European Journal of Pharmaceutics and Biopharmaceutics (v.68, #3)
Bioavailability and bioequivalence of topical glucocorticoids
by Sandra Wiedersberg; Claudia S. Leopold; Richard H. Guy (pp. 453-466).
Dermatological therapy depends significantly upon the use of topical glucocorticoids (TG). While effective, and often remarkably so, their delivery into the skin is sometimes rather inefficient. The factors which determine drug efficacy, and the various methodologies, by which the rate and extent of TG transport to their sites of action may be assessed, are the foci of this review.
Keywords: Topical glucocorticoids; Bioavailability; Bioequivalence; Percutaneous absorption; Side-effects; Vehicle effects; Potency
Use of cyclodextrins in topical formulations: Practical aspects
by Krzysztof Cal; Katarzyna Centkowska (pp. 467-478).
Cyclodextrins are promising, but using them as an excipients can sometimes be difficult. The aim of this review is to summarize the results of recent studies on the application of parent cyclodextrins (α-, β-, γ-) and some of their derivatives in topical formulations. General properties, current legal status, and toxicological aspects of cyclodextrins are briefly described. The goal of using cyclodextrins to create new formulations with well-known actives, advantages, and limitations in topical formulations is presented, and possible applications in such preparations are also discussed.
Keywords: Cyclodextrins; Topical preparations; Skin penetration; Mucosal delivery; Ocular delivery; Toxicity
Biocompatible poly(methylidene malonate)-made materials for pharmaceutical and biomedical applications
by Pascal Breton; Virginie Larras; Didier Roy; Serge Sagodira; David Limal; David Bonnafous; Nathalie Colin; Nicole Bru; Elias Fattal; Patrick Couvreur (pp. 479-495).
In the past 20 years, mainly with the sponsorship of Laboratoires UPSA (France) and, afterwards, its spin-off company Virsol (France), several authors have studied methylidene malonate-based polymers used in drug delivery approaches and in the development of novel biomaterials. The present paper aims at summing up the preparation of methylidene malonate monomers, and essentially a novel asymmetric diester structure: 1-ethoxycarbonyl-1-ethoxycarbonylmethylenoxycarbonyl ethene named methylidene malonate 2.1.2. Their polymeric and copolymeric derivatives and a few of their applications which were reported in the literature are also presented. It encompasses the manufacturing of particulate systems such as nano- and macroparticles designed for the delivery of hydrophilic or hydrophobic drugs and biomolecules. This review article also describes their use as biomaterials of interest in the fields of tissue repair, as drug reservoirs or ophthalmology, as implants. Copolymers based on these monomers offer a large range of properties and could be used as new surfactants, micellar vectors, or particulate systems for gene delivery. Therefore, this review, certainly the first dedicated exclusively to methylidene malonate-based materials, highlights the great biomedical and pharmaceutical technology potential of these new materials.
Keywords: Methylidene malonate; Polymers; Polymeric materials; Cationic polymers; Drug delivery; Biomaterials; Implants; Particles; Encapsulation
Approval of new biopharmaceuticals 1999–2006: Comparison of the US, EU and Japan situations
by Kaori Tsuji; Kiichiro Tsutani (pp. 496-502).
Biopharmaceuticals, defined as either proteins derived from recombinant DNA technology (rDNAs) or therapeutic monoclonal antibodies (mAbs), have become the therapeutics of significance in the 21st century. This article identifies the new biopharmaceuticals approved in the three major pharmaceutical markets (US, EU and Japan) and analyzes the so-called “drug lag” in said regions. Between 1999 and 2006, a total of 65 new biopharmaceuticals were approved. Of this total, 59 (90.8%) were approved in the US, 52 (80.0%) in EU and 22 (33.8%) in Japan. The mean approval lag was 3.7 months in the US, 7.5 months in EU and 52.6 months in Japan. The US was ahead of the two other regional markets in approvals of biopharmaceuticals, while there was a significant drug lag in Japan. The authors also found that US companies were the licensors of 42 out of 65 new biopharmaceuticals, followed by European companies with 21 licensors and Japanese companies with only 2 licensors. These figures suggest that Japanese companies are still weak in biopharmaceuticals innovation and licensing, and this weakness appears to be a major contributing factor to the drug lag in the country.
Keywords: Drug lag; Three regions (US, EU, Japan); Drug approval; Biopharmaceuticals
Characterization of cisplatin cytotoxicity delivered from PLGA-systems
by Daniel Moreno; Conchita Tros de Ilarduya; Eva Bandrés; María Buñuales; María Azcona; Jesús García-Foncillas; María J. Garrido (pp. 503-512).
Biodegradable lactic acid-glycolic acid copolymer (PLGA) formulations incorporating cisplatin have been developed to evaluate the cytotoxicity of this agent in cultured cells. Two different W/O/W protocols were used to formulate micro- (MP) and nanoparticles (NP) under the solvent evaporation method. Although the amount of cisplatin encapsulated was higher in the MP, the efficiency of encapsulation was similar: 10.33% vs. 11.23%, for both MP and NP, respectively. The “in-vitro” release profiles displayed a significant difference in the initial burst effect, which had a significant impact in the antiproliferative effect of cisplatin. In addition, a duality in the cell cycle distribution was found for both formulations and low doses of free cisplatin (2.5, 10μM) in comparison with the high doses of free cisplatin. The 50μM caused a rapid inhibition of cells growth followed by a significant loss of cells in phases G0/G1 and G2/M which correlated with an increase in the number of cells in sub-G1. However, cisplatin released from controlled formulations induced an accumulation of cells in the phase G2/M. These results led to enhance the caspase-3 activity for MP and NP. These findings indicate that controlled release formulations of cisplatin are able to induce a more effective apoptosis than free cisplatin.
Keywords: Poly(; d; ,; l; -lactic-co-glycolic acid) (PLGA); Microparticles; Nanoparticles; Cisplatin; Drug release; Cytotoxicity; Apoptosis
Chitosan–sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery: Formulation, optimisation and in vitro characterisation
by Sanjay K. Motwani; Shruti Chopra; Sushma Talegaonkar; Kanchan Kohli; Farhan J. Ahmad; Roop K. Khar (pp. 513-525).
Management of extraocular disease is mainly limited by the inability to provide long-term extraocular drug delivery without avoiding the systemic drug exposure and/or affecting the intraocular structures and poor availability of drugs, which may be overcome by prolonging the contact time with the ocular surface, for instance with bioadhesive polymers. In the present study, mucoadhesive chitosan (CS)-sodium alginate (ALG) nanoparticles were investigated as a new vehicle for the prolonged topical ophthalmic delivery of antibiotic, gatifloxacin. A modified coacervation or ionotropic gelation method was used to produce gatifloxacin-loaded submicroscopic nanoreservoir systems. It was optimised using design of experiments by employing a 3-factor, 3-level Box-Behnken statistical design. Independent variables studied were the amount of the bioadhesive polymers: CS, ALG and the amount of drug in the formulation. The dependent variables were the particle size, zetapotential, encapsulation efficiency and burst release. Response surface plots were drawn, statistical validity of the polynomials was established and optimised formulations were selected by feasibility and grid search. Nanoparticles were characterised by FT-IR, DSC, TEM and atomic force microscopy. Drug content, encapsulation efficiency and particle properties such as size, size distribution (polydispersity index) and zetapotential were determined. The designed nanoparticles have average particle size from 205 to 572nm (polydispersity from 0.325 to 0.489) and zetapotential from 17.6 to 47.8mV. Nanoparticles revealed a fast release during the first hour followed by a more gradual drug release during a 24-h period following a non-Fickian diffusion process. Box-Behnken experimental design thus facilitated the optimisation of mucoadhesive nanoparticulate carrier systems for prolonged ocular delivery of the drug.
Keywords: Optimisation; Response surface methodology; Box-Behnken design; Formulation; Mucoadhesion; Nanoparticles
Nasal absorption enhancement of insulin using PEG-grafted chitosan nanoparticles
by Xinge Zhang; Huijie Zhang; Zhongming Wu; Zhen Wang; Haimei Niu; Chaoxing Li (pp. 526-534).
The objective of this work was to explore the potential of polyethylene glycol-grafted chitosan (PEG- g-chitosan) nanoparticles as a system for improving the systemic absorption of insulin following nasal administration. Insulin-loaded PEG- g-chitosan nanoparticles were prepared by the ionotropic gelation of PEG- g-chitosan solution using tripolyphosphate ions as the crosslinking agent. The nanoparticles were in the size range 150–300nm, had a positive electrical charge (+16 to +30mV) and were associated with insulin (loading efficiency 20–39%). The physicochemical properties of nanoparticles were affected by the composition of the copolymer. In vitro insulin release studies showed an initial burst followed by a slow release of insulin. Intranasal administration of PEG- g-chitosan nanoparticles in rabbits enhanced the absorption of insulin by the nasal mucosa to a greater extent than a suspension of insulin-PEG- g-chitosan and control insulin solution. PEG- g-chitosan nanoparticles are promising vehicles for insulin transport through the nasal mucosa.
Keywords: PEG-; g; -chitosan; Nanoparticles; Nasal delivery; Insulin; Blood glucose; Absorption enhancement
Cyclosporine-loaded solid lipid nanoparticles (SLN®): Drug–lipid physicochemical interactions and characterization of drug incorporation
by R.H. Müller; S.A. Runge; V. Ravelli; A.F. Thünemann; W. Mehnert; E.B. Souto (pp. 535-544).
Solid lipid nanoparticles (SLN) were produced loaded with cyclosporine A in order to develop an improved oral formulation. In this study, the particles were characterized with regard to the structure of the lipid particle matrix, being a determining factor for mode of drug incorporation and drug release. Differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS) measurements were employed for the analysis of the polymorphic modifications and mode of drug incorporation. Particles were produced using Imwitor®900 as lipid matrix (the suspension consisted of 10% particles, 8% Imwitor®900, 2% cyclosporine A), 2.5% Tagat S, 0.5% sodium cholate and 87% water. DSC and WAXS were used to analyse bulk lipid, bulk drug, drug incorporated in the bulk and unloaded and drug-loaded SLN dispersions. The processing of the bulk lipid into nanoparticles was accompanied by a polymorphic transformation from the β to the α-modification. After production, the drug-free SLN dispersions converted back to β-modification, while the drug-loaded SLN stayed primarily in α-modification. After incorporation of cyclosporine A into SLN, the peptide lost its crystalline character. Based on WAXS data, it could be concluded that cyclosporine is molecularly dispersed in between the fatty acid chains of the liquid-crystalline α-modification fraction of the loaded SLN.
Keywords: Solid lipid nanoparticles; Cyclosporine A; Characterization; Oral bioavailability; Lipid polymorphism
Novel cationic solid lipid nanoparticles enhanced p53 gene transfer to lung cancer cells
by Sung Hee Choi; Su-Eon Jin; Mi-Kyung Lee; Soo-Jeong Lim; Jeong-Sook Park; Byung-Gyu Kim; Woong Shick Ahn; Chong-Kook Kim (pp. 545-554).
Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in lung cancer. However, there have been few reports of nonviral vector-mediated p53 gene delivery in lung cancer. A new formulation of cationic solid lipid nanoparticles (SLNs) for gene delivery was produced by the melt homogenization method with slight modification, and the SLNs were formulated by mixing tricaprin (TC) as a core, 3β[ N-( N′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidylethanolamine (DOPE) and Tween 80 in various ratios. Plasmid DNA (pp53-EGFP)/SLNs complexes were transfected into human non-small cell lung cancer cells (H1299 cells) and transfection efficiency was determined by FACS analysis. The gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The cellular growth inhibition and apoptosis of treated cells with pp53-EGFP/SLNs complexes were assessed by trypan blue exclusion assay and annexin V staining, respectively. In vivo biodistribution of plasmid DNA was investigated by PCR and RT-PCR. The transfection efficiency of SLN1 (TC:DC-Chol:DOPE:Tween 80=0.3:0.3:0.3:1), which showed the highest transfection efficiency among the SLN formulations, was higher than that of commercially available Lipofectin®. The SLNs-mediated transfection of the p53 gene resulted in efficient high levels of wild-type p53 mRNA and protein expression levels in H1299 cells. The efficient reestablishment of wild-type p53 function in lung cancer cells restored the apoptotic pathway. Taken together, our results reveal that cationic SLN-mediated p53 gene delivery may have potential for clinical application as a nonviral vector-mediated lung cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.
Keywords: p53; Nonviral vector; Cationic SLN; Lung cancer
Grafting of abciximab to a microbubble-based ultrasound contrast agent for targeting to platelets expressing GP IIb/IIIa – Characterization and in vitro testing
by A. Della Martina; E. Allémann; T. Bettinger; P. Bussat; A. Lassus; S. Pochon; M. Schneider (pp. 555-564).
Abciximab-grafted ultrasound sensitive microbubbles were developed for the diagnosis of stroke. The antibody fragment abciximab, which binds to the GP IIb/IIIa and αvβ3 receptors expressed by activated platelets, was chosen because most ischemic strokes are due to arterial thrombi that are mainly composed of platelets. The abciximab antibody fragment was activated by reduction of the disulfide bond for grafting on the microbubbles. The suspension was freeze-dried after the grafting was performed directly on the formed microbubbles. Quantification of the amounts of abciximab present on the surface of the microbubbles was assessed semi-quantitatively by flow cytometry, and quantitatively using radio-labeled abciximab. A protocol for human and rat platelets deposition and fixation was implemented and the expression of the GP IIb/IIIa receptor was validated by immunostaining. The abciximab-grafted microbubbles showed high static and dynamic binding to fixed platelets. Detection by ultrasonography of microbubbles bound on white and red clots gave higher signals compared to SonoVue microbubbles.
Keywords: Abbreviations; AEC Kit; aminoethyl carbazole staining kit; BAC-SulfoNHS; biotinamido hexanoic acid 3-sulfo-; N; -hydroxysuccinimide ester sodium salt; BSA; bovine serum albumin; DMSO; dimethyl sulfoxide; DPPE-MPB; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine-; N; -[4-(; p; -maleimido-phenyl)butyramide] sodium salt; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)] sodium salt; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; EDTA; ethylenediaminetetraacetic acid tetrasodium salt dihydrate; Extravidin-HRP; horseradish peroxidase-labeled extravidin; Fab; antigen-binding fragment; Go Anti-Hu FITC; anti-human IgG FITC-labeled goat IgG; GVA; glycerol vinyl alcohol aqueous mounting solution; Hu NonSpe Fab; non-specific human Fab; PBS; phosphate-buffered saline; RtS; normal rat serum; TCEP; tris(2-carboxyethyl)-phosphine hydrochlorideTargeted microbubbles; Abciximab (ReoPro); Ultrasound contrast agents; Activated platelets; GP IIb/IIIa receptor; Static binding; Dynamic binding; Binding on clots
Chitosan coated Ca–alginate microparticles loaded with budesonide for delivery to the inflamed colonic mucosa
by Maja Simonoska Crcarevska; Marija Glavas Dodov; Katerina Goracinova (pp. 565-578).
Using a novel one-step spray-drying process uncoated and Eudragit® S 100 coated chitosan–Ca–alginate microparticles efficiently loaded with budesonide (BDS), with bioadhesive and controlled release properties in GIT, were prepared. Microparticles were spherical with mean particle size of 4.05–5.36μm, narrow unimodal distribution and positive surface charge. A greater extent of calcium chloride limited the swelling ratio of beads, while swelling behaviour of coated beads was mainly determined by properties of enteric coating. Comparing the release profiles of formulations, under different pH conditions, influence of polymer properties and concentration of cross-linker on the rate and extent of drug release was evident. Coating has successfully sustained release of BDS in buffers at pH 2.0 and 6.8, while providing potential for efficient release of BDS at pH 7.4. Release data kinetics indicated influence of erosion and biodegradation of polymer matrix on drug release from microparticles. Prepared formulations were stable for 12months period at controlled ambient conditions.In conclusion coated microparticles prepared by one-step spray-drying procedure could be suitable candidates for oral delivery of BDS with controlled release properties for local treatment of inflammatory bowel diseases.
Keywords: Alginate; Chitosan; Budesonide; Spray-drying; Controlled release
Characterization and physical stability of fast-dissolving microparticles containing nifedipine
by Francesco Cilurzo; Francesca Selmin; Paola Minghetti; Chiara G.M. Gennari; Francesco Demartin; Luisa Montanari (pp. 579-588).
The suitability of a poly(sodium methacrylate, methyl methacrylate) (NaPMM), a novel mucoadhesive material, to prepare fast-dissolving microparticles containing nifedipine (NIF) in the range of 25–75% w/w was verified. Microparticles made of a low-viscosity hydroxypropylmethylcellulose (HPMC), were also prepared to compare the NIF release profile and bioadhesive properties. The release test was carried out in oversaturation conditions. The physical state of microparticles was also investigated. The formulation stability was evaluated over a 3-month period in long-term and accelerated conditions. The presence of amorphous NIF within freshly prepared microparticles was attributed to interactions between the drug and both polymers. NaPMM conferred to microparticles suitable mucoadhesive properties and significantly increased NIF dissolution rate in comparison to HPMC. Nevertheless, NIF apparent solubilities obtained by NaPMM microparticles were lower than those obtained by the HPMC set. After 3-month storage in the case of HPMC microparticles, NIF dissolution rate and supersaturation degree significantly decreased due to drug crystallization. As far as NaPMM microparticles are concerned, neither NIF dissolution rate nor apparent solubility significantly changed.
Keywords: Nifedipine; Mucoadhesion; Solid state stability; Microparticles; Poly(sodium methacrylate, methyl methacrylate); Supersaturation
Controlled release of PEI/DNA complexes from PLGA microspheres as a potent delivery system to enhance immune response to HIV vaccine DNA prime/MVA boost regime
by Xianfeng Zhou; Bin Liu; Xianghui Yu; Xiao Zha; Xizhen Zhang; Xueyun Wang; Yinghua Jin; Yongge Wu; Yue Chen; Yaming Shan; Yan Chen; Junqiu Liu; Wei Kong; Jiacong Shen (pp. 589-595).
A novel approach involving the preparation of biodegradable PLGA microspheres containing entrapping complexes of DNA and polyethylenimine was developed to improve the delivery of DNA into antigen-presenting cells after intramuscular injection. Compared to the traditional biodegradable microspheres which release naked DNA, these microspheres released intact and penetrative PEI/DNA complexes over a period of 2 weeks in vitro. In addition, the DNA was not degraded during encapsulation in the PLGA microspheres, owing to the protection of polyethylenimine. After i.m. immunization, the microspheres induced significantly enhanced serum antibody responses 2–3 orders of magnitude greater than naked DNA. Additionally, in contrast to naked DNA, the microspheres induced potent CTL responses at a low dose.
Keywords: Abbreviations; PLGA; poly(; d; ,; l; -lactide-co-glycolide); APCs; antigen-presenting cells; i.m.; intramuscular; DCs; dendritic cells; CTL; cytotoxic T lymphocyte; HIV; human immunodeficiency virus; MΦs; macrophages; PEI, polyethylenimine; DLS; dynamic light scattering; SEM; Scanning electron microscopy; TA; anterior tibialis; LDH; lactate dehydrogenasePLGA microspheres; Solvent emulsification-diffusion; DNA vaccine; PEI/DNA complexes; Controlled release; Immunogenicity
The effect of core composition in biodegradable oligomeric micelles as taxane formulations
by Myrra G. Carstens; Pascal H.J.L.F. de Jong; Cornelus F. van Nostrum; Johan Kemmink; Ruud Verrijk; Leo G.J. de Leede; Daan J.A. Crommelin; Wim E. Hennink (pp. 596-606).
Docetaxel (DCTX) and paclitaxel (PTX) are very potent anti-cancer drugs, but the currently marketed formulations, Taxotere® and Taxol®, respectively, are associated with vehicle-related toxicity. An attractive alternative to formulate these hydrophobic cytotoxic agents are polymeric micelles. In this study, the loading of taxanes into oligomeric micelles composed of mPEG750- b-oligo(ε-caprolactone)5 (mPEG750- b-OCL5) with a hydroxyl (OH), benzoyl (Bz) or naphthoyl (Np) end group was investigated. Next, the release characteristics and cytotoxicity of the loaded micelles were studied. MPEG750- b-OCL5-OH micelles loaded with taxanes formed unstable particles with rapid leakage of the drug. In contrast, the presence of an aromatic end group (Bz or Np) resulted in the formation of small (10nm), almost monodisperse micelles with stable encapsulation of 10% (w/w) of PTX or DCTX. This was ascribed to a better compatibility between the micellar core and the drug as compared to the oligomers with the hydroxyl end group.1H NMR studies showed that the micellar core was liquid, and that PTX was molecularly dissolved in the core. The in vitro stability was studied in PBS at 37°C, which showed that leakage of PTX from 10% and 5% (w/w) loaded mPEG750- b-OCL5-Bz micelles started after 8 and 24h, respectively. The presence of albumin did not affect the stability, suggesting that the micelles are not destabilised and the drug was not extracted from the micellar core by this protein. The in vitro cytotoxic effect of the taxane-loaded micelles on C26 carcinoma cells was comparable to that of the commercial formulations, but the empty micelles were far less toxic than the Cremophor EL® vehicle. The results show that mPEG- b-oligo(ε-caprolactone) micelles hold good promise for the formulation of taxanes.
Keywords: Formulation; Polymeric drug carrier; Micelle; Poly(ethylene glycol)-oligo(ε-caprolactone); Paclitaxel; Docetaxel; Particle size; Particle stability; Drug solubilisation; Core composition
A novel liposomal irinotecan formulation with significant anti-tumour activity: Use of the divalent cation ionophore A23187 and copper-containing liposomes to improve drug retention
by Euan Ramsay; Jehan Alnajim; Malathi Anantha; Jason Zastre; Hong Yan; Murray Webb; Dawn Waterhouse; Marcel Bally (pp. 607-617).
We determined whether the method used to encapsulate irinotecan into 1,2-distearoyl- sn-glycero-phosphocholine/cholesterol (DSPC/Chol; 55:45mol%) liposomes influenced: (i) irinotecan release rate and (ii) therapeutic efficacy. DSPC/Chol (55:45mol%) liposomes were prepared with: (i) unbuffered CuSO4; (ii) buffered (pH 7.5) CuSO4; (iii) unbuffered MnSO4 and the ionophore A23187 (exchanges internal metal2+ with external 2H+ to establish and maintain a transmembrane pH gradient); and (iv) unbuffered CuSO4 and ionophore A23187. All formulations exhibited >98% irinotecan encapsulation (0.2 drug-to-lipid molar ratio; 10min incubation at 50°C). Following a single intravenous injection (100mg/kg irinotecan) into Balb/c mice, the unbuffered CuSO4 plus A23187 formulation mediated a half-life of irinotecan release of 44.4h; a ⩾4-fold increase compared to the other liposome formulations. This surprising observation demonstrated that the CuSO4 plus A23187 formulation enhanced irinotecan retention compared to the MnSO4 plus A23187 formulation, indicating the importance of the divalent metal. A single dose of the CuSO4 plus A23187 formulation (50mg/kg irinotecan) mediated an 18-fold increase in median T− C (the difference in days for treated and control subcutaneous human LS 180 adenocarcinoma xenografts to increase their initial volume by 400%) when compared to a comparable dose of Camptosar®. Improved irinotecan retention was associated with increased therapeutic activity.
Keywords: Copper; Liposome; Irinotecan; A23187; In vivo
Folate-tethered emulsion for the target delivery of retinoids to cancer cells
by Sung-Hoon Kim; Jin-Ki Kim; Soo-Jeong Lim; Jeong-Sook Park; Mi-Kyung Lee; Chong-Kook Kim (pp. 618-625).
Folic acid, conjugated to poly(ethylene glycol)-distearoylphosphatidylethanolamine (folate-PEG-DSPE), was used to target emulsions of all- trans retinoic acid (ATRA) to folate receptor-overexpressing tumor cells. Two kinds of ATRA-incorporated folate-tethered emulsions (ATRA-FTE2000/3400) were prepared using soybean oil, egg phosphatidylcholine and folate-PEG-DSPE with different PEG length. As a control, ATRA-incorporated non-tethered emulsion (ATRA-NTE) was prepared by using PEG2000-DSPE without folate instead of using folate-PEG-DSPE. The mean particle diameters of ATRA-FTE2000/3400 were about 100–130nm. The cellular uptake in KB cells of fluorescence-labeled ATRA-FTE3400 was determined with HPLC (for ATRA) and confocal microscopy (for lipid emulsion). The growth inhibitory activity of ATRA was evaluated by MTT assay. The folate ligands in emulsion increased the cellular uptake of ATRA about 3-fold and 1.6-fold in ATRA-FTE3400 and ATRA-FTE2000, respectively. Growth inhibitory activity of ATRA-FTE3400 in KB cells was higher than that of ATRA-NTE at the same dose. Whereas the growth inhibitory effect in MCF-7 cells of ATRA was similar between ATRA-NTE and ATRA-FTE3400. The addition of free folate significantly reduced the uptake of ATRA regardless of the length of PEG attached to folate. Folate-tethered lipid emulsion showed effective and selective delivery to the folate receptor-abundant carcinomas, suggesting a potential for targeted delivery of anticancer agents.
Keywords: Folic acid; Folate receptor; All-; trans; retinoic acid; Folate-tethered emulsion
Temperature-sensitive hydrogels composed of chitosan and hyaluronic acid as injectable carriers for drug delivery
by Jia-You Fang; Jyh-Ping Chen; Yann-Lii Leu; Jiuan-Wen Hu (pp. 626-636).
Temperature-sensitive hydrogels composed of poly( N-isopropylacrylamide) (PNIPAAm) with chitosan (CPN) and chitosan+hyaluronic acid (CPNHA) were grafted in order to examine their physicochemical characteristics, in vitro drug release, and in vivo pharmacodynamics. The sol–gel transition behavior was investigated by UV/visible spectrophotometry, differential scanning calorimetry, and viscometry. A slight difference in the transition temperatures was observed among these polymer systems, with CPN and CPNHA exhibiting higher temperatures compared with PNIPAAm. A zeta potential determination revealed a positive charge for the CPN hydrogel, whereas no or only a negligible charge was observed for PNIPAAm and CPNHA. The entanglement of CPN hydrogels observed using scanning electronic microscopy showed the densest cross-linkage structure, followed by CPNHA and PNIPAAm. Both hydrophilic and lipophilic drugs, including nalbuphine, indomethacin, and the nalbuphine prodrug, were used as model drugs in an in vitro drug release experiment. All 3 hydrogels significantly prolonged drug release. The release rate of hydrophilic nalbuphine increased in the order CPN
Keywords: Temperature-sensitive hydrogel; Chitosan; Hyaluronic acid; Controlled release; Nalbuphine
Dexamethasone-loaded poly(ε-caprolactone) intravitreal implants: A pilot study
by Silvia Ligório Fialho; Francine Behar-Cohen; Armando Silva-Cunha (pp. 637-646).
Purpose: Poly(ε-caprolactone) (PCL) is a biodegradable and biocompatible polymer that presents a very low degradation rate, making it suitable for the development of long-term drug delivery systems. The objective of this pilot study is to evaluate the feasibility and characteristics of PCL devices in the prolonged and controlled intravitreous release of dexamethasone. Methods: The in vitro release of dexamethasone was investigated and the implant degradation was monitored by the percent of mass loss and by changes in the surface morphology. Differential scanning calorimetry was used to evaluate stability and interaction of the implant and the drug. The short-term tolerance of the implants was studied after intravitreous implantation in rabbit eye. Results: PCL implant allows for a controlled and prolonged delivery of dexamethasone since it releases 25% of the drug in 21 weeks. Its low degradation rate was confirmed by the mass loss and scanning electron microscopy studies. Preliminary observations show that PCL intravitreous implants are very well tolerated in the rabbit eye. Conclusion: This study demonstrates the PCL drug delivery systems allowed to a prolonged release of dexamethasone in vitro. The implants demonstrated a strikingly good intraocular short-term tolerance in rabbits eyes. The in vitro and preliminary in vivo studies tend to show that PCL implants could be of interest when long-term sustained intraocular delivery of corticosteroids is required.
Keywords: Poly(ε-caprolactone); Implant; Prolonged release; In vitro; study; Short-term tolerance
Guanidinium-grafted polyethylenimine: An efficient transfecting agent for mammalian cells
by Surendra Nimesh; Ramesh Chandra (pp. 647-655).
Polyethylenimine (PEI) is one of the most efficient polycationic non-viral gene delivery vectors. Its efficiency and cytotoxicity depends on molecular weight, with the 25-kDa PEI being most efficient but accompanied with cytotoxicity. In the present study, enhancement in gene delivery efficiency along with reduction in cytotoxicity by attachment of guanidinium side group was explored. The hypothesis was that the guanidination would lead to the delocalization of charge present on primary amines of the polymer thereby leading to enhancement in gene delivery efficiency along with reduction in cytotoxicity. The polymer was guanidinated using O-methylisourea hemisulfate and the chemical linkage characterized by FTIR spectroscopy. The hydrodynamic diameter of guanidinated PEI–DNA complexes was determined using DLS. Subsequently, these complexes were used for DNA binding assay and zeta-potential measurements, taking native PEI as reference. Further, guanidinated PEI–DNA complexes were investigated for their gene delivery efficacy on HEK 293 cells. The hydrodynamic diameter of guanidinated PEI–DNA complexes was found to be in the range of 176–548nm. As expected, the zeta potential values increased, on increasing the N/P ratios. It was found that guanidinated PEI had higher transfection efficiency at the majority of the N/P ratios tested as compared to commercially available transfecting agent lipofectin and native PEI itself. The toxicity of guanidinated PEI–DNA complexes was also reduced considerably in comparison to PEI polymer, as determined by MTT colorimetric assay. Out of the various derivatives prepared, gPEI 56% was found to be the most efficient in in vitro transfection.
Keywords: Polyethylenimine; Guanidination; Polyplexes; Transfection; Cytotoxicity
Supramolecular association of recombinant human growth hormone with hydrophobized polyhydroxyethylaspartamides
by Stefano Salmaso; Rodolfo Schrepfer; Gennara Cavallaro; Sara Bersani; Francesca Caboi; Gaetano Giammona; Giancarlo Tonon; Paolo Caliceti (pp. 656-666).
The protein delivery properties of polymer supramolecular assemblies were investigated by using recombinant human growth hormone (rh-GH) and two polyhydroxyethylaspartamide (PHEA) derivatives: (a) PHEA-C16 obtained by PHEA random grafting with hexadecylalkylamine; (b) PHEA-PEG5000-C16 obtained by PHEA random co-grafting with hexadecylalkylamine and 5kDa poly(ethylene glycol). The two polymers possessed similar self-assembling properties: critical micelle concentration (CMC) and particle size. The protein loading (protein/polymer, w/w, %) was 12.1±1.3% and 8.5±0.4% with PHEA-C16 and PHEA-PEG5000-C16, respectively. The rh-GH/polymer association constant calculated by Scatchard analysis was 1.87×105M−1 with PHEA-C16 and 0.27×105M−1 with PHEA-PEG5000-C16. The Klotz analysis showed that 5 PHEA-C16 and 9 PHEA-PEG5000-C16 polymer chains associated with one protein molecule. The protein dissociation from the PHEA-C16 and PHEA-PEG5000-C16 supramolecular complexes was complete in about 350–450 and 450–550h, respectively. With both polymers, the protein release was faster as the protein/polymer ratio increased. Pharmacokinetic studies were performed by subcutaneous administration to rats of protein/polymer solutions at different w/w ratios (1:75 and 1:150). Both polymer formulations slowed the protein absorption. The protein bioavailability increased as the protein/polymer complex stability decreased and the protein/polymer w/w ratio increased indicating that efficient protein delivery can be achieved by proper polymer choice and formulation composition.
Keywords: Protein delivery; Supramolecular assembly; Growth hormone; Polyhydroxyethylaspartamide
Improved peroral delivery of glucagon-like peptide-1 by site-specific biotin modification: Design, preparation, and biological evaluation
by Yu Seok Youn; Su Young Chae; Seulki Lee; Min Jung Kwon; Han Jong Shin; Kang Choon Lee (pp. 667-675).
Peptide oral delivery is still a significant challenge because of two major impediments, low absorption efficiency and instability in gastrointestinal tract. The aim of this study was to design, prepare, and evaluate an intestine enzyme-resistant, superior intestine absorptive, and biologically preserved glucagon-like peptide-1 (GLP-1) derivative using the site-specific modification of biotin, which can take advantage of the carrier-mediated active intestine transport. Two series of site-specific Lys34- and Lys26,34–biotin–GLP-1 derivatives were prepared, and their intestine membrane permeabilities, proteolytic stabilities against the intestine enzymes, and bioactivities were then evaluated. Especially, Lys26,34–biotin–GLP-1 was found to have the most promising scores: (i) it displayed a 5.6-fold higher Caco-2 cell monolayer permeability than GLP-1; (ii) it showed an 8.5- and 3.5-fold longer half-life than GLP-1 in rat intestine fluid and homogenate, respectively; and interestingly (iii) it had a well-preserved insulinotropic activity (94.5% vs. GLP-1) in the rat islets. Finally, Lys26,34–biotin–GLP-1 showed a 9-fold higher oral hypoglycemic efficacy (25.3%) than native GLP-1 (2.7%) ( P<0.005) after direct peroral administration into type 2 diabetic db/db mice. This study highlights the oral hypoglycemic potential of site-specific Lys26,34-biotinylated GLP-1, and this orally available analogue would find a role in the treatment of type 2 diabetes.
Keywords: Glucagon-like peptide-1; Oral peptide delivery; Biotin; Site-specific modification; Oral hypoglycemic efficacy; Type 2 diabetes
Cell line-dependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors
by Kimberly L. Douglas; Ciriaco A. Piccirillo; Maryam Tabrizian (pp. 676-687).
It has been suggested that cell physiology may affect the internalization pathways of non-viral vectors, leading to cell line-dependent transfection efficiency. To verify this hypothesis, fluorescently labeled alginate–chitosan nanoparticle complexes were used as non-viral vectors to transfect 293T, COS7, and CHO cells and to observe the cellular interactions and internalization mechanisms of the complexes in each cell line. 293T cells, which demonstrate the highest transfection efficiency, internalize complexes primarily through clathrin-mediated processes. COS7 cells also demonstrate some internalization of complexes through the clathrin-dependent pathway, explaining the moderate transfection exhibited. In contrast, CHO cells internalize complexes predominantly through caveolin-mediated pathways and are not transfected. Results suggest that following clathrin-mediated endocytosis, complexes are trafficked to the endo-lysosomal pathway, where the proton-sponge effect leads to their release into the cytosol. Contrarily, the absence of trafficking to this pathway following caveolin-mediated endocytosis results in vesicle-entrapped complexes that become transfection-incompetent. These results demonstrate that cell physiology is a critical factor in efficient transfection, and that trafficking to the endo-lysosomal pathway through specific internalization mechanisms is essential for transfection with alginate–chitosan nanoparticle complexes.
Keywords: Non-viral vector; Internalization; Transfection; Intracellular trafficking; Caveolin; Clathrin
Liver targeting and anti-HBV activity of reconstituted HDL–acyclovir palmitate complex
by Meiqing Feng; Qinsheng Cai; Hai Huang; Pei Zhou (pp. 688-693).
High-density lipoprotein (HDL) particles deliver cholesterol from periphery tissues to liver in human body through specifically binding to a receptor on the surface of hepatocytes. Therefore, HDL particles can be potentially used to target drugs to liver. We synthesized the acyclovir palmitate as the pro-drug of acyclovir and incorporated it into recombinant HDL to form reconstituted HDL (rHDL)–acyclovir palmitate complex. The efficiency of the encapsulation of acyclovir palmitate was high, reached 97% when the acyclovir palmitate and phosphatidylcholine (PC) ratio was 1:20. In an in vitro HBV inhibition assay, 0.0022μmol/ml rHDL–acyclovir palmitate showed 20% inhibition of HBV. To reach the same level of inhibition, 20 times, 40 times, and 200 times more acyclovir palmitate liposome, acyclovir liposome, and free acyclovir were needed, respectively. Rat body distribution study showed that 71.2% of the dose was recovered in the liver, 10.2% of the dose was in the plasma, and 18.6% was in the rest of the body at 30min after injection. These results indicated that rHDL–acyclovir palmitate complex had strong liver targeting property, suggesting that reconstituted HDL could be used to deliver drugs to treat liver diseases.
Keywords: Apolipoprotein A-I; HDL; Liver targeting; Acyclovir; Acyclovir palmitate
Evaluation of brain-targeting for the nasal delivery of ergoloid mesylate by the microdialysis method in rats
by Jian Chen; Xiaomei Wang; Juan Wang; Guangli Liu; Xing Tang (pp. 694-700).
The aim of this study was to quantify the nasal delivery of ergoloid mesylate (EM) to the brain by comparing cerebrospinal fluid (CSF) and plasma EM levels after nasal administration at a dose of 4mg/kg with those after intravenous administration. Following nasal delivery, EM reached a Cmax value (mean±SD) in plasma of 348.41±19.47ng/ml and in CSF of 87.35±6.37ng/ml after 107 and 20min, respectively, while after intravenous injection, EM reached a Cmax value (mean±S.D.) in CSF of 54.81±4.92ng/ml at 60min and the Cmax in plasma was 1255.51±133.59ng/ml. The AUCCSF/AUCplasma ratio (0.48±0.05) after intranasal delivery differed greatly from the ratio (0.14±0.04) observed after intravenous injection ( P<0.05). The further analyzed data demonstrated a statistically significant distribution advantage of EM to the brain via the nasal route, and further suggesting that nasal administration can be a promising alternative for EM that undergoes first-pass metabolism following oral administration.
Keywords: Ergoloid mesylate; Nasal delivery; Microdialysis; Brain-targeting; RP-HPLC with fluorescence detection
Solubilization of indomethacin using hydrotropes for aqueous injection
by Akhilesh Kumar Jain (pp. 701-714).
Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) that exhibits analgesic, antipyretic and anti-inflammatory activities. It is practically insoluble in water. The effect of various hydrotropes such as urea, nicotinamide, resorcinol, sodium benzoate and sodium p-hydroxy benzoate on the solubility of indomethacin was investigated. The solubility enhancement of indomethacin by the hydrotropes was observed in decreasing order as sodium p-hydroxy benzoate>sodium benzoate>nicotinamide>resorcinol>urea. In order to elucidate the probable mechanism of solubilization, various solution properties of hydrotropes such as viscosity, specific gravity, surface tension, refractive index and specific conductance of hydrotropic solutions were studied at 25±2°C. Each solubilized product was characterized by ultraviolet, infrared, powder X-ray diffraction and differential scanning calorimetry techniques. The hydrotropic solubilization of indomethacin at lower hydrotrope concentration may be attributed to weak ionic interactions while that at higher hydrotrope concentration may be due to molecular aggregation. Aqueous injectable formulations using sodium p-hydroxy benzoate, sodium benzoate and nicotinamide as hydrotropes were developed and studied for physical and chemical stability.
Keywords: Hydrotropic solubilization; Indomethacin; Aqueous injection; Cyclo-oxygenase (COX)
A novel approach for the control of drug release rate through hydrogel membrane: I. Effect of drug immobilization on drug release rate by copolymerization method
by Lei Shang; Sam Zhang; Hejun Du; Subbu S. Venkatraman (pp. 715-723).
Precise control of drug release rate in hydrogel drug delivery systems to better mimic physiological condition is a challenging research topic in development of Advanced Drug Delivery Systems. One of the major issues with bioresponsive drug delivery systems is the excessive ‘leakage’ of drug while the system is in the ‘off’ state, which leads to shortening of the device life-time and potential overdose problem for the patient. In the present study, a new approach, based on partition effects, termed drug immobilization via copolymerization, is proposed to control the drug release rate of membrane-based drug delivery systems. In this method, a certain level of drug is pre-immobilized in the membrane through copolymerization. The immobilized drug contributes to the overall chemical potential of drug molecules in the membrane but their mobility is restricted, hence will not be released. At equilibrium, the amount of drug from donor that dissolved in the membrane is reduced due to contribution of immobilized drug, resulting in an effective reduction in partition coefficient and hence the release rate. The testing of the method by bovine serum albumin (BSA) as a model drug confirmed the controllability of the method: almost 35% reduction of the drug leakage in the ‘off-state’ was observed when 20mg BSA was immobilized in the pH-sensitive hydrogel membrane. The mathematical model of the drug partition in the membrane was modified to describe the new partition phenomenon (mobile drug and immobilized drug in the membrane) in this study.
Keywords: Controlled drug delivery; Hydrogel; Release rate control; Burse release; Partition coefficient
Effect of abrasion induced by a rotating brush on the skin permeation of solutes with varying physicochemical properties
by Franklin K. Akomeah; Gary P. Martin; Andy G. Muddle; Marc B. Brown (pp. 724-734).
A transient reduction in the barrier nature of the skin can be a pre-requisite for successful (trans)dermal delivery of some drugs. The aim of this present study was to investigate and effect of a dermal abrading “rotating brush” device on percutaneous absorption and skin integrity. In vitro experiments were conducted using excised human epidermal membrane. The effect of device parameters (bristle type, treatment duration and applied pressure) on skin permeability of model solutes (methyl paraben, butyl paraben, caffeine, acyclovir and angiotensin II) with varying physicochemical properties was examined and compared to established methods of skin penetration enhancement (positive controls). The device parameter which was found to have the most marked effect on permeability of the compounds was bristle type. Profound changes (2- to 100-fold increase) were observed in the epidermal permeability of the hydrophilic penetrants (caffeine, acyclovir and angiotensin II), when the brush device was employed compared to positive controls (ethanol enhancement, delipidisation, iontophoresis and tape-stripping). Findings from this present study support the effectiveness of a rotating brush applied to the skin in enhancing epidermal permeability. Further optimization of operational parameters is required to exploit this simple and effective delivery device.
Keywords: Abrasion; Brush; Human skin; Transdermal delivery; In vitro; diffusion
Synergistically enhanced transdermal permeation and topical analgesia of tetracaine gel containing menthol and ethanol in experimental and clinical studies
by Chao Fang; Yi Liu; Xun Ye; Zheng-xing Rong; Xue-mei Feng; Chan-bing Jiang; Hong-zhuan Chen (pp. 735-740).
The aim of this study is to observe the synergistically enhanced percutaneous penetration and skin analgesia of tetracaine gel containing menthol and ethanol through experimental and clinical studies. Four anesthetic gels containing 4% tetracaine in carbomer vehicle named T-gel (containing no menthol or ethanol), 5%M/T-gel (containing 5% menthol), 70%E/T-gel (containing 70% ethanol, an optimal concentration for antiseptic), and 5%M+70%E/T-gel (containing both 5% menthol and 70% ethanol), respectively, were fabricated. The in vitro mouse skin permeation was investigated using a Franz diffusion cell. The mouse skin morphology was examined by a scanning electron microscope. The in vivo skin analgesic effect in mice was evaluated using the von Frey tests. To determine the efficacy of tetracaine gels for managing the pain in human volunteers, a paralleled, double-blinded, placebo-controlled, randomized controlled trial design combined with verbal pain scores (VPS) was performed. The combination of menthol and ethanol (5%M+70%E/T-gel) conferred significantly higher tetracaine diffusion across full-thickness mouse skin than 5%M/T-gel, 70%E/T-gel, and T-gel. The ultra structure changes of mouse skin stratum corneum treated with 5%M+70%E/T-gel were more marked compared with those of any other tetracaine gel. von Frey tests in mice showed a synergistically enhanced effect of menthol and ethanol on the analgesia of tetracaine gel. The mean VPS were significantly lower for volunteers treated with 5%M+70%E/T-gel than those receiving other gels or the EMLA cream. 5%M+70%E/T-gel possessed the shortest anesthesia onset time, the longest anesthesia duration and the strongest anesthesia efficacy. Seventy percent ethanol in 5%M+70%E/T-gel not only improved the analgesic efficacy of the tetracaine gel through synergistically enhanced percutaneous permeation with menthol but also served as an antiseptic agent keeping drug application site from infection. 5%M+70%E/T-gel is a potential topical anesthesia preparation for clinical use.
Keywords: Menthol; Ethanol; Tetracaine gel; Synergistic effect; Transdermal permeation
Formulation of an intermediate product from human serum albumin for the production of a solid dosage form
by Katalin Kristó; János Bajdik; István Erős; Imre Dékány; Zsolt Pallai; Klára Pintye-Hódi (pp. 741-746).
The main objective of this study was to evaluate and to increase the processibility of a model protein (human serum albumin (HSA)) for preparation of an intermediate for a solid dosage form. The applicability of the solid forms is easier, and therefore their formulation is a promising method for the application of proteins. The layering of powdered cellulose with HSA solutions of different concentrations in a fluid bed apparatus with the top spray method was applied. The yield of this technique was very good, independently of the concentration of the applied solution. The HSA covered the particles (the HSA layer formed was smooth), but it caused aggregation of the cellulose particles, and spray-dried microparticles also formed. The proportion of optimum-sized particles (200–315μm) decreased. The largest amount was detected for the samples prepared with liquid containing 15% HSA (about 2 times higher than the second best). Not only the size, but also the shape of the particles was changed. The alteration in this parameter caused a change in the flowability. This was likewise the best for the samples prepared with the liquid containing 15% HSA. The concentration of HSA in the fraction containing smaller particles was higher, because of the abrasion of the particles and the enrichment of the spray-dried HSA. The distribution of HSA in the large particles was uneven. The layering of powder cellulose can be applied to produce an intermediate from HSA for solid dosage forms, but the appropriate concentration of this protein solution must be optimized previously because HSA can act as a binder. The formation of large agglomerates must be eliminated, because the distribution of the active agent in these is very inhomogeneous. The present results indicated that the best value can be achieved with liquid containing between 12.5% (most homogeneous distribution of HSA) and 15% HSA (best flowability).
Keywords: Human serum albumin; Fluidized bed technology; Layering; Powdered cellulose; Solid dosage form
Design and study of ibuprofen disintegrating sustained-release tablets comprising coated pellets
by M.R. Abbaspour; F. Sadeghi; H. Afrasiabi Garekani (pp. 747-759).
One challenge in tableting of sustained-release multiparticulates is maintaining the desired drug release after compaction. The aim of this study was to design sustained-release ibuprofen tablets which upon oral ingestion rapidly disintegrate into sustained-release pellets in which the integrity of the pellet core and/or coat is preserved.First free films composed of Eudragit RS 30D and RL 30D in 4:1 ratio and containing different levels of triethyl citrate (TEC) were prepared and tested to optimize the plasticizer level. Cured Eudragit based pellets with 60% ibuprofen loading which in our previous study showed proper mechanical properties for compression were coated with Eudragit RS 30D/RL 30D (4:1) containing 20% triethyl citrate at different coating levels. The mechanical properties of the coated pellets were tested. Polymer coated pellets were compacted into tablets either alone or with a blend of excipients comprising Avicel, PEG 4000, cross-linked PVP. A 32 full factorial design was used to optimize the filler blend composition. Effects of pellet to filler ratio, compression force and granulation of filler on tablet characteristics were investigated.Results of mechanical test showed that the coating of cured pellets had no significant effect on yield point and elastic modulus of the pellets. In the case of 5% coating level sustained release of ibuprofen over a period of 24h was achieved. The results obtained from tableting procedure showed that by selecting suitable filler blend (60% Avicel, 10% cross-linked PVP and 30% PEG 4000), compression force, and granulation of filler it was possible to prepare sustained-release tablets containing high ratio of coated pellets (even 80%) with desirable strength, disintegration time, and drug release rate. It was observed that compression force, pellet to filler ratio, composition of filler blend and granulation of fillers had no effect on drug release rate from compacted pellets but had significant influence on tablet strength, friability, and disintegration time. SEM graphs and in vitro release profiles for compacted pellets showed no apparent damage to the coated pellets as a result of the compaction process.
Keywords: Ibuprofen; Eudragit; Pellet; Multiparticulate tablet; Sustained release
Process design applied to optimise a directly compressible powder produced via a continuous manufacturing process
by Y. Gonnissen; S.I.V. Gonçalves; B.G. De Geest; J.P. Remon; C. Vervaet (pp. 760-770).
Manufacturing of ‘ready-to-compress’ powder mixtures for direct compression was performed by spray drying, without granulation, milling and/or blending steps in between spray drying and compaction. Powder mixtures containing acetaminophen, mannitol, erythritol, maltodextrin, crospovidone, colloidal silicon dioxide and polyoxyethylene 20 sorbitan monooleate were prepared via co-spray drying. A feed suspension having a solid content of 27.2% w/w was selected for further process optimisation because of its high process yield, excellent flowability and short tablet disintegration time. Experimental design was applied to evaluate processibility, physico-chemical properties and compactability of the spray dried powder mixtures. Significant and adequate regression models were developed for powder flowability, median particle size, bulk density, residual moisture content and process yield. An increasing inlet and outlet drying air temperature improved process yield. However, a higher inlet drying air temperature had a negative influence on density and moisture content, while the latter decreased at higher outlet drying air temperatures. Median particle size increased with a higher inlet temperature, while the outlet temperature had the opposite affect. Numerical optimisation determined the optimal spray drying process (inlet temperature: 221°C, outlet temperature: 81°C and atomisation pressure: 6bar) in order to produce ‘ready-to-compress’ powder mixtures.
Keywords: Co-spray drying; Process design; Continuous processing; Acetaminophen; Carbohydrates; Compression
Formation and physical stability of the amorphous phase of ranitidine hydrochloride polymorphs prepared by cryo-milling
by Norman Chieng; Thomas Rades; Dorothy Saville (pp. 771-780).
The effect of cryo-milling on ranitidine hydrochloride polymorphs form 1 and 2 was investigated with particular interest in the formation and the stability of the amorphous phase. Cryo-milling was carried out using an oscillatory ball mill for periods up to 60min, with re-cooling of the milling chamber with liquid nitrogen at 15min intervals. Results showed that both ranitidine hydrochloride form 1 and form 2 could be fully converted to the amorphous form as determined by XRPD within 30min. Upon 14 days storage, the amorphous samples crystallized back to their original forms. In the stability studies of amorphous drug with seeds, significant polymorphic transformation from form 1 to form 2 was not found when amorphous form prepared from form 1 was seeded with form 2 crystals by gentle physical mixing. In contrast, amorphous form prepared from form 1 seeded with form 2 crystals by ball milling for 1min and simultaneous cryo-milling methods were found to transform amorphous form prepared from form 1 to crystalline form 2 under some storage conditions. The transformation was thought to be facilitated by interaction between seed crystals and amorphous drug and a storage temperature above the Tg. Amorphous form prepared from form 2 did not transform to crystalline form 1 under any conditions used in this study.
Keywords: Ranitidine hydrochloride; Cryo-milling; Amorphous; Stability; Polymorphic transformation; Seeding; Glass transition; Crystallization temperature
Supercritical fluid drying of carbohydrates: Selection of suitable excipients and process conditions
by Andréanne Bouchard; Nataša Jovanović; Gerard W. Hofland; Wim Jiskoot; Eduardo Mendes; Daan J.A. Crommelin; Geert-Jan Witkamp (pp. 781-794).
The processibility of 15 carbohydrates, more or less commonly used, was investigated as excipients in supercritical fluid drying. The focus was on the ability to produce amorphous powder, the stability of the powders towards crystallisation, and the residual water and ethanol content. The aqueous solutions were sprayed into a pressurised carbon dioxide–ethanol mixture flowing cocurrently through a coaxial two-fluid nozzle. The powder characteristics appeared to be influenced by the supersaturation level reached during the SCF-drying process and by the properties of the sugar species, such as water solubility and glass transition temperature, or the solution viscosities. The stability and the residual solvent content of a selected set of sugars and some mixtures were further analysed. The stability of amorphous powders was investigated at 4°C, room temperature, 40 and 50°C. Lactose, maltose, trehalose, raffinose, cyclodextrin, low-molecular-weight dextran and inulin could form free-flowing powders that remained amorphous during the 3-month stability study. Sucrose had to be mixed with other sugars to form a stable amorphous powder. Ethanol could be entrapped in supercritical fluid dried low-molecular-weight sugars, whereas polysaccharide powders were free of ethanol. Measures to prevent or overcome the presence of ethanol are discussed.
Keywords: Aqueous solution; Amorphous powder; Carbohydrates; Excipients; Supercritical carbon dioxide; Crystallisation; Ethanol
The role of solution calorimetry in investigating controlled-release processes from polymeric drug delivery systems
by Stefania Conti; Simon Gaisford; Graham Buckton; Lauretta Maggi; Ubaldo Conte (pp. 795-801).
In this paper we investigate the potential role of solution calorimetry measurements in aiding the formulation of swellable matrices containing a mixture of HPMC and NaCMC, an ionic cellulose derivate. These polymers show a synergistic effect in their ability to modulate drug delivery rates; a matrix containing a 1:1 mixture of NaCMC and HPMC exhibits a significantly slower drug release rate than either polymer shows alone. The exact cause of this synergism is not clear and it is not an easy effect to examine using conventional means (such as dissolution testing). Here, we used solution calorimetry to study the system holistically. By comparing the measured response of a physical blend with a theoretical one (obtained by summation of the power–time data for each material), it was possible to assess if there was/was not any interaction which may explain the synergism. Furthermore, since a thermodynamic quantity was returned it was possible to establish if the interaction was favourable or unfavourable and so to obtain useful information to understand and predict the dissolution behaviour of polymeric systems containing the same materials.An unfavourable interaction was noted between NaCMC and the model drug (Diltiazem HCl); no interaction was seen between HPMC and the drug; and a favourable interaction was recorded when both polymers were formulated with the drug. The trend was mirrored by the t90 (the time required for 90% drug release) values determined from dissolution testing; NaCMC 10.8h, HPMC 16.4h, NaCMC and HPMC 19.1h. The data suggest that solution calorimetry measurements can be used to aid the selection of polymeric excipients to design controlled-release drug delivery systems.
Keywords: Matrix tablets; Drug release kinetics; Controlled release; Solution calorimetry; Drug–polymer interactions
Compaction of lactose drug mixtures: Quantification of the extent of incompatibility by FT-Raman spectroscopy
by Anett Flemming; Katharina M. Picker-Freyer (pp. 802-810).
It is well known that lactoses interact with drugs containing amino groups and undergo the Maillard reaction. Lactose monohydrate may also interact with moisture sensitive drugs and affect the stability of the drug. These interactions were analyzed using three model drugs – Thiaminchloride hydrochloride, Nicotinamide and Acetylsalicylic acid – which interact with spray-dried lactose or anhydrous lactose. FT-Raman spectroscopy was used for the first time to qualitatively and quantitatively analyze these excipient drug interactions in powders and tablets. Both lactoses undergo the Maillard reaction with Thiaminchloride hydrochloride. Nicotinamide did not react with the lactoses because the amide group is protected against the reaction with the lactoses. Only a transition from β- to α-lactose was noticed. The moisture sensitive drug Acetylsalicylic acid remained stable even when the tablets were stored under accelerated conditions (40°C and 75% RH). The crystal water of lactose monohydrate (spray-dried lactose) had no influence on the drug stability but a transition from β- to α-lactose was noticed. In conclusion, FT-Raman spectroscopy is a fast and valuable tool for a quantitative determination of the extents of incompatibility in solid dosage forms.
Keywords: FT-Raman spectroscopy; Maillard; Moisture; Lactose; Thiaminchloride hydrochloride; Nicotinamide; Acetylsalicylic acid
Use of dye as tracer of drug release from medicated chewing gums
by Evelyn Ochoa; Lauretta Maggi; Stefania Conti; Ubaldo Conte; Guy Vergnault; Pascal Grenier (pp. 811-817).
The evaluation of the potential use of a dye as indicator of in vivo drug release from a medicated chewing gum is described. The device is a three-layer tablet obtained by direct compression consisting of a gum core and two external protective soluble layers to prevent gum adhesion to the punches of the tableting machine. The active ingredient and a colour are contained in the gum core. To evaluate the drug and the dye release from the formulations, a chew-out study was performed by a panel of volunteers. The results obtained suggest that the use of a dye could be useful to indicate the chewing time necessary to complete drug delivery from medicated chewing gums.
Keywords: Medicated chewing gum; Three-layer tablet; Dye; Chew-out study; In vivo; drug release
Standardization, evaluation and early-phase method validation of an analytical scheme for batch-consistency N-glycosylation analysis of recombinant produced glycoproteins
by Stefan Zietze; Rainer H. Müller; René Brecht (pp. 818-827).
In order to set up a batch-to-batch-consistency analytical scheme for N-glycosylation analysis, several sample preparation steps including enzyme digestions and fluorophore labelling and two HPLC-methods were established. The whole method scheme was standardized, evaluated and validated according to the requirements on analytical testing in early clinical drug development by usage of a recombinant produced reference glycoprotein (RGP). The standardization of the methods was performed by clearly defined standard operation procedures. During evaluation of the methods, the major interest was in the loss determination of oligosaccharides within the analytical scheme. Validation of the methods was performed with respect to specificity, linearity, repeatability, LOD and LOQ. Due to the fact that reference N-glycan standards were not available, a statistical approach was chosen to derive accuracy from the linearity data. After finishing the validation procedure, defined limits for method variability could be calculated and differences observed in consistency analysis could be separated into significant and incidental ones.
Keywords: Glycosylation; Biotechnology; Validation; Quality control; Pharmaceutical analysis
Monitoring galenical process development by near infrared chemical imaging: One case study
by C. Gendrin; Y. Roggo; C. Spiegel; C. Collet (pp. 828-837).
The objective of this study is to evaluate by NIR imaging the homogeneity of process intermediates obtained with different process parameters during the development of a new pharmaceutical solid form. The process under investigation is a solid dosage form based on extrusion. The parameters are two kinds of crystallizations, two sizes of particle of API, two screw speeds during the extrusion and two milling screens used to reduce the extrudates into a granulate form. Two kinds of intermediates are evaluated: the extrudates and the cores. Two approaches are used to analyze the data: the univariate NIR analysis which consists in wavelength selection and multivariate analysis, i.e., Classical Least Squares (CLS), which takes into account the whole spectra. The univariate method reveals good chemical homogeneity of the extrudates but differences in their physical aspect. CLS shows well-distributed excipients for all the cores; differences in the sizes of the granules have also been revealed. The univariate method can be applied on simple chemical systems such as binary mixtures. When complex samples are analyzed, multivariate analysis is the method of choice. This study demonstrates that NIR imaging can be a useful tool for the optimization of the process and for the selection of the final parameters of the process.
Keywords: Near infrared imaging; Process Analytical Technology; Galenical development; Multivariate analysis; Extrusion; Homogeneity
A novel method for preparing immune stimulating complexes (ISCOMs) by hydration of freeze-dried lipid matrix
by Mingtao Liang; Istvan Toth; Nigel M. Davies (pp. 840-845).
The purpose of this study was to investigate the application of hydration of freeze-dried lipid monophase matrices as a novel technique to produce immune stimulating complexes (ISCOMs) encapsulating lipopeptides as potential sub-unit antigens. Size, polydispersity and morphology of the resulting colloidal particles were measured and characterized by photon correlation spectroscopy and transmission electron microscopy. The homogeneity of ISCOM preparations produced by this method was found to be influenced by the amount of matrix-forming material as well as the ratio of phospholipid:Quil A:cholesterol used for ISCOM preparation. Further, it was observed that more homogeneous ISCOM dispersions were produced if Quil A was included in the hydrating solution compared to incorporating Quil A in the lipid matrix. Entrapment of lipopeptide within ISCOMs was not affected by chain length (C12–C16) or the number of alkyl chains (1–3) and was greater than 80% when loaded at 5% w/w of total lipid. Entrapment efficiency was noted to decrease dramatically on increasing amount of lipopeptide in the ISCOMs from 5% to 10% of total lipid, decreasing to around 40%. All lipopeptide-loaded ISCOMs were observed to aggregate upon storage.
Keywords: Vaccine; ISCOMs; Lipopeptide; Lipid matrix; Lyophilization