International Journal of Pharmaceutics (v.275, #1-2)

Title page (iii).

Early development candidates are often selected for pre-clinical and clinical development based primarily on pharmacological and toxicological data. In order to choose the best compounds from a biopharmaceutical point of view, physicochemical parameters such as solubility, dissolution rate, hygroscopicity, lipophilicity, pK a, stability, polymorphism and particle characteristics need to be evaluated as early as possible and above all with the highest accuracy. However, the low amounts of drug substance available in early development often compromise data quality, and therefore, hamper an early pharmaceutical assessment. This article summarises the Aventis approach on early pharmaceutical compound profiling with the aim of providing a high quality assessment requiring not more than 100 mg of drug substance. In particular, the evaluation criteria, process and miniaturised analytical technology that can be applied for this purpose are discussed.
Keywords: Physicochemical characterisation; Pharmaceutical evaluation; Salt selection; Miniaturisation; Pre-formulation;

The aim of this study was to investigate the effect of size and charge on the permeation of nanoparticles through the skin as the first step in designing a transdermal vaccine delivery system. Fluorescent particles ranging in size and charge were applied to the surface of full thickness pig skin in a diffusion chamber and the receptor fluid was assayed to determine permeation. Fluorescence microscopy was used to visualise the skin after experiments. The results showed that only 50 and 500 nm particles that were negatively charged were able to permeate the skin. This provides evidence of the potential of nanoparticles as delivery vectors for antigens and DNA for the purpose of transdermal vaccination protocols. The results would indicate that negative particles with sufficient charge may be ideal carriers for this purpose.
Keywords: Skin; Transdermal; Permeation; Nanoparticles; Vaccine; Adjuvant;

Influence of physicochemical properties and intestinal region on the absorption of 3-fluoro-2-pyrimidylmethyl 3-(2,2-difluoro-2-(2-pyridyl)ethylamino)-6-chloropyrazin-2-one-1-acetamide, a water insoluble thrombin inhibitor, in dogs by Danielle Euler; Patricia Frech; Shyam Karki; Cameron Cowden; Gareth Pearce; Pratik Mehta; Christopher Lindemann; Paul Byway; Michael Wang; Todd Gibson; Yu Cheng; Gloria Kwei; Jayna Rose (19-27).
In this paper, we describe the physicochemical and biopharmaceutical properties of 3-fluoro-2-pyrimidylmethyl 3-(2,2-difluoro-2-(2-pyridyl)ethylamino)-6-chloropyrazin-2-one-1-acetamide, a direct thrombin inhibitor (1, Fig. 1). Three crystalline forms were characterized and studies were planned to investigate the absorption characteristics of the three selected crystalline forms. Due to the short half-life observed in preclinical species, regional absorption studies were also conducted to support potential controlled release formulation development. Results showed that the absorption of 1 was dependent on the surface area of the particles administered as suspensions and was independent of the crystal forms. From Caco-2 cell transport studies, it was determined that the permeability of 1 was high. Based on the low aqueous solubility it would be classified as a class 2 compound in the Biopharmaceutics Classification System. Regional absorption results suggested that the compound was absorbed along the gastrointestinal tract in Beagle dogs, however colonic absorption appeared to be reduced by slower dissolution.
Keywords: Surface area; Polymorphism; Intestinal absorption;

Evaluation of drug physical form during granulation, tabletting and storage by Adrian C. Williams; V.Brett Cooper; Lisa Thomas; Letecia J. Griffith; Catherine R. Petts; Steven W. Booth (29-39).
An active pharmaceutical ingredient (API) was found to dissociate from the highly crystalline hydrochloride form to the amorphous free base form, with consequent alterations to tablet properties. Here, a wet granulation manufacturing process has been investigated using in situ Fourier transform (FT)-Raman spectroscopic analyses of granules and tablets prepared with different granulating fluids and under different manufacturing conditions. Dosage form stability under a range of storage stresses was also investigated.Despite the spectral similarities between the two drug forms, low levels of API dissociation could be quantified in the tablets; the technique allowed discrimination of around 4% of the API content as the amorphous free base (i.e. less than 1% of the tablet compression weight). API dissociation was shown to be promoted by extended exposure to moisture. Aqueous granulating fluids and manufacturing delays between granulation and drying stages and storage of the tablets in open conditions at 40 °C/75% relative humidity (RH) led to dissociation. In contrast, non-aqueous granulating fluids, with no delay in processing and storage of the tablets in either sealed containers or at lower temperature/humidity prevented detectable dissociation.It is concluded that appropriate manufacturing process and storage conditions for the finished product involved minimising exposure to moisture of the API. Analysis of the drug using FT-Raman spectroscopy allowed rapid optimisation of the process whilst offering quantitative molecular information concerning the dissociation of the drug salt to the amorphous free base form.
Keywords: Active pharmaceutical ingredient; Granulation; Tabletting; Storage; Raman spectroscopy; Process control;

The influence of the incorporation of two oppositely charged hydrophilic natural polymers, chitosan and sodium alginate, alone and in combination, on the ability of formulations containing a model drug (paracetamol) to form spherical pellets by the process of extrusion/spheronisation and the properties of the pellets, has been undertaken. A statistically experimental design was employed to allow the major factors which determined the properties of the pellets, to be identified. A standardised procedure was used to prepare the pellets with a ram producing the extrudate for spheronisation. Statistical analysis of the results indicated that the formulation variables of the type and level of the polymer, the proportion of the model drug, and the proportion of the microcrystalline cellulose influenced (a) the quantity of liquid binder required to produce a good formulation (narrow size range and high value for the shape factor indicating sphericity), (b) the steady-state extrusion force, (c) the pellet perimeter, (d) the apparent pellet density and (e) the porosity of the pellets. The median size of the pellets of the “good formulation” could only be related to the chitosan and sodium alginate content of the formulations. The proportion of the drug, chitosan and sodium alginate content of the formulation significantly influenced the in vitro dissolution of the model drug (paracetamol). The drug release mechanism differed with the formulation variables, although if the pellets remained intact during the dissolution test, diffusion was the controlling mechanism. There was no significant advantage to be gained by using a mixture of the two polymers in terms of retarding drug release.
Keywords: Drug release; Extrusion/spheronisation; Hydrophilic polymers (chitosan, sodium alginate); Pellet properties;

Leucinostatin-A loaded nanospheres: characterization and in vivo toxicity and efficacy evaluation by Maurizio Ricci; Paolo Blasi; Stefano Giovagnoli; Luana Perioli; Claudia Vescovi; Carlo Rossi (61-72).
Leucinostatin A (Leu-A) is a nonapeptide exerting a remarkable activity especially against Candida albicans and Cryptococcus neoformans; nevertheless, its employment is limited due its toxicity. Therefore, we recently developed liposomal formulations, as suitable delivery systems, in order to increase its therapeutic index. However, liposomes present disadvantages related to their long-term instability. For this reason poly(lactic-co-glycolic) nanospheres (NS) were chosen as alternative colloidal carriers for Leu-A delivery. NS were formulated by spontaneous emulsification solvent diffusion method. This study investigates the effects of different parameters on drug encapsulation efficiency and particle size as well. The best preparation obtained was also characterized for its in vitro release, in vivo acute toxicity (LD50), and effectiveness against C. albicans in mice. In vitro release was performed over 100 h and resulted sufficiently sustained with more than 93% of the peptide released. Acute toxicity showed that the LD50 was increased more than 18-fold and the study on systemic candidiasis models revealed high effectiveness of the NS in reducing either the growth of fungal colonies in infected mice liver or in the mortality index. In conclusion, we can propose that Leu-A loaded NS could represent a new promising therapeutic system against Candida infection.
Keywords: Leucinostatin A; Nanospheres; Candida albicans infection;

Microscopic molecular mobility of amorphous AG-041R measured by solid-state 13C NMR by Akiko Koga; Etsuo Yonemochi; Minoru Machida; Yoshinori Aso; Hidetoshi Ushio; Katsuhide Terada (73-83).
Purpose: AG-041R is characterized to be stable in amorphous state and difficult to crystallize at normal period of time. In order to investigate the molecular mobility in microscopically, the spin–lattice relaxation time (T 1) of AG-041R was investigated by solid-state CP/MAS 13 C NMR at temperature below and above glass transition temperature (T g). Method: CP/MAS measurement and T 1 measurement were performed by means of 13 C NMR, where the measurement temperatures were 60, 70, 80, 100, and 110 °C. The spin–lattice relaxation time (T 1) of AG-041R was calculated from the relaxation curves. Results: From the analysis of T 1 of amorphous AG-041R, it was clarified that all of the carbons did not start moving drastically at T g and there were some groups of carbon in terms of temperature dependency of T 1. One is a type, such as the carbons in benzene ring: their T 1 was drastically changed at T g. On the other hand, T 1 of carbonyl carbons gradually decreased, and above T g their T 1 was still higher than that of the other carbons. There was no significant change of T 1 in the methyl carbons around T g. From the study of IR and 1 H NMR in solution, the inter- and intramolecular hydrogen bondings between NH and CO were found in AG-041R. Due to hydrogen bonding, the inter- and/or intramolecular interaction is considered to retain even at supercooled liquid state. Conclusion: The structure that contributes glass transition is the main skeleton structure, such as benzene ring, while small group, like methyl, start to move at lower temperature than T g. On the other hand, for the carbons, such as carbonyl, their structure was restricted by inter- and/or intramolecular interaction, therefore, their molecular mobility was significantly low above T g.
Keywords: Amorphous; Solid-state 13 C NMR; Relaxation; Molecular mobility;

Preparation of nimodipine-loaded microemulsion for intranasal delivery and evaluation on the targeting efficiency to the brain by Qizhi Zhang; Xinguo Jiang; Wenming Jiang; Wei Lu; Lina Su; Zhenqi Shi (85-96).
The purpose of this study was to improve the solubility and enhance the brain uptake of nimodipine (NM) in an o/w microemulsion, which was suitable for intranasal delivery. Three microemulsion systems stabilized by the nonionic surfactants either Cremophor RH 40 or Labrasol, and containing a variety of oils, namely isopropyl myristate, Labrafil M 1944CS and Maisine 35-1 were developed and characterized. The nasal absorption of NM from microemulsion formulation was investigated in rats. The optimal microemulsion formulation consisted of 8% Labrafil M 1944CS, 30% Cremophor RH 40/ethanol (3:1) and water, with a maximum solubility of NM up to 6.4 mg/ml, droplet size of 30.3±5.3 nm, and no ciliotoxicity. After a single intranasal administration of this preparation at a dose of 2 mg/kg, the plasma concentration peaked at 1 h and the absolute bioavailability was about 32%. The uptake of NM in the olfactory bulb from the nasal route was three folds, compared with intravenous (i.v.) injection. The ratios of AUC in brain tissues and cerebrospinal fluid to that in plasma obtained after nasal administration were significantly higher than those after i.v. administration. These results suggest that the microemulsion system is a promising approach for intranasal delivery of NM for the treatment and prevention of neurodegenerative diseases.
Keywords: Nimodipine; Microemulsion; Intranasal delivery; Solubilization; Olfactory pathway; Brain targeting;

Microballoons (MB) possessing a spherical cavity enclosed within a hard polymer shell have been developed as a dosage form characterized by excellent buoyancy in the stomach. MB were prepared by the emulsion solvent diffusion method using enteric acrylic polymers dissolved in a mixture of dichloromethane and ethanol. Riboflavin-containing MB were administered orally to each of three healthy volunteers. The pharmacokinetics of riboflavin was investigated by analysis of the urinary excretion. Prolongation of the urinary excretion of riboflavin could be obtained by ingestion of water as well as “fed” conditions. This phenomenon was attributable to the buoyancy properties of MB in the stomach and an increase in the gastric residence time (GRT). The excretion half-life time (t 1/2) following administration of MB (particle size: 500–1000 μm) exhibiting high buoyancy was longer than that of MB (particle size: <500 μm) displaying low buoyancy. Therefore, the intragastric floating properties of MB are potentially beneficial as far as a sustained pharmacological action is concerned. MB prepared by mixing it with hydroxypropylmethylcellulose (HPMC) in different ratio, results in improved riboflavin-release properties. These MB were evaluated in vivo by analysis of the urinary excretion of riboflavin. As a result, strong correlations were observed between the buoyancy and excretion half-life (t 1/2) and between the riboflavin release from the MB and total urinary excretion.
Keywords: Floating controlled drug delivery system; Hollow microsphere (microballoon); Emulsion solvent diffusion method; Riboflavin; Gastric residence time (GRT); Urinary excretion;

The purpose of this study was to examine the effects of various additives on the profiles of rhBMP-2 release from minipellet, which is a sustained release formulation for protein drugs using collagen as a carrier, and to examine the influence of varying release profiles on ectopic bone formation. When the amount of rhBMP-2 remaining in the preparation after subcutaneous implantation to mice was examined, it was found that the addition of sucrose, glucose, PEG4000, alanine (Ala) or acacia in a concentration of 20% (w/w) to the minipellet with 5% (w/w) of rhBMP-2 did not accelerate the drug release in a noticeable manner, while the addition of sodium chondroitin sulfate, glutamic acid (Glu) or citric acid accelerated the release of rhBMP-2 markedly. When two types of minipellets (a fast release type added with 20% Glu and 20% Ala and a slow release type without additives) containing varying amounts of rhBMP-2 were implanted subcutaneously to mice, the soft X-ray observation, histological examination and measurement of calcium formation 3 weeks after implantation revealed extensive ectopic bone formation in mice implanted with the fast release type preparation. Ectopic bone formation was dose-dependent. The result of this study exhibited that the effects of controlled release formulation of rhBMP-2 on bone formation vary depending on their release profiles, and suggested that combination of initial burst and sustained release was effective for bone formation. It was also shown that minipellet is useful as a controlled release formulation which can release rhBMP-2 to areas around the implanted site with various release profiles.
Keywords: Bone morphogenetic protein (BMP); Collagen; Minipellet; Controlled release; Ectopic bone formation;

Use of an impinging jet for dispersion of dry powder inhalation aerosols by Zhaolin Wang; Carlos F. Lange; Warren H. Finlay (123-131).
The dispersion of Ventodisk® (salbutamol sulphate with lactose) from different drug reservoirs by an air jet at normal impingement is examined experimentally. The effect on dispersion efficiency of jet velocity, nozzle location, reservoir size and shape, and the loaded dose is investigated for possible design of new dosing methods or inhalers. Results show that higher jet velocity (as high as feasible), lower drug loading (2 mg or smaller), a cylindrical hole reservoir (6 mm in diameter and 3 mm in depth) and a medium distance (approximately 5 jet diameters) from the nozzle to the reservoir yield optimum dispersion. The dispersed fine particle dose improves by a factor of 2–3 times between optimized conditions and poor conditions.
Keywords: Aerosol; Dry powder inhaler; Impinging jet; Powder dispersion; Powder deagglomeration; Cascade impactor;

Organic salts of diclofenac were predicted by using computed molecular descriptors and multivariate partial least squares (PLS). The molecular descriptors including binding energy and surface area of salts were calculated by the use of Hyperchem and ChemPlus QSAR programs for Windows. Other physicochemical properties such as hydrogen acceptor for oxygen atoms, hydrogen acceptor for nitrogen atoms, hydrogen bond donors, hydrogen bond-forming ability, molecular weight, and log partition coefficient (log  P) of bases were also used as descriptors. Good statistical models were derived that permit simple computational prediction of salt solubility of a same parent structure. The final models derived had R 2 value=0.96 and root mean square error for prediction (RMSEP) values ranging from 0.021 to 0.054 (log scale). Preferably all utilized descriptors in the final models can readily obtain from the chemical structure of salt and base. Molecular weight of base is one of the important factors associated with salt solubility. While increased molecular weight of base, surface area of salt and hydrogen bonding ability of base increase solubility, and increased binding energy and log  P of base have negative effect on salt solubility.
Keywords: Salt; Solubility; Partial least squares; Diclofenac; Binding energy;

Melt pelletization with polyethylene glycol in a rotary processor by Thomas Vilhelmsen; Jakob Kristensen; Torben Schæfer (141-153).
The purpose of this study was to investigate the effect of the airflow, the binder concentration, the massing time, the friction plate rotation speed, and the surface structure of the friction plate on melt pelletization in a laboratory scale rotary processor. Lactose monohydrate was melt agglomerated with polyethylene glycol (PEG) 3000 as meltable binder. The study was performed as a full factorial design. An increase in agglomerate size was found when the binder concentration, the massing time, or the friction plate rotation speed was increased. The agglomerate size was also increased when increasing the shearing forces by using a friction plate with a different surface structure. The size distribution of the agglomerates was significantly narrowed when the binder concentration or the shearing forces caused by the friction plate were increased. An increase in the adhesion of material to the friction plate was found when the shearing forces of the friction plate were increased either by the rotation speed or by the surface structure. Generally, the rotary processor was found to be a suitable alternative to melt pelletization in a high shear mixer.
Keywords: Rotary processor; Melt agglomeration; Polyethylene glycol; Spheronization; Friction plate;

The present study illustrates and evaluates the capability of the double-barrier-desiccant system to regulate the humidity inside a medicament powder chamber. The model medicament chamber chosen was the reservoir-based dry powder inhaler Taifun®. The study was focussed to screen the critical parameters affecting the function of the desiccant system. For this purpose a novel simulation method was developed and applied to predict the progress of the internal relative humidity of the powder chamber. The simulation method showed good correlation with the measurement results and, hence, it was concluded that it was valid for the system. According to the simulations, effective regulation of the humidity inside a closed powder chamber with the double-barrier-desiccant system is possible for a prolonged time. However, this can be achieved only if the critical parameters, such as the permeabilities of the desiccant container and the powder chamber, the amount of the desiccant and the initial moisture content of the desiccant, can be accurately specified and strictly controlled in the production of the components comprising the medicament chamber.
Keywords: Dry powder inhalers; Modeling; Moisture permeation; Packaging; Relative humidity; Stability;

Enhanced bioavailability of paclitaxel after oral coadministration with flavone in rats by Jun-Shik Choi; Hoo-Kyun Choi; Sang-Chul Shin (165-170).
The purpose of this study was to investigate the effect of flavone on the bioavailability of paclitaxel orally coadministered in rats. Paclitaxel (40 mg/kg) and flavone (2, 10, 20 mg/kg) were orally administered to rats orally. The plasma concentration of paclitaxel with flavone increased significantly (P<0.01) compared to that of paclitaxel control. Area under the plasma concentration–time curve (AUC) of paclitaxel with the dose of 2–20 mg/kg flavone was significantly (P<0.05 at 10 mg/kg, P<0.01 at 20 mg/kg) higher than that of control. AUCs of paclitaxel were increased dose-dependently in the dose range of flavone. The absorption rate constant (K a) of paclitaxel with the dose of 10–20 mg/kg flavone was significantly increased (P<0.05 at 10 mg/kg, P<0.01 at 20 mg/kg) compared to that of control. Peak concentration (C max) of paclitaxel with the dose of 10–20 mg/kg flavone were significantly increased (P<0.05 at 10 mg/kg, P<0.01 at 20 mg/kg) compared to that of control. Half-life (t 1/2) of paclitaxel with the dose of 10–20 mg/kg flavone was significantly prolonged (P<0.05 at 10 mg/kg, P<0.01 at 20 mg/kg) compared to that of control. The relative bioavailability increased significantly by about 2.38- or 3.10-fold (P<0.05 at 10 mg/kg, P<0.01 at 20 mg/kg) compared to that of paclitaxel control. Based on these results, It might be considered that the bioavailability of paclitaxel coadministered with flavone was significantly enhanced by the both inhibition of cytochrome P-450 and the P-gp efflux pump in the intestinal mucosa. It could be possible to administer paclitaxel orally besides the established IV route.
Keywords: Paclitaxel; Flavone; Pharmacokinetics; Bioavailability;

Factorial design, physicochemical characterisation and activity of ciprofloxacin-PLGA nanoparticles by Kathleen Dillen; Jo Vandervoort; Guy Van den Mooter; Loes Verheyden; Annick Ludwig (171-187).
Poly(lactide-co-glycolide) nanoparticles incorporating ciprofloxacin HCl were prepared by means of a W/O/W emulsification solvent evaporation method. The stabiliser selected was poly(vinylalcohol). A 24 full factorial design based on four independent variables was used to plan the experiments and the variable parameters were the number of homogenisation cycles, addition of boric acid to the inner water phase containing the drug, ciprofloxacin concentration in the inner water phase and oil:outer water phase ratio. The effects of these parameters on the particle size, zeta potential, drug loading efficiency and drug release were investigated. Also the effect of gamma irradiation on the particle size and drug release was evaluated and DSC and XRD analyses of the compounds and the nanoparticles were performed. The activity on two series of microorganisms, Pseudomonas aeruginosa and Staphylococcus aureus, was examined.
Keywords: PLGA; Nanoparticles; W/O/W emulsification solvent evaporation method; Ciprofloxacin; Experimental design;

The objective of this study was to investigate the presence of a large neutral amino acid transporter on the ARPE-19 cell line. ARPE-19 cells were grown on 24-well plates for uptake studies. Uptake characteristics of [ 3 H ]l-phenylalanine (l-Phe) were determined at various concentrations and pH at 37 °C. Inhibition studies were conducted in presence of l- and d-amino acids, metabolic inhibitors, like ouabain, sodium azide, and in presence of sodium-free medium, to delineate the mechanism of uptake. RT–PCR was carried out on total RNA isolated from the ARPE-19 cells. Presence of Na+-free buffer did reduce the uptake rate. Hence, all experiments were carried out in Na+-free medium to delineate the sodium-independent uptake mechanism. Uptake of l-Phe on ARPE cells was found to be saturable with a K m=89.35±14 μM, V max=58.9±2.5 pmol min−1  mg protein−1, and K d=0.108±0.04 μl min−1  mg protein−1. Dose-dependent inhibition was observed with increasing concentrations of unlabeled l-Phe. Uptake also was found to be energy independent. Significant inhibition of [ 3 H ]l-Phe was observed with large neutral aromatic and aliphatic amino acids as well as small neutral amino acids. System L-specific inhibitor BCH produced partial inhibition of uptake. Neither acidic nor basic amino acids altered the uptake rate. Results obtained were predominantly characteristic of LAT2, particularly with respect to substrate selectivity and pH dependence. Bands for LAT2 were detected by RT–PCR in the ARPE cell line. This study provides biochemical evidence of the presence of a Na+-independent, facilitative transport system, LAT2, on the ARPE-19 cells.
Keywords: ARPE-19; Amino acid transporter; Phenylalanine; LNAA; LAT2;

The aim of this study was to establish a correlation between carrier characteristics and the dispersibility of drug from the blend. The influence of the roughness of a commonly used carrier material, lactose monohydrate, on the adhesion, dose uniformity, and aerodynamic properties of a model drug, terbutaline sulphate was investigated. Evaluation of adhesion was carried out with a mechanical sieve and an Alpine air-jet sieve. For the characterisation of lactose roughness, we used image analysis software. Aerodynamic evaluation of fine particle dose and emitted dose was obtained using a twin stage impinger. The study with the mechanical sieve demonstrated that at least 60% of drug adheres to lactose. The Alpine air-jet sieve assays showed there was a correlation between drug separation from a carrier by sieving and that obtained from longer in vitro deposition studies. Adhesion, blend homogeneity and stability are related to the surface roughness of the lactose used as carrier. There is a linear relationship between the parameters “fine particle fraction” and “roughness”. A compromise between homogeneity and drug liberation must be found: a certain roughness is necessary to allow for drug adhesion and blend homogeneity, but if too high it will prevent drug liberation after inhalation.
Keywords: Terbutaline sulphate; Dry powder inhaler; Roughness; Adhesion; Drug delivery;

α-Lactose monohydrate is the standard carrier used for dry powder inhalation drug products. The physico-chemical characteristics of this carrier material need to be monitored and specified carefully in order to guarantee functionality of the powder formulation. But also microbiological acceptance criteria need to be considered during development and routine testing. In this study, the endotoxin content of 19 batches of α-lactose monohydrate provided by two different manufacturers was determined with a semi-quantitative LAL assay. The endotoxin content was found to be less than 18 endotoxin units (EU)/g lactose in all cases and less than 3 EU/g in most cases. Assuming that the typical lactose has an endotoxin content of 10 EU/g and that a patient inhales six times daily 25 mg of lactose and that the total amount of lactose reaches the lung, this translates to an endotoxin exposure of 1.5 EU per day. On the other hand, the proposal for endotoxins in air limits the endotoxin concentration to 50 EU/m3 which corresponds to approximately 3333 EU inhaled endotoxins a day during normal breathing (breathing at rest conditions). The maximum endotoxin exposure by dry powder inhalations is thus two log steps lower than the recommended acceptable daily intake.
Keywords: Dry powder inhaler; Interactive powder mixtures; Lactose; Endotoxins; Inhalation exposure;

Transdermal delivery of phosphorodiamidate Morpholino oligomers across hairless mouse skin by Angela K Pannier; Vikram Arora; Patrick L Iversen; Rhonda M Brand (217-226).
The skin is the largest organ in the body and an obvious route for both local and systemic drug delivery. Antisense oligomers have tremendous potential as therapeutic agents for numerous diseases. The objective of this study was to investigate the influence of vehicle on the transdermal delivery of several phosphorodiamidate Morpholino oligomers (PMOs) with different sizes, lengths, base compositions, sequences, and lipophilicities. Eleven different PMOs were synthesized complementary to biologically relevant gene targets and delivered across hairless mouse skin in vitro using vehicles composed of 95% propylene glycol, 5% linoleic acid (PG/LA), water, 50% water:50% PG/LA, and 75% water:25% PG/LA. The data suggest that size, sequence and guanine composition all influence transdermal penetration. There was an inverse linear relationship between size and penetration for a given sequence when the PG/LA formulation was used (r 2=0.94), but this trend was not evident when the vehicle contained water. An oligomer targeted to the gene p53 had lower than expected transdermal penetration based on its size, but was shown to localize within the skin, demonstrating that sequence and thus target will impact transdermal delivery. The presence of G-quartets correlated with better PMO penetration from a water vehicle. Overall, the data suggest that some oligomers and vehicles would be better for transdermal delivery and others for topical applications.
Keywords: Transdermal drug delivery; Antisense; Oligonucleotides; Penetration enhancers; Hairless mouse skin;

Pharmacokinetic changes of oltipraz after intravenous and oral administration to rats with liver cirrhosis induced by dimethylnitrosamine by Soo K. Bae; Shin J. Lee; Jang Y. Lee; Youngsoo Lee; Inchul Lee; Sang G. Kim; Myung G. Lee (227-238).
Pharmacokinetic changes of oltipraz were investigated after intravenous and oral administration at a dose of 30 mg/kg to control Sprague–Dawley rats and rats with liver cirrhosis induced by dimethylnitrosamine. After intravenous administration in rats with liver cirrhosis, the area under the plasma concentration–time curve from time zero to time infinity (AUC) was significantly greater (1490 μg min/ml versus 2840 μg min/ml) than that in control rats. This was due to significantly slower total body clearance (CL) (20.2 ml/(min kg) versus 10.6 ml/(min kg)) in the rats. The slower CL was due to significantly slower CLNR (20.1 ml/(min kg) versus 10.5 ml/(min kg)) in rats with liver cirrhosis. The significantly slow CLNR was due to slower hepatic blood flow rate and significantly slower in vitro intrinsic oltipraz disappearance clearance (CLint, 77.2 ml/min per whole liver versus 11.5 ml/min per whole liver) because the free (unbound in serum proteins) fraction of oltipraz was significantly greater (15.1% versus 31.3%) in the rats. After oral administration in rats with liver cirrhosis, the AUC was also significantly greater (354 μg min/ml versus 812 μg min/ml) and this was not due to increased absorption in the rats. This also could be due to slower hepatic blood flow rate and significantly slower CLint in the rats.
Keywords: Oltipraz; Pharmacokinetics; Liver cirrhosis; Dimethylnitrosamine; Rats;

Lipid transfer protein transports compounds from lipid nanoparticles to plasma lipoproteins by Junzo Seki; Satoru Sonoke; Akira Saheki; Tomohiro Koike; Hiroshi Fukui; Masaharu Doi; Tadanori Mayumi (239-248).
Nanometer-sized lipid emulsion particles with a diameter of 25–50 nm, called Lipid Nano-Sphere (LNS®), are expected as a promising drug carrier to show prolonged plasma half-life of an incorporating drug. In terms of successful drug delivery using LNS, a drug should be incorporated into the lipid particles and remain within the particle, not only in the formulation in vitro but also after administration into the systemic blood circulation. In this study, we showed that phospholipids and some water-insoluble molecules also moved from lipid particles to plasma lipoproteins or albumin in serum and plasma half-lives of these compounds did not reflect that of the drug carriers. It was suggested that phospholipid or its derivative were transferred from LNS particles to plasma lipoproteins by lipid transfer proteins (LTP) in the circulation. These phenomena leaded to unsuccessful delivery of the drug with lipid–particulate drug carriers. On the other hand, lipophilic derivatives with cholesterol pro-moiety tested in this study were not released from LNS particles and showed prolonged plasma half-lives. Lipophilicity is known to be an important parameter for incorporating drugs into lipid particles but substrate specificity for LTP seems to be another key to success promising drug design using lipid emulsion particulate delivery system.
Keywords: Lipid emulsion; Nanoparticle; Lipid transfer protein; LNS; Drug delivery;

The CAG is being developed for pulmonary drug delivery. Liquids are pumped, heated and vaporized by the CAG, whence they nucleate and condense to form aerosols. This study characterized the effect of energy on the aerosolization process. With increasing energy, the CAG produced an increasing fine particle fraction (FPF) until “optimal aerosolization” was achieved between 40 and 45 J; this energy range agreed with that theoretically required to vaporize the dose of PG. Further increases in energy above this optimal range did not improve PG’s aerosolization efficiency. Based on the energy, FPF and temperature profiles, it was possible to deduce the nature of the liquid flow-boiling during aerosol generation. The aerosol particle size went through a minimum, as energy was increased through the “optimal range.” In the “energy excess” region, where additional energy increased PG vapor temperature and velocity, droplet sizes were increased primarily due to changes in the nucleation rates and supersaturation ratios affecting the nucleation and condensation processes occurring within the vapor jet. The in vitro MMAD of the PG aerosol changed as a function of the applied energy, suggesting that for any pharmaceutical application, the choice of applied energy is critical to deposition profile of the aerosol.
Keywords: Aerosol; Propylene glycol; Vaporization; Energy;

Physically cross-linked hydrogel had been investigated in order to make use of oral polypeptide drug delivery carrier. By using 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer, we had prepared a spontaneously forming hydrogel showing controllable dissociation via pH changes. In this study, the dissociation and release of polypeptide drugs from the MPC polymer hydrogel loaded with polypeptide drugs, which had been prepared from aqueous solutions containing water-soluble poly[MPC-co-methacrylic acid (MA)] (PMA) and poly[MPC-co-n-butyl methacrylate (BMA)] (PMB) had been executed. The polymer concentration was 10 wt.% and PMA/PMB feed ratio (A/B feed ratio) was 5/5. Insulin labeled with fluorescein-4-isothiocyanate (FITC) and cytochrome c had been loaded for the examination of the release behavior. The hydrogel in pH 1.8 aqueous solution would be swelling, for the hydrogel would absorb outside water. However, during this process, the polymer is dissoluting out from the hydrogel due to the widening of the polymer network. The cytochrome c followed anomalous transport while insulin depended on the swelling and dissolution of the polymer chains. The hydrogel showed surface erosion in neutral condition, although the hydrogel is composed of two different polymer possessing divergent physical properties. The release followed anomalous transport, but the erosion rate slightly changed with as the hydrophobicity of the loaded drugs. The total amount of the drugs released in neutral condition was larger compared to the acidic condition. When the eroded percentage and the release percentage were compared with each other, it showed that release was slightly slower than erosion, indicating that the erosion was controlling the release phenomenon.
Keywords: Swelling; Dissociation; 2-Methacryloyloxyethyl phosphorylcholine polymer; Physically cross-linked hydrogel; Polypeptide delivery; Controlled release; Erosion mechanism;

Permeation characteristics of a hydrophilic basic compound across a bio-mimetic artificial membrane by Kiyohiko Sugano; Yoshiaki Nabuchi; Minoru Machida; Yoshinori Asoh (271-278).
In the present study, the permeation characteristics of a hydrophilic basic compound (HBC) in a bio-mimetic parallel artificial membrane permeability assay (bio-mimetic PAMPA) were investigated in detail. The bio-mimetic PAMPA membrane was constructed on a hydrophobic filter by impregnating a lipid solution consisting of phosphatidylcholine (0.8%, w/w), phosphatidylethanolamine (0.8%, w/w), phosphatidylserine (0.2%, w/w), phosphatidylinositol (0.2%, w/w), cholesterol (1.0%, w/w), and 1,7-octadiene (97.0%, w/w). The pH–permeability curve (pH 3–10), the effect of lipid composition, concentration dependency (0.02–2.00 mM), and inhibition by other cationic compounds, were investigated for several HBCs. Ketoprofen and methylchlorpromazine were also employed as an acidic and a quaternary ammonium compound, respectively. At pH 3–6, the permeability of timolol, a HBC, was higher than expected from the pH-partition hypothesis, especially in the PI-containing membrane, whereas the pH–permeability curve of ketoprofen followed the pH-partition hypothesis. Permeation of HBC was saturable and inhibited by basic and quaternary ammonium compounds. Similar results were also found for methylchlorpromazine. The permeation characteristics of HBC observed in the present study are not usually expected in a passive permeation process across an artificial membrane. The participation of facilitated permeation of cationic species was suggested, in addition to a simple passive diffusion of un-dissociated species. Ion pair transport was suggested as a possible permeation mechanism of cationic species. However, further investigation is necessary to clarify the reason for the permeation characteristics of HBC.
Keywords: Permeability; Phospholipid; Physicochemical properties; Hydrophilic basic compound;

A dosage design of mitomycin C tablets containing finely powdered green tea by Takurou Kurita; Atsuo Miyagishima; Yasuo Nozawa; Yasuyuki Sadzuka; Takashi Sonobe (279-283).
We previously reported a method of preparing finely powdered green tea (PT), powder characteristics and release profiles of green tea components from PT. In this study, we performed formulation studies of PT tablets containing mitomycin C (MMC), expecting its combined antitumor effects with mitomycin C and green tea components. The hardness of PT tablets was low (22–50 N) and the disintegration time was about 180 min regardless of hardness or tabletting pressure (15–200 MPa). Perfiller®-101 improved tablet characteristics practically into 90 N of hardness and 18.5 min of disintegration time. Release rates of MMC, caffeine and EGCG from the tablets were similar, and depended on the disintegration time. PT and epigallocatechin gallate (EGCG) increased significantly in MMC uptake in Ehrlich ascites carcinoma cells as compared with the control dose-dependently in vitro.
Keywords: Green tea; Epigallocatechin gallate; Caffeine; Mitomycin C; Tablet characteristics; Ehrlich ascites carcinoma cells;

Noticeboard (285-287).