Discussion of EMA Draft Guideline on Quality and Equivalence of Topical Products Based on Comparison of Approved Mometasone Furoate Drugs.

INTRODUCTION: Today, the approval for a generic topical product includes the presentation of therapeutic equivalence to the originator based on clinical trials. To facilitate this procedure, in 2018 the European Medicines Agency (EMA) published a draft guideline on quality and equivalence of topical products, which includes request parameters regarding the quality of the newly developed generic product and test protocols for the implementation of equivalence tests regarding efficacy. METHODS: To date, no data are available on the quality and evidence of the proposed test conditions. In this study, we performed an in vitro penetration test (IVPT) following the terms of the EMA draft guideline on two authorized topical products for which therapeutic equivalence was already proven during the approval process. RESULTS: The complex biometric data processing revealed that in vitro equivalence could not be observed for all skin sections for either originator or generic product. Moreover, the necessity of the negative control proposed in the draft guideline is more than questionable. From the results presented, there were indications that a reduced number of skin donors would be sufficient to achieve statistically significant equivalence in the comparison of all applied formulations. Here, n = 7 donors was proposed instead of n ≥ 12 as the EMA draft guideline demands, decreasing the degree of biodiversity simultaneously. Moreover, a higher number of independent replicates (n > 2) was proposed for proper statistics. CONCLUSION: This bioequivalence study shows insufficient parameters, which should be discussed together with the EMA draft guideline.

Investigation of ex vivo Skin Penetration of Coenzyme Q10 from Microemulsions and Hydrophilic Cream.

INTRODUCTION: Coenzyme Q10 (CoQ10) has been widely used in topical and cosmeceutical products due to its cutaneous antioxidant and energizer effects. CoQ10 is found in a higher concentration in the epidermis compared to dermis. The epidermal level of CoQ10 can be reduced due to several factors such as skin UV irradiation and photoaging. Various dermal nano-formulations have been investigated to overcome the skin barrier and enhance the poor penetration of CoQ10. The nanocarriers are designed to target and concentrate the CoQ10 in the viable epidermis. Most of these studies, however, failed to show the depth and extent of penetration of CoQ10 from the various carrier systems. OBJECTIVE: The distribution of CoQ10 across the various skin layers has to be shown using skin slices representing the different skin layers. METHODS: To realize this objective, a sensitive and selective HPLC method was developed and validated for the quantification of CoQ10 in the different skin slices. The method applicability to skin penetration (using excised human skin) as well as stability studies was investigated using CoQ10-loaded lecithin-based microemulsion (ME) and hydrophilic cream formulations. RESULTS: It could be shown that the highest concentration of CoQ10 in the viable epidermis, the target skin layer for CoQ10, was observed after application of the CoQ10 in the hydrophilic cream. This cream contains 10% of 2-ethylhexyl laurate which works obviously as a penetration enhancer for CoQ10. In contrast, the penetration of CoQ10 was lower from the ME. Just in the deeper dermis, a certain amount of CoQ10 could be detected. CONCLUSIONS: The HPLC method quantified the trace quantities of the CoQ10 distributed across the various skin layers and, hence, can be used to investigate the skin penetration of CoQ10 from various dermal standard and nano-formulations.

Development and characterization of microemulsions containing hyaluronic acid.

Tween80 and Span20 were used as surfactant mixture for developing non-ionic microemulsions (MEs) containing hyaluronic acid 22 kDa (HA). The effect of Tween80:Span20 ratio (T:S ratio) on microemulsion (ME) water intake and stability was studied. Moreover, the effect of HA on the consumed surfactant amount which is for stabilizing the MEs, for reducing water intake was investigated. Two W/O MEs containing HA were optimized. The first ME was composed of 2% HA, 13.8% Tween:80:Span20 (2:3), 4.2% water and 79.9% isopropylpalmitate (IPP). The second was composed of 2% HA, 16% Span20, 9.6% water:dimethyl sulfoxide (W:DMSO) (6:3.6) and 72.4% medium chain triglycerides (MCTG). The droplet sizes of MEs were determined using dynamic light scattering (DLS). The multilayer membrane system (MLMS) was used for testing the release of HA from both MEs and the released amount of HA was quantified using capillary zone electrophoresis (CZE). Furthermore, three phase diagrams and relevant rheological characteristics were generated. The droplet size of the ME without HA decreased and increased with increasing the temperature. Furthermore, the droplet size of the IPP-ME and MCTG-ME without HA and of the MCTG-ME with HA decreased with increasing temperature. In contrast to this results, the droplet size of the IPP-ME with HA increased with increased temperature. This ME belongs to the Newtonian fluids. Compared to the first ME, the second ME shows droplet sizes at 25 °C of 6.5 nm without and 37 nm with HA. The droplet size in the second ME decreased proportionally with an increase of the temperature with and without HA. The release of HA was faster from the IPP ME compared to the MCTG-ME. The two developed MEs were stable, isotropic and their properties comply with ME properties concerning the droplet size and viscosity.

Drug Release from ß-Cyclodextrin Complexes and Drug Transfer into Model Membranes Studied by Affinity Capillary Electrophoresis.

PURPOSE: Is to characterize the drug release from the ß-cyclodextrin (ß-CD) cavity and the drug transfer into model membranes by affinity capillary electrophoresis. Phospholipid liposomes with and without cholesterol were used to mimic the natural biological membrane. METHODS: The interaction of cationic and anionic drugs with ß-CD and the interaction of the drugs with liposomes were detected separately by measuring the drug mobility in ß-CD containing buffer and liposome containing buffer; respectively. Moreover, the kinetics of drug release from ß-CD and its transfer into liposomes with or without cholesterol was studied by investigation of changes in the migration behaviours of the drugs in samples, contained drug, ß-CD and liposome, at 1:1:1 molar ratio at different time intervals; zero time, 30 min, 1, 2, 4, 6, 8, 10 and 24 h. Lipophilic drugs such as propranolol and ibuprofen were chosen for this study, because they form complexes with ß-CD. RESULTS: The mobility of the both drug liposome mixtures changed with time to a final state. For samples of liposomal membranes with cholesterol the final state was faster reached than without cholesterol. CONCLUSIONS: The study confirmed that the drug release from the CD cavity and its transfer into the model membrane was more enhanced by the competitive displacement of the drug from the ß-CD cavity by cholesterol, the membrane component. The ACE method here developed can be used to optimize the drug release from CD complexes and the drug transfer into model membranes.

Optimization of ion-pair formation between glycopyrronium bromide and different ion-pair agents using ACE.

Glycopyrronium bromide (GLB) is an anticholinergic drug. Its highly polar quaternary ammonium group could limit the skin permeation of the topical hydrophilic drug. The ion pair formation with suitable anionic molecules was suggested to play a role in enhancing the cationic drug transport to the target site. The interactions between GLB and different ion-pair agents (IPAs) were investigated using ACE. The changes in the effective mobility of 0.05 mM GLB were correlated with the increasing concentrations of IPAs in 20 mM BGE of pH 6.2, and successfully fitted into a nonlinear binding isotherm equation assuming 1:1 stoichiometric interaction. The formation constants (Kf ) were 74.33, 28.5, 18.17, 8.2, 7.6, 5.69, 4.76, and 3.96 M-1 for sodium salts of dodecyl sulfate, taurodeoxycholate, taurocholate, glycodeoxycholate, glycocholate, salicylate, quinolone sulfonate, and p-toluene sulfonate; respectively. Surfactant's and bile salts' concentrations were below CMCs. The partition coefficient of GLB between buffer phase and n-octanol was determined successfully in the absence and presence of different IPAs. The study confirmed the linear correlation (R2 = 0.907) between the affinity strength of ion pair and the partition behavior of GLB in the presence of anionic molecules at 1:1 molar ratio.

Controlled nail delivery of a novel lipophilic antifungal agent using various modern drug carrier systems as well as in vitro and ex vivo model systems.

The penetration behavior into human nails and animal hoof membranes of a novel antifungal agent (EV-086K) for the treatment of onychomycosis was investigated in this study. The new drug provides a high lipophilicity which is adverse for penetration into nails. Therefore, four different formulations were developed, with particular focus on a colloidal carrier system (CCS) due to its penetration enhancing properties. On the one hand, ex vivo penetration experiments on human nails were performed. Afterwards the human nail plates were cut by cryomicrotome in order to quantify the drug concentration in the dorsal, intermediate and ventral nail layer using high-performance liquid chromatography (HPLC) with UV detection. On the other hand, equine and bovine hoof membranes were used to determine the in vitro penetration of the drug into the acceptor compartment of an online diffusion cell coupled with Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy. In combination, both results should exhibit a correlation between the EV-086K penetration behavior in human nail plates and animal hoof membranes. The investigations showed that the developed CCS could increase drug delivery through the human nail most compared to other formulations (nail lacquer, solution and hydrogel). Using animal hooves in the online diffusion cell, we were able to calculate pharmacokinetic data of the penetration process, especially diffusion and permeability coefficients. Finally, a qualitative correlation between the penetration results of human nails and equine hooves was established.

Microemulsion and mixed micelle for oral administration as new drug formulations for highly hydrophilic drugs.

Microemulsions (MEs) and mixed micelles (MMs) have been used as new drug formulations for high hydrophilic drugs such as cefpirom and cefodizim for oral administration. Cefpirom and cefodizim are neither actively nor passively transported across cell membranes. Up to date, they can be only administrated intravenously (i.v.) or intramuscularly (i.m.). The rabbit (Chinchilla) in vivo model was used in the present work to investigate ways of overcoming the poor oral absorption of these cephalosporins. The cephalosporins at 100mg/kg were formulated in MEs and MMs and administered intraduodenally (i.d.). Very low bioavailability (2.5-3.0%) was observed, if cefpirom or cefodizim i.d. were applied without colloidal vehicle. However, the addition of the cephalosporins to ME or MM is shown to be highly effective in increasing the bioavailability values (up to 64% absolute bioavailability) of the model drugs. In conclusion, MEs and MMs improve essentially the oral bioavailability of the high hydrophilic drugs.

Preparation, physicochemical characterization and biological evaluation of cefodizime metal ion complexes.

OBJECTIVES: Cefodizime is a broad spectrum cephalosporin belonging to the third generation agents. In this study, attention has been paid to the preparation, physicochemical characterization and biological evaluation of new Cu2+, Zn2+, Fe3+, Co2+ and Al3+ complexes of cefodizime. METHODS: The stoichiometrics and the mode of bonding of the complexes were deduced from their elemental and metal analysis, electrical conductivity measurements, UV-vis, infrared and Raman spectroscopic investigations. Study of the stoichiometry of these complexes referred to the formation of 1 : 1 ratios of metal to ligand. Antimicrobial activity of the complexes was determined using two strains of Gram-positive (Bacillus subtilis and Proteus vulgaris) and two strains of Gram-negative (Escherichia coli W3110 and Pseudomonas putida) bacteria. The minimal inhibitory concentration was determined as the lowest concentration inhibiting bacterial growth on solid Luria Bertani medium. KEY FINDINGS: The spectra gave evidence as to the position of binding. In addition, the aqueous solubility of cefodizime was strongly reduced by complexation. CONCLUSIONS: The antibacterial activity of cefodizime was not affected by complexation with Al3+ but it was reduced by complexation with the other tested metal ions against the bacteria under study.

Characterization of the interaction of cefadroxil with different metal ions using CE.

The present study describes the application of CE to investigate the interaction between a cephalosporin, cefadroxil and different metal ions. This study aims at quantifying the interaction of cefadroxil with Zn(2+), Al(3+), Fe(3+), Cu(2+) and Co(2+) ions using the effective electrophoretic mobility as a parameter for calculation of the reaction association constant (K). For this purpose, the electrophoretic mobilities of cefadroxil at different metal ion concentrations in citrate buffer pH 3.6 were determined. A mathematical model to calculate the association constant between cefadroxil and metal ions was used. The results showed that a strong change in the cefadroxil electrophoretic mobility was observed by increasing the metal ion concentrations. The results revealed that by increasing the concentration of metal ions in the running buffer the degree of complexation increases too. On the basis of the results, the strength of the interaction of cefadroxil with the investigated metal ions follows the order Zn(2+)>Cu(2+)>Co(2+)>Fe(3+)>Al(3+). The association constant of the investigated reactions ranged from 451.57 to 1546.52 L/mmol. The results indicate that it is possible to characterize the interaction between cefadroxil and metal ions quantitatively using CE.

Effect of Different Metal Ions on the Biological Properties of Cefadroxil.

The effect of different metal ions on the intestinal transport and the antibacterial activity of cefadroxil [(6R,7R)-7-{[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino}-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid] was investigated. The [14C]Gly-Sar uptake via PEPT1 was inhibited by Zn2+ and Cu2+ treatment in a concentration-dependent manner (Ki values 107 ± 23 and 19 ± 5 µM, respectively). Kinetic analysis showed that the Kt of Gly-Sar uptake was increased 2-fold in the presence of zinc sulphate (150 µM) whereas the Vmax value were not affected suggesting that zinc ions inhibited Gly-Sar uptake by PEPT1 in a competitively manner. Ni2+ exhibited moderate inhibitory effect, whereas Co2+, Mg2+, Al3+ ions showed no inhibitory effect on Gly-Sar uptake via PEPT1. Subsequently, we examined the effect of Zn2+ and Al3+ ions on the transepithelial transport of cefadroxil across Caco-2 cells cultured on permeable supports. The results showed that zinc ions inhibited the transepithelial flux of cefadroxil at Caco-2 cell monolayers while Al3+ ions had no effect. The interaction of cephalosporins with the metal ions could suggest negative effects of some metal ions on the clinical aspects of small intestinal peptide and drug transport. Finally, the effect of Zn2+, Cu2+ and Al3+ ions on the antibacterial activity of cefadroxil was tested. It was found that there is no significant difference between the activity of cefadroxil and the cefadroxil metal ion complexes studied against the investigated sensitive bacterial species.

Chiral separation of the plant lignan matairesinol by capillary electrophoresis.

Lignans are dimeric phenylpropanoid compounds in plants that enjoy increasing medicinal interest because of their phytoestrogen activity. Lignans are chiral compounds and for most natural occurring lignans, chirality is not known. Separation of racemic matairesinol by CE in a non-coated silica capillary with carboxymethyl-beta-cyclodextrin as chiral selector in phosphate buffer was successful. Electrolyte and selector concentrations and pH were systematically optimized in order to obtain baseline separation and short analysis times. Matairesinol from safflower fruit was determined as (-)-enantiomer. Quantitation results for matairesinol with the optimized method after calibration with authentic lignan were very similar to those by HPLC. The limit of detection is 2 microg/mL sample by DAD detection.

CE characterization of potential toxic labile iron in colloidal parenteral iron formulations using off-capillary and on-capillary complexation with EDTA.

The present study describes the application of CZE to investigate the portion of labile iron in the following parenteral formulations: iron gluconate, iron saccharate, and iron dextran. Labile iron was detected as Fe(III)-chelate of EDTA at 246 nm. When EDTA was incubated with the formulations before electrophoresis, labile iron, or chelatable iron, respectively, was detected in all formulations, mostly in iron gluconate and iron saccharate. It was observed that the amount of iron released is time- and pH-dependent. In contrast, when EDTA was separately injected before the formulation sharp peaks of the Fe(III)-chelate were detected only after injection of iron gluconate. This type of labile iron can be described as "electrophoretically free" iron. CE provides thus a new method to characterize potential toxic, labile iron in parenteral iron formulations.

Influence of absorption enhancers on the pharmacokinetic properties of non-oral beta-lactam-cefpirom using the rabbit (Chinchilla) in vivo model.

The oral application is the application of the first choice for drug administration. A lot of drugs exhibit relatively low bioavailability. This may be caused by binding of the drug in the gastro-intestinal tract, by poor penetration of the intestinal mucose or by highly hydrophilic properties. Therefore, problem drugs were only used for i.v. administration (intravenously) or for i.m. administration (intramuscularly). In the present study, cefpirom was investigated as a model substance. Cefpirom (Cp) is a semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin. It exhibits highly hydrophilic properties (P(ow)=0.02+/-0.01) and a very low bioavailability (AUC=524+/-403 microg min/ml). It was only applied i.v. or i.m. In this work, the influence of absorption enhancers (aggregation and ion-pair formation) on the bioavailability and on the hydrophilic properties of Cp was investigated. The bioavailability of cefpirom was improved through the combination with absorption enhancers (hexadecyldimethylbenzylammonium chloride, BAC; hexylsalicylic acid, HSA). The absolute bioavailability of the Cp combination with absorption enhancers was 21 times larger for BAC and 15 times larger for HSA than in the case when Cp was used alone.

Improvement of lipophilicity and membrane transport of cefuroxime using in vitro models.

Most beta-lactam antibiotics cannot be absorbed orally and, therefore, must be administered intravenously (i.v.) or intramuscularly (i.m.). Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefuroxime exhibiting significant advantages in the parental treatment of common infections, was used as model drug in the present study. The effect of the cationic absorption enhancers (four quaternary ammonium salts) on the lipophilicity of cefuroxime was investigated by means of the n-octanol/water system. The results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in vitro transport model with artificial (dodecanol collodium membrane) and biological membranes (Charles-River guinea pig).

Influence of enhancers on the absorption and on the pharmacokinetics of cefodizime using in-vitro and in-vivo models.

In the development of novel antibiotics, more and more compounds have been found that cannot be absorbed orally and, therefore, must be administered intravenously or intramuscularly. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability has received much attention in pharmaceutical research. Cefodizime, a novel third-generation cephalosporin with significant advantages in the parenteral treatment of common infections, was used as a model drug. Cefodizime behaves as a highly hydrophilic compound, as shown from its extremely low partition coefficient. The effect of cationic absorption enhancers (hexadecyldimethylbenzylammonium chloride, N-hexadecylpyridinium bromide, dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) on the lipophilicity of cefodizime was investigated by means of the n-octanol/water system. Results showed that the counter-ions had a positive influence on the solubility of cefodizime. These results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in-vitro transport model with artificial and biological membranes (Caco-2-cells). Furthermore, the physiological compatibility of the absorption enhancers was investigated using the active D-glucose transport. The pharmacokinetic profile of cefodizime was evaluated in rabbits after intraduodenal administration with and without an absorption enhancer.

The release profiles of intact and enzymatically digested hyaluronic acid from semisolid formulations using multi-layer membrane system.

A multi-layer membrane system was used to measure in vitro release of hydrophilic macromolecules such as hyaluronic acid (HA) from semisolid formulations. One enzymatically digested HA-derivative with molecular mass of 22 kDa (HA-D) and 1200 kDa intact HA (HA) were incorporated into three semisolid formulations: water-containing hydrophilic ointment (WHO), amphiphilic cream (AC) and water-containing wool wax alcohol ointment (WWO). Because of the high hydrophilic properties of HA-D and HA, the artificial model membranes consisted of collodion as the matrix and glycerol as the hydrophilic acceptor phase. The area under the concentration-time curve and the mean dissolution time were used as a quantitative parameter to characterise the rate and extent of release in vitro. This study showed that the HA-D and HA release as hydrophilic substances from WHO was higher than both from AC and WWO. It was observed that 83% of HA-D1 was released from WHO after 2 h; in contrast, only 10% was released from 2% HA from the same vehicle during the same time. In conclusion, the in vitro availability of enzymatically digested HA-D was higher for WHO than for the other formulations, AC and WWO. Similarly, the availability of HA-D was higher than that of HA from the same formulations.

Characterization of enzymatically digested hyaluronic acid using NMR, Raman, IR, and UV-Vis spectroscopies.

Hyaluronic acid (HA) is a linear polysaccharide formed from disaccharide units containing N-acetylglucosamine and glucuronic acid. When HA was digested with the enzyme hyaluronidase, a double bond is formed. It is known that this double bond forms a complex (radical scavenger) with the radicals (ROO, HO) during UV irradiation, and reduced the toxicity of the radicals before they are absorbed in the human skin. Therefore, the characterization of the double bond formed after the enzymatic digestion of HA is very important. In this study, 1H NMR, 13C NMR, Raman, infrared (IR), and UV-Vis spectroscopies were used for characterization of the double bond of HA after enzymatic digestion. HA derivatives in shape of films were tested using Raman and infrared (IR) spectroscopies and the wavenumber of the double bond and some other assignment were determined. The 1H and 13C NMR spectra were measured for HA derivatives in D(2)O solutions. The chemical shifts and coupling constant of 1H and 13C were assigned to the CH=C fragment. The relative amount of olefinic proportion in the mixture was obtained from 1H and 13C NMR spectra. The spectroscopy measurement showed an increase in the double bond amount with increasing enzymatic digestion time.

In-vitro and in-vivo studies of cefpirom using bile salts as absorption enhancers.

Cephalosporins have to be administered by injection because of the poor intestinal absorption of the orally delivered drugs. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefpirom (Cp) is a new semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin that has been substituted in position 3 with a cyclopenteno-pyridinium group in order to create a zwitterionic compound. It exhibits highly hydrophilic properties, as shown from its extremely low partition coefficient, and therefore its lipophilicity was increased using bile salts. The effect of this on the partition coefficients determined in the n-octanol/buffer system was confirmed using an in-vitro transport model with artificial and biological membranes. The pharmacokinetic properties of Cp were investigated in rabbits after intraduodenal administration with and without bile salts. Furthermore, the physiological compatibility of the bile salts was investigated using active D-glucose transport.

Determination of CMC of sodium glucocorticides hemisuccinates by CE.

The capillary zone electrophoresis (CZE) method was applied to the determination of the critical micelle concentration (CMC) and the anionic mobilities (mu(e)) for sodium glycocorticides hemisuccinates (Urbason solubile forte 1000, Hydrocortison 100 Rotexmedica, Prednisolut 100) in phosphate solution at pH 7.2. The CZE enables an efficient and very fast determination of parameters for characterizing physicochemical properties of micelles.

Characterization of interaction between protein and carbohydrate using CZE.

The present study describes the application of the capillary zone electrophoresis (CZE) to investigate interactions between Concanavalin A (Con A) and the one of their major components (L-asparagin) with a variety of carbohydrates (D-glucose, D-fructose, D-mannose D-galactose, maltose and lactose). It was observed that the carbohydrates show different interactions with Con A and L-asparagin. Addition of carbohydrates to the electrolyte buffer and different concentrations of carbohydrates led to a change of the migration time and the ionic mobility of Con A and L-asparagin. A mathematical model for quantitative evaluation to calculate the aggregation constants (K) was used. Our investigations show that it is possible to characterize the interaction between carbohydrates and proteins or amino acid quantitatively using CZE.