European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V

Published by Elsevier
Print ISSN: 0939-6411
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The hepatic disposition of a new analgesic, SCP-1, a derivative of acetaminophen, was studied in the isolated perfused rat liver using a recirculating system. The aim of this study was to compare the kinetic parameters of this molecule with those of acetaminophen. Sprague-Dawley rat (230-330 g) livers were perfused for 2 h with 250 ml Krebs-Henseleit bicarbonate buffer containing SCP-1 or acetaminophen, 0.07 mmol l(-1) (n=4), 0.28 mmol l(-1) (n=4), and 0.8 mmol l(-1) (n=4) (approximately one, four and ten times the therapeutic doses in man, respectively). Perfusate samples were collected from the efflux at various times. The SCP-1 and acetaminophen perfusate concentrations were assayed by a HPLC method. Pharmacokinetic analysis was carried out using a computer program. There were significant differences between the hepatic kinetics of SCP-1 and those of acetaminophen. Thus, SCP-1 elimination half-life (mean 14.8+/-10.0 min) was shorter than that of the acetaminophen (186.1+/-27.7 min) (t=11.6, P=0.0001). While the half-life of SCP-1 increases with concentration, the half-life of acetaminophen remains constant as the concentration increases. The hepatic clearance was higher for SCP-1 than acetaminophen (mean 19.01+/-14.5 ml min(-1) vs. 1.29+/-0.08 ml min(-1), respectively) (t=2.44, P<0.05), and it behaved according to dose-dependent kinetics. The SCP-1 extraction ratio was higher (mean 0.63+/-0.49) than for acetaminophen (0.04+/-0.01) (t=2.41, P<0.05) and this parameter tended to decrease as the perfusate concentrations of SCP-1 increased. It was concluded that the hepatic kinetics of SCP-1 behaved according to dose-dependent kinetics, and statistically significant differences were found between pharmacokinetics parameters of both drugs studied.
 
If a semisolid vehicle does not allow for the sufficient penetration of the incorporated drug, the addition of enhancers, e.g. glycols, is an option. Propylene glycol is most frequently applied in dermal products. Other 1,2-alkanediols like pentylene glycol were found to exhibit moisturizing effects and good anti-microbial activity. In the present study, the influence of propylene glycol and mainly butylene glycol (BuG) and pentylene glycol (PeG) on release and skin penetration of Dihydroavenanthramide D (DHAvD) was investigated. DHAvD release increased twice up to fourfold within 30 min if 2% of a mixture of BuG and PeG was added to a lipophilic as well as to a hydrophilic cream. Incorporation of single 1,2-alkanediols into the hydrophilic cream resulted in a linear slope of the released DHAvD amount with increasing chain length of the glycol. Trends found in the release model were also reflected in penetration studies on full thickness human breast skin using Franz diffusion cells. Here, the hydrophilic cream containing the BuG/PeG mixture was compared to the glycol-free reference. Already within 30 min the amount that penetrated into the viable skin layers doubled using the glycol-containing vehicle. After 300 min 12% of the applied dose was detected in the viable epidermis and dermis following application of the pure cream compared to 41% from the improved formulation. Dermal availability was further enhanced by administration of a polymer-stabilized hydrodispersion gel which also contained the glycol mixture. Due to their favorable biopharmaceutical and technological properties, longer chain 1,2-alkanediols represent a valuable class of ingredients for dermal products.
 
5-Acetoxyacetylimino-4-methyl-delta2-1,3,4,-thiadiazoline -2-sulfonamide (compound (1)) is an ester prodrug that lowered intraocular pressure (IOP) in albino New Zealand rabbits, but was found to be inactive in pigmented Dutch Belt rabbits. In order to explain the differences in pharmacological activity for the two rabbit species, metabolism and melanin binding were studied. Depending on the initial concentration, the binding of compound (1) to natural melanin (Sepia officinalis) was 20-60%. The binding constant, K, at 37 degrees C was 4.32 x 10(5) M(-1) and the maximum moles bound to melanin, r(max), was 4.5 x 10(-7) mol/mg of melanin. From a determination of binding at temperatures between 25 degrees C and 47 degrees C, a van't Hoff plot was constructed to determine enthalpy and entropy changes accompanying the binding process, deltaH and deltaS, respectively. Values calculated from the plot were -12.7 and -15.4 kcal/(mol deg), respectively. Negative values for these parameters are consistent with charge transfer interactions and therefore suggest that this may be an operative mechanism between compound (1) and melanin. The in vitro incubation of compound (1) was also studied with various ocular tissues from both albino and pigmented rabbits which were iris-ciliary body, intact cornea, stroma/endothelium and aqueous humor. A major metabolite, MET 1, was identified and also observed from in vivo analyses of the same tissues following topical application. The metabolite was isolated and subjected to mass spectroscopy and proton nuclear magnetic resonance spectroscopy analysis. From these analyses, it was hypothesized that the formation of MET 1 involved a GSH conjugation mechanism which displaced the sufonamide (-SO2NH2) group. The metabolism was found to be less extensive in the pigmented rabbit than in the albino rabbit and suggested that the binding affinity of compound (1) for melanin was a better explanation for the lack of IOP activity in the pigmented rabbit than differences in metabolism.
 
Both d-alpha-tocopheryl polyethylene glycol 1000 (TPGS 1000) and polyvidone-vinylacetate 64 (PVPVA 64) provided an increase in the degree of supersaturation and stability of supersaturated Itraconazole solutions, compared to a blanc without excipient. Therefore, both components were combined as carrier in order to make ternary solid dispersions of Itraconazole by spray drying. This way, TPGS 1000 could be incorporated into a powder. Dissolution experiments on the ternary solid dispersions revealed that during the first hour the release was much higher than for the binary Itraconazole/PVPVA 64 solid dispersions. For some compositions a release of more than 80% was reached after 10min. However, after the first hour the drug started to precipitate. The ternary solid dispersions were all XRD amorphous, but MDSC revealed the coexistence of multiple amorphous phases and a crystalline Itraconazole phase, depending on the composition. Therefore the burst effect during the first hour can be ascribed to an accelerated dissolution of the amorphous Itraconazole fraction in the presence of TPGS 1000. The precipitation after 1h, however, is probably due to the combination of the surfactant properties of TPGS and the small crystalline Itraconazole fraction.
 
The present research deals with the improvement of the dissolution properties of the anti-HIV drug UC 781. A ternary solid dispersion consisting of a high amount of TPGS 1000 and exhibiting good powder properties with respect to flowability was developed. Eudragit E100 was selected as a polymer based on supersaturation studies. DSC analysis of solid dispersions containing drug doses from 0 to 80% w/w revealed eutectic phase behaviour of the ternary TPGS 100-Eudragit E100-UC 781 mixture. The release of UC 781 in a medium simulating the gastrointestinal lumen was markedly enhanced, reaching a release of 70% w/w after 4h. XRD results pointed to the presence of crystalline drug in the solid dispersion. The presence of UC 781 in the dispersion had an influence on the TPGS 1000-Eudragit E100 carrier, favoring folding of the polyethylene glycol chains in TPGS 1000. Moreover, the addition of UC 781 to the binary polymer-surfactant mixture was physically expressed by an increase in fluidity of the samples up to a drug load of 50% w/w. NMR was used to investigate this phenomenon, revealing a shielding and/or deshielding effect of the carrier on aromatic C atoms and methyl groups in UC 781. Polyethylene glycol chains present in TPGS 1000 seemed to play a role in this process. In addition, combining UC 781 with the TPGS 1000-Eudragit E100 mixture led to the appearance of TPGS 1000 clusters with a glass transition temperature well below the T(g)'s of the pure compounds.
 
The treatment of iron deficiency anemia with polynuclear iron formulations is an established therapy in patients with chronic kidney disease but also in other disease areas like gastroenterology, cardiology, oncology, pre/post operatively and obstetrics' and gynecology. Parenteral iron formulations represent colloidal systems in the lower nanometer size range which have traditionally been shown to consist of an iron core surrounded by a carbohydrate shell. In this publication, we for the first time describe the novel matrix structure of iron isomaltoside 1000 which differs from the traditional picture of an iron core surrounded by a carbohydrate. Despite some structural similarities between the different iron formulations, the products differ significantly in their physicochemical properties such as particle size, zeta potential, free and labile iron content, and release of iron in serum. This study compares the physiochemical properties of iron isomaltoside 1000 (Monofer) with the currently available intravenous iron preparations and relates them to their biopharmaceutical properties and their approved clinical applications. The investigated products encompass low molecular weight iron dextran (CosmoFer), sodium ferric gluconate (Ferrlecit), iron sucrose (Venofer), iron carboxymaltose (Ferinject/Injectafer), and ferumoxytol (Feraheme) which are compared to iron isomaltoside 1000 (Monofer). It is shown that significant and clinically relevant differences exist between sodium ferric gluconate and iron sucrose as labile iron formulations and iron dextran, iron carboxymaltose, ferumoxytol, and iron isomaltoside 1000 as stable polynuclear formulations. The differences exist in terms of their immunogenic potential, safety, and convenience of use, the latter being expressed by the opportunity for high single-dose administration and short infusion times. Monofer is a new parenteral iron product with a very low immunogenic potential and a very low content of labile and free iron. This enables Monofer, as the only IV iron formulation, to be administered as a rapid high dose infusion in doses exceeding 1000 mg without the application of a test dose. This offers considerable dose flexibility, including the possibility of providing full iron repletion in a single infusion (one-dose iron repletion).
 
The recent rapid progress of molecular biology together with the steady progress of genome projects has given us some essential and revolutionary information about DNA and RNA to elucidate various biological phenomena at a genetic level. Under these circumstances, the technology and methodology of gene transfection have become more and more important to enhance the efficacy of gene therapy for several diseases. In addition, gene transfection is a fundamental technology indispensable to the further research development of basic biology and medicine regarding stem cells. Stem cells genetically manipulated will enhance the therapeutic efficacy of cell transplantation. In this paper, the carrier and technology of gene delivery are briefly overviewed while the applications to the basic researches of biology and medicine as well as regenerative medical therapy are introduced. A new non-viral carrier and the cell culture system are described to efficiently manipulate stem cells.
 
Irganox 1076(R) (octadecyl-3,5-di-tert-butyl-4-hydroxyhydrocinnamate) is a common phenolic antioxidant used in many polymer-based medical devices. As with many organic compounds, several polymorphs exist. However, in literature, only two forms of Irganox 1076(R) have been mentioned. In this study, we were able to produce, by crystallization in different solvents, three distinct polymorphs, which were characterized by DSC, FTIR and PXRD. Moreover, the three polymorphs have long-time stability at ambient pressure and temperature, meaning that they can potentially be present in or on polymeric devices. During DSC measurements, a fourth polymorph, which was only stable at low temperature, was evidenced. Thanks to Raman microspectroscopy, Irganox 1076(R) was identified directly on commercial polyurethane catheters which exhibited a blooming phenomenon. This study proves that the polymorph identified on the surface is different from the commercially available Irganox 1076(R). These results emphasize the importance of the screening of polymorphs before any study of the biocompatibility of antioxidants used in medical devices.
 
Two synthesis methods of poly(methyl vinyl ether-co-maleic anhydride) (Gantrez AN 119) nanoparticles (NP) (used for oral vaccination) were compared. Wheat germ agglutinin (WGA) was used as ligand to enhance the bioadhesive properties of NP and beta-galactosidase as antigen. The first method encapsulated beta-galactosidase in NP by co-precipitation in an acetone/water mixture containing 44% acetone. In the second method, antigen addition occurred in 100% acetone. To improve stability, NP were crosslinked with 1,3-diaminopropane. The stability of WGA-conjugated NP with encapsulated antigen diminished at lower pH and when decreasing the amount of crosslinker. The binding type between WGA and polymer depended on the synthesis method: predominantly ionic bonds were formed using the 44% acetone method, whereas synthesis via the 100% acetone method resulted in covalent bonds. The biological activity of the WGA coating, evaluated via a pig gastric mucin binding test, was lower in NP prepared via the 100% acetone method. No release of native antigen was detected after hydrolysis of NP, due to the covalent antigen binding during antigen encapsulation and the high reactivity of the polymer. Moreover, the mucosal irritation capacity was evaluated upon nanoparticle hydrolysis using a slug mucosal irritation assay. Herein, hydrolysed NP of the 44% acetone method were classified as mild irritative.
 
Target of the study was to characterize crystals which had grown in steroid-containing matrix patches during short-term storage and to thereby establish a rationale for the inhibition of crystal formation in those patches in general. Matrix type transdermal drug delivery systems (TDDS) containing either 2.2% gestodene or 3.3% estradiol were free of crystals directly after their production. However, crystals of up to 800 microns in length grew during 3 months of storage at ambient temperature. The application of several analytical methods did not help to identify the crystals. This was mainly due to the fact that the adhesive matrix surrounding the crystals could not be fully removed in the course of sample preparation with routine laboratory methods and thus impaired DSC, FTIR microscopy and hot stage polarized microscopy. However, within X-ray diffractometry, the residual amorphous patch matrix did not hamper the measurement of the crystals. Thus, they were identified as estradiol hemihydrate and gestodene form I, respectively. These results suggest that steroid-containing matrix TDDS should be stabilized against drug recrystallization e.g. by the addition of suitable crystallization inhibitors. Furthermore, systems containing estradiol may be stabilized by efficient removal water.
 
Poly(ortho esters) have been under development since the early 1970s and four families of such polymers have been described. Of most interest are poly(ortho ester) III and poly(ortho ester) IV. Poly(ortho ester) III is a semisolid material that has been shown to be highly biocompatible and is currently being investigated as an adjunct to glaucoma filtering surgery and other ocular applications. However, the polymerization is difficult to control and is not readily scaled up. Poly(ortho ester) IV can be easily prepared in a highly reproducible manner, is very stable provided moisture is rigorously excluded and has also been shown to be highly biocompatible. It is currently under development for a variety of applications, such as ocular delivery, protein release, post-operative pain treatment and post-operative cancer treatment.
 
A series of diblock copolymers based on methoxypolyethylene glycol-block-poly(caprolactone) (MePEG-b-PCL) was synthesized and evaluated for enhancing the cellular accumulation of a P-glycoprotein (P-gp) substrate, rhodamine-123 (R-123), into caco-2 cells. Altering MePEG:caprolactone feed weight ratio allowed diblocks with varying PCL lengths to be synthesized onto MePEG of molecular weight 750 or 2000. The critical micelle concentration (CMC) and the hydrophilic-lipophilic balance all decreased with increasing degree of polymerization of PCL. R-123 accumulation by caco-2 cells increased to a maximum in the presence of increasing concentrations of MePEG-b-PCL diblock copolymers (compared to R-123 alone) beginning at concentrations at or above the CMC, with little or no R-123 accumulation enhancement observed below the CMC. Further increases in MePEG-b-PCL concentration resulted in a decrease in R-123 uptake back to baseline levels. It is suggested that the higher concentrations of diblock above the CMC were required to serve as a 'depot' for free unimer partitioning into the cell membrane in order to obtain a critical concentration of diblock in the membrane for P-gp modulation. Alternatively, MePEG-b-PCL micelles may increase R-123 accumulation via endocytosis of micellized R-123.
 
(13)C NMR spectroscopy provides insight into the chemistry of carbohydrate-based ferric preparations. Specifically, it reveals whether oxygen atoms of the carbohydrate are directly bonded to the preparations' ferric centres or whether more distant interactions are present. After having validated the method by investigating the ferric solutions of low-molecular complexes as well as polynuclear ferric samples, it is demonstrated that common constituents of medically used ferric preparations such as sucrose and other glucose-based saccharides do not support ferric carbohydrate chelates. Instead, these carbohydrates reside outside the NMR-spectroscopically 'blinded' region about the ferric centres and experience the so-called Evans effect that can be used to measure the magnetic moment of the solutions. As a result, an easily accessible physicochemical parameter is provided to characterise commercial iron(III) preparations, namely the samples' magnetism in terms of the in-situ-measured spin-normalised effective Bohr magneton number μeff(2)/35. The procedure can, moreover, be combined with a facile NMR-spectroscopic iron assay.
 
A number of systems were prepared at five compositions (5, 10, 20, 30 and 40% w/w) of diclofenac/N-(2-hydroxyethyl) pyrrolidine salt and acidic diclofenac in PEG6000 and Gelucire 50/13, as physical mixtures and as solid dispersions. Powder X-ray diffractograms for the systems examined show shifted and normal peaks, suggesting that the drug is present inside the samples in different physical states. Differential scanning calorimetry does not offer important information, since drug solubility into the carriers increases with temperature and thermograms show only the melting point peak of the carriers. Hot-stage microscopy examination explains that, in high concentration samples, the drug is present either dissolved into the carriers, or precipitated as microcrystals, or undissolved crystals of larger size. Gelucire 50/13 allows the formation of larger crystals than PEG, using both the chemical forms of the drug. The release percentage of the drug from PEG6000/acidic diclofenac reaches 50% after few minutes in the most favourable case and appears to be dependent on the composition of the samples: the more diclofenac is present as dissolved in the pre-treated samples, the higher is the release. The optimum composition was found in the range of 5-10% w/w.
 
The objective of this study was to determine the influence of the formulation technique for 2-hydroxypropyl-beta-cyclodextrin (HP beta CD) on the stability of aspirin in a suspension-based pressurized metered-dose inhaler (pMDI) formulation containing a hydrofluoroalkane (HFA) propellant. HP beta CD was formulated in a pMDI as a lyophilized inclusion complex or a physical mixture with aspirin. A pMDI formulation containing aspirin alone was used as the control. The chemical stability of aspirin in each pMDI formulation was determined over 6-months storage at 5, 25 and 40 degrees C. The quantity of water taken up into the pMDI canister was determined by Karl Fisher titration after storage for 6 months. Differential scanning calorimetry (DSC) was used to confirm the formation of a complex between HP beta CD and aspirin. Aspirin in the lyophilized inclusion complex exhibited the most significant degree of degradation during the 6-months storage, while aspirin alone in the pMDI demonstrated a moderate degree of degradation. Aspirin formulated in the physical mixture displayed the least degree of degradation. The water uptake study showed that water ingress occurred to the greatest extent for formulations containing aspirin and HP beta CD physical mixture, and to the least extent for formulations containing aspirin alone. Finally, the DSC study indicated that an inclusion complex was formed in situ in the pMDI formulations containing the HP beta CD and aspirin physical mixture. In conclusion, HP beta CD may be used to enhance the stability of a chemically labile drug, but the drug stability may be affected by the method of preparation of the formulation.
 
Metered-dose inhalers (MDI) are the most widely prescribed devices in the treatment of lung diseases but the continued use of chlorofluorocarbons (CFC) as propellants has made them unpopular due to their influence on the stratospheric ozone layer. The purpose of this study was to show possibilities of formulating beclomethasone-17,21-dipropionate (BDP) with the alternative propellant R 134a as a solution or as a suspension-type metered-dose inhaler. Influencing factors such as surfactant concentration, cosolvent content and actuator tube design were investigated. Metered-dose inhaler formulations were manufactured using a pressure filling technique. The resulting formulations were characterized with regard to their emitted fine particle fraction using the two-stage impinger, BP 93. Fine particle fraction was found to be independent on the surfactant concentration but highly dependent on the cosolvent content and the actuator tube design. In vitro fine particle fractions of 50% were obtained with solution phase MDIs. Formulating BDP as a suspension resulted in unstable dispersions in most cases because of the partial solubility of the drug in the liquified propellant. Stable suspension formulations gave an in vitro fine particle fraction of about 30%. A comparison with established marketed BDP suspension formulations which were found to emit a fine particle fraction in the range 10-50% showed the equivalence of the new CFC-free formulations.
 
A series of homogeneous Eudragit RS100 matrix microspheres containing molecularly dispersed acylated esterified homologues of salicylic acid, (acetylsalicylic acid, valerylsalicylic acid, or caprylsalicylic acid) were prepared in order to investigate the effect of encapsulation on solid-state orientation of the encapsulated molecule. Electrostatic association of the drug with the charged quaternary residues in the polymer may be responsible for the previously observed stability of acetylsalicylic acid (ASA) in aqueous swollen ASA-loaded Eudragit RS100 microspheres. Evaluation of the 13C nuclear magnetic resonance spectra for evidence of structural association of the incorporated probe molecules indicated that alteration of the microenvironment of the incorporated solutes had occurred. For instance, increasing the aliphatic character of the acyl side chain resulted in an increase in the upfield shift of the acyl bearing aromatic ring carbon, (C2), in the incorporated probe molecule as compared to the unincorporated probe molecule. Similarly, a downfield perturbation in the chemical shift of the free acid bearing aromatic ring carbon, (C1), was also observed. This microenvironment electrostatic shielding in the proximity of the ester carbonyl is attributed to an increase in the association of the probe molecule with the polymer subunits. Thereby, it is postulated that the matrix incorporated probe molecule is essentially shielded from hydrolytic attack until it is liberated into the external aqueous environment.
 
In vitro and in situ intestinal transport of beta-lactam antibiotics in the presence of two novel pharmaceutical excipients, caprylocaproyl and lauroyl macrogolglycerides (Labrasol and Gelucire 44/14), is described. The objective was to compare the effects of both macrogolglycerides on the intestinal transport of cephalexin, a substrate of oligopeptide transporters, and cefoperazone, a non-substrate of them. The in vitro transport studies were performed using a sheet of rat jejunum mounted in Ussing-type diffusion chambers. The in situ studies used an isolated internal loop model in the rat. Labrasol and Gelucire 44/14 were used as the excipients at low concentrations (0.01-0.5%, w/v). The membrane permeability of both drugs was compared by apparent permeability coefficients (P(app)) determined from changes in the amount of permeation vs. time in in vitro studies and by apparent absorptive clearance (CL(app)) determined from changes in the steady state drug concentration of perfusate in in situ studies. The P(app) value of cephalexin increased with an increase in the concentration of Labrasol (0.05-0.5%) compared to the value without Labrasol. The enhancing effect of Labrasol on cephalexin transport was similarly observed in in situ studies, and when 0.5% Labrasol was used in the presence of glycyl-L-leucine or L-alanyl-L-alanine, 60 or 46% enhancement of the active transport of cephalexin by Labrasol was obtained. On the other hand, Gelucire 44/14 did not affect the P(app) and CL(app) of either drug. The different effects of the excipients on cephalexin transport were thought to be due to the influences of size parameters such as a polydispersity index and particle size, and the change in the short-circuit current of jejunum by the addition of the excipient.
 
Piroxicam is a non-steroidal anti-inflammatory drug that is characterized by low solubility and high permeability. The purpose of the study was to investigate the in vitro and in vivo performance of the semi-solid dispersion prepared with Gelucire 44/14 and Labrasol into hard gelatin capsules (GL) for enhancing the dissolution rate of the drug. The results were evaluated by comparing with pure piroxicam filled into hard gelatin capsules (PP) and a commercially available tablet dosage form containing a piroxicam:beta-cyclodextrin complex (CD). The in vitro dissolution testing of the dosage forms was performed in different media (simulated gastric fluid, pH 1.2; phosphate buffers, pH 4.5 and 6.8; and water). Amongst the dosage forms, GL provided at least 85% piroxicam dissolution within 30 min in each of the media, behaving like a fast-dissolving immediate release drug product. Oral bioavailability of 20 mg piroxicam in GL, CD, and PP was compared after administration of a single dose to eight healthy volunteers. Three treatments were administered in crossover fashion, separated by a washout period of 2 weeks. Piroxicam was monitored in plasma by high-performance liquid chromatography. The apparent rate of absorption of piroxicam from GL (Cmax=2.64 micrograms/ml, tmax=82.5 min) was significantly higher than that of the PP (Cmax=0.999 micrograms/ml, tmax=144 min) (P<0.05) and similar to that of CD (Cmax=2.44 micrograms/ml, tmax=120 min) (P>0.05). The relative bioavailability values as the ratios of mean total AUC for GL relative to PP and CD, were 221 and 98.6%. Piroxicam is characterized by a slow and gradual absorption via the oral route and this causes a delayed onset of therapeutic effect. Thus, plain piroxicam preparations are not indicated for analgesia. The results of the in vivo study revealed that the GL dosage form would be advantageous with regards to rapid onset of action, especially in various painful conditions where an acute analgesic effect is desired.
 
We have investigated the distribution of hydrolytic enzymes which metabolize beta-estradiol 17-acetate (EA) to beta-estradiol (E) in man and animal skins in vitro. The distribution of hydrolytic enzymes in human cadaver, hairless dog, rat and hairless mouse skin, was investigated by a skin-slicing technique. We performed histological studies with hematoxylin and eosin stain. The highest amount of metabolite (E) appeared in the layers of 80-120 microm from the skin surface, the basement layer in human skin, while the amount of metabolite was distributed evenly in the hairless dog skin from 0 to 180 microm. In the rat and hairless mouse skin, on the other hand, peak levels of metabolite were observed in the basement layer of dermis, the surrounding area of the cutaneous plexus. The total metabolic activities in the area of epidermis in human, hairless dog and hairless mouse skin were 2.59, 8.03 and 0.33 x 10(-4) microg/ml/microm/h, respectively. The values in whole skin layers in the hairless dog and hairless mouse skin were 3.35 and 1.85 x 10(-4)microg/ml/microm/h, respectively. EA transported across the human and hairless dog skin can be effectively metabolized before entering the capillary. Among animal models investigated, hairless dog skin might be the most facile model in simulating drug metabolism for human skin under the clinical (in vivo) conditions. Hairless mouse skin, on the other hand, was also an excellent model in excised human skin under in vitro conditions.
 
17Beta-estradiol hemihydrate (17beta-E2) is a poorly water-soluble drug. Physical methods for improving the solubility and dissolution rate, e.g. micronization, have certain inherent disadvantages. The method of choice in this study, melt extrusion, proved to overcome many of the shortcomings of conventional methods. Different compositions of excipients such as PEG 6000, PVP (Kollidon 30) or a vinylpyrrolidone-vinylacetate-copolymer (Kollidon VA64) were used as polymers and Sucroester WE15 or Gelucire 44/14 as additives during melt extrusion. The solid dispersions resulted in a significant increase in dissolution rate when compared to the pure drug or to the physical mixtures. For example, a 30-fold increase in dissolution rate was obtained for a formulation containing 10% 17beta-E2, 50% PVP and 40% Gelucire 44/14. The solid dispersions were then processed into tablets. The improvement in the dissolution behavior was also maintained with the tablets. The USP XXIII requirement for estradiol tablets reaching greater than 75% drug dissolved after 60 min was obtained in this investigation.
 
The high loading efficiency and controlled release of hydrophobic drugs is still an unmet goal in the development of drug delivery systems. In the present study, liposomes were developed to encapsulate 17β-estradiol, which is a sex steroid shown to confer protective effects in the cardiovascular system. Egg phosphatidylcholine (EPC), 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were used to prepare liposomes by thin film hydration and tested for their ability to load E2 with a high efficiency. DOPC-based liposomes were found to improve E2 encapsulation efficiency and loading capacity compared to those composed of EPC and DPPC. In addition, neutral liposomes, liposomes prepared with the cationic charging agent DDAB, and liposomes prepared with the anionic charging agent DMPG, were characterized with regards to their E2 encapsulation efficiency, loading capacity, particle size, zeta potential, and in vitro drug release. A human coronary artery endothelial (HCAE) cell model was used to further evaluate effects on cytotoxicity and relative cellular uptake efficiency of each formulation. Results showed that DOPC liposomes composed of DDAB had the highest E2 loading capacity and improved cellular uptake compared to uncharged and DMPG-based liposomes, demonstrating the greatest potential to be used in future cardiovascular therapeutic applications.
 
In [(18)F]fluoride chemistry, the minute amounts of radioactivity taking part in a radiolabeling reaction are easily outnumbered by other reactants. Surface areas become comparably larger and more influential than in standard fluorine chemistry, while leachables, extractables, and other components that normally are considered small impurities can have a considerable influence on the efficiency of the reaction. A number of techniques exist to give sufficient (18)F-tracer for a study in a pre-clinical or clinical system, but the chemical and pharmaceutical understanding has significant gaps when it comes to scaling up or making the reaction more efficient. Automation and standardization of [(18)F]fluoride PET tracers is a prerequisite for reproducible manufacturing across multiple PET centers. So far, large-scale, multi-site manufacture has been established only for [(18)F]FDG, but several new tracers are emerging. In general terms, this transition from small- to large-scale production has disclosed several scientific challenges that need to be addressed. There are still areas of limited knowledge in the fundamental [(18)F]fluoride chemistry. The role of pharmaceutical factors that could influence the (18)F-radiosynthesis and the gaps in precise chemistry knowledge are discussed in this review based on a normal synthesis pattern.
 
Brain tumors such as glioblastoma reappear in their original location in almost 50% of cases. To prevent this recurrence, we developed a radiopharmaceutical system that consists of a gel applied immediately after surgical resection of a brain tumor to deliver local radiation booster doses. The gel, which strongly adheres to tissue in the treatment area, consists of fibrin glue containing the beta-emitters rhenium-188 and rhenium-186 in microsphere-bound form. Such microspheres can be prepared by short (2 h or less) neutron activation even in low neutron flux reactors, yielding a mixture of the two beta-emitters rhenium-188 (E(max)=2.1 MeV, half life=17 h) and rhenium-186 (E(max)=1.1 MeV, half life=90.6h). The dosimetry of this rhenium-188/rhenium-186 fibrin glue system was determined using gafchromic film measurements. The treatment efficacy of the radioactive fibrin glue was measured in a 9L-glioblastoma rat model. All animals receiving the non-radioactive fibrin glue died within 17+/-3 days, whereas 60% of the treated animals survived 36 days, the final length of the experiment. Control animals that were treated with the same amount of radioactive fibrin glue, but had not received a previous tumor cell injection, showed no toxic effects over one year. The beta-radiation emitting rhenium-188/rhenium-186-based gel thus provides an effective method of delivering high doses of local radiation to tumor tissue, particularly to wet areas where high adhesive strength and long-term radiation (with or without drug) delivery are needed.
 
Drug permeability of the model drugs ketoprofen and nadolol across Caco-2 cell monolayers was determined in the absence and presence of the non-ionic surfactant Poloxamer 188 (Pluronic® F68, P-188). Stringent controls confirmed that P-188 in concentrations up to 50 mg/ml did not adversely affect cell viability or monolayer integrity. Equilibrium experiments confirmed that the drugs were merely passively transported. Caco-2 permeability of both drugs was found to be decreased by the surfactant in a concentration-dependent manner. Ultrafiltration revealed that both drugs were associated with surfactant micelles. The systematic investigation of micellization on passive absorption showed that association of drugs with P-188 micelles appears to depress their passive permeability under conditions where other transport mechanisms can be neglected.
 
The European Union's (EU) centralized procedure for new drug review was implemented in 1995 to unify the regulatory process and provide EU-wide marketing authorizations for innovative medicinal products. Goals were instituted to ensure the timeliness of the various steps of the process. The EU approved 27 biopharmaceutical products through the centralized procedure during 1995-1999. This study documents the success of the EU in meeting the timeline goals for the group and for separate categories of biopharmaceuticals (recombinant proteins, monoclonal antibodies, and antisense oligonucleotides). A subset of the 27 biopharmaceuticals approved in the EU were also approved in the United States (US). We compared EU and US approval times for these products by product category and by review status (exceptional/non-exceptional circumstance in the EU and priority/standard in the US).
 
Development status of unapproved biopharmaceuticals in the US, EU and Japan. Source: IMS R&D Focus (accessed March 2007) Reprint with permission. 
Relative drug lag -the number of first approval and approval lag
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.
 
The organic anion transporting protein 1B3 (OATP1B3), formerly termed OATP8, is responsible for uptake and subsequent elimination of multiple amphipathic drugs by the liver. In silico methods for the prediction of transport rates for drugs and drug-like molecules might provide an important tool in drug development. Most prediction methods however require a large training set of in vitro experimental data in order to yield reliable results. To obtain these data, we have developed a fluorescence-based assay that allows screening a relatively high number of substances for their transporter affinity. HEK293 cells overexpressing OATP1B3 (HEK-OATP8) [Y. Cui, J. Konig, D. Keppler, Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2, Mol. Pharmacol. 60 (2001) 934-943.] were tested for transport of Fluo-3. Fluo-3 uptake could be seen in a concentration-dependent manner. Uptake can be inhibited completely by the addition of the known OATP1B3-inhibitor rifampicin proving that Fluo-3 is transported by OATP1B3. To verify the suitability of the system to identify modulators of OATP1B3, we tested known substrates for competitively inhibiting the Fluo-3 transport by giving them simultaneously with a 2muM Fluo-3-solution to the cells. The transport of Fluo-3 was decreased by all test substrates in a concentration dependent manner.
 
1 H NMR spectra of benzoic acid and sodium benzoate in 0.2% chitosan solution (pH 3.3).  
IR spectra of benzoic acid (F) and benzoic-chitosan nanoparticle with different concentrations of benzoic acid solution in the formulations. (A) 0.5 mg/ml, (B) 1 mg/ml, (C) 1.5 mg/ml, (D) 2.0 mg/ml, (E) 2.5 mg/ml.  
Heat flow versus time profiles for the sequential titrations of insulin solution into chitosan solution (A), phosphate buffer solution into chitosan solution as a blank experiment (B), benzoic acid solution into chitosan solution (C) and benzoic acid into dilute acetic acid solution as a blank experiment (D).  
Integrated data obtained from the previous raw data for the titration of insulin solution into pH 5 chitosan solution (A) and benzoic acid solution into pH 3.3 chitosan solution (B) in the reaction cell after subtracting the blank experiments.  
Integrated data obtained from the titration of tripolyphosphate solution into pH 5 chitosan solution and pH 3.3 chitosan solution after subtracting the blank experiments.  
Electrostatic interaction between opposite charge of drugs (insulin and benzoic acid) and chitosan was studied by 1H NMR, FTIR and isothermal titration calorimetry (ITC). No ionic interaction between the carboxyl group of benzoic acid and the amine group of chitosan could be detected. There was a minor change in the FTIR spectra of insulin-chitosan microparticles made of different concentrations of insulin. Exothermic heat of reaction between insulin and chitosan was obtained by ITC. However, the measured interaction enthalpy change (delta H) was possibly due to the conformational changes and the adsorption phenomena of insulin onto the surfaces of the particles but not to a binding interaction. The binding of tripolyphosphate, a widely used cross-linking agent, to pH 3.3 and pH 5 chitosan was also studied by ITC. The interaction enthalpy change of the binding between tripolyphosphate and chitosan indicated that tripolyphosphate provided a stronger interaction to pH 5 chitosan than to pH 3.3 chitosan. However, it can be stated that the electrostatical interaction forces between the tested molecules insulin, benzoic acid, and tripolyphosphate and chitosan are found to be very weak.
 
Zein is a protein based natural biopolymer containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids to form stable protein-drug complexes. In this work π-A isotherm, NMR and Dynamic light scattering were used to detect the formation of protein aggregates and the affinity between zein and two different drugs: tetracycline and indomethacin. An effective interaction of zein and the two drugs was evidenced by means of liquid NMR reinforced by means of changes in the surface pressure by π-A isotherm. The effective interactions zein/drugs under air/water interface were evidenced as a change in the surface pressure of the π-A isotherm of zein in the presence of drug solutions. The presence of tetracycline in the subphase decreased the area occupied by the monolayer at the expanded region until pressures of 12 mN/m were the areas became similar, but indomethacin produces an increment of the area in both expanded and collapsed region. The feasible methodology employed, focused in the functionality of the protein-drug interaction, can be very promising in the drug delivery field.
 
In order to treat malignant brain tumors by local delivery of antineoplastic agents, the feasibility of 5-fluorouracil (5-FU)-sustained release biodegradable microspheres with a novel material, poly(methylidene malonate 2.1.2), was investigated using an emulsion/extraction method. This polymer was expected to present a slow degradation rate, thus leading to a long term local delivery system. Microparticles were successfully obtained and characterized in terms of drug loading, size, morphology and release profile. The size of the particles was between 40 and 50 microm, which was compatible with a stereotactic injection through a needle. Sufficient drug loadings were obtained (i.e. compatible with the preparation of therapeutic 5-FU doses in a minimal volume of injection), and perfectly spherical microspheres were observed. The respective influences of the polymer molecular weight, the polymer concentration, and the emulsion time on the release profiles were studied using a 2(3) factorial design. In the same objective, the solvent extraction time was extended while keeping all the previous parameters fixed at their optimal values. The in vitro study of these different parameters allowed a reduction of the initial burst release, with a percentage of 5-FU released after 24 h that was lowered from 90 to 65%, and the achievement of a long term drug delivery system, since the release was still ongoing after 43 days. Moreover, the microparticles could be gamma-sterilized (25 kGy) without modification of the release kinetics. Thus, the requested specifications to perform animal experiments were attained.
 
Single-walled carbon nanotubes (SWNTs) have been already used as drug carriers. In this study, we introduced sgc8c aptamer (this aptamer targets leukemia biomarker protein tyrosine kinase-7) to complex between Dau (daunorubicin) and SWNT to enhance targeted delivery of Dau to acute lymphoblastic leukemia T-cells (Molt-4). Dau-aptamer-SWNTs tertiary complex formation was analyzed by visible spectroscopy and spectrofluorophotometric analysis. Dau release profiles from the complex were investigated in pH 7.4 and 5.5. For cytotoxic studies (MTT assay), Molt-4 (target) and U266 (B lymphocyte human myeloma, non-target) cells were treated with Dau, Dau-aptamer-SWNTs tertiary complex. Internalization was analyzed by flow cytometry. Targeted delivery of Dau was antagonized using antisense of aptamer. Dau was efficiently loaded onto SWNTs (efficiency ∼ 157%). Dau was released from Dau-aptamer-SWNTs tertiary complex in a pH-dependent manner (higher release rate at pH 5.5). Flow cytometric analysis showed that the tertiary complex was internalized effectively to Molt-4 cells, but not to U266 cells. Cytotoxicity of Dau-aptamer-SWNTs tertiary complex also confirmed internalization data. Dau-aptamer-SWNTs tertiary complex was less cytotoxic in U266 cells when compared to Dau alone. No significant change in viability between Dau- and complex-treated Molt-4 cells was observed. Cytotoxicity of Dau-aptamer-SWNTs complex was efficiently and quickly reversed using antisense in Molt-4 cells. Dau-aptamer-SWNTs complex is able to selectively target Molt-4 cells. The other advantages of this system are reversibility and pH-dependent release of Dau from its complex.
 
A photosensitizer, meso-tetra(4-hydroxyphenyl)porphyrin, was incorporated into sub-150 nm nanoparticles using the emulsification-diffusion technique in order to perform sterilization by filtration using 0.22 microm membranes. The three selected polyesters (poly(D,L-lactide-co-glycolide), (50:50 PLGA, 75:25 PLGA) and poly(D,L-lactide (PLA)) for the nanoparticle production were all amorphous in nature and have similar molecular weights but different copolymer molar ratios. The influence of the copolymer molar ratio and the theoretical drug loading was investigated in terms of particle size, drug loading, entrapment efficiency and surface characteristics. With all the polymers used, sub-150 nm nanoparticles were produced with good reproducibility and narrow size distributions irrespective of both the polymer nature and the theoretical drug loading. After purification by cross-flow filtration, the nanoparticle suspensions were sterilized by membrane filtration and freeze-dried in the presence of a lyoprotectant (trehalose). For all types of nanoparticles, complete redispersion in various media could be obtained. All final freeze-dried products were refiltrable on a 0.22 microm membrane and were stable in terms of mean particle size and drug loading over a period up to 6 months. The effective drug loading increased at higher theoretical drug loading, the entrapment efficiency was however decreased. The same trend was observed with the three polyesters. The sterility of the final freeze-dried nanoparticles was confirmed by the results of the sterility testing which showed no bacterial contamination.
 
The incorporation of a drug in a carrier by melt embedding may either result in a solid solution or in a solid suspension of the active ingredient within the carrier material. As the dispersivity of the drug is of outstanding importance for its dissolution characteristics, parameters which are supposed to influence crystallinity and dispersivity, e.g. cooling rate during preparation and storage conditions like temperature and relative humidity are investigated. It is found that the absence of crystalline drug material in solid dispersions containing nimodipine and polyethylene glycol 2000 is the prerequisite for a high dissolution rate and a remarkable supersaturation in the dissolution medium. Shock freezing during the preparation process, low storage temperatures and low relative humidities are identified to prevent recrystallisation. Furthermore, emphasis is put on the physico-chemical characterisation of solid dispersions. It is shown that the determination of crystallinity and dispersivity of the drug in solid dispersions can only be successful by combining different investigation methods like differential scanning calorimetry, hot stage microscopy, X-ray diffraction as well as macroscopic observation.
 
The therapeutic biologics market is currently dominated by recombinant protein products. However, many of these products are mature, and growth of the biologics market will increasingly rely on the expansion of the therapeutic monoclonal antibody sector. Successive technology waves have driven the growth of the monoclonal antibody sector, which is currently dominated by chimeric antibodies. Chimeric products, led by Remicade and Rituxan, will continue to drive market share through to 2008. However, over the forecast period, humanized and fully human monoclonal antibodies, together with technologies such as Fabs and conjugated antibodies, will play an increasingly important role, driving monoclonal antibody market growth at a forecast compound annual growth rate of 20.9%, to reach $16.7 billion by 2008. In terms of therapeutic focus, the monoclonal antibody market is heavily focused on oncology and arthritis, immune and inflammatory disorders, and products within these therapeutic areas are set to continue to be the key growth drivers over the forecast period. Underlying the growth of the market is the evolution of the monoclonal antibody company business model, set to transition towards the highly successful innovator model.
 
The presence of tight barriers, which regulate the environment of ocular tissues in the anterior and posterior part of the eye, is essential for normal visual function. The development of strategies to overcome these barriers for the targeted ocular delivery of drugs, e.g. to the retina, remains a major challenge. During the last years numerous cell culture models of the ocular barriers (cornea, conjunctiva, blood-retinal barrier) have been established. They are considered to be promising tools for studying the drug transport into ocular tissues, and for numerous other purposes, such as the investigation of pathological ocular conditions, and the toxicological screening of compounds as alternative to in vivo toxicity tests. The further development of these in vitro models will require more detailed investigations of the barrier properties of both the cell culture models and the in vivo ocular barriers. It is the aim of this review to describe the current status in the development of cell culture models of the ocular barriers, and to discuss the applicability of these models in pharmaceutical research.
 
Water containing hydrophilic ointment DAB 1997 was modified by the incorporation of ethanol and the effects of ethanol on the evaporation, the drug liberation and the permeation through human excised stratum corneum were investigated. Creams with 10%, 20%, 30% (v/v) and without ethanol were produced. As a model drug 2% (w/w) hydrocortisone-21-acetate was suspended in the o/w cream. The evaporation of the creams decreased with an increasing amount of ethanol which was unexpected because the vapor pressure of ethanol is higher than that of water. From this result it was concluded that ethanol might be interlamellarly fixed in the mixed crystal of the polyhydrate of the emulsifier to a higher extent than it is distributed within the aqueous bulk phase. In context with the liberation studies, ethanol decreased the drug liberation from the cream. This is in accordance with the above hypothesis of the ethanol partitioning within the cream, because the solubility of the drug in ethanol is higher than that in water. Therefore the interlamellar drug concentration should be higher than the solubility of the drug in the bulk phase, with the assumption that the gel network of the emulsifier polyhydrate is finally responsible for the delay in drug liberation. The permeation through stratum corneum showed no significant differences between the alcohol-free and the alcohol-loaded formulations. Obviously the decrease in drug liberation by ethanol was compensated for by the penetration enhancing effect.
 
Systemic delivery of amphiphobic drugs (insoluble in both water and oil) represents a formidable challenge in drug delivery. This work aimed to engineer a functional mesoporous carbon material to efficiently load SNX-2112, an amphiphobic anticancer agent, and to evaluate its performance in tumor-targeting delivery. Hydrothermal reaction combined with high-temperature activation was used to fabricate glucose-based mesoporous carbon nanospheres (MCNs). SNX-2112-loaded MCNs stabilized by phospholipid (SN-PMCNs) were prepared by the absorption/solvent diffusion/high-pressure homogenization method. The obtained SN-PMCNs were 180 nm around in particle size, showing a high drug load (42.7%) and acceptable physical stability. SN-PMCNs demonstrated an enhanced in vitro antitumor effect and increased uptake into cancer cells in comparison with the formulation of SNX-2112 solution (SN-Sol). The in vivo antitumor effect and biodistribution in 4T1 xenograft tumor mice, a breast cancer model, were also significantly improved through SN-PMCNs. It was shown that specific clathrin-dependent and nonspecific caveolae-dependent endocytosis were involved in the cellular trafficking of SN-PMCNs. Glucose transporter-mediated transport, prolonged body residence time and improved biodistribution via EPR effect were the main mechanisms of enhanced antitumor effect. SN-PMCNs have presented excellent tumor targeting properties and should be a promising carrier to address the systemic delivery of SNX-2112. Copyright © 2015. Published by Elsevier B.V.
 
Oligonucleotides are promising tools for in vitro studies where specific downregulation of proteins is required. In addition, antisense oligonucleotides have been studied in vivo and have entered clinical trials as new chemical entities with various therapeutic targets such as antiviral drugs or for tumour treatments. The formulation of these substances were widely studied in the past. With this review we will focus on peptides used as drug delivery vehicles for oligonucleotides. Different strategies are summarised. Cationically charged peptides from different origins were used e.g. as cellular penetration enhancers or nuclear localisation tool. Examples are given for Poly-L-lysine alone or in combination with receptor specific targeting ligands such as asialoglycoprotein, galactose, growth factors or transferrin. Another large group of peptides are those with membrane translocating properties. Fusogenic peptides rich in lysine or arginine are reviewed. They have been used for DNA complexation and condensation to form transport vehicles. Some of them, additionally, have so called nuclear localisation properties. Here, DNA sequences, which facilitate intracellular trafficking of macromolecules to the nucleus were explored. Summarizing the present literature, peptides are interesting pharmaceutical excipients and it seems to be feasible to combine the specific properties of peptides to improve drug delivery devices for oligonucleotides in the future.
 
Anionic pH-sensitive membrane-disruptive polymers have evolved as a new class of bioactive excipients for the cytosolic delivery of therapeutic macromolecules. A large variety of anionic copolymers and analogues of poly(acrylic acid) (PA) was investigated and compared to a cationic PA copolymer. The pH-responsive membrane-disruptive properties were characterized by employing three in vitro models, such as pH dependent shift of pyrene fluorescence, liposome leakage and lysis of red blood cells. The pH-dependent increase of polarity and membrane disruption in the different model systems was in good agreement for all tested PA polymers. The efficacy of polymer-induced membrane disruption was concentration-dependent and significantly affected by the composition of the membrane. The sensitivity of relatively complex membranes of mammalian cells can be ranked between plain diphosphatidylcholine (DPPC) liposomal membranes and the more rigid cholesterol-containing DPPC membranes. Among the various studied PA polymers, medium and low molecular poly(ethacrylic acid) (PEA) and poly(propacrylic acid) (PPA) were identified as displaying significant pH-dependent disruptive activity. Relative to the disruptive cationic PA polymer (PDMAEM) the ranking is PEA < PPA < PDMAEM. The fine tuning of the pH-responsive hydrophilic-hydrophobic balance is likely to be responsible for the superior effect of PEA and PPA compared to other anionic PA polymers. This thorough investigation of a large variety of different anionic PA polymers and the comparison with an efficient, although rather toxic cationic PA polymer provides a good assessment for further therapeutic applications.
 
Solid lipid nanoparticles (SLN) carrying cholesteryl butyrate (chol-but), doxorubicin and paclitaxel had previously been developed, and the antiproliferative effect of SLN formulations versus conventional drug formulations was here evaluated on HT-29 cells. The 50% inhibitory concentration (IC(50) values were interpolated from growth curves obtained by trypan blue exclusion assay. In vitro cytotoxicity of SLN carrying chol-but (IC(50 72 h) 0.3 +/- 0.03 mM vs >0.6 mM) and doxorubicin (IC(50 72 h) 81.87 +/- 4.11 vs 126.57 +/- 0.72 nM) was higher than that of conventional drug formulations. Intracellular doxorubicin was double after 24 h exposure to loaded SLN versus the conventional drug formulation, at the highest concentration evaluated by flow cytometry. In vitro cytotoxicities of paclitaxel-loaded SLN and conventional drug formulation (IC(50 72 h) 37.36 +/- 6.41 vs 33.43 +/-1.17 nM) were similar. Moreover, the combination of low concentrations of chol-but SLN (0.1-0.2 mM) and doxorubicin (1.72 nM) or paclitaxel (1.17 nM) exerted a greater-than-additive antiproliferative effect at 24 h exposure, while the combination of Na-but and doxorubicin or paclitaxel did not. These preliminary in vitro results suggest that SLN could be proposed as alternative drug delivery system.
 
It is common practice to coat oral solid dosage forms with polymeric materials for controlled release purposes or for practical and aesthetic reasons. Good knowledge of thermo-mechanical film properties or their variation as a function of polymer grade, type and amount of additives or preparation method is of prime importance in developing solid dosage forms. This work focused on the dynamic mechanical thermal characteristics of free films of hypromellose 2910 (also known as HPMC), prepared using three grades of this polymer from two different manufacturers, in order to assess whether polymer chain length or origin affects the mechanical or thermo-mechanical properties of the final films. Hypromellose free films were obtained by casting their aqueous solutions prepared at a specific concentrations in order to obtain the same viscosity for each. The films were stored at room temperature until dried and then examined using a dynamic mechanical analyser. The results of the frequency scans showed no significant differences in the mechanical moduli E' and E″ of the different samples when analysed at room temperature; however, the grade of the polymer affected material transitions during the heating process. Glass transition temperature, apparent activation energy and fragility parameters depended on polymer chain length, while the material brand showed little impact on film performance.
 
A novel modified film-hydration-dilution method was employed to prepare highly encapsulated interferon-alpha-2b containing liposomes for intramuscular sustained release. The liposomes produced by this technique were a mixture of mainly unilamellar vesicles and a small number of multilamellar vesicles. The trapping efficiency was above 80%. With at least 60-fold dilution, Triton X-100 at the concentration of 0.3% (w/v) in phosphate buffered saline (PBS) was able to solubilize phospholipids without denaturing the protein and/or interfering with the enzyme-linked immunoassay (ELISA). After three homogenization cycles under a pressure of 70 MPa the size of liposomes was reduced from 978 to 101 nm while the activity of interferon-alpha-2b decreased by 9.9% compared to the control. Although liposomes were physically stable for 22 months at 4 degrees C the mean size of the liposomes increased slightly from 101 to 122 nm. The levels of free interferon-alpha-2b at the site of intramuscular injection decreased rapidly with only 4.15% of initial dose retained at the injection site after 0.33 h following injection of an interferon-alpha-2b solution (nonencapsulated). In contrast, interferon-alpha-2b encapsulated in liposomes was retained at the site of intramuscular injection at higher levels than free interferon-alpha-2b (p < 0.05). Larger liposomes containing interferon-alpha-2b (978 nm) were the most effective for local retention because 27.8% of interferon-alpha-2b was retained after 24 h. These liposomes have the potential to be topically injected for treating genital herpes with prolonged interferon levels at the local injection site. Since the smaller liposomes (75.8 and 101 nm) retained interferon-alpha-2b at the injection site for shorter times while enhancing the blood circulation of the drug, they are potentially good carriers for systemic therapy with higher bioavailability and liver targeting.
 
Cyclosporine is a potent immunosuppressive drug that undergoes extensive hepatic metabolism catalyzed primarily by the cytochrome P450 (P450) 3A enzyme family. Cyclosporine alters its own metabolism by selective suppression of specific P450 isoforms after chronic therapy in rats. Modulation of hepatic P450 by chronic cyclosporine dosing is associated with increased blood concentrations leading to nephropathy. However, the relationship between cyclosporine dose and hepatic enzyme suppression is not known. The purpose of this study was to examine the effect of escalating doses of cyclosporine on P450 regulation and metabolic activity in the rat. Following 1 week of a low-salt diet, rats were given cyclosporine 5, 15, 30 or 50 mg/kg per day or an equal volume of vehicle for 2 weeks via oral gavage. At the end of the dosing period, livers were removed and hepatic microsomes prepared. Hepatic P450 proteins were measured using Western blot analysis and catalytic activity determined by in vitro testosterone hydroxylation. Cyclosporine dosing suppressed both P450 3A2 and 2C11 protein expression and catalytic activity in a dose-dependent manner. Catalytic activity of two other P450 isoforms, 2A1 and 2B1, were unchanged by cyclosporine administration. Thus, the selective suppression of hepatic microsomal P450 by cyclosporine is not only dependent on the length of therapy, but also the dose administered.
 
(2R)-(3-amino-2-fluoropropyl)sulphinic acid (AFPSiA) is a potent GABAB agonist, which makes it a possible alternative in future GERD treatment. The degradation of AFPSiA was investigated to support the drug-development effort. The compound is too polar to be compatible with regular reversed-phase LC. Moreover, the compound and the degradation products cannot be detected by UV due to low absorption. Instead, the degradation of AFPSiA was followed by two different capillary electrophoresis methods with indirect UV detection and 1H NMR and 19F NMR. AFPSiA was very unstable in basic conditions and at temperatures above room temperature. The corresponding sulphonic acid and allylamine are formed via two separate degradation routes. Both these degradation products may cause unwanted side-effects in vivo. Aqueous solutions of AFPSiA were found to be more stable at pH between 1 and 3. It was suggested that AFPSiA should be stored frozen, preferably at -70 degrees C. In solid state, the compound can be stored at ambient conditions in closed vials with low relative humidity. A solid oral dosage form should be kept in a blister package.
 
We report oxabicyclo[3.3.1]nonanones as inhibitors of key redox enzymes, trypanothione synthetase (TryS) and trypanothione reductase (TryR) of Leishmania. Further, detailed cellular effects of 4-(4,4,8-Trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester, a oxabicyclo[3.3.1]nonanones, on the parasite were investigated. As these compounds inhibit key redox enzymes (TryR amd TryS), treatment of these compounds resulted in increased reactive oxygen species (ROS), mitochondrial membrane damage, activation of caspase like proteases and DNA damage that finally leads to apoptosis. Although the compound has modest IC50 value against parasite (4.9 ± 0.4 μM), they identify a novel chemical space to design and develop drugs based on these compounds against the Leishmania parasite. This is first report of oxabicyclo[3.3.1]nonanones as antileishmanial.
 
Film forming emulsions containing Eudragit® NE 30D (NE) and/or Eudragit® RS 30D (RS) were developed and investigated. The influences of surfactant (polysorbate 80), thickeners (hydroxypropylmethylcellulose (HPMC), polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA)) and varying compositions of NE and RS on glass transition temperature, mechanical properties and water resistance of free films were investigated. Elongation was found to increase with increasing NE fraction or addition of polysorbate 80 and to decrease with the addition of PVP. Adhesion to polycarbonate was generally higher than to glass. Adhesion to glass and polycarbonate was found to be influenced by NE/RS ratio, choice of thickener and addition of surfactant. An increase in RS led to stronger adherence to glass and to higher water resistance. Desired properties were obtained from films containing PVA, polysorbate 80 and 0-40% NE. These formulations were used in in vitro release and permeation experiments. In vitro release of nonivamide was found to rise with increasing amount of NE in the formulation. Permeation through pig ear skin was found to be independent of NE-ratio but was clearly slower than from a standard formulation.
 
The activity of the renal peptide transporters PEPT2 and PEPT1 determines-among other factors such as metabolic stability in liver and plasma-the circulatory half-life of penicillins and cephalosporins during therapy. This study was initiated to examine systematically the interaction of beta-lactam antibiotics with PEPT2. Interaction of 31 cephalosporins and penicillins with the carrier protein was characterized by measuring their ability to inhibit the uptake of [(14)C]Gly-Sar into renal SKPT cells. Cefadroxil, cefaclor, cyclacillin, cephradine, cephalexin and moxalactam were recognized by PEPT2 with very high affinity comparable to that of natural dipeptides (K(i)=3-100microM). Ceftibuten, dicloxacillin, amoxicillin, metampicillin, cloxacillin, ampicillin, cefixime, cefamandole, oxacillin and cefmetazole interacted with PEPT2 with medium affinity (K(i)=0.1-5mM). For the other beta-lactam antibiotics studied interaction was very low or not measurable (K(i)>5mM). The affinity constants of beta-lactam antibiotics at rPEPT2 and hPEPT1 are significantly correlated, but the rank orders are not identical. Decisive differences between PEPT1 and PEPT2 recognition of the N-terminal part of the compounds became evident. Moreover, this large data set of affinity constants of beta-lactam antibiotics will be useful for structure-transport (binding) analyses of PEPT2.
 
The main objective of the current study was to investigate penetration of cell penetrating peptides (CPPs: TAT, R8, R11 and YKA) through skin intercellular lipids using (31)P magic angle spinning (MAS) solid-state NMR. In vitro skin permeation studies were performed on rat skin, sections (0-60, 61-120 and 121-180 μm) were collected and analyzed for (31)P NMR signal. The concentration dependent shift of 0, 25, 50, 100 and 200 mg/ml of TAT on skin layers, diffusion of TAT, R8, R11 and YKA in the skin and time dependent permeation of R11 was measured on various skin sections using (31)P solid-state NMR. Further, CPPs and CPP-tagged fluorescent dye encapsulate liposomes (FLip) in skin layers were tagged using confocal microscopy. The change in (31)P NMR chemical shift was found to depend monotonically on the amount of CPP applied on skin, with saturation behavior above 100 mg/ml CPP concentration. R11 and TAT caused more shift in solid-state NMR peaks compared to other peptides. Furthermore, NMR spectra showed R11 penetration up to 180 μm within 30 min. The results of the solid-state NMR study were in agreement with confocal microscopy studies. Thus, (31)P solid-state NMR can be used to track CPP penetration into different skin layers.
 
Because of the immunogenicity and toxicity in vivo of large molecules such as lectins, the application of these molecules is remarkably restricted in drug delivery systems. In this study, to improve the brain drug delivery and reduce the immunogenicity of traditional lectin modified delivery system, Odorranalectin (OL, 1700 Da), a novel non-immunogenic small peptide, was selected to establish an OL-modified cubosomes (Cubs) system. The streptavidin (SA)-conjugated Cubs were prepared by incorporating maleimide-PEG-oleate and taking advantage of its thiol group binding reactivity to conjugate with 2-iminothiolane thiolated SA; mono-biotinylated OL was then coupled with the SA-modified Cubs. The OL-decorated Cubs (OL-Cubs) devised via a non-covalent SA-biotin "bridge" made it easy to conjugate OL and determine the number of ligands on the surface of the Cubs using sensitive chemiluminescent detection. Retention of the bio-recognitive activity of OL after covalent coupling was verified by hemagglutination testing. Nose-to-brain delivery characteristic of OL-Cubs was investigated by in vivo fluorescent biodistribution using coumarin-6 as a marker. The relative uptake of coumarin carried by OL-Cubs was 1.66- to 3.46-fold in brain tissues compared to that incorporated in the Cubs. Besides, Gly14-Humanin (S14G-HN) as a model peptide drug was loaded into cubosomes and evaluated for its pharmacodynamics on Alzheimer's disease (AD) rats following intranasal administration by Morris water maze test and acetylcholinesterase activity determination. The results suggested that OL functionalization enhanced the therapeutic effects of S14G-HN-loaded cubosomes on AD. Thus, OL-Cubs might offer a novel effective and noninvasive system for brain drug delivery, especially for peptides and proteins.
 
Top-cited authors
Robert Gurny
  • University of Geneva
Sven Gohla
  • University of Zurich
Hideki Ichikawa
  • Kobe Gakuin University
Thomas Kissel
  • Philipps University of Marburg
Jerome Berger
  • Center for primary care and public health, University of Lausanne (Unisanté)