questions. Mechanistic diffusion models describe skin transport by solving Fick’s 2nd law of diffusion in time and space; however models relying entirely on a consistent experimental data set are missing. For a two-dimensional model membrane consisting of a biphasic stratum corneum (SC) and a homogeneous epidermal/dermal compartment (DSL) methods are presented to determine all relevant input parameters.The data were generated for flufenamic acid ( MW 281.24g/mol;logKOct/H2O 4.8; p Ka 3.9) and caffeine ( MW 194.2g/mol;logKOct/H2O −0.083; p Ka 1.39) using female abdominal skin. Klip/don (lipid-donor partition coefficient) was determined in equilibration experiments with human SC lipids. Kcor/lip (corneocyte-lipid) and KDSL/lip (DSL-lipid) were derived from easily available experimental data, i.e. KSC/don (SC-donor), Klip/don and KSC/DSL (SC-DSL) considering realistic volume fractions of the lipid and corneocyte phases. Lipid and DSL diffusion coefficients Dlip and DDSL were calculated based on steady state flux. The corneocyte diffusion coefficient Dcor is not accessible experimentally and needs to be estimated by simulation.Based on these results time-dependent stratum corneum concentration-depth profiles were simulated and compared to experimental profiles in an accompanying study.
Keywords: Partition coefficient; Diffusion coefficient; Modeling skin penetration; Tape-stripping; Flufenamic acid; Caffeine
In-silico model of skin penetration based on experimentally determined input parameters. Part II: Mathematical modelling of in-vitro diffusion experiments. Identification of critical input parameters
by Arne Naegel; Steffi Hansen; Dirk Neumann; Claus-Michael Lehr; Ulrich F. Schaefer; Gabriel Wittum; Michael Heisig (pp. 368-379).
This work describes a framework for in-silico modelling of in-vitro diffusion experiments illustrated in an accompanying paper [S. Hansen, A. Henning, A. Naegel, M. Heisig, G. Wittum, D. Neumann, K.-H. Kostka, J. Zbytovska, C.M. Lehr, U.F. Schaefer, In-silico model of skin penetration based on experimentally determined input parameters. Part I: experimental determination of partition and diffusion coefficients, Eur. J. Pharm. Biopharm. XX (2007) xx–xx]. A mathematical model of drug permeation through stratum corneum (SC) and viable epidermis/dermis is presented. The underlying geometry for the SC is of brick-and-mortar character, meaning that the corneocytes are completely embedded in the lipid phase. The geometry is extended by an additional compartment for the deeper skin layers (DSL). All phases are modelled with homogeneous diffusivity. Lipid-donor and SC–DSL partition coefficients are determined experimentally, while corneocyte–lipid and DSL–lipid partition coefficients are derived consistently with the model. Together with experimentally determined apparent lipid- and DSL-diffusion coefficients, these data serve as direct input for computational modelling of drug transport through the skin. The apparent corneocyte diffusivity is estimated based on an approximation, which uses the apparent SC- and lipid-diffusion coefficients as well as corneocyte–lipid partition coefficients. The quality of the model is evaluated by a comparison of concentration–SC-depth-profiles of the experiment with those of the simulation. Good agreements are obtained, and by an analysis of the underlying model, critical parameters of the models can be identified more easily.
Keywords: Skin; Drug diffusion; Concentration–depth-profiles; Mathematical modelling; Numerical simulation
In vitro skin absorption and drug release – A comparison of six commercial prednicarbate preparations for topical use
by S. Lombardi Borgia; P. Schlupp; W. Mehnert; M. Schäfer-Korting (pp. 380-389).
Reconstructed human epidermis is a useful tool for in vitro skin absorption studies of chemical compounds. If this may hold true also for topical dermatics, we investigated the glucocorticoid prednicarbate applied by two sets (innovator and generic) of cream, ointment and fatty ointment using the commercially available EpiDerm™ model. Moreover, stability and local tolerability of the preparations as well as drug release were studied, to estimate an influence on prednicarbate absorption and metabolism. While release ranked in the order cream
Keywords: Prednicarbate; Topical dermatics; Drug release; Skin absorption; Skin metabolism; Reconstructed human epidermis
Establishment and validation of an ex vivo human cervical tissue model for local delivery studies
by Christian Hiller; Udo Bock; Sigrid Balser; Eleonore Haltner-Ukomadu; Michael Dahm (pp. 390-399).
The objective of this study was to establish and validate an ex vivo human cervical tissue model appropriate for transport studies of molecular and especially nucleic acid based drugs. For that purpose conditions had to be established for a standardized tissue handling and preparation following hysterectomy to allow an immediate experimental use of fresh tissue samples. Samples of the ectocervical, endocervical and the transition zone representing the entire cervix organ were characterized in Franz diffusion cells by the determination of the in vitro permeation of low and high molecular weight markers (propanolol, mannitol, dextran 4000, 10,000, 20,000 and 40,000Da). Additionally, the permeability of mannitol and dextran 4000 across fresh and frozen cervical tissue was compared. The apparent permeability coefficients ( Papp) of the various markers demonstrated (i) that with increasing molecular weight the marker permeability decreases, (ii) an upper permeability limit between 10,000 and 20,000Da, (iii) no significant difference of the permeability across the three cervical tissue zones, (iv) a statistically significant but effectively small variation of the permeability among different patient samples. A continuous difference of approximately two log values between the Papp values of mannitol and dextran 4000 makes them suitable as an internal marker control pair for each biopsy. Moreover, the Papp values of both markers across fresh and frozen tissue are comparable. According to the presented data we conclude that the human cervical tissue model has been well characterized and is therefore suitable for local delivery and permeation studies.
Keywords: Cervix; Macromolecules; Morphology; Permeability; Uptake
Distribution coefficient of rifabutin in liposome/water system as measured by different methods
by Vitaly V. Vostrikov; Alla A. Selishcheva; Galina M. Sorokoumova; Yulia N. Shakina; Vitaly I. Shvets; Oleg Yu. Savel’ev; Vladimir I. Polshakov (pp. 400-405).
Distribution coefficient ( D) of rifabutin in liposome/water system was measured by phase separation and fluorescence probe quenching techniques. D values were identical suggesting that rifabutin is fully immersed into lipid bilayer. Structural studies of phospholipid bilayer employing31P NMR spectroscopy demonstrated that introduction of rifabutin does not alter the bilayer structure. A scheme of the rifabutin position in lipid bilayer based on the calculated size of rifabutin molecule is proposed.
Keywords: Rifabutin; Distribution coefficient; 31; P NMR; Liposomes; Fluorescence
Preparation and pre-vivo evaluation of no-carrier-added, carrier-added and cross-complexed [68Ga]-EDTMP formulations
by Stefan Toegel; Wolfgang Wadsak; Leonhard K. Mien; Helmut Viernstein; Rainer Kluger; Harald Eidherr; Daniela Haeusler; Kurt Kletter; Robert Dudczak; Markus Mitterhauser (pp. 406-412).
The present study aimed to develop convenient preparation and quality control protocols for [68Ga]-EDTMP, a potential radiotracer for skeletal PET imaging. Furthermore, bone binding characteristics with special focus on the influence of carrier addition were evaluated.No-carrier-added (nca), carrier-added and novel cross-complexed [68Ga]-EDTMP formulations were prepared using [68Ga]-gallium chloride and a commercial EDTMP kit. Respective bone binding characteristics were determined on the basis of an established in-vitro method using hydroxyapatite and human bone powders as binding matrices.Pre-vivo evaluation of nca [68Ga]-EDTMP yielded irreversible binding on the mineral bone phase characterised by fast binding kinetics. Generally, nca [68Ga]-EDTMP showed low uptake values comparable to nca [99mTc]-EDTMP. Interestingly, the bone binding affinity of [68Ga]-EDTMP could be increased by the addition of carriers, presumably by changing the complex structure.This fast and reliable preparation protocol could enable small PET facilities without onsite cyclotron to perform PET bone scans. A comparison of all cross-complexed [68Ga]-EDTMP preparations further strengthens the recently presented “foreign carrier theory”, which highlights carrier addition as a factor strongly affecting bone uptake of radiolabelled polyphosphonates. The clinical applicability of [68Ga]-EDTMP – particularly with respect to lesion specificity and sensitivity – should be clarified in forthcoming in-vivo studies.
Keywords: Gallium-68; EDTMP; PET; Bone imaging; Carrier; Targeting; Pharmacokinetics; Binding studies
Pharmacoscintigraphic and pharmacokinetic evaluation of tobramycin DPI formulations in cystic fibrosis patients
by Gabrielle Pilcer; Jonathan Goole; Bernard Van Gansbeke; Didier Blocklet; Christiane Knoop; Francis Vanderbist; Karim Amighi (pp. 413-421).
Tobramycin dry powder formulations were evaluated by gamma scintigraphy and pharmacokinetic methods. In an open single-dose, three-treatment, three-period, cross-over study, nine cystic fibrosis patients received both the two test products and the reference product Tobi® (nebulizer solution) in order to assess lung deposition and systemic comparative bioavailability of the two investigational inhaled products versus the marketed inhaled comparator product. The percentage of dose (mean±SD) in the whole lung was 53.0±10.0% for the tobramycin Form 1, 34.1±12.4% for the tobramycin Form 2 and 7.6±2.7% for the comparator product Tobi®. Lung deposition expressed as a percentage of the nominal dose was thus estimated to be 7.0 and 4.5 times higher for the Tobra Form 1 and Tobra Form 2 than for the Tobi®, respectively. Furthermore, the systemic bioavailability (adjusted to correspond to the same drug dose as that of the comparator product deposited in the lung) was found to be 1.6 times higher for the comparator product Tobi® than for the two DPI formulations.The principal advantages of the DPI formulations include reduced systemic availability and thus, side effects, and higher dose levels of the drug at the site of drug action.
Keywords: Dry powder inhaler; Cystic fibrosis; Tobramycin; Gamma scintigraphy; Pharmacokinetics
In vivo evaluation of risperidone-SAIB in situ system as a sustained release delivery system in rats
by Yaxin Lu; Xing Tang; Yue Cui; Yu Zhang; Feng Qin; Xiumei Lu (pp. 422-429).
The objective of this study was to evaluate a sustained release sucrose acetate isobutyrate (SAIB) in situ system formulation of risperidone (RSP) in vivo. The formulation contained SAIB, ethanol, and polylactic acid (PLA) as a release regulator. In vivo pharmacokinetics (PK) studies have shown that PLA is effective in reducing the burst effect. After a 12.5mg/kg IM injection of a 25mg/g RSP-SAIB in situ system, the Cmax was markedly reduced from 944.1±80.2 to 330.4±33.6ng/ml by increasing PLA from 1% to 10% (w/w), the Tmax were prolonged from 2 to 4.3±2.0h, and the area under the curve from day 0 to 2 (AUC0–2day) was reduced significantly from 16294.8±3946.4 to 7025.3±1979.2ngh/ml. For the RSP-SAIB in situ system including 10% PLA, the high release rates over a short period allowed therapeutic plasma concentrations to be achieved in the initial stages after activation, and sustained release of the drug led to a stable plasma concentration (by day 25, the plasma concentration was 8% of the Cmax). These initial in vivo studies suggest that RSP-SAIB in situ system is effective as a sustained delivery system.
Keywords: SAIB; In situ system; Risperidone; Sustained release delivery system; In-vivo pharmacokinetics
Method transfer for fast liquid chromatography in pharmaceutical analysis: Application to short columns packed with small particle. Part II: Gradient experiments
by Davy Guillarme; Dao T.T. Nguyen; Serge Rudaz; Jean-Luc Veuthey (pp. 430-440).
Liquid chromatography (LC) is currently considered as the gold standard in pharmaceutical analysis. Today, there is an increasing need for fast and ultra-fast methods with good efficiency and resolution for achieving separations in a few minutes or even seconds. A previous article (i.e. method transfer for fast LC in pharmaceutical analysis. Part I: isocratic separation) described a simple methodology for performing a successful method transfer from conventional LC to fast and ultra-fast LC in isocratic mode. However, for performing complex separations, the gradient mode is often preferred. Thus, this article reports transfer rules for chromatographic separations in gradient mode. The methodology was applied for the impurity profiling of pharmaceutical compounds, following two strategies.A first approach, using short columns (20–50mm) packed with 3.5μm particles and optimized HPLC instrumentation (with reduced extra-column and dwell volumes), was applied for the separation of a pharmaceutical drug and eight related impurities. Special attention was paid to the dwell (gradient delay) volume, which causes the most detrimental effect for transferring a gradient method. Therefore, the dwell volume was simultaneously decreased with the column dead volume. Under optimal conditions, it was possible to reduce the analysis time by a factor of 10, with an acceptable loss in resolution since the column length reduction is less critical in gradient than isocratic mode.The second tested approach was Ultra Performance Liquid Chromatography (UPLC), where sub-2μm particles were used simultaneously with very high pressures (up to 1000bar). A complex pharmaceutical mixture containing 12 compounds was separated in only 1.5min allowing a reduction of the analysis time by a factor of 15 in comparison to a conventional method, with similar peak capacity.
Keywords: High speed liquid chromatography; Method transfer; Gradient mode; Short columns; UPLC; Fast separation
Enhanced skin penetration of P20 phosphopeptide using protein transduction domains
by Luciana B. Lopes; Elizabeth Furnish; Padmini Komalavilas; Brandon L. Seal; Alyssa Panitch; M. Vitória L.B. Bentley; Colleen M. Brophy (pp. 441-445).
Protein transduction domains (PTDs) were recently demonstrated to increase the penetration of the model peptide P20 when the PTD and P20 were covalently attached. Here, we evaluated whether non-covalently linked PTDs were capable of increasing the skin penetration of P20. Two different PTDs were studied: YARA and WLR. Porcine ear skin mounted in a Franz diffusion cell was used to assess the penetration of P20 in the stratum corneum (SC) and viable skin (VS); VS consists of dermis and epidermis without SC. The transdermal delivery of P20 was also assessed. At 1mM, YARA promoted a 2.33-fold increase in the retention of P20 in the SC but did not significantly increase the amount of P20 that reached VS. WLR significantly increased (2.88-fold) the penetration of P20 in VS. Compared to the non-attached form, the covalently linked WLR fragment was two times more effective in promoting the penetration of P20 into VS. None of the PTDs promoted transdermal delivery of P20 at 4h post-application. It was concluded that selected non-covalently linked PTDs can be used as a penetration enhancer, but greater skin penetration efficiency can be achieved by covalently binding the PTD to the therapeutic agent.
Keywords: Protein transduction domains; Skin penetration; Topical delivery; Peptides
Percutaneous release of caffeine from microemulsion, emulsion and gel dosage forms
by M.-A. Bolzinger; S. Briançon; J. Pelletier; H. Fessi; Y. Chevalier (pp. 446-451).
The transport of caffeine to the hypodermis by an alcohol-free o/w microemulsion was investigated and compared with an aqueous gel and an o/w emulsion. The microemulsion was well characterized and in vitro diffusion measurements through pig skin having the hypodermis either kept or removed were performed in static Franz cells. The microemulsion allowed delivery of a large fraction of the caffeine in the hypodermis: 23% of caffeine reached the hypodermis after 24h diffusion, 1.3-fold larger than from the emulsion and gel dosage forms. Half this amount was stored in the hypodermis, the other half continuing its diffusion to the receptor compartment of the Franz cell.
Keywords: Microemulsion; Caffeine; Percutaneous penetration; Hypodermis; Topical delivery