Ketotifen-loaded microspheres prepared by spray-drying poly(D,L-lactide) and...

Ketotifen-Loaded Microspheres Prepared by Spray-DryingPoly(D,L-Lactide) and Poly(D,L-Lactide-co-Glycolide)Polymers: Characterization and In Vivo Evaluation

SANDRA GUERRERO,1 ENRIQUETA MUNIZ,2 CESAR TEIJON,1 ROSA OLMO,1

JOSE M. TEIJON,1 M. DOLORES BLANCO1

1Departamento de Bioquımica y Biologıa Molecular, Facultad de Medicina,Universidad Complutense de Madrid, Madrid, Spain

2Departamento de Biologıa Celular, Facultad de Biologicas, Universidad Complutense de Madrid, Madrid, Spain

Received 28 February 2007; accepted 1 October 2007

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21241

Corresponde913941447; FaxE-mail: mdb523

Journal of Pharm

� 2007 Wiley-Liss

ABSTRACT: Ketotifen (KT) was encapsulated into poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA 50/50) by spray-drying to investigate the use of biodegrad-able drug-loaded microspheres as delivery systems in the intraperitoneal cavity. Keto-tifen stability was evaluated by HPLC, and degradation was not observed. Drugentrapment efficiency was 74� 7% (82� 8 mg KT/mg for PLA) and 81� 6% (90�7 mg KT/mg for PLGA 50/50). PLA microspheres released ketotifen (57% of encapsulatedKT) in 350 h at two release rates (221 mg/h, 15 min to 2 h; 1.13 mg/h, 5–350 h). A quickerrelease of ketotifen took place from PLGA 50/50 microspheres (67.4% of encapsulatedKT) in 50 h (322 mg/h, 15 min to 2 h; 16.18 mg/h, 5–50 h). After intraperitonealadministration (10 mg KT/kg b.w.), microsphere aggregations were detected in adiposetissue. Ketotifen concentration was determined in plasma by HPLC. The drug releasedfrom PLA and PLGA 50/50 microspheres was detected at 384 and 336 h, respectively.Noncompartmental analysis was performed to determine pharmacokinetic parameters.The inclusion of ketotifen in PLGA and PLA microspheres resulted in significantchanges in the plasma disposition of the drug. Overall, these ketotifen-loaded micro-spheres yielded an intraperitoneal drug release that may be suitable for use as deliverysystems in the treatment of inflammatory response in portal hypertension. � 2007 Wiley-

Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3153–3169, 2008

Keywords: ketotifen; poly(D,L-lactide);
poly(D,L-lactide-co-glycolide); microspheres;spray-drying; in vitro–in vivo correlation
INTRODUCTION

Allergic symptoms can be treated with first-generation antihistamines, lipid-soluble drugsthat block peripheral H1 receptors. Among them,ketotifen fumarate (KT), 4-(methyl-4-piperidyli-

nce to: M. Dolores Blanco (Telephone: 34-: 34-913941691;[email protected])

aceutical Sciences, Vol. 97, 3153–3169 (2008)

, Inc. and the American Pharmacists Association

JOURNAL OF PH

dene)-4H-benzo[4,5]cyclophepta[1,2-b]thiophen-10(9H)-one hydrogen fumarate, has been widelyused as an antiallergic and antianaphylacticagent in adults and children.1 It is availableorally and as eye drops for the treatment ofallergic symptoms.2 Since ketotifen is a mast cellstabilizer, it can prevent local tissue damage andmultiorgan dysfunction due to vasoactive and pro-inflammatory mediators derived from these cells,particularly histamine, after intestinal ischemia/reperfusion.3 At clinically relevant drug concen-trations, ketotifen also induces primary necrosis

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3154 GUERRERO ET AL.

in human eosinophils. Ketotifen and other hista-mine H1-receptor antagonists freely cross theblood–brain barrier (BBB) and can affect thecentral nervous system (CNS); therapeutic dosescan lead to drowsiness and a worsening ofepileptic symptoms, and overdoses may causefatal seizures. Ketotifen has also been investi-gated in multidrug resistance in human breastcancer cells and doxorubicin toxicity in mice.4 Ithas been demonstrated that this drug reversesmultidrug resistance due to P-glycoprotein over-expression and provides cardioprotection againstdoxorubicin. There is not much published phar-macokinetic data on ketotifen,1,5–7 most of whichis in humans receiving an oral route of adminis-tration. The dosage of ketotifen administered toadult patients with bronchial asthma and allergicresponses is 1 mg orally taken twice daily.1 Dosesof 2, 10 and 50 mg/kg body weight have been orallyadministered to rats for 2 weeks to study theireffect on fertility and pregnancy.8 To study theepileptogenic activity of ketotifen, doses of 20,30 and 40 mg/kg body weight were intraperito-neally administered to amygdala-kindled andsham rats.9 Pharmacokinetic studies of ketotifenafter intravenous, intranasal and rectal adminis-tration in rabbits have been carried out with dosesof 1 mg/kg body weight, and 5 mg/kg body weightfor oral administration.7

The diverse administration routes of ketotifenand the possible advantages of loading the drug indrug delivery systems have led to designingdifferent approaches in modulating ketotifendelivery. Thus, for topical and transdermaladministration ketotifen has been included indeformable liposomes and ethosomes,10 in pres-sure sensitive adhesives (PSA) matrices11 and inpolyisobutylene patches.12 Due to its use in thetreatment of bronchial asthma, particularly of anallergic origin, dry powder inhalation formula-tions of liposomally entrapped drug have beenprepared10 for direct ketotifen delivery in therespiratory tract. Since ketotifen is used in thetreatment of allergic eye disease, the drug hasbeen loaded in contact lenses.13

Microparticles represent drug delivery systemssuitable for diverse administration routes. Overthe years, a variety of natural and syntheticpolymers have been explored for the preparationof microparticles, of which poly(lactic acid) and itscopolymers with poly(glycolic acid) have beenextensively investigated because of their biocom-patibility and biodegradability.14,15 We havecarried out degradation studies of microspheres

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008

prepared by spray-drying poly(D,L-lactic acid)(PLA) and poly(D,L-lactide-co-glycolide) (PLGA)polymers.16 Significant differences in decreasingaverage molecular weight were observed betweenPLA and PLGA 50/50 microspheres from thebeginning of the degradation process; the rateconstant of the first stage of degradation waslarger for PLGA 50/50 (k¼ 14.3� 10�3 day�1)than for PLA microspheres (k¼ 2.1� 10�3

day�1).16 Thus, this study focuses on the prepara-tion of ketotifen-loaded biodegradable micro-spheres based on PLA and PLGA 50/50polymers, their morphology and size character-ization as well as drug release experiments. Inthis study in vivo evaluation of the systems hasbeen carried out by intraperitoneal administra-tion in rats. This administration route has beenchosen due to the possible use of these polymericsystems in the treatment of the inflammationobserved in portal hypertension, a clinical syn-drome that is frequently studied using partialportal vein-ligated (PVL) rats.17 Thus, as a priorand necessary step, ketotifen-loaded PLA andPLGA microspheres were administered to non-operated rats to evaluate plasma levels of thedrug.

EXPERIMENTAL

Materials

PLA (Sigma–Aldrich, Barcelona, Spain), PLGA[lactide:glycolide 50:50] (Sigma–Aldrich), dichlor-omethane (Panreac, Barcelona, Spain), acetoni-trile (Panreac), chloroform (Panreac), potassiummonohydrogen phosphate (K2HPO4) (Panreac),potassium dihydrogen phosphate (KH2PO4) (Pan-reac), heparin (Analema, Vigo, Spain), Tween80 (Panreac) were used as received. Milli-Q1

water (Millipore, Madrid, Spain) was used.Ketotifen hydrogen fumarate was kindly suppliedby Novartis (Madrid, Spain).

Methods

Preparation of Microspheres

Preparation of microspheres was carried out by aspray-drying process (Mini Spray-dryer B-191,Buchi, Flawil, Switzerland). To obtain micro-spheres without drug, PLA or PLGA 50/50 wasdissolved in dichloromethane (2 wt%).18–21 Micro-spheres with KT were prepared from 1.8 wt%

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KETOTIFEN-LOADED MICROSPHERES PREPARED BY PLA AND PLGA POLYMERS 3155

of polymer and 0.2 wt% of ketotifen dissolved indichloromethane. The polymeric solutions (100mL) were maintained under constant stirring (900rpm) and sprayed through the nozzle of the spray-dryer. Assay conditions were: inlet air tempera-ture 63–668C, outlet air temperature 51–538C,spray flow 5 mL/min, and compressed spray airflow (represented as the volume of the air input)700 L/h. Microspheres were collected from thespray-dryer cyclone separator, and were thenplaced in a vacuum oven (Bioblock Scientifics,Illkirch, Strasbourg) for 24 h at 100 mBar ofpressure and at 378C. Microspheres were stored ina dessicator under vacuum conditions.

Particle Size and Appearance

The size and appearance as well as the sizedistribution of PLA- and PLGA50/50-based micro-spheres and KT-loaded microspheres were char-acterized by scanning electron microscopy (JeolJSM-6400 Electron Microscope, resolution 36 mmfrom Centro de Microscopıa Electronica Luis Bru,UCM). The samples were fixed to an adhesivesheet on a rigid support and coated with gold fortheir later visualization. Micrographs (Fig. 1)were recorded in randomly selected particlepopulations. For each of the SEM micrographs250 particles were measured using micrographenlargements. The counted particles were used tocheck the convergence of the polydispersity index.The combined diameters were used to calculatethe number-average diameter Dn and the weight-average diameter Dw using the following equa-tions:22

Dn ¼P

NiDiPNi

Dw ¼P

NiD4iP

NiD3i

U ¼ Dw

Dn

Ni is the number of particles measured, and Di thediameter of the measured particle. By using Dn

and Dw the polydispersity index U was calculated.The particle distribution is considered to bemonodisperse when the polydispersity index isbetween 1.0 and 1.1.23

Ketotifen Stability in Different Drug Release Media

Stability of ketotifen in different drug releasemedia, phosphate buffer 1 mM pH 7.4 and pH 2 as

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well as chloroform was studied by HPLC. Aketotifen solution was prepared (60 mg/mL) ineither phosphate buffer 1 mM pH 7.4 or pH 2 andwas maintained at 378C and at a constant stirringrate (200 rpm) for 20 days. At intervals, 100 mLsamples were withdrawn from the solution inorder to determine ketotifen concentration byHPLC (Spectra-Physics SP8800 HPLC pump, SP100 UV absorbance detector and SP 4400 comput-ing integrator). Furthermore, ketotifen stabilityin chloroform was determined; a ketotifen solution(60 mg/mL) in chloroform was maintained for 2 hat room temperature and then aliquots of 100 mLwere evaporated under N2 stream to obtain aresidue. The residue was reconstituted withmobile phase and ketotifen concentration wasdetermined by HPLC. The HPLC method is basedon that developed by Yagi et al.7 The stationaryphase was Spherisorb ODS2, C18, 5 mm(25� 0.46 cm; Waters). The mobile phasewas KH2PO4 0.1 M, pH 4 with 40% (v/v) ofacetonitrile. The flow rate was set at 1 mL/minand the detector wavelength was 297 nm. Thecalibration curve was obtained from KT solutionsbetween 0.1 and 100 mg/mL and a good linearcorrelation (r2¼ 0.99) was obtained. The valida-tion of the HPLC method was demonstrated by theprecision, calculating the coefficient of variation(CV).24 The CV for intra-run and inter-run of KTwas calculated from concentrations of 100, 1 and0.1 mg/mL. The ketotifen retention time was6.0� 0.2 min. Stability of ketotifen in the afore-mentioned solvent media was also studied by UV/V spectra (188–600 nm) (UNICAM 8700 Spectro-photometer).

Estimation of Drug Content

PLA and PLGA 50/50 as well as ketotifen aresoluble in chloroform. UV/V spectra (188–600 nm)of the polymer solutions and the drug in chloro-form were carried out. UV/V spectrum of ketotifenshows a maximum absorbance at 297 nm; how-ever, the polymer solutions did not absorb at thiswavelength. Thus, to determine the amount ofketotifen (KT) included in the polymeric micro-spheres, 10 mg of the drug-loaded microsphereswas dissolved in 1 mL of chloroform. The amountof ketotifen was determined by UV/V spectroscopyat 297 nm using a microcell (50 mL). The absorb-ance of the samples was interpolated in a cali-bration curve, which was obtained from ketotifensolutions in the range of 0.1–100 mg/mL KT. It was

NAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008

Figure 1. Scanning electron micrographs (SEM) of PLA microspheres (A), ketotifen-loaded PLA microspheres (B), PLGA 50/50 microspheres (C) and ketotifen-loaded PLGA50/50 microspheres (D). SEM of ketotifen-loaded PLA microspheres (E) and ketotifen-loaded PLGA 50/50 (F) microspheres after 350 h in the release medium (phosphate buffer1 mM, pH 7.4 at 378C).


previously verified that the absorbance of KTwas not modified when the polymers and thedrug were dissolved in freshly prepared solutions.The experiment was carried out in triplicate.The amount of drug entrapped per weight ofmicrospheres was calculated [Drug loading¼(weight of ketotifen in microspheres/microspheresample weight)]. The percentage of entrapmentefficiency was expressed by relating the


actual drug entrapment to the theoretical drugentrapment.25

In Vitro Drug Release Studies

For drug release studies, 30 mg of KT-loadedmicrospheres was added to 50 mL of phosphatebuffer (1 mM; pH 7.4), which was placed in avessel covered with Parafilm1 at 378C and at a

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constant shake (200 rpm) in an orbital incubator(Ecotron1 Inforts AG CH-4103). At intervals,100 mL samples were withdrawn from the solutionin order to follow the change in ketotifenconcentration by UV/V spectroscopy at 297 nm.In order to confirm the stability of ketotifenreleased from PLA and PLGA microspheres, theconcentration of the drug in the release mediumwas also evaluated by HPLC. The sampling timepoints were: a sample every 15 min during thefirst hour; a sample every 20 min duringthe second hour; a sample every 30 min duringthe following 2 h; a sample every hour during thefollowing 3–4 h and then one or two samples a dayfor 13 days. The volume removed from the vesselwas replaced with phosphate buffer. The concen-tration of the pharmaceutical formulation ofophthalmic solution of ketotifen (ZADITORTM;Novartis Ophthalmics) is 0.25 mg/mL. Solubilityof ketotifen in phosphate buffer pH 7 at 268Cis 10.75 mg/mL.26 In drug release experimentsketotifen concentration was always very muchlower than its solubility in phosphate buffer; thus,sink conditions were maintained. The experi-ments were carried out in triplicate.

From plots of cumulative ketotifen releasedversus time, two release rates were calculated.The first release rate was determined between15 min and 2 h; a straight line was obtained byusing a least square fit and the release rate wasdetermined from the slope of this line. The secondrelease rate was determined between 5 and 350 hfor KT-loaded PLA microspheres, and from 5 to50 h for KT-loaded PLGA microspheres by usingthe same procedure.

In Vivo Ketotifen Administration

Male Wistar rats, weighing 227� 8 g, wereobtained from the Animalario of the UniversidadComplutense de Madrid (Spain) (DC 86/609/CEE;OM 13/X/1989, RD 1201/2005). Guidelines con-tained in the NIH publication on the principles oflaboratory animal care, 85–23 revised in 1985,were followed throughout. Experiments wereapproved by the Animal Care Committee ofUniversidad Complutense de Madrid. The ani-mals were housed in cages under environmentallycontrolled conditions of light (12:12 h light:darkcycle), temperature (22� 28C) and were fedstandard rat food and water ad libitum. Justbefore injection, the dissolution solvent was putunder ultraviolet light (Ecogen Lamp, ViberLourmat, Intensity 7 mW/cm2) at 254 nm for 4 s


because of the germicidal action of this wave-length.

Different groups of animals were established.Before the drug release experiments were carriedout, a group of animals (three rats) was adminis-tered the vehicle used for microsphere adminis-tration; thus, 1 mL of saline solution (0.9% NaCl)containing 0.06 wt% of Tween-80 was intraper-itonealy injected for 1 min. This solution wasbiocompatible. The animals that were adminis-tered the drug were divided into three groups.GROUP 1: Animals injected with KT-loadedmicrospheres. The microspheres were dispersedin 1 mL of saline solution (0.9% NaCl) containing0.06 wt% of Tween-80. The animals were anaes-thetized with halothane (Burtons Series 5 T.C.V.,Kent, United Kingdom) and the microspheredispersion was then intraperitonealy injected inthe rat for 1 min using a sterile syringe with a1.2� 40 mm nozzle (Microlance 3). Group 1A (sixrats): 27 mg of PLA microspheres, whose KTcontent was 2.21 mg. Group 1B (six rats): 26 mgof PLGA 50/50 microspheres, whose KT contentwas 2.34 mg. GROUP 2 (six rats): Animalsintraperitonealy injected with 1 mL of an aqueoussolution of KT of 2 mg/mL for 1 min. Furthermore,a control group of animals was intraperitonealyinjected with PLA (three rats) or PLGA 50/50(three rats) microspheres without drug. Themicrospheres were dispersed in 1 mL of salinesolution (0.9% NaCl) containing 0.06 wt% ofTween-80.

Determination of Ketotifen in Plasma

At predetermined times after the injection of KT-loaded microspheres and KT solution, animalswere anaesthetized with halothane. Blood(0.2 mL) was collected by puncturing the jugularvein in heparinized (15 units¼ 3 mL) polypropy-lene tubes. In animals administered KT-loadedmicrospheres, blood samples were taken 6, 24 and30 h after the injection and at 24-h intervalsthereafter. In animals administered a KT solu-tion, blood samples were taken 20, 40 and 84 minand then every hour up to 4.4 h and every 2 h up to8.4 h after the injection. The heparinized bloodwas centrifuged at 11000g for 10 min in a Sigma202 M centrifuge immediately after collection soas to obtain plasma. Plasma samples were thenstored at �208C.

Ketotifen was precipitated from plasma sam-ples by acetonitrile according to a modification ofthe method proposed by Yagi et al.:7 250 mL



of acetonitrile was added to 100 mL of plasma.After vigorous shaking for 5 min and centrifuga-tion (3500g, 10 min), the supernatant wasevaporated with N2 to remove the organic solvent,and the sample was then placed in a vacuum oven(Bioblock Scientifics) for 12 h at 300 mBar ofpressure and at 308C, to obtain a residue. Theresidue was reconstituted with 12.5 mL of anaqueous solution with 50 v% of acetonitrile, andketotifen concentration in the sample was deter-mined by the HPLC system described above. Forcalibration, drug-free plasma pooled with knownamounts of KT, to obtain a KT concentrationbetween 0.1 and 100 mg/mL, was used afterundergoing the same extraction procedure. KTstandards were run for external standardizationand a linear curve with a correlation coefficient of0.989 was generated from the area under the peakmeasurements. The validity of the method wasinvestigated by the determination of precision ofthe assay based on the reported guidelines.24 Fivereplicates of control samples at each concentrationof 100, 5 and 0.1 mg/mL were used to determinethe inter- and intra-run validity. The precisionwas demonstrated by the CV values. The ketotifenretention time was 6.3� 0.2 min.

Pharmacokinetic Parameters

Noncompartmental analysis was performed. Theterminal elimination rate constant (Ke) wasestimated from the log-linear portion of theplasma concentration-time courses. The absorp-tion rate constant (Ka) was estimated from the logplasma concentration-time course; the elimina-tion slope was back-extrapolated to the ordinate,and using the intercept, the differences betweenthe actual blood level points during the absorptivephase and the concentration on the back-extra-polated line at the same time were plotted as afunction of time. By combining these differentpoints, a straight line was obtained by using aleast square fit, and the absorption rate constantwas determined from the slope of this line.27 Thearea under the curve (AUC) of plasma concentra-tion-time was estimated from the plasma con-centrations at different time points using thelinear trapezoidal rule with extrapolation toinfinity. The area under the concentration timestime versus time curve (AUMC) was also calcu-lated by the trapezoidal rule. From the AUC andAUMC values, the mean residence time (MRT)was calculated (MRT¼AUMC/AUC).28 Apparent


total body clearance (Cl) and volume of distribu-tion at steady-state (Vss) were also estimated.Data analysis of the pharmacokinetic parameterswas performed by using unpaired Student’s t-test.A value of p< 0.05 was considered significant.

In Vivo/In Vitro Correlation (IVIVC)

In order to establish level B IVIVC,29 the meanin vitro dissolution time (MDT) was compared tothe MRT in vivo.30 The MDT was calculated usingthe following equation:

MDT ¼ ABCin vitro

M1

where ABCin vitro is the area between the releasecurve and its asymptote, calculated by thetrapezoidal rule from time zero to the lastmeasured time point, and M1 is the total amountof released drug at this time point.

Histological Studies

Animals were sacrificed in a CO2 atmosphere17 days after the intraperitoneal injection ofthe microspheres. An incision was made on theperitoneal region of the rat to examine tissues.Tissues where the presence of microspheres wasdetected were removed. A piece of the removedtissue, fixed with formol (10%, v/v), was immersedin paraffin. Cuts (10 mm) were carried out with aparaffin microtome (Minot type). Samples weredyed using the hematoxilin-eosin method.31

Statistical Analysis

Statistical comparisons were performed withunpaired Student’s t-test. A value of p< 0.05 wasconsidered significant.

RESULTS

Preparation of ketotifen-loaded microspheres wascarried out using spray-drying technology.21

Polymer concentration and solvent as well asassay conditions were chosen based on previouslypublished data on preparation and optimization ofPLA and PLGA microspheres by spray drying.18–21

The polymers and the drug were dissolved indichloromethane, which allowed obtaining outlettemperatures between 50 and 538C. The tempera-ture of microspheres obtained by the spray-dryingtechnique used were about 15–208C lower thanthe outlet temperature;32 thus, it is very probable

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that the temperature of ketotifen in the experi-ment was only around 30–358C.

SEM studies indicated that PLA and PLGA 50/50 microspheres were small in size and theirsurface was smooth and slightly porous. Somehollows or deformations, which are commoncharacteristics of microspheres obtained by thisspray-drying process,33 were observed in PLAmicrospheres (Fig. 1A and B). This appearance ismainly determined by the solvent evaporationprocess.20 The solvent evaporation rate of dichlor-omethane is quick, and this can allow achievingmore spherical microspheres than those obtainedusing solvents with a higher boiling point.20

Significant differences in appearance betweenketotifen-loaded microspheres and unloaded-microspheres were not observed (Fig. 1C andD). The size distribution of microspheres withoutdrug was similar to that of ketotifen-loaded

Figure 2. Size distribution of PLA (A), ketotketotifen-loaded PLGA 50/50 (D) microsphere


microspheres, and most of the particles werethe same size in both groups (Fig. 2). Microsphereswithout drug reach average diameters of 1.2 mm(PLA) and 0.9mm (PLGA 50/50), whereas the sizeswere 1.2 mm (PLA) and 0.8 mm (PLGA50/50) forketotifen-loaded microspheres. All the types ofparticles are considered polydisperse, with apolydispersity index over 1.123 (Tab. 1). Thepresence of the drug in the feed mixture did notinfluence the size of the ketotifen-loaded micro-spheres.

Stability of ketotifen was determined by HPLC.The precision of the HPLC assay was demon-strated by the coefficients of variation. Thecoefficients of variation for intra-run of KT at0.1, 1 and 100 mg/mL were 8.9%, 6.1% and 0.5%,respectively, and 7.0%, 7.3% and 1.0% for inter-run at the same concentrations. Figure 3 showschromatograms of ketotifen in phosphate buffer

ifen-loaded PLA (B), PLGA 50/50 (C) ands. Average diameter: solid line.


Table 1. Particle Size and Polydispersity Index (U) ofMicrospheres

Sample Dn (mm) U

PLA microspheres 1.2 2.3Ketotifen-loaded PLA microspheres 1.2 1.6PLGA 50/50 microspheres 0.9 1.4Ketotifen-loaded PLGA 50/50

microspheres0.8 1.7

Dn, number-average diameter.


1 mM pH 7.4 (Fig. 3A) and pH 2 (Fig. 3B). Thepeak of KT was observed in the chromatograms at6.0� 0.2 min, retention time of the drug stan-dards. Differences as a function of the incubationtime were not observed. Ketotifen was also stableafter dissolution in chloroform, evaporationunder N2 and then reconstitution with mobilephase. Thus, the drug was stable in the assayconditions. The drug stability in the above-mentioned solvent media was also observed by

Figure 3. Chromatograms of ketotifen (KT) in phos-phate buffer 1 mM, pH 7.4 (A) and pH 2 (B) at 378C, andchromatograms of KT released from PLA (C) and PLGA50/50 (D) microspheres after 250 h of drug release inphosphate buffer 1 mM, pH 7.4 at 378C.


UV/V spectra, which showed two characteristicpeaks at 190 and 297 nm.

The percentage of entrapment efficiency ofketotifen was 74� 7% for PLA microspheres and81� 6% for PLGA 50/50 microspheres. Drug-loaded microspheres included between 82�8 and 90� 7 mg KT/mg microspheres for PLAand PLGA 50/50 microspheres, respectively.

Stability of ketotifen released from KT-loadedPLA- and PLGA- microspheres was confirmed byHPLC. Figure 3C and D shows chromatograms ofketotifen released from KT-loaded microspheresafter 250 h. Degradation of the drug was notobserved. The cumulative release of ketotifen didnot reach 100% (Fig. 4). A burst effect wasobserved in both types of microspheres, 9%(7.4� 3.0 mg/mg microspheres) and 10%(8.6� 3.4 mg/mg microspheres) of the loaded KTwas released from PLA and PLGA 50/50 micro-spheres, respectively, during the first 15 min. Aquick release of the most external ketotifen fromPLA microspheres was observed; it took place at arate of 221 mg/h [KT (mg)¼ 322þ 221 time (h),r2¼ 0.98] from 15 min to 2 h, and then a slowerrate of 1.13 mg/h [KT (mg)¼ 1048þ 1.13 time (h),r2¼ 0.90] from 5 to 350 h was determined. Therelease rate of ketotifen from PLGA 50/50 micro-spheres was 322 mg/h [KT (mg)¼ 114þ 322 time(h), r2¼ 0.98] from 15 min to 2 h, and 16.18 mg/h[KT (mg)¼ 1044þ 16.18 time (h), r2¼ 0.96] from5 to 50 h. Thus, the release of ketotifen took placeat two rates from both types of microspheres;however, the different composition of the polymerof the microspheres determined the value of thoserates, which were higher in PLGA 50/50 micro-spheres. The maximum release of ketotifen fromPLA microspheres was 57% of the loaded drug at350 h (Fig. 4A). The release of the drug from PLGA50/50 microspheres reached the maximum (67.4%of the loaded drug) at 50 h (Fig. 4B); from thispoint a decrease in drug concentration in therelease medium was observed. In fact, ketotifen-loaded PLA microspheres maintained their mor-phology after 350 h of incubation in phosphatebuffer (Fig. 1E). However, at that time thedegradation of ketotifen-loaded PLGA 50/50 microspheres was evident (Fig. 1F). Thus,the release of ketotifen from these microspheresmust be related to their degradation character-istics.

In this study 10 mg KT/kg body weight was thedose used to evaluate the intraperitoneal admin-istration of ketotifen-loaded PLA and PLGA 50/50microspheres as well as a solution of the drug. The

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Figure 4. In vitro release kinetic of ketotifen (KT) from ketotifen-loaded PLA (A) andketotifen-loaded PLGA 50/50 (B) microspheres. Inset: KT release during the first hours.Phosphate buffer (1 mM, pH 7.4) at 378C. Each point shows average values� standarddeviation (n¼ 3).


coefficients of variation of HPLC assay for intra-run of ketotifen at 0.1, 5 and 100 mg/mL were5.0%, 6.1% and 1.8%, respectively. The coefficientsof variation for inter-run at the same concentra-tions were 8.0%, 5.2% and 12%. After adminis-tration of ketotifen-loaded PLA microspheres(Fig. 5A), the drug started to be detected inplasma at 30 h, and the maximum drug concen-tration (571 ng/mL) was reached at 169 h (Tab. 2);then, the drug concentration began to decrease upto hour 384. The ketotifen concentration inplasma decreased at 120 h and then increasedagain to reach the maximum; thus, the presence ofthe drug in plasma during the first 120 h can be

Figure 5. Plasma concentration of ketotifenfen-loaded PLA microspheres (A) and ketotifeEach point shows average values� standard


due to the initial quick release of ketotifenobserved in vitro. Plasma levels of ketotifen afterdrug-loaded PLGA 50/50 microspheres alsoshowed two peaks (Fig. 5B); the maximum ofthe first peak (177 ng/mL) took place at 24.4 h andthe maximum of the second peak (102 ng/mL) wasdetected at 169 h (Tab. 2), where there was adecrease in plasma concentration of the drug upuntil hour 336. The quicker release of ketotifenfrom PLGA 50/50 microspheres observed in vitro(Fig. 4B) caused a lower drug plasma concentra-tion when microspheres were administeredin vivo. The drug is probably protected in themicrosphere, and ketotifen is metabolized when it

after intraperitoneal injection of ketoti-n-loaded PLGA 50/50 microspheres (B).

deviation (n¼ 6).


Table 2. Pharmacokinetic Parameters of Ketotifen (KT) after Intraperitoneal Injection of the Drug-LoadedMicrospheres and a Solution of the Drug

Parameter

KT Formulation

KT-Loaded PLGA 50/50 Microspheres KT-Loaded PLA Microspheres KT Solution

Ka (h�1) 0.0199� 0.0070a 0.0125� 0.0038a 2.29� 0.34Ke (h�1) 0.0194� 0.0085ab 0.0058� 0.0003a 1.23� 0.02AUC (mg h/mL) 20� 6ab 142� 61a 51� 16AUMC (mg h2/mL) 641� 238ab 10044� 4771a 51� 17Cmax (ng/mL)

1st peak 177� 14 102� 11 39.4� 103� 11.7� 103

2nd peak 352� 258 571� 217Tmax (h)

1st peak 24.4 169 2.42nd peak 97.4 169

MRT (h) 31� 2ab 71� 4a 1.00� 0.02Cl (L/h kg) 0.53� 0.13ab 0.081� 0.031a 0.21� 0.06Vss (L/kg) 16.43� 3.3ab 5.75� 1.79a 0.21� 0.04

KT dose: 10 mg KT/Kg body weight.aSignificant difference with respect to KT solution group (p< 0.05).bSignificant difference with respect to KT-PLA microsphere group (p< 0.05).


is released; the slower release of ketotifen fromPLA microspheres seems to cause longer andhigher levels of the drug. When ketotifen wasintraperitoneally administered at the same dosein solution (Fig. 6) the maximum drug concentra-tion (39.4 mg/mL) was observed at 2.4 h, and thedrug was not detected starting at 4.4 h.

The inclusion of ketotifen in PLGA and PLAmicrospheres resulted in significant changes inthe plasma disposition of the drug. The pharma-cokinetic parameters are summarized in Table 2.The absorption constant (Ka) was slower and Cmax

Figure 6. Plasma concentration of ketotifen afterintraperitoneal injection of a KT solution (dose 10mg KT/kg body weight). Each point shows averagevalues� standard deviation (n¼ 6).


was lower when ketotifen was released from bothtypes of microspheres. The steady-state volume ofdistribution (Vss) of ketotifen was 78- and 27-foldlarger when the drug was administered by KT-loaded PLGA and PLA microspheres, respec-tively, than the corresponding value of the drugadministered in the form of a solution. Theelimination rate constant (Ke) was 63- and 212-fold lower when the drug was administered by KT-loaded PLGA and PLA microspheres, respec-tively. Total body clearance (Cl) depended onAUC values, which were 2.5-fold lower for KTreleased from PLGA microspheres and 2.7-foldlarger when the drug was released from PLAmicrospheres in comparison with the administra-tion of ketotifen in the form of a solution. Thevalue of the MRT of ketotifen in plasma wasbetween 30 and 70 times larger when ketotifenwas administered by drug-loaded PLA or PLGA50/50 microspheres.

Although at least three different formulationsare necessary to establish an in vitro/in vivo levelB correlation, MDT values were calculated from invitro ketotifen release from microspheres: 307�6 h for KT-loaded PLA microspheres; 63.0� 0.7 hfor KT-loaded PLGA 50/50 microspheres. Thelinear relationship for in vitro MDT and in vivoMRT was: MRT¼ 20.93þ 0.16 MDT. Further-more, a linear relationship was establishedbetween the absorption constant (Ka) and thein vitro release rates of the drug (Fig. 7); thus, thein vitro release rates of KT from KT-loaded PLGA

DOI 10.1002/jps

Figure 7. Relationship between the absorption con-stant (Ka) of ketotifen in plasma and in vitro releaserates of ketotifen from ketotifen-loaded PLA and PLGA50/50 microspheres. Each point shows averagevalues� standard deviation (n¼ 6).


microspheres were larger, and the absorptionconstant of the drug was also larger than thecorresponding values of the drug released fromKT-loaded PLA microspheres.

To examine the peritoneal region, animals weresacrificed 17 days after the injection, whenketotifen was not detected in plasma. From amacroscopic point of view, microspheres were notobserved in the liver, kidney, pancreas, intestinesor tegument. However, unloaded and ketotifen-loaded microspheres were detected in adiposetissue. In Figure 8 an example of ketotifen-loadedPLGA 50/50 microspheres included in adiposetissue is shown. Connective tissue surrounding

Figure 8. Photomicrograph of microspheres and thesurrounding tissue after 17 days of intraperitonealinjection of ketotifen-loaded PLGA 50/50 microspheres.Microspheres (1), connective tissue (2), adipose tissue(3).


groups of microspheres is observed. This fact canbe considered in accordance with the normal bodyreaction to a biocompatible material, whichconsists of walling it off in an avascular,collagenous bag.

DISCUSSION

Antihistaminic drugs used in the treatment ofasthma have been included in drug deliverysystems34,35 to improve their pharmacologicalefficiency. Ketotifen36 is one of these types ofdrug. Furthermore, this drug has been used in thetreatment of some inflammatory intestinal dis-eases caused by parasite infections,37 allergy tomilk proteins38 or eosinophil gastroenteritis.39

From an experimental point of view, ketotifen canbe an interesting drug in decreasing the inflam-matory response caused in the endothelium of ratsused in experimental models of prehepatic portalhypertension.17 In this case, ketotifen adminis-tration in the intraperitoneal cavity using sus-tained drug delivery systems could be useful inimproving the antiinflammatory effect of thedrug.

Different studies have evaluated the effect ofvarious processing parameters on the preparationof PLA and PLGA using spray-drying technology.Thus, the effect of the solvent on PLA micro-spheres was studied by Bain et al.,18 and theyconcluded that microspheres prepared by dichlor-omethane were technologically superior. Further-more, the lowest solvent residue was obtainedwhen dichloromethane was used in microspherepreparation, and its amount significantlydecreased as a function of in vacuo storage time.18

Dichloromethane achieved more spherical PLGAand PLA microspheres due to its rapid solventevaporation rate.20 Although dichloromethane isa class 2 solvent according to the ICH classifica-tion, and its administration should be limited(PDE 6 mg/day; concentration limit 600 ppm), it isconsidered one of the halogen solvents that causesthe least toxicity.40 Studies on particle size anddistribution, and morphology of PLA and PLGAmicrospheres prepared by spray drying based onpolymer concentration and solvent used werecarried out by Wang and Wang.20 Their resultsshowed that the viscosity of the liquid exerts asignificant influence on the average size ofatomized droplets. They demonstrated that theviscosities of the polymer solutions were severaltimes the viscosity of dichloromethane solvent,



and increased as the percentage of lactic acidincreased in the copolymer; furthermore, a higherpolymer concentration produces more particleswith a larger particle size.20 Low PLA concentra-tion (<1.5%, w/v) tended to form poor sphericalparticles; in addition, high PLA concentration(>3%, w/v) produced fibrin products.19 Regardingthe drug load of microspheres, studies carried outwith chlorambucil (25–75%, w/w of polymer)indicated that higher drug loading decreasedthe encapsulation efficiency of the drug in PLAmicrospheres.19 PLGA microspheres were loadedwith paclitaxel and the drug load was kept at 1%(w/w) of polymer.20 Thus, the parameters selectedfor the preparation of ketotifen-loaded PLA andPLGA microspheres were chosen considering theabove mentioned data as well as our previousstudies on 5-fluorouracil-loaded PLA and PLGAmicrospheres21 and bupivacaine-loaded poly(e-caprolactone) microspheres.41 Unloaded and KT-loaded PLA and PLGA microspheres were small insize and polydisperse, and their average diameterand appearance were similar to those obtained inequivalent experimental designs.18,20

Microspheres prepared by spray-drying tech-nology allow obtaining high percentages of drugentrapment efficiency when the drug and thepolymer can be dissolved in a solvent or in misciblesolvents.41 Thus, the high entrapment efficiencyobtained for ketotifen in PLA and PLGA 50/50microspheres was similar to that reported for theencapsulation of drugs that were soluble in thesame solvent as polymers using the spray-dryingtechnique.41 The entrapment of ketotifen inliposomes42 used to obtain a dry powder inhala-tion formulation took place with a maximumefficiency of 97.92� 0.54% starting from 200 mgof ketotifen per 200 mg of formulation. Lowerentrapment efficiency of ketotifen was obtainedfor deformable liposomes and ethosomes,74.51� 0.86% and 43.98� 0.96%, respectively,prepared for topical delivery.10 In the preparationof liposomes and ethosomes with ketotifen, drugand lipids were dissolved in the same solvent.However, in order to remove the unentrappedketotifen from liposomal dispersion dialysis42 andultracentrifugacion10 were used. These proce-dures can contribute to decreasing the percentageof entrapped drug. This is a significant differenceregarding the spray-drying procedure used in thiscase, since ketotifen and each one of the polymerswere dissolved in dichloromethane, and thisdissolution was used to obtain drug-loaded micro-spheres. Furthermore, ketotifen has been


included in PSA matrices (maximum drug content313.1� 19.7 mmol/cm3) for skin permeation11 aswell as in silicone-containing and p-HEMAhydrogel contact lens materials (maximum druguptake 227� 9 mg/lens for Vifilcon, a p-HEMAbased contact lens with methacrylic acid).13 Thehigh ketotifen content of silicone-typed PSA wasdue to the casting method used in their prepara-tion, in which the amount of ketotifen dependedon its solubility in the organic solvent used.42 Onthe other hand, the uptake of ketotifen in thecontact lens depended on the concentration ofthe drug solution used (222 mg/mL). The uptakeincreased with high water content materials,p-HEMA containing materials and with ionicmaterials.13 Thus, the amount of ketotifen loadedwas small (approximately 8 mg of ketotifen per mgof lens).

Drug release experiments indicated that themost external ketotifen was released at thebeginning of the process. This burst effectobserved during the first 15 min of ketotifenrelease is usual in drug release from microparti-culate systems due to the quick diffusion of thedrug from the external part of the microspheres.20

The burst effect is not an advantage or disadvan-tage of the formulation; it depends on the type ofdrug entrapped and also on the type of applicationof the microspheres.43,44 Since ketotifen is anantihistaminic drug, a quick release of the drugcould be an advantage in the control of histaminedelivery from cells during the first stage of thetreatment. Then, a slow release of the drug allowsmaintaining the antihistaminic effect, where themicrospheres probably act as a reservoir toprevent enzymatic metabolism of the drug.

Furthermore, a significant interaction betweenketotifen and the polymers seems to exist, sincetotal release of the loaded drug from micro-spheres did not take place. In previous studies wehave observed that degradation of PLA micro-spheres was slow;16 mass loss was not observedduring the 150 days of incubation in phosphatebuffer and the average molecular weight of themicrospheres decreased only 12% during the first2 months. Thus, the slow hydrolysis of backboneester groups due to the hydrophobic character ofPLA microspheres makes the release of the mostinternal ketotifen difficult. KT-loaded PLAmicrospheres maintained their morphology after350 h of drug release (Fig. 1E), which was notobserved in KT-loaded PLGA 50/50 microspheresat the same time (Fig. 1F). Two considerationsmust be taken into account in the release kinetics

DOI 10.1002/jps


of the drug from PLGA 50/50 microspheres. Onthe one hand, the percentage of ketotifenreleased was larger than that from PLA micro-spheres in a shorter period of time. This fact isvery likely related with its quicker degradationand with the larger hydrophilic nature of thepolymer, which facilitates the uptake of aqueoussolution. The hydrolytic effect of the aqueousmedium on polymeric chains allows the forma-tion of larger pores and channels inside themicrospheres, which makes drug release morefavourable.45 A decrease of 94% in molecularweight during the first 2 months of incubation inphosphate buffer has been observed in thedegradation process of PLGA 50/50 micro-spheres.16 Decrease in molecular weight of PLGA50/50 microspheres took place in two phases; thelength of the first one was the first 22 days.16 Thesecond consideration in the in vitro kineticrelease of ketotifen was the decrease in the drugconcentration observed starting at 50 h ofincubation (data not shown), which led to a KTconcentration of 28% of the loaded drug at 350 h.The link breaks that took place in PLGA 50/50microspheres very likely causes the release ofsmall polymer chains as well as glycolic and lacticacid, which probably interact with the drug in thesolvent medium. Ketotifen has a carbonyl group.The carbonyl group is polar because of thegreater electronegativity of oxygen, and thepresence of oxygen with its nonbonding electronpairs turns aldehydes and ketones into hydro-gen-bond acceptors. Thus, hydrogen bonds maybe established between the carbonyl group ofketotifen and the hydroxyl of the carboxylicgroup of the polymer chains. The degradation ofthe PLGA 50/50 microspheres increases theamount of small polymer chains and as aconsequence the amount of carboxylic groups toform hydrogen bonds. Furthermore, ketotifen is aweak base (pKa ¼ 8.5),11 which is ionized at acidpH (pH<pKa). Thus, the protoned nitrogen of thedrug may also establish interactions with thecarboxylic group of the polymer chains. This way,ketotifen released from PLGA 50/50 micro-spheres may interact with the carboxylic groupsgenerated by degradation of the polymer and beremoved from the release medium, with the drugconcentration decreasing. Another possibilitythat could explain the decrease in ketotifenconcentration after 50 h of release from PLGA50/50 microspheres is the degradation of thedrug. However, chromatograms of ketotifen insolution at pH 2 and pH 7.4 and of the drug


released from PLGA 50/50 microspheres (Fig. 3)showed a peak at the retention time of ketotifenstandards. Thus, degradation does not seem to bethe best option to explain the decrease in drugconcentration.

The pathophysiology of prehepatic portal hyper-tension has been studied using the experimentalmodel of partial PVL rat. One of the character-istics of PVL rats is an increased infiltration of theintestinal mucosa and submucosa by mast cells,whose inflammatory mediators could producevasodilatation and angiogenesis.46 Thus, theadministration of ketotifen in the intraperitonealcavity could exert its antihistaminic activity,decreasing the level of inflammatory mediators.

Considering previous studies,1,7–9 the dose ofKT intraperitonealy administered to rats in thisstudy was 10 mg KT/Kg body weight. Whenketotifen was administered by drug-loaded micro-spheres, they were in the adipose tissue. Thus,ketotifen was detected in plasma 336 and 384 hwhen it was administered by drug-loaded PLGA50/50 and PLA microspheres, respectively (Fig. 5).The values of the pharmacokinetic parameters ofketotifen (Tab. 2) showed the effect of the differentpolymer formulations of the drug. Except whenthe drug is administered intravenously in theform of a solution, the drug has to be released fromthe dosage form and then be absorbed intosystemic circulation by passing through variousmembranes.27 A drug given in different dosageforms will yield varying amounts of drug absorbedand, hence, differences in onset, intensity andduration of the pharmacologic or clinical effect.27

Thus, the administration of the KT-loaded PLAmicrospheres produced a maximum drug plasmaconcentration between 112 and 69 times lowerthan the maximum drug plasma concentrationobtained when the KT solution was administered;regarding KT-loaded PLGA 50/50 microspheres,the maximum drug plasma concentration wasbetween 386 and 222 times lower. The polymercomposition of KT-loaded microspheres influ-enced their in vitro drug release characteristics(Fig. 4), and it determined not only the maximumplasma KT concentration but also the time thedrug was present in plasma. Significant differ-ences in MRT and AUC, pharmacokinetic para-meters reflecting the residence time and amountof free KT, respectively, in systemic circulation,between the i.p. administration of the drug by KT-loaded microspheres and KT solution, as well asbetween different types of microspheres intraper-itonealy administered were observed. Thus, the



relative bioavailability,27 determined from AUCvalues, was very different for both types ofmicrospheres (Tab. 2). Whereas it was 39% forKT-loaded PLGA 50/50 microspheres, the relativebioavailability of the drug was 278% when KT-loaded PLA microspheres were administered. Onthe other hand, the higher MRT values of KTsuggest a more prolonged action when KT isformulated with PLA or PLGA 50/50. In general,polymer drug delivery systems exert protection onthe loaded drug against its degradation ormetabolism; thus, the drug absorption from thistype of systems after i.p. administration dependson the release of the drug from the polymersystem, its permeability through tissue barriers,and its dissolution under physiological conditions.Studies carried out with rabbits7 have shown thatthe intravenous administration of KT in solutionat a dose of 1 mg/kg body weight produced an AUCvalue of 0.514� 0.048 mg h/mL, and the AUCvalue was 0.338� 0.069 mg h/mL when the drug insolution was administered intranasally. Thus,these AUC values are about 10 times lower thanthose obtained in our experiments when KTsolution was intraperitonealy administered torats using a dose of 10 mg/kg body weight.Transdermal delivery studies with ketotifenpatches of polyisobutylene and fatty acids12

containing 10 mg of ketotifen were carried outin rabbits (2.5–3.5 kg); the maximum concentra-tion of ketotifen (0.0552 mg/mL) was obtained at6 h, the drug was detected in plasma for 72 h andthe AUC was 2.56 mg h/mL. The bioavailability ofthe ketotifen patch, based on AUC data, indicatedthat about 0.6 mg of KT was absorbed intosystemic circulation.

On the other hand, ketotifen was absorbedrapidly after the i.p. administration of the drugsolution to rats (Ka¼ 2.29� 0.34 h�1) (Tab. 2). Asimilar value of absorption constant has beendetermined for ketotifen after rectal administra-tion in rabbits (Ka¼ 2.45� 0.47 h�1).7 The admin-istration of KT-loaded microspheres significantlydecreased the absorption constant of ketotifenand, as a consequence, the Cmax. Thus, Cmax wasproportional to the rate and quantity of absorbeddrug. Furthermore, since tmax is related to theabsorption rate, it was longer when the drug wasreleased from the microspheres. In a similar way,the value of the elimination constant of ketotifenadministered in the form of a solution to rats(Tab. 1) was similar to the value determined forthe intravenous and oral administration to rabbits(1.25� 0.16 h�1 and 1.27� 0.10 h�1, respec-


tively).7 The administration of the drug by KT-loaded microspheres caused a significant decreasein the elimination constant; the lowest value wasfor the KT released from PLA microspheres,which was in accordance with the slowerin vitro release of KT from PLA microspheres.The total body clearance of ketotifen administeredin the form of a solution to rats was 10 times lowerthan the value determined in rabbits afterintravenous administration (2.04� 0.23 L/h kg);7

this was in accordance with the correspondingAUC value, which was 10 times lower for rats incomparison with the values obtained in rabbits, ashas been indicated above. The slow release of thedrug from KT-loaded PLA microspheres caused alow clearance, 2.6 times lower than the clearanceof the drug administered in the form of a solution,and a slow elimination (Ke¼ 0.0058� 0.0003 h�1).However, the release of ketotifen from PLGAcaused a very small plasma concentration of thedrug, which allowed a quick clearance in spite ofthe slow elimination.

The steady-state volume of distribution (Vss) ofketotifen when it was administered by intraper-itoneal injection to rats in the form of a solution(Tab. 2) suggests that the distribution of the drugwas mainly into extra-cellular fluids. In rats, themean circulating blood volume is 64 mL/kg,47 andthe mean extra-cellular fluid volume is 200 mL/kg,48 a value very similar to Vss value determinedfor the intraperitoneal administration of the drugin solution (Vss¼ 0.21� 0.04 L/kg). The intraper-itoneal administration of ketotifen by KT-loadedPLA and PLGA microspheres significantlyincreased the steady-state volume of distributionof the drug, and it suggests that these micro-spheres facilitate the distribution of the drug intotissues, which may be related to the hydrophobiccharacteristics of the polymers and the interactionof the microspheres with peritoneal cavity tissues.The binding of a drug to polymers may modify itsdistribution; thus, conjugation of methylpredni-solone with dextran-70 drastically altered thedistribution of the steroid by converting methyl-prednisolone from a large volume of distributiondrug (2290 mL/kg) to a prodrug with a smallvolume of distribution (102 mL/kg), probablybecause the hydrophilic carrier decreased thelipophilicity of the drug.49

From in vitro KT release studies and in vitrodegradation of PLA and PLGA microspheres,16 itcan be deduced that KT release from PLA andPLGA microspheres occurs mainly by dissolutionof the drug inside the microspheres, and then by

DOI 10.1002/jps


the diffusion of the drug from them, even though acombination of drug diffusion and microsphereerosion processes also takes place. A level B IVIVCcan be established not only when drug dissolutionis the rate-limiting step for the in vivo ADME,30

but also when drug release occurs by a combina-tion of diffusion and erosion.50 In this study withKT-loaded PLA and PLGA microspheres, arelationship has been established between meanin vitro dissolution time (MDT) and in vivo MRT.Although only two KT formulations have beenstudied, which questions the possibility of pre-dicting MRT values of KT from in vitro experi-ments, a clear relationship exits between bothparameters. The largest AUC value of KT when itwas released from PLA microspheres corre-sponded with the longer release observedin vitro, whereas the lower AUC of the drugwas obtained when KT was released from PLGA50/50. Another relationship has also been observedbetween the in vitro release rates of ketotifen fromthe microspheres and the in vivo absorptionconstant (Fig. 7). A faster absorption of the drugwas observed when the drug was quickly releasedfrom PLGA 50/50 microspheres, and this took placeat each one of the two release rates. On thecontrary, the slower release of the drug from PLAmicrospheres caused a lower absorption constant.Thus, a correlation seems to exist between therelease rate and the absorption rate of the drug.

The presence of unloaded or KT-loaded PLA andPLGA 50/50 microspheres was observed in theadipose tissue after i.p. injection (Fig. 8). Theimplantation of biocompatible and biodegradablemicrospheres induces the activation of humoraland cellular mechanisms to produce inflammatoryand healing responses of the material.16 The acuteand chronic inflammatory responses are of shortduration, 1–2 weeks, regardless of polymercomposition of the biodegradable microspheres.Polymorphonuclear leukocytes, monocytes andlymphocytes are the cell types associated withthis event. However, the presence of lymphocytesor mast cells in the adipose tissue close to theinjected microspheres was not observed. Groups ofmicrospheres surrounded by very active connec-tive tissue were observed, with the large amountof nuclei around the polymer indicating theactivity of this tissue. The formation of the fibrouscapsule is one of the local events following theimplantation of microspheres. On the other hand,microspheres were not observed in internalorgans. Thus, the formation of fibrous capsulein metabolically essential tissues did not take


place, and possible undesired consequences werenot detected.

In conclusion, the preparation of ketotifen-loaded PLA and PLGA 50/50 microspheres byspray-drying technology allows obtaining a highpercentage of entrapped efficiency. A quickrelease of the drug takes place during the first2 h and then a slower release rate is observed. Theintraperitoneal administration of ketotifen-loaded microspheres allows detecting the drugin plasma between 336 and 384 h, and the MRT ofthe drug increases between 30 and 70 times withrespect to the i.p. administration of ketotifen insolution at the same dose. The concentration andthe presence of KT in plasma can be modulateddepending on the composition of the polymer.These systems may be used to improve thetreatment of the inflammation observed in portalhypertension, a clinical syndrome that is fre-quently studied using partial portal vein-ligatedrats.

ACKNOWLEDGMENTS

The financial support of the Health Ministry ofSpain, Instituto de Salud Carlos III (FIS ref.PI050385), of the UCM-CAM for Research Groups(Group 920613), and the FPI grant from Comuni-dad de Madrid and FSE to S. Guerrero are grate-fully acknowledged.

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