Evaluation of solid carvedilol-loaded SMEDDS produced by the spray drying method and a study of related substances.

In this study, various formulations of solidified carvedilol-loaded SMEDDS with high SMEDDS loading (up to 67% w/w) were produced with the spray drying process using various porous silica-based carriers. The process yield was improved with higher atomization gas flow rate during the spray drying process and with prolonged mixing time of dispersion of liquid SMEDDS and solid porous carriers prior to the spray drying process. Depending on the choice of the carrier and the SMEDDS:carrier ratio in solid SMEDDS, different drug loading, self-microemulsifying properties, drug release rates, and released drug fractions were obtained. The products exhibited fast drug release due to preserved self-microemulsifying properties and the absence of crystalline carvedilol, which was confirmed with XRD and Raman mapping. A decrease in drug content during the stability study was observed and investigated. This was at least partially attributed to the chemical degradation of the drug. Key degradation products determined by the LC-MS method were amides formed by in situ reaction of carvedilol with fatty acids present in the oily phase of SMEDDS.

Quality improvement of melt extruded laminar systems using mixture design.

This study investigates the application of melt extrusion for the development of an oral retard formulation with a precise drug release over time. Since adjusting the formulation appears to be of the utmost importance in achieving the desired drug release patterns, different formulations of laminar extrudates were prepared according to the principles of Experimental Design, using a design for mixtures to assess the influence of formulation composition on the in vitro drug release from the extrudates after 1h and after 8h. The effect of each component on the two response variables was also studied. Ternary mixtures of theophylline (model drug), monohydrate lactose and microcrystalline wax (as thermoplastic binder) were extruded in a lab scale vertical ram extruder in absence of solvents at a temperature below the melting point of the binder (so that the crystalline state of the drug could be maintained), through a rectangular die to obtain suitable laminar systems. Thanks to the desirability approach and a reliability study for ensuring the quality of the formulation, a very restricted optimal zone was defined within the experimental domain. Among the mixture components, the variation of microcrystalline wax content played the most significant role in overall influence on the in vitro drug release. The formulation theophylline:lactose:wax, 57:14:29 (by weight), selected based on the desirability zone, was subsequently used for in vivo studies. The plasma profile, obtained after oral administration of the laminar extruded system in hard gelatine capsules, revealed the typical trend of an oral retard formulation. The application of the mixture experimental design associated to a desirability function permitted to optimize the extruded system and to determine the composition space that ensures final product quality.

Immunomodulation mediated by a herbal syrup containing a standardized Echinacea root extract: a pilot study in healthy human subjects on cytokine gene expression.

In this study, the immunomodulatory effect of a triply standardized Echinacea angustifolia root extract (Polinacea(®)) was evaluated in 10 healthy subjects. Ten ml of syrup containing one hundred mg of extract (corresponding to 4.7 mg of Echinacoside and 8.0mg of a high molecular weight-20,000 Da- polysaccharide) were administered as a herbal syrup once a day for one month. The immunomodulatory effect was evaluated before and after herbal syrup administration evaluating the expression levels of the cytokines IL-2, IL-8, IL-6 and TNF-α. Cytokine expression was studied in lympho-monocytes and in plasma samples measuring the mRNA and protein levels, respectively. The results were analysed by ANOVA and non-parametric Friedman rank sum tests; when possible it was adopted a pair-wise comparisons at different post-treatment times, using the paired t-tests with Holm correction. The correlation between the variations of cytokine plasma levels and the respective mRNA was carried out using a linear regression model. In lympho-monocytes our data indicate the up-regulation of the mRNA levels of IL-2 and IL-8 and the down regulation of the mRNA levels of the pro-inflammatory cytokines TNF-α and IL6. The differential regulation was maximal after 14 days of treatment. IL-2 up-regulation and IL-6 down-regulation were also confirmed at the protein level in plasma. Finally, the up-regulation of the mRNA of IL-2/IL-8 and the down-regulation of IL-6 positively correlated with the protein levels detected in the plasma. In conclusion, this pilot study suggests a relevant role for the standardized Echinacea angustifolia root extract in the control of cytokine expression. This first demonstration of the immuno-modulating activity of Echinacea angustifolia root extract in the healthy subject, supports at least in part the common use of such products as health promoting supplement.

A new approach to enhance oral bioavailability of Silybum Marianum dry extract: association of mechanochemical activation and spray congealing.

The aim of the work was to produce a delivery system for Silybum Marianum dry extract with enhanced oral bioavailability by combining two technologies (mechanochemical activation and spray congealing). Initially, the active was coground with sodium croscarmellose in a planetary mill in order to reach an activated state more prone to dissolution. DSC, XRD, FT-IR and LD analyses showed the formation of nanosized particles of dry extract, with reduced degree of crystallinity of the main crystalline flavolignans (silybine A and B). Then, microparticles containing the activated coground and, as comparison, the corresponding physical mixture of extract and polymer and the dry extract alone were produced by spray congealing technology using Gelucire(®) 50/13 as a hydrophilic low m.p. carrier. Microparticles containing the activated coground were produced spherical in shape, achieved satisfactory yield and high encapsulation efficiency. These microparticles, in addition to a favourable in vitro solubilisation kinetic, in a preliminary in vivo study in five rats demonstrated their ability to improve very significantly the oral bioavailability of the main flavolignans of Silybum Marianum dry extract (silybin A and B). These results suggested that the association of mechanochemical activation and spray congealing could be considered an innovative and very useful approach to the oral delivery of Silybum Marianum. Furthermore, for the first time the possibility of successfully applying the spray congealing technology for the preparation of a herbal drug delivery system was shown.

Oral bioavailability of silymarin phytocomplex formulated as self-emulsifying pellets.

The objective of this study was to develop new solid self-emulsifying pellets to deliver milk thistle extract (silymarin). These pellets were prepared via extrusion/spheronisation procedure, using a self-emulsifying system or SES (Akoline MCM®, Miglyol®, Tween 80®, soy lecithin and propylene glycol), microcrystalline cellulose and lactose monohydrate. To select the most suitable formulations for extrusion and spheronisation, an experimental design of experiences was adopted. The screening amongst formulations (13 different blends) was performed preparing pellets and evaluating extrusion profiles and quality of the spheronised extrudates. The pellets were characterised for size and shape, density, force required to crush them. Although more than one type of pellets demonstrated adequate morphological and technological characteristics, pellets prepared from formulation 7 revealed the best properties and were selected for further biopharmaceutical investigations, including in vitro dissolution and in vivo trials on rats to study serum and lymph levels after oral administration of the pellets. These preliminary technological and pharmacokinetic data demonstrated that extrusion/spheronisation is a viable technology to produce self-emulsifying pellets of good quality and able to improve in vivo oral bioavailability of main components of a phytotherapeutic extract of more than 100 times by enhancing the lymphatic route of absorption.

Solid state mechanochemical activation of Silybum marianum dry extract with betacyclodextrins: Characterization and bioavailability of the coground systems.

Silybum marianum dry extract, whose therapeutic use is partially restricted by the insolubility in water of its main flavonolignans, was subjected to a mechanochemical activation process in planetary mill using betacyclodextrins as carriers. After optimization of the operating conditions according to an established theoretical model, the best active-to-carrier proportion was selected from the preliminary trials. When using the optimized conditions, the mechanochemical process permits an improvement of the physico-chemical properties of the active, which reaches an "activated" solid state, that is stable for at least 1 year. In fact, XRD, DRIFT and Raman spectroscopy analyses showed that the main extract component, Silybin, completely lost its crystalline structure after co-grinding with betacyclodextrins and formed weak interactions with the carrier. The powder characteristics remarkably changed after co-grinding, leading to a sample with a very small mean diameter and with a twofold increase of the specific surface area in comparison to the dry extract. The activated solid state of the coground systems remarkably enhanced the in vitro drug dissolution kinetics with consequent improved oral bioavailability. Furthermore, the in vivo studies on rats revealed a 6.6-fold bioavailability increase respect to the S. marianum Italian commercial product used as reference (Silirex 200 capsules).

Solid state mechanochemical simultaneous activation of the constituents of the Silybum marianum phytocomplex with crosslinked polymers.

Simultaneous improvement of solubilization kinetics of main flavolignans of Silybum marianum extract was obtained cogrinding with two crosslinked polymers: micronized crospovidone, PVP-CL(R) and sodium carboxymethylcellulose, Ac-Di-Sol(R) in the 1:3 active-to-polymer weight ratio. By this process it was assessed that the main extract components lost its crystalline structure, and the powder surface area was increased by 2.1- and 1.7-fold in the coground products with Ac-Di-Sol(R) and PVP-CL(R), respectively. This activated status of the dry extract remained stable over a period of 2 years. Solubilization kinetics resulted ameliorated both in terms of entire dry extract and in terms of single components. When the 1/3 coground systems with PVP-CL(R) and Ac-Di-Sol(R) were dissolved in saturated conditions they gave a concentration improvement compared to the native product of 8 and 31 times of silybin A, 7 and 27 times of silybin B, whereas in the case of silychristin a double concentration was obtained only using Ac-Di-Sol(R). The in vivo studies on rats confirmed that this solubilization improvement corresponded to an effective oral bioavailability enhancement. The highest bioavailability improvement was obtained with Ac-Di-Sol(R), with a relative bioavailability of 88.6, 17.96, and 16.4 compared to the extract for silybin A, silybine B, and silychristine, respectively.

Pharmacokinetics and immunomodulatory effects of phytotherapeutic lozenges (bonbons) with Echinacea purpurea extract.

The relative bioavailability of the major alkamides, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides, from Echinacea purpurea phytotherapeutic lozenges at three different dose levels (0.07, 0.21 and 0.9 mg) was evaluated in a pharmacokinetic study in humans and the possible effects on the immunological system were measured. Alkamides were found to be rapidly absorbed and measurable in plasma 10 min after administration of 0.21 and 0.9 mg lozenges and remained detectable for 3h for the 0.21 mg lozenges and for more then 3h for the 0.9 mg lozenges; 0.07 mg lozenges were measurable 20 min after administration and remained detectable for only 2h after the administration. A significant dose-independent down-regulation of the pro-inflammatory cytokines IL-12p70, IL-8, IL-6, IL-10 and TNF was observed 24h after oral administration. The results of non-compartmental pharmacokinetic analysis revealed that a C(max) of (0.65+/-0.41 ng/ml) was reached at 32 min with the 0.07 mg lozenges, (1.00+/-0.21ng/ml) at 25 min with the 0.21 mg lozenges and (8.88+/-5.89 ng/ml) at 19 with the 0.9mg lozenges. As evidenced by the dose-exposure relationship, no significant departure from dose proportionality was observed, indicating linearity in pharmacokinetics. To get a further insight in pharmacokinetics of dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides a compartmental population pharmacokinetic model was developed applying mixed effect modelling procedure. The results demonstrate that within the dose range studied pharmacokinetics of dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides are linear and that absorption is very rapid (t(1/2)=6 min) with apparently no lag time, thus indicating the possibility that a fraction of the drug is absorbed through the oral mucosa.

Preparation of sustained release co-extrudates by hot-melt extrusion and mathematical modelling of in vitro/in vivo drug release profiles.

Aim of this work was to develop a cylindrical co-extrudate characterised by an in vivo sustained release profile by means of a hot-melt extrusion process. Co-extrudate was made up of two concentric extruded matrices: an inner one having a hydrophilic character, based on polyethylene glycol, and an outer one with lipophilic character, based on microcrystalline wax. Both segments contained theophylline as a model drug. A screening between several devices differing for dimensions (diameter and length) and relative proportions of the inner and outer part was carried out on the basis of their in vitro drug release and the release mechanism was studied by means of a mathematical model. The co-extrudate exhibiting the desired sustained release was selected for in vivo bioavailability studies. In vivo studies confirmed the achievement of the purpose of the research, demonstrating the desired release of theophylline on four healthy volunteers. Accordingly, hot-melt extrusion process is a viable method to produce in a single step co-extrudates showing a sustained release. In addition, the developed mathematical model proved to be a reliable descriptor of the both in vitro and in vivo experimental data.

Supercritical antisolvent precipitation of nimesulide: preliminary experiments.

The purpose of this preliminary study was to investigate the physico-chemical properties of nimesulide precipitated by continuous supercritical antisolvent (SAS) from different organic solvents like acetone, chloroform and dichloromethane at 40 degrees C and 80, 85 and 88 bar, respectively. Scanning electron microscopy, differential scanning calorimetry, X-Ray diffractometry and in vitro dissolution tests were employed to study how the technological process and the solvent nature would affect the final product. SAS-processed nimesulide particles showed dramatic morphological change in crystalline structure if compared to native nimesulide, resulting in needle and thin rods shaped crystals. The solid state analysis showed that using chloroform or dichloromethane as a solvent the drug solid state remained substantially unchanged, whilst if using acetone the applied method caused a transition from the starting form I to the meta-stable form II. So as to identify which process was responsible for this result, nimesulide was further precipitated from the same solvent by conventional evaporation method (RV-sample). On the basis of this comparison, the solvent was found to be responsible for the re-organization into the different polymorphic form and the potential of the SAS process to produce micronic needle shaped particles, with an enhanced dissolution rate if compared to the to the pure drug, was ascertained. Finally, the stability of the nimesulide form II, checked by DSC analysis, was ruled on over a period of 15 months.

Preparation of theophylline-hydroxypropylmethylcellulose matrices using supercritical antisolvent precipitation: a preliminary study.

Several controlled release systems of drugs have been elaborated using a supercritical fluid process. Indeed, recent techniques using a supercritical fluid as a solvent or as an antisolvent are considered to be useful alternatives to produce fine powders. In this preliminary study, the effect of Supercritical Anti Solvent process (SAS) on the release of theophylline from matrices manufactured with hydroxypropylmethylcellulose (HPMC) was investigated. Two grades of HPMC (HPMC E5 and K100) as carriers were considered in order to prepare a sustained delivery system for theophylline which was used as a model drug. The characterization of the drug before and after SAS treatment, and the coprecipitates with carriers, was performed by X-ray Diffraction (XRD) and Differential Scanning Calorimetry (DSC). The dissolution rate of theophylline, theophylline-coprecipitates, and matricial tablets prepared with coprecipitates were determined. The physical characterizations revealed a substantial correspondence of the drug solid state before and after supercritical fluid treatment while drug-polymer interactions in the SAS-coprecipitates were attested. The dissolution studies of the matrices prepared compressing the coprecipitated systems showed that the matrices based on HPMC K100 were able to promote a sustained release of the drug. Further, this advantageous dissolution performance was found to be substantially independent of the pH of the medium. The comparison with the matrices prepared with untreated substances demonstrated that matrices obtained with SAS technique can provide a slower theophylline release rate. A new mathematical model describing the in vitro dissolution kinetics was proposed and successfully tested on these systems.

Spray-dried chitosan/ethylcellulose microspheres for nasal drug delivery: swelling study and evaluation of in vitro drug release properties.

The aim of this study was to develop spray-dried chitosan-based microspheres, suitable for nasal delivery of loratadine, and to evaluate their potential of modifying loratadine release. The microspheres were composed with ethylcellulose (EC) and chitosan (CM) in two different weight ratios, 1:2 and 1:3. One-phase systems (dispersions) and two-phase systems (emulsions and suspensions) were subjected to spray-drying, resulting in conventional and composed microspheres, respectively. The microspheres were evaluated with respect to the yield, particle size, encapsulation efficiency, physical state of the drug in the polymer matrix, swelling properties and in vitro drug release profile. It was shown that particle size, swelling ability and loratadine release from spray-dried microspheres were significantly affected by the polymeric composition and feed concentration in spray-drying process. Emulsifying method to produce composed EC/CM microspheres resulted in improved loratadine entrapment and moderate swelling, when compared to conventional chitosan microspheres. It seems like better formation of EC cores and chitosan coating were obtained when higher feed concentration and ultrasonic homogenization were employed in the preparation of emulsion systems and when EC to CM weight ratio was 1:3.

Development and bioadhesive properties of chitosan-ethylcellulose microspheres for nasal delivery.

Loratadine-loaded microspheres were prepared by spray-drying of dispersions, emulsions and suspensions differing in polymeric composition and solvents used. Conventional microspheres were obtained by spray-drying of dispersions composed of chitosan (CM) as only polymer, while composed microspheres were obtained by spray-drying of two-phase systems composed of chitosan and ethylcellulose (EC). Microspheres differed in EC/CM weight ratio (0:1, 1:2 and 1:3) and in loratadine/polymers weight ratio (1:6 and 1:8). The entrapment efficiencies were between 67.9 and 86.1%; less loratadine was entrapped as polymer/drug ratio decreased. In comparison to one-phase systems composed of CM as only polymer, spray-drying of two-phase systems composed of both, CM and EC resulted in improved loratadine entrapment (80.1-86.1%). All microspheres were positively charged, indicating the presence of chitosan at the surface, regardless of the drug content and the type of spray-dried system. The highest zeta-potential was measured for loratadine-free conventional microspheres, consisting of chitosan only (32.7+/-1.3 mV). Tensile studies showed that both, EC/CM ratio and the type of spray-dried system influenced the bioadhesive properties of the microspheres in a way that the microspheres with higher chitosan content were more bioadhesive and microspheres prepared from suspensions were more bioadhesive than those prepared from emulsions, regardless of the same polymeric composition. The results suggested that the spray-drying method is useful to produce bioadhesive loratadine-loaded microspheres.

Characterisation of nimesulide-betacyclodextrins systems prepared by supercritical fluid impregnation.

The purpose of this study was to apply the supercritical CO(2) impregnation process for preparing solvent-free nimesulide (NMS)-betacyclodextrins (BCD) association systems with enhanced drug dissolution rate. Several drug-to-carrier molar ratios were tested (1:1; 1:2.5; 1:3.5) at different conditions of temperatures (40, 100, and 130 degrees C) and pressures (140, 190 or 220 bar). The physical and morphological characterisation of the systems using powder X-ray diffraction, thermal analysis, diffuse reflectance Fourier transform-infrared spectroscopy and scanning electron microscopy was carried out to understand the influence of this technological process on the physical status of single components and binary systems and to detect possible interactions between drug and carrier. These analyses provided no evidence of a complete inclusion of NMS in the carrier but the existence of interactions between drug and carrier together with a partial dehydration of the BCD and the formation of drug crystallites with lower melting point and heat of fusion than the native NMS. These phenomena were more intense when severe conditions of pressure and temperature (220 bar and 130 degrees C) were used during impregnation trials and when the amount of BCD augmented in the systems. These activated solid state of the impregnated systems promoted an enhancement of drug dissolution rate that, in keeping with the results of the physical characterisation, was function of the process conditions and BCD content.

Study of the solid state of carbamazepine after processing with gas anti-solvent technique.

The purpose of this study was to investigate the influence of supercritical CO2 processing on the physico-chemical properties of carbamazepine, a poorly soluble drug. The gas anti-solvent (GAS) technique was used to precipitate the drug from three different solvents (acetone, ethylacetate and dichloromethane) to study how they would affect the final product. The samples were analysed before and after treatment by scanning electron microscopy analysis and laser granulometry for possible changes in the habitus of the crystals. In addition, the solid state of the samples was studied by means of X-ray powder diffraction, differential scanning calorimetry, diffuse reflectance Fourier-transform infrared spectroscopy and hot stage microscopy. Finally, the in vitro dissolution tests were carried out. The solid state analysis of both samples untreated and treated with CO2, showed that the applied method caused a transition from the starting form III to the form I as well as determined a dramatic change of crystal morphology, resulting in needle-shaped crystals, regardless of the chosen solvent. In order to identify which process was responsible for the above results, carbamazepine was further precipitated from the same three solvents by traditional evaporation method (RV-samples). On the basis of this cross-testing, the solvents were found to be responsible for the reorganisation into a different polymorphic form, and the potential of the GAS process to produce micronic needle shaped particles, with an enhanced dissolution rate compared to the RV-carbamazepine, was ascertained.

Melt pelletization in high shear mixer using a hydrophobic melt binder: influence of some apparatus and process variables.

The effects of process conditions and the apparatus variables on the granulometric characteristics of a formulation containing a hydrophobic binder (stearic acid), lactose and paracetamol prepared by melt pelletization process were investigated in a 10-litre high shear mixer. The factors under investigation were: impeller speed, massing time, type of impeller blades and presence of the deflector and their reciprocal interactions. Two granule characteristics were analysed: the percentage of aggregates larger than 3000 microm (Y(1)) and the yield of the 2000-microm pellet size fraction (Y(2)). In order to estimate simultaneously the above-mentioned factors, a particular experimental design was adopted, that allowed the reduction of the number of trials from 378 to 35 and took into consideration other uncontrolled factors with the aid of a block variable. Using the postulated model, we found the optimal operating conditions to minimize Y(1) and increase Y(2) by selecting the type of impeller, and by using an impeller speed lower than 300 rpm, a massing time of 8-9 min and by not using the deflector. Finally, the validity of the adopted strategy has been proved with an additional check point.

Processing of carbamazepine-PEG 4000 solid dispersions with supercritical carbon dioxide: preparation, characterisation, and in vitro dissolution.

The purpose of this study was to apply the attractive technique of the supercritical fluid to the preparation of solvent-free solid dispersions. In particular, the gas antisolvent crystallisation technique (GAS), using supercritical carbon dioxide as processing medium, has been considered to prepare an enhanced release dosage form for of the poorly soluble carbamazepine, employing PEG 4000 as a hydrophilic carrier. The physical characterisation of the systems using laser granulometer, powder X-ray diffraction, thermal analyses, and scanning electron microscopy was carried out in order to understand the influence of this technological process on the physical status of the drug. The results of the physical characterisation attested a substantial correspondence of the solid state of the drug before and after treatment with GAS technique, whereas a pronounced change in size and morphology of the drug crystals was noticed. The dramatic reduction of the dimensions and the better crystal shape, together with the presence of the hydrophilic polymer determined a remarkable enhancement of the in vitro drug dissolution rate.

Preparation in high-shear mixer of sustained-release pellets by melt pelletisation.

The preparation of sustained-release pellets by melt pelletisation was investigated in a 10-l high shear mixer and ternary mixtures containing stearic acid as a melting binder, anhydrous lactose as a filler and theophylline as a model drug. A translated Doehlert matrix was applied for the optimisation of process variables and quality control of pellets characteristics. After determination of size distribution, the pellets were characterised with scanning electron microscopy, X-ray photoelectron spectroscopy and porosimetric analysis. Finally, the in vitro release from every single size fraction was evaluated and the release mechanism was analysed. Since the drug release rate decreased when enhancing the pellet size fraction, the 2000-microm fraction, exhibiting a substantially zero-order release, was selected for further in vivo biovailability studies. These data demonstrated that pellets based on the combination of stearic acid and lactose can be used to formulate sustained release pellets for theophylline.