International Journal of Pharmaceutics (v.314, #1)

Formulation of proteins/peptides for therapeutic uses has often posed some challenges to drug formulators. The main problem is the relatively weak forces involved in the native conformation of these proteins and so making them quite labile. Furthermore, their susceptibility to proteolytic enzymes in the gut makes oral administration quite challenging. While various routes like, ocular, transdermal, nasal and buccal have been tried, none of these routes has proved to be a potential alternative to the invasive injection. However, various studies have been performed on the formulation of these proteins as aerosols for pulmonary delivery and promising results have been obtained. This article looks at the prospects of inhaled proteins as a delivery route for systemic activity.
Keywords: Proteins and peptides; Aerosols; Metered dose inhalers; Dry powder inhalers; Nebulizers; Pulmonary;

Mechanical property anisotropy of pharmaceutical excipient compacts by Matthew P. Mullarney; Bruno C. Hancock (9-14).
The mechanical property anisotropy of compacts made from six commercially available pharmaceutical excipient powders was evaluated. Uni-axially compressed cubic compacts of each excipient were subjected to pendulum impact testing and transverse tensile testing in several orientations. The pendulum impact test was used to measure the dynamic indentation hardness of each compact face (side, top, and bottom). Transverse tensile testing was utilized to determine the compact axial and radial tensile strength values. The indentation hardness (top > bottom > side) and tensile strength tests (radial > axial) revealed mechanical property anisotropy in all the compacts. The extent of mechanical property anisotropy was quantified by using dimensionless ratios and was found to be significantly different for each material. In general, compacts with a higher degree of compact mechanical anisotropy also exhibited a higher brittle fracture index (BFI). This suggests that the macroscopic flaws intentionally made in the compact for the BFI measurement were similar to the flaws induced in highly anisotropic materials during uni-axial compaction. These results are consistent with the practical observation that brittle materials are more likely to exhibit failure in a plane normal to the compaction axis, i.e. experience tablet capping and lamination phenomena.
Keywords: Compact; Anisotropy; Indentation hardness; Tensile strength; Excipient;

In order to prepare a liquid formulation for a weak electrolyte, micellization or complexation is often applied with the solution pH controlled to have some of the drug molecules ionized. The efficiency of the micellization is evaluated by either the micellar solubilization capacities, κ u, and κ i or the micellar partition coefficients, K u m and K i m , for the unionized and ionized drug species. Similarly, the efficiency of complexation is evaluated by either the complex solubilization capacities, τ u and τ i or the drug–ligand binding constants, K u 1 : 1 and K i 1 : 1 . In this study, the experimental values of these descriptors were generated for seven ionizable drugs. The relationships of the logarithms of each descriptor to the logarithm of the octanol-water partition coefficient of the unionized drug (log  P u) and ionized drug species (log  P i) were evaluated. Although κ and τ cannot be predicted, this study shows that K m and K 1:1 are dependent on log  P for both the unionized and ionized drug species. Thus, the total drug solubility for a weak electrolyte solubilized by micellization or complexation can be predicted at any pH.

Modelling intestinal absorption of salbutamol sulphate in rats by B. Valenzuela; E. López-Pintor; J.J. Pérez-Ruixo; A. Nácher; A. Martín-Villodre; V.G. Casabó (21-30).
The objective was to develop a semiphysiological population pharmacokinetic model that describes the complex salbutamol sulphate absorption in rat small intestine. In situ techniques were used to characterize the salbutamol sulphate absorption at different concentrations (range: 0.15–18 mM). Salbutamol sulphate at concentration of 0.29 mM was administered in presence of verapamil (10 and 20 mM), grapefruit juice and sodium azide (NaN3) (0.3, 3 and 6 mM). Different pharmacokinetic models were fitted to the dataset using NONMEM. Parametric and non-parametric bootstrap analyses were employed as internal model evaluation techniques. The validated model suggested instantaneous equilibrium between salbutamol sulphate concentrations in lumen and enterocyte, and the salbutamol sulphate absorption was best described by a simultaneous passive diffusion (k a  = 0.636 h−1) and active absorption (V Max  = 0.726 mM/h, K m  = 0.540 mM) processes from intestinal lumen to enterocyte, together with an active capacity-limited P-gp efflux ( V ′ Max = 0.678 mM / h , K ′ m = 0.357 mM ) from enterocyte to intestinal lumen. The extent of salbutamol sulphate absorption in rat small intestine can be improved by NaN3, grapefruit juice and verapamil.
Keywords: Salbutamol sulphate; Intestinal absorption; Active transport; Oral bioavailability; P-glycoprotein inhibitors; Kinetic modelling;

Enhancing effect of alpha-hydroxyacids on “in vitro” permeation across the human skin of compounds with different lipophilicity by A. Copoví; O. Díez-Sales; J.V. Herráez-Domínguez; M. Herráez-Domínguez (31-36).
The percutaneous penetration-enhancing effects of glycolic acid, lactic acid and sodium lauryl sulphate through the human epidermis was investigated using 5-fluorouracil as a hydrophilic model permeant and three compounds belonging to the phenylalcohols: 2-phenyl-ethanol, 4-phenyl-butanol and 5-phenyl-pentanol. The lipophilicity values of the compounds ranged from log  P oct −0.95 to 2.89. The effect of the enhancer concentration was also studied. Skin pretreatment with aqueous solutions of the three enhancers did not increase the permeability coefficient of the most lipophilic compound (log  P oct  = 2.89). For the other compounds assayed, the increase in the permeability coefficients depended on the concentration used in skin pretreatment, and on the lipophilicity of the compounds tested—and was always greater for the most hydrophilic compound (5-fluorouracil), for which lactic acid exerted a greater enhancer effect than glycolic acid or sodium lauryl sulphate. Primary irritation testing of the three enhancers was also carried out at the two concentrations used in skin pretreatment for diffusional experiments (1% and 5%, w/w). The least irritant capacity corresponded to lactic acid; consequently, this alpha-hydroxyacid could be proposed as a percutaneous penetration enhancer for hydrophilic molecules that are of interest for transdermal administration.
Keywords: Alpha-hydroxyacids; Sodium lauryl sulphate; Percutaneous enhancers; Lipophilicity; Skin irritation;

Influence of modified cyclodextrins on solubility and percutaneous absorption of celecoxib through human skin by C.A. Ventura; S. Tommasini; A. Falcone; I. Giannone; D. Paolino; V. Sdrafkakis; M.R. Mondello; G. Puglisi (37-45).
We evaluated the ability of two modified cyclodextrins, hydroxypropyl-β-cyclodextrin (HP-β-Cyd) and 2,6-di-O-methyl-β-cyclodextrin (DM-β-Cyd), to influence the percutaneous absorption through isolated human stratum corneum and epidermis (SCE) of celecoxib (CCB). Previous studies demonstrated that DM-β-Cyd includes the drug, producing a significant increase of water solubility (0.5 mg/ml at 25 °C) and dissolution rate of CCB. In this work chemical-physical characterization studies were performed to evaluate the ability of HP-β-Cyd to include CCB. We showed that only an external interaction could exist between CCB and HP-β-Cyd that positively influences the water solubility of the drug (0.12 mg/ml at 25 °C for CCB-HP-β-CyD system and 4.12 × 10−3  mg/ml at 25 °C for free CCB). In vitro percutaneous experiments were performed using samples in solution and in suspension containing different Cyd concentrations. Both HP-β-Cyd and DM-β-Cyd enhanced drug flux through SCE by means of an increase of dissolution rate of the drug as well as a direct action on the stratum corneum (SC). Histological analysis of treated SCE showed a protective effect of the two Cyds towards an invasive action shown by CCB on SC.
Keywords: Celecoxib; Modified cyclodextrins; In vitro percutaneous studies; Isolated human skin; Histological analysis;

Poly(lactic-co-glycolic acid) (PLGA) nanospheres containing protease inhibitors, camostat mesilate (CM) and nafamostat mesilate (NM), were prepared by the emulsion solvent diffusion methods in water or in oil, and the w/o/w emulsion solvent evaporation method. The average diameter of PLGA nanospheres prepared in the water system were about 150–300 nm, whereas those prepared in the oil system were 500–600 nm. Among the three methods, these drugs were the most efficiently encapsulated up to 60–70% in PLGA nanospheres in the oil system. Other factors that may influence drug encapsulation efficiency and in vitro release such as drug load, molecular weight of polymer were also investigated. Both the CM- and NM-loaded nanospheres prepared in the water system immediately released about 85% of the drug upon dispersed in the release medium while the drug initial burst of nanospheres prepared by the emulsion solvent diffusion in oil method reduced to 30% and 60% for CM and NM, respectively. Poly(aspartic acid) (PAA), a complexing agent for cationic water soluble drugs, showed little effect on the encapsulation efficiency and release behavior for CM and NM. The DSC study and AFM pictures of nanospheres demonstrated that temperature-dependent drug release behavior was ascribable to the glass transition temperature of the polymer, which also affected the morphology of nanospheres upon dispersed in the release medium and influenced the drug release consequently.
Keywords: Camostat mesilate; Nafamostat mesilate; Poly(lactic-co-glycolic acid); Nanospheres; Emulsion solvent diffusion in oil method; In vitro release;

Recently, colloidal dispersions based on solid lipids (solid lipid nanoparticles, SLN) and mixtures of solid and liquid lipids (nanostructured lipid carriers, NLC) were described as innovative carrier systems. A spherical particle shape is the basis of features such as a high loading capacity and controlled drug release characteristics due to smaller lipid–water interfaces and longer diffusion pathways when compared to thin platelets. The structures of SLN and the influence of oil load (NLC) on particle properties were investigated by photon correlation spectroscopy (PCS), laser diffractometry (LD), cryo-field emission scanning electron microscopy (cryo-FESEM), Raman spectroscopy and infrared spectroscopy (IR), and compared to a conventional nanoemulsion. PCS and LD data show similar size and size distribution for SLN and NLC (approximately 210 nm, polydispersity index approximately 0.15) and suggested a long term physical stability for the dispersions which had been stored for up to 12 months at different temperatures. Using cryo-FESEM droplets (for the nanoemulsion) and almost spherical particles for SLN and NLC were observed. Raman spectroscopy resulted in spectra for NLC that are weighted to the SLN spectra, suggesting an undisturbed crystal structure. Infrared spectra of the NLC are predominantly SLN in nature. Importantly the SLN bands are unshifted in the NLC spectrum indicating that the crystalline structure is unaffected by the presence of the oil.
Keywords: Solid lipid nanoparticles; Nanostructured lipid carriers; Cryo-FESEM; Raman spectroscopy;

Pre-deliver chitosanase to cells: A novel strategy to improve gene expression by endocellular degradation-induced vector unpacking by Dong Chun Liang; Wen Guang Liu; Ai Jun Zuo; Shu Jun Sun; Nan Cheng; Gang Guo; Jing Yu Zhang; Kang De Yao (63-71).
A radio-labeled plasmid pTracer/Bsd/LacZ containing LacZ reporter gene was complexed with different molecular weights of chitosans (CS). Mouse myoblast cell line C2C12 was transfected by these chitosan–plasmid DNA complexes, and lipofectamine 2000 was used as control. Forty-eight hours after transfection, the activity of β-galactosidase and radioactive count of cell lysis were determined. It was found that chitosan, especially low molecular weight species, had a surprising ability to deliver DNA into cells, since the radioactive count of cells transfected by chitosan–DNA complexes was even two times that of cells transfected by lipofectamine 2000. But the β-galactosidase activity of chitosan/DNA complexes was much lower compared to that of lipofectamine 2000. Chitosanase which could degrade chitosan in specific mode was transported into C2C12 cells by osmotic lysis prior to gene delivery. Then these chitosanase-modified cells were transfected by CS–DNA complexes. The results indicated that β-galactosidase activity in these cells increased markedly to 425.4 ± 45.1 U/mg protein, nearly two-fold as that of cells transfected by liposome. This transfection protocol was also applied to 3T3 mouse fibroblast, 2T3 mouse osteoblast and MG63 human osteosarcoma cell lines, and an increased gene expression level was observed without exception. It is thought that the incorporated chitosanase could aid in chitosan degradation, which would promote gene unpacking, consequently increasing gene expression.
Keywords: Chitosan; Non-viral vector; Gene transfection; Unpacking;

Evaluation of ciprofloxacin-loaded Eudragit® RS100 or RL100/PLGA nanoparticles by Kathleen Dillen; Jo Vandervoort; Guy Van den Mooter; Annick Ludwig (72-82).
The objective of present study was to prepare positively charged ciprofloxacin-loaded nanoparticles providing a controlled release formulation. The particles were prepared by water-in-oil-in-water (w/o/w) emulsification and solvent evaporation, followed by high-pressure homogenisation. Two non-biodegradable positively charged polymers, Eudragit® RS100 and RL100, and the biodegradable polymer poly(lactic-co-glycolic acid) or PLGA were used alone or in combination, with varying ratios. The formulations were evaluated in terms of particle size and zeta potential. Differential scanning calorimetry measurements were carried out on the nanoparticles and on the pure polymers Eudragit® and PLGA. Drug loading and release properties of the nanoparticles were examined. The antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus was determined. During solvent evaporation, the size and zeta potential of the nanoparticles did not change significantly. The mean diameter was dependent on the presence of Eudragit® and on the viscosity of the organic phase. The zeta potential of all Eudragit® containing nanoparticles was positive in ultrapure water (around +21/+25 mV). No burst effect but a prolonged drug release was observed from all formulations. The particles’ activity against P. aeruginosa and S. aureus was comparable with an equally concentrated ciprofloxacin solution.
Keywords: Eudragit® RS100; Eudragit® RL100; PLGA; Nanoparticles; Ciprofloxacin;

Preparation and characteristics of monostearin nanostructured lipid carriers by Fu-Qiang Hu; Sai-Ping Jiang; Yong-Zhong Du; Hong Yuan; Yi-Qing Ye; Su Zeng (83-89).
Nanostuctured lipid carriers (NLC) consisted of solid lipid and liquid lipid are a new type of lipid nanoparticles, which offer the advantage of improved drug loading capacity and release properties. In this study, solvent diffusion method was employed to produce NLC. Monostearin (MS) and caprylic/capric triglycerides (CT) were chosen as the solid lipid and liquid lipid. Clobetasol propionate used as a model drug was incorporated into the NLC. The influences of preparation temperature and CT content on physicochemical properties of the NLC were characterized. As a result, monostearin solid lipid nanoparticles (without CT content, SLN) obtained at higher temperature (70 °C) exhibited slightly higher drug loading capacity than that of 0 °C (P  < 0.05). In contrast, the production temperature made little effect on NLC drug loading capacity (P  > 0.05). The improved drug loading capacity was observed for NLC and it enhanced with increasing the CT content in NLC. The results were explained by differential scanning calorimetry (DSC) measurement for NLC. The incorporation of CT to NLC led to crystal order disturbance and thus left more space to accommodate drug molecules. NLC displayed a good ability to reduce the drug expulsion in storage compared to SLN. The in vitro release behaviors of NLC were dependent on the production temperature and CT content. NLC obtained at 70 °C exhibited biphasic drug release pattern with burst release at the initial 8 h and prolonged release afterwards, whereas NLC obtained at 0 °C showed basically sustained drug release throughout the release time. The drug release rates were increased with increasing the CT content. These results indicated that the NLC produced by solvent diffusion method could potentially be exploited as a carrier with improved drug loading capacity and controlled drug release.
Keywords: Nanostructured lipid carriers; Monostearin; Caprylic/capric triglycerides; Entrapment efficiency; In vitro release; Preparation temperature;

Synthesis and characterization of biocompatible poly(organophosphazenes) aiming for local delivery of protein drugs by Ji-Yeon Seong; Yong Joo Jun; Byong Moon Kim; Yong Man Park; Youn Soo Sohn (90-96).
Biocompatible and thermosensitive poly(organophosphazenes) with a lower critical solution temperature (LCST) below body temperature have been designed with the aim for the local delivery of peptide and protein drugs. These polymers could be synthesized by introducing short chain tri- or tetraethylene glycol as a hydrophilic group and a dipeptide, GlyGluEt2 as a hydrophobic group into the polyphosphazene backbone. The local tolerance tests using rabbits have shown that our polymers are biocompatible. Using the amphiphilic properties of these polymers, in vitro studies were performed for loading and releasing of a human growth hormone (hGH) as a model drug. The entrapment efficiency (%) of hGH by the polymer decreased as its polymer concentration increased, but exhibited high efficiency of more than 95% even at 20% hGH concentration in the polymer. The entrapped hGH has shown to be controlled releasing for 3–4 days.
Keywords: Polyphosphazene; Drug delivery; Thermosensitive polymer; Protein drug;

Noticeboard (97-98).