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Encapsulation and modified-release of thymol from oral microparticles as adjuvant or substitute to current medications.

ABSTRACT

The aim of this study was to encapsulate, thymol, in natural polymers in order to obtain (i) taste masking effect and, then, enhancing its palatability and (ii) two formulations for systemic and local delivery of herbal drug as adjuvants or substitutes to current medications to prevent and treat several human and animal diseases. Microspheres based on methylcellulose or hydroxypropyl methylcellulose phthalate (HPMCP) were prepared by spray drying technique. Microparticles were in vitro characterized in terms of yield of production, drug content and encapsulation efficiency, particle size, morphology and drug release. Both formulations were in vivo orally administered and pharmacokinetic analysis was carried out. The polymers used affect the release and, then, the pharmacokinetic profile of thymol. Encapsulation into methylcellulose microspheres leads to short half/life but bioavailability remarkably increases compared to the free thymol. In contrast, enteric formulation based on HPMCP shows very limited systemic absorption. These formulations could be proposed as alternative or adjuvants for controlling pathogen infections in human or animal. In particular, methylcellulose microspheres can be used for thymol systemic administration at low doses and HPMCP particles for local treatment of intestinal infections.

Keywords:

Thymol

Herbal drug encapsulation

Anti-infective formulations

Microparticles

Modified-release formulation

In vivo studies

Introduction

Essential oils and their volatile constituents are used widely to prevent and treat human and animal diseases. Essential oils rich in phenolic constituents such as eugenol and thymol (e.g. Thymus serpyllum or spathulifolius and Origanum vulgare L.) possess an antioxidant activity against free radicals and other reactive oxygen species as well as against oxidation of low density lipoproteins (LDL), causative agents of cardiovascular diseases (Kulisic et al., 2004; Sokmen et al., 2004; Tepe et al., 2005). Recently, because of its inhibitory effect against various inflammatory cytokines, thymol is proposed as potential anti-inflammatory drug, especially in the case of lipopolysaccharide induced inflammation (Chauhan et al., 2014).

Thymol, and essential oils rich in thymol, have proven benefits in medical, food, agricultural, veterinarian and pest control applications because of their antibacterial and antifungal properties (Ulbricht, 2004; Sacchetti et al., 2005; Oussalah et al., 2006; Lazar-Baker et al., 2010). In fact, thymol inhibits Gram-positive and Gram-negative pathogenic bacteria; activity against some pathogenic bacterial strains such as Escherichia coli, Salmonella enteritidis, Salmonella choleraesuis and Salmonella typhimurium, Bacillus subtilis, Klebsiella pneumoniae and Staphylococcus aureus is reported (Edris, 2007). Thymol, like other phenolic compounds, is hydrophobic and is likely to dissolve in the hydrophobic domain of the cytoplasmic membrane of bacterial cells, between the lipid acyl chains, thus, altering the fluidity and permeability of cell membranes (Trombetta et al., 2005). Due to this ability, many studies demonstrated an additively or moderate synergism of essential oil components in combination with antibiotics, indicating that they may offer possibilities for reducing antibiotic use (Hamoud et al., 2014; Langeveld et al., 2014).

Moreover, thymol interferes more than eugenol on the envelope of Candida albicans and thus its colonization and infectiousness (Braga et al., 2007) and reduces in vitro viability of the causative agent of cystic echinococcosis (Elissondo et al., 2013).

Unfortunately, while thymol provides beneficial therapeutic effects, it also provides the consumer with a flavour perception that can be described as unpleasant, harsh or medicinal in taste. Thus, taste-masked compositions would provide the consumer with a product with pleasant, acceptable taste that increases the patient compliance towards this herbal drug.

Moreover, the use of natural essential oils or compounds extracted from essential oils as the main therapeutic agents still faces the problems of (i) the easiness of degradation or chemical reactivity, (ii) the limited water solubility of these materials which limits thymol administration and/or oral bioavailability (Shoji and Nakashima, 2004) and (iii) high volatile character (Wattanasatcha et al., 2012).

On the basis of these remarks, aim of this work was the encapsulation of thymol in microspheres based on natural polymers in order to obtain (i) taste masking effect and, then, enhancing its palatability and (ii) two formulations for systemic and local delivery herbal drug as adjuvants or substitutes to current anti-infective medications to prevent and treat human and animal infections. Furthermore, a simple preparative method is proposed for easy scale up for rapid industrial production.

Microencapsulation provides an important tool for food, pharmaceutical, cosmetic and agrochemical industries, enabling protection and controlled release of several active agents. The encapsulation of essential oils in core-shell or matrix particles has been investigated for various reasons, e.g., protection from oxidative decomposition and evaporation, odour masking or merely to act as support to ensure controlled release (Martins et al., 2014). Spray-drying is the most common and cheapest technique to produce microencapsulated materials. It results in powders with good quality, low water activity, easier handling and storage (Favaro-Trindade et al., 2010; Carneiro et al., 2013). Equipment is readily available and production costs are lower than most other methods (Gharsallaoui et al., 2007). In this work methylcellulose and hydroxylpropyl methylcellulose phthalate were chosen as polymers to produce microspheres. Methylcellulose swells in water and reproduces a clear to opalescent, viscous, colloidal solution (Kamel et al., 2008). Hydroxypropyl methylcellulose phthalate is a pH-sensitive polymer which is stable in acidic conditions of the stomach but degradable in enteric conditions. It is often used as an enteric drug carrier (Pal et al., 2009).

Materials and methods

Materials

Thymol (T) (purity [greater than or equal to] 99.5%) and aniline (IS) were obtained from Sigma Aldrich (Italy). Methylcellulose (Methocel) (MC) and hydroxylpropyl methylcellulose phthalate NF (HPMCP) were purchased from BioChemika FlukaChemie (Buchs, Switzerland) and Eastman Chemical Company (Kingsport, Tennessee, USA), respectively. Dichloromethane (C[H.sub.2][Cl.sub.2], Riedel-de-Haen, Milan, Italy) and methanol (C[H.sub.3]OH, Normapur p.a., BDF1 Prolabo, Paris, France) were of analytical grade. Ultra-pure water is prepared with the MilliQ R4 system Millipore (Milan, Italy).

Pre-formulation study

Determination of free thymol solubility in gastric and intestinal simulated fluid

The solubility of T was determined in two different pH values (1.2 and 6.8), representative of main sections of the gastrointestinal tract. Drug powder was added in excess in a flask, which contained 50 ml of the medium studied in each case. The flasks were placed in a thermostated water bath at 37.0 [+ or -] 0.5 [degrees]C, under magnetic stirring, for 24 h. Afterwards, 1 ml was withdrawn, filtered and injected in capillary electrophoresis-diode array detection system (Agilent Technologies).

An uncoated fused-silica capillary (50 cm x 50 [micro]m ID) was used for the capillary electrophoresis separation. The running buffer consisted of lOOmM sodium phosphate pH 7.4. A separation voltage of 20 kV was applied. Samples were injected hydrodynamically with a pressure of 50 mbar for 20 s. The detection was made at 210nm. Standard curves show linearity in the range 0.01-1 mg/ml with [r.sup.2] = 0.999.

Ex vivo permeation capability of free thymol through colonic pig mucosa

Experiments for testing the permeation capability of T to cross the intestinal mucosa were carried out by ex vivo permeation tests.

Experiments were performed using a new modified Franz diffusion system incorporating three in-line flow-through diffusion cells (Gavini et al., 2011). Each cell consisted of a donor compartment and a receptor compartment. The diffusion membrane was placed between the cell compartments; the diffusional area was 3.14 [cm.sup.2]. The receptor solution was continuously stirred by means of a spinning bar magnet.

A fragment of colon was excised from intestine of pigs obtained from a local slaughterhouse, gently washed with PBS, stored in ice-cold PBS for transport to the laboratory, and finally deprived of the serosal mucosa. The mucosa was positioned to ensure that the mucosal portion was in contact with the microparticles and the muscular mucosa was in contact with the pH 6.8 buffer, in the receptor chamber thermostated at 37[degrees]C. An exactly weighed amount of T powder (9 mg) was uniformly dispersed on the superior portion of the mucosa. The flux of liquid was set at 6.8 ml/min and saturated with 5% of C[O.sub.2].

The amount of buffer employed as acceptor medium was 250 ml. At selected time points (0-6 h), the amount of drug permeated was determined by capillary electrophoresis analysis. The acceptor medium withdrawn was refilled in order to maintain sink condition.

At the end of test, the surface of excised mucosa was washed with 10 ml of pH 6.8 buffer to remove T which eventually did not permeate; thus, the tissue was frozen at -20[degrees]C before the T extraction process which was performed by placing the pieces of mucosa in 2 ml of methanol under constant stirring for 24 h at room temperature. Thereafter, the suspensions were stirred in vortex and centrifuged for 10 min at 3000 rpm; supernatant was analysed by capillary electrophoresis. Experiment was performed in triplicate.

Preparation of spray-dried microspheres

Two formulations containing T were produced using a co-current spray-dryer apparatus, Mini Spray Dryer, model B-191 (BiichiLabortechnik AG, Flawil, Switzerland).

Microspheres were prepared by spraying solutions obtained by dissolving T and MC (T-MC) or HPMCP (T-HPMCP) polymers in a methanol and dichloromethane mixture (50-50 v/v); concentrations of total solid in solutions were 2% and 5% (wt/v) for T-MC and T-HPMCP, respectively. The drug to polymer ratio was 1:2 (wt/wt).

The following conditions were used during spray drying: drying airflow, 31.3 [m.sup.3]/h; spraying airflow, 5001/h; solution feed rate, 2.9 [+ or -] 0.2 ml/min; nozzle size, 0.7 mm; the inlet temperature was established at 50 [degrees]C and the outlet temperature was 42-45[degrees]C.

After production, microspheres were stored in desiccators at 20[degrees]C before continuing with the experiments.

In vitro characterization of microparticles

Yield of production, drug content and encapsulation efficiency

Dried microspheres were accurately weighed, and considering the total amount of drug and polymers used for preparing the feed solution, the yield of production (YP) was calculated, as a percentage, obtained by dividing the amount of microspheres produced (MS and the weight of drug and polymer solubilized in the feed solutions (T + P), as follows:

YP = MS/T+P x 100 (1)

In order to determine the exact amount of T loaded into polymer matrix, 10 mg of microspheres were solubilized in 10 ml MilliQ water, 18 h after production. The concentration of drug in solution was determined by capillary electrophoresis analysis (see Section 2.3). The amount of drug loaded into the polymer matrix (DL(%)) was calculated applying the following equation:

DL(%)(rT/tT) x tDL (2)

where rT is the detected amount of T in microspheres, tT is the T amount solubilized in feed solution and tDL the percentage of the expected theoretical value. Then, encapsulation efficiency (EE(%)) was calculated:

EE(%)= (DL/tDL) x 100 (3)

Drug content and encapsulation efficiency values were determined by performing experiments in triplicate for all batches.

Moreover, the content of T in the produced formulations was determined after 1 year, in order to verify the long-term stability of the drug into the polymeric matrix.

Particle size and morphology

Measurements of particle size of microparticles were performed using a Coulter Laser Diffraction, model LS 100Q (Beckman Coulter, Miami, FL). Microspheres were suspended in 2-propanol and sonicated for about 2 s. The analyses were performed at room temperature under gentle magnetic stirring of the dispersion medium.

Shape and surface characteristic of microspheres were analysed by Scanning Electron Microscopy (SEM) (Zeiss DMS 962, Carl Zeiss NTS GmbH, Oberkochen, Germany). The samples were placed on double-sided tape that had previously been secured on aluminium stubs and then analysed at 20 kV acceleration voltage after gold sputtering, under argon atmosphere.

Thymol release in gastrointestinal environment

The release of T from microspheres in pH-simulated gastric fluid followed by pH-simulated intestinal fluid was investigated. Tests were performed using a USP dissolution apparatus 1 (Erweka DT70, Erweka GmbH, Heusenstamm, Germany) at 37 [+ or -] 0.5[degrees]C and 100 rpm. Experiments were carried out by putting an amount of microspheres containing 10 mg of T into 300 ml simulated gastric fluid (Eur. Pharm. 7.0 (2011). After 2 h, 0.2 M trisodium phosphate dodecahydrate solution (82 ml) was added to the acidic medium in order to reach pH 6.8 and the test was continued for further 4h. At appropriate intervals, 1 ml of sample was taken and fresh medium was added to maintain a constant volume and sink condition. Then, collected samples were analysed by electrophoresis and the amount of T released was calculated by referring to the calibration curves previously prepared.

The drug dissolution rate was determined as comparison. All experiments were performed in triplicate.

In vivo pharmacokinetic studies

Twelve pigs, weaned at 24 days of age (10 kg average live weight), were taken from a commercial piggery. The piglets were moved to an experimental farm, housed in pens with a mesh floor and kept at a controlled temperature (28[degrees]C at the beginning and 25[degrees]C at the end of the experiment). The procedures carried out on the pigs were conducted in compliance with Italian laws on experimental animals and were approved by the Ethic-Scientific Committee for Experiments on Animals of the University of Bologna (n. 19413-X/10 of 04 May 2011).

The piglets were divided into two groups, six subjects each, balanced for litter and weight. Piglets assigned to T-MC or T-HPMCP received the product mixed in 50 g of feed, to reduce the feeding time and ensure the complete ingestion of the thymol dose. The exact amount of microspheres and, thus, of T was determined. The administered doses were 0.03 (T-MC) and 0.035 g/Kg (T-HPMCP).

Blood samples were collected by vene puncture of a vena cava of each pig for pharmacokinetic studies. The timing of sampling ranged between 0.5 and 10 h.

The blood samples were centrifuged to obtain plasma ad stored at -20[degrees]C until analysis.

Faecal samples were collected at 12 and 24 h. After 50 h, the pigs were anaesthetized with sodium thiopental (10mg/kg body weight, Zoletil 100, Virbac, Milano, Italy) and euthanized by an intracardiac injection of Tanax[R] (0.5 ml/kg BW; Intervet Italia, Peschiera Borromeo, Italy); samples of kidney, liver, lung and muscle were picked up to determine the amount of T eventually accumulated.

One ml of plasma was deproteinized with 2 ml of acetonitrile: the suspension obtained was mixed and centrifuged at 1000 x g for 10 min, and the supernatant was then transferred into vials.

One g of tissues and faeces was extracted with methanol (2 ml), stirred in a US-apparatus for 30 min and centrifuged at 1000 x g for 10 min. The supernatant was filtered with syringe filters 0.45 [micro]m and transferred into vials for GC/MS analysis.

The GC/MS analysis was performed on an Agilent 6850 gas chromatograph coupled with an Agilent 5973 N mass spectrometer. The GC column was HP-5 (30 m x 0.25 mm ID, film thickness 0.15 [micro]m). The temperature of the column was kept at70[degrees]Cfor4minand was then raised to 180[degrees]C (20[degrees]C/min). The ions were scanned in the selected ion-monitoring (SIM) mode: main fragments are 91,135 and 150 m/z for thymol ([t.sub.R] = 8.00 min) and 52,66 and 93 for anyline ([t.sub.R] = 3.40 min). The results were compared with the in vivo pharmacokinetic behaviour of free T, previously tested (Nieddu et al., 2014).

Statistical analysis

Statistical analysis was performed with GraphPad Prism 4.0 for Windows. Unpaired t-test was applied to test the significance of the effect of the kind of polymer on characteristics of spray-dried microspheres. Analysis of dissolution data was performed using the One-way Anova test following which individual differences between treatments were identified using non-parametric post-hoc tests (Turkey's or Dunnett's test). The significant level was set at p < 0.05. Pharmacokinetic analysis was performed with a PK Solutions software (version 2.0, Summit Research Services, Montrose, CO).

Results

Pre-formulation study

Determination of free thymol solubility in gastric and intestinal simulated fluid

Thymol is poorly water soluble compound; its [pK.sub.a] and log P are 10.6 and 3.4, respectively. In order to assure sink conditions during release test, solubility value of T in gastrointestinal simulated fluids was assayed.

The pH of the solution used has an effect on the solubility values of the drugs: in fact, solubility decreases from 0.5 mg/ml at pH 1.2 to 0.34 mg/ml at pH 6.8.

Ex vivo permeation capability of free thymol through colonic pig mucosa

In order to verify the ability of T to permeate across colonic pig mucosa, ex vivo permeation studies of thymol were carried out. Results show that only 9.3 [+ or -] 2.1% of T permeates the mucosa after 360 min, whereas 53.3 [+ or -] 0.5% of T is extracted from the tissue and 9.7 [+ or -] 3.8% is detected on colonic pig mucosa. Data reported in literature show that the in vivo absorption of thymol is fast and occurs mainly and nearly completely in stomach. Nevertheless, thymol shows low plasma concentrations compared to the dose administered and part of EO is not absorbed from stomach and reach the small intestine. The amounts that are recovered in small intestine represented only a minor fraction of the ingested quantity of EO that probably is the part of the EO had escaped solubilization and absorption in stomach due to adsorption to organic matter (Michiels et al., 2008). Ex vivo permeation results confirm, thus, that thymol has not high capability to cross the colon mucosa and reach the bloodstream but probably it remains on the tissue by exerting a topical action; this behaviour could be useful during Salmonella infection as therapeutic approach.

Preparation and in vitro characterization of microparticles

Thymol microspheres were produced by spray-drying technique using MC and HPMCP as polymers, spray-drying appears to be a suitable method for the preparation of T loaded microspheres; yield of production depends on polymer used and it decreases significantly when MC is used as well as the encapsulation efficiency (p < 0.0001) (Table 1).

After 1 year, the drug loading and encapsulation efficiency are reduced of about 10.5% for T-MC with respect to 6.9% of T-HPMCP (p < 0.05). This behaviour can be due to the slow evaporation of T from the polymer matrix and HPMCP is more able to retain T in the microspheres than MC.

All prepared microspheres are characterized by small [d.sub.vs] values ranging from 5.19 to 5.86 [micro]m and unimodal particle size distributions (Fig. 1), regardless the polymer and manufacturing parameters used. Likewise, T-MC and T-HPMCP microspheres show similar morphology: they have smooth surface, almost spherical shape and several invaginations (Fig. 2). No free drug crystals are found outside the microspheres.

Results from in vitro release tests are illustrated in Fig. 3. A targeted release can be achieved with regard to polymer used: as expected, T-HPMCP releases drug mainly in the enteric simulated fluid.

In particular, T dissolves quickly in acidic medium: 70% of the amount tested is already estimated in 0.1 M HCl medium after 120 mi; thereafter, following the change of pH, further 20% of T is found in phosphate buffer, pH 6.8. Therefore, about 90% of free T totally dissolves at the end of the test.

By encapsulating T into T-MC microspheres the dissolution behaviour does not change and drug release profile is almost super-imposable to the dissolution curve of free T (p > 0.05). On the contrary, only 20% of loaded drug is released after 120 min at pH 1.2 from T-HPMCP (p < 0.001), while the remainder 50% of T is released within 30 min from the change of pH.

In vivo pharmacokinetic studies

The pharmacokinetic profile of drug after in vivo oral administration of formulations T-MC and T-HPMCP has been evaluated. T plasma concentration profile vs time is shown in Fig. 4, and pharmacokinetic parameters are reported in Table 2. The results were compared with the data of free T previously obtained (Nieddu et al., 2014), re-calculated here on the basis of the observed area (0-10 h).

Free T shows [T.sub.max] and [C.sub.max] values of 1.3 h and 3.6 [micro]g/ml, respectively; the AUC in the time range considered (0-1 Oh), is 17.3 [micro]g h/ml.

When T is loaded into microspheres based on methylcellulose (T-MC), which are designed for the gastric release of the drug, an improvement of the absorption amount and rate can be obtained because of the dosage form; in fact, [T.sub.max] of microspheres is 0.5 h compared to [T.sub.max] of drug which is 1.3 h (p < 0.05) and [C.sub.max] related to T-MC is about 5 folds higher then [C.sub.max] of free T (p < 0.05). Moreover, the AUC value also vastly increases (p < 0.05) (Table 2).

As already pointed out, microspheres are used in the field of drug delivery studies to improve solubility of poorly water-soluble drugs not well-absorbed after oral administration: by reducing drug particle size to the absolute minimum, and hence improving drug wet-tability, bioavailability may be significantly improved (Wong et al., 2006; Paudel et al., 2013). In fact, results show that bioavailability of T encapsulated into microspheres T-MC remarkably increases even if the half-life halved. The high blood concentration and bioavailability would consent dosage decrease. As consequence of these results this formulation seems to be suitable for the systemic administration of T. Data from in vitro release experiments above discussed, find a evidence in obtained in vivo results: encapsulated T into T-MC is released into the stomach and mainly absorbed leading to a short [T.sub.max] and high [C.sub.max]. The improvement in the absorption rate of T is due to the encapsulation into microspheres; it is known, indeed, that by loading a drug into microspheres, its dissolution rate can be improved and this could also increase drug bioavailability (Gavini et al., 2006; Le-Ngoc Vo et al., 2013). Drug dissolution rate is affected by effective surface area that is highly increased by reducing particle size. The inclusion of T into a solid dispersion matrix of hydrophilic compound such as MC, should give a large effective surface area and ensure wetting of the drug particle. In our previous work thymol-[beta]-cyclodextrin complex was prepared; the improvement in the absorption rate of T is achieved due to [beta]-CD; it is known, indeed, that CDs interact with compounds that are poorly water soluble to increase their apparent solubility and enhance transmucosal absorption. Increasing the apparent solubility of a drug can increase the drug dissolution rate. Bioavailability of T, however, is not improved with respect to free T (Nieddu et al., 2014).

The effect of the polymer used on in vivo behaviour of T is not negligible. Microspheres T-MC and T-HPMCP have different plasma profiles (Fig. 4), characterized by a rapid absorption ([T.sub.max] 0.5 h) even if with highly diverse concentrations ([C.sub.max] of 14.9 and 3.0 [micro]g/ml, respectively) as illustrated in Table 2: even in case of T-HPMCP, the microencapsulation process leads to the increase of absorption rate; nevertheless the amount of T dissolved into the stomach is low compared to T-MC due to polymer property and, thus the [C.sub.max] is still low. Although the rapid absorption time, indeed, T-HPMCP shows pharmacokinetic behaviour similar to free T and drug bioavailability is comparable to the free T. Data are in agreement with results from the in vitro dissolution test: T-HPMCP is able to retain T in gastric environment; It is released later into the enteric tract where scarce absorption occurs giving low [C.sub.max] and bioavailability. Thus, it can exert topically antimicrobial activity making this formulation useful for local administration of T.

Encapsulation of T in microspheres leads to reduction of drug half/life which is almost two times lower than free T (3.2 and 3.8 h in case of T-MC and T-HPMCP, respectively, and 6.1 h in case of T) (p < 0.05). These data are confirmed by the mean residential time (MRT) values which are higher in case of T than T-MC and T-HPMCP meaning that free T elimination slowly occurs.

As consequence of the reduction of drug half/life observed, higher administration number with respect to T can be assumed. Nevertheless, as already pointed out, low dosage can be used. On the basis of the in vivo results, T-MC formulation can be proposed for the systemic administration of T.

Scarce drug absorption from microspheres T-HPMCP is observed as they release thymol in the intestinal tract where the absorption process of T seems to be very limited. Therefore, this formulation could be useful for the topical administration of thymol in the intestine. Here, thymol released from T-HPMCP could have local effect against Gram-positive and Gram-negative pathogenic bacteria or Candida in human infected or to prevent microorganisms development during the early phases of the diseases.

In all cases, no residual drug is recovered in tissues analysed (kidney, muscles, lung and liver). Small amounts of T are found in faeces after 12 and 24 h: differences among T-MC and T-HPMCP are observed. According to the highest quantities absorbed, T-MC shows the lowest T residual in faeces. Thymol amount recovered in faeces after 12 and 24 h are 0.175 [+ or -]0.05 jig/g and just traces in case of T-MC, and 2.02 [+ or -] 0.21 and 0.55 [+ or -] 0.11 in case of T-HPMCP.

Conclusion

Thymol can be loaded into microspheres by spray drying. By using different polymers is possible to affect the release and, then, the pharmacokinetic profiles of Thymol. Encapsulation into methylcellulose microspheres leads to short half/life but remarkably bioavailability increases. Therefore, it can be proposed for thymol systemic administration at low doses. On the contrary, enteric formulation based on HPMCP shows very limited systemic absorption and therefore it can be proposed for local treatment of intestinal infections.

Conflict of interest statement

The authors declare that there is no conflict of interests regarding the publication of this article.

http://dx.doi.org/10.1016/j.phymed.2014.07.017

Acknowledgments

This work was supported by "Regione Autonoma della Sardegna" (Grant number: CRP1.404; Legge Regionale 7 August 2007, no. 7).

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ARTICLE INFO

Article history:

Received 7 May 2014

Received in revised form 17 June 2014

Accepted 29 July 2014

G. Rassu (a), M. Nieddu (a), P. Bosi (b), P. Trevisi (b), M. Colombo (b), D. Priori (b), P. Manconi (a), P. Giunchedi (a), E. Gavini (a), G. Boatto (a,*)

(a) Department of Chemistry and Pharmacy, University ofSassari, 07100 Sassari, Italy

(b) Department of Agri-Food Science and Technologies (DISTAL), University of Bologna, 42123 Reggio Emilia, Italy

* Corresponding author. Tel.: +39 079228725; fax: +39 079228727.

E-mail address: gboatto@uniss.it (G. Boatto).

Table 1
Results of characterization of spray-dried microspheres.

         Formulation                  T-MC

         Yield of production          37.14  [+ or -]  4.22
         (% [+ or -] SD)
18 h     Drug loading                 22.25  [+ or -]  0.64 ($, #)
         (% [+ or -] SD)
         Encapsulation                64.76  [+ or -]  1.87 ($, #)
         efficiency (% [+ or -] SD)
1 year   Drug loading                 19.92  [+ or -]  0.99 (,#)
         (% [+ or -] SD)
         Encapsulation                57.96  [+ or -]  2.89 (,#)
         efficiency (% [+ or -] SD)

         Formulation                  T-HPMCP

         Yield of production          52.75  [+ or -]  3.17
         (% [+ or -] SD)
18 h     Drug loading                 26.25  [+ or -]  0.00 ($, *)
         (% [+ or -] SD)
         Encapsulation                78.74  [+ or -]  0.00 ($, *)
         efficiency (% [+ or -] SD)
1 year   Drug loading                 24.45  [+ or -]  0.00 (,*)
         (% [+ or -] SD)
         Encapsulation                73.33  [+ or -]  0.00 (,*)
         efficiency (% [+ or -] SD)

Unpaired t-test: T-MC vs T-HPMCP.

($) p < 0.001.

([section]) p < 0.005.

# T-MC 18 h vs T-MC 1 y p < 0.05.

* T-HPMCP 18 h vs T-HPMCP 1 y p < 0.0001.

Table 2
Pharmacokinetic parameters.

Parameters                Units           T          T-MC     T-HPMCP

Half-life                 h                    6.1      3.2       3.8
[C.sub.max] (obs)         [micro]g/ml          3.6     14.9       3.0
[T.sub.max] (obs)         h                    1.3      0.5       0.5
AUC (0-10 h) (obs area)   [micro]g-h/ml       17.3     47.4       9.8
MRT (area) (a)            h                    8.2      3.8       3.7
Vd (obs area) (b)         ml              255596.1   3025.9   20258.7
CL (obs area) (c)         ml/h             28831.0   6495.6    3676.7

(a) .MRT: mean residence time (time for 63.2% of administered
dose to be eliminated).

(b) Vd: volume of distribution.

(c) CL: systemic clearance based on observed data points: AUC
(0-10 h).
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Article Details
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Author:Rassu, G.; Nieddu, M.; Bosi, P.; Trevisi, P.; Colombo, M.; Priori, D.; Manconi, P.; Giunchedi, P.; G
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Oct 15, 2014
Words:5624
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