o_196105cle1ebl9qs18ch1gaadrma.pdf

European Journal of Pharmaceutical Sciences 16 (2002) 53–61
www.elsevier.nl/locate/ejps
®
Eudragit RS100
nanosuspensions for the ophthalmic controlled delivery
of ibuprofen
a, b a b a
Rosario Pignatello *, Claudio Bucolo , Piera Ferrara , Adriana Maltese , Antonina Puleo ,
a
Giovanni Puglisi
Dipartimento di Scienze Farmaceutiche, Universita`
degli Studi di Catania, Citta`
Universitaria, viale A. Doria, 6- 95125 Catania, Italy
b
Bausch & Lomb - Fidia Oftal Pharmaceuticals, corso Italia, 141, 95127 Catania, Italy
a
Received 4 January 2002; received in revised form 23 April 2002; accepted 1 May 2002
Abstract
Topical application of non-steroidal anti-inflammatory drugs on the eye is a common treatment used to contrast the miosis induced by
surgical traumas, such as cataract extraction. With the aim of improving the availability of sodium ibuprofen (IBU) at the intraocular
®
level, IBU-loaded polymeric nanoparticle suspensions were made from inert polymer resins (Eudragit RS100 ). The nanosuspensions
were prepared by a modification of the quasi-emulsion solvent diffusion technique using variable formulation parameters (drug-topolymer
ratio, total drug and polymer amount, stirring speed). Nanosuspensions had mean sizes around 100 nm and a positive charge
(z-potential of 140/160 mV), this makes them suitable for ophthalmic applications. Stability tests (up to 24 months storage at 4 8C orat
room temperature) or freeze-drying were carried out to optimize a suitable pharmaceutical preparation. In vitro dissolution tests indicated
a controlled release profile of IBU from nanoparticles. In vivo efficacy was assessed on the rabbit eye after induction of an ocular trauma
(paracentesis). An inhibition of the miotic response to the surgical trauma was achieved, comparable to a control aqueous eye-drop
formulation, even though a lower concentration of free drug in the conjunctival sac was reached from the nanoparticle system. Drug levels
in the aqueous humour were also higher after application of the nanosuspensions; moreover, IBU-loaded nanosuspensions did not show
toxicity on ocular tissues. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Ibuprofen; Eudragit RS100; Nanoparticles; Ophthalmic delivery; Paracentesis
1. Introduction despite the instillation of topical mydriatic agents (Sabiston
et al., 1987; Gimbel, 1989; Brown and Roberts, 1996).
The inflammatory response of ocular tissues is a com- Arylpropionic-type NSAI drugs, such as ibuprofen
mon side effect associated with ophthalmic surgery. It can (IBU), can antagonize the pupillary constriction during
interfere with the normal function of the eye, whose optic intra-ocular surgery, by blocking the cyclooxygenase pathtransparency
must be maintained. Along with cortico- way and reducing polymorphonuclear leukocyte (PMNs)
steroids, non-steroidal anti-inflammatory drugs (NSAIDs) infiltration in the aqueous humor (Murray and Leopold,
are used during eye surgery therapy. 1982; Bucolo and Spadaro, 1995; Samiy and Foster, 1996).
Surgical or mechanical traumas of the anterior segment Most ocular diseases are treated with a topical applicaof
the eye cause a vascular inflammatory reaction due to tion of drug solutions administered as eye-drops; however,
the disruption of the blood–aqueous barrier, with a marked they often require frequent instillation of highly concenrise
in protein content of the aqueous humour, a transient trated solutions, due to the rapid pre-corneal loss from the
eye. A significant effort towards new drug delivery sys-
tems (DDS) for ophthalmic administration has then been
seen over the last few decades. Hydrogels, micro- and
nanoparticles, liposomes and collagen shields have been
investigated (Le Bourlais et al., 1998). Among them,
nanoparticles have shown to give efficient ocular DDS
ocular hypertension and miosis (Kulkarni, 1991). Miosis is
a frequent problem during extracapsular cataract surgery,
*Corresponding author. Tel.: 139-0957-384-021; fax: 139-095-222-
239.
E-mail address: pignatel@unict.it (R. Pignatello).
(Harmia et al., 1986; Li et al., 1986; Losa et al., 1991;
Marchal-Heussler et al., 1993; Zimmer et al., 1994; Calvo
0928-0987/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S0928-0987(02)00057-X

54 R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61
et al., 1997); moreover, nanoparticle technologies are at Table 1
present catalyzing increasing efforts in many pharma- Formulative variables of IBU-loaded Eudragit RS100 nanosuspensions.
Stirring speed was 20 500 rpm, 13 500 rpm and 9500 rpm, for batches A,
ceutical areas (Kawashima, 2001).
B and C, respectively
In a previous research we studied a nanoparticle colloi-
Batch Percent IBU versus RS Initial IBU1RS
dal system for flurbiprofen (FLU) delivery into the eye
(w/w)
amount (mg)
(Pignatello et al., 2002). Eudragit RS100 (RS) and RL100
(RL) nanosuspensions were prepared by means of an A1 10 100
A2 20 100
opportunely modified solvent evaporation method (QESD) A3 33 100
(Kawashima et al., 1989), to achieve particles with size, A4 50 100
charge and stability features suitable for ocular applica- A5 10 200
tions.
A6 20 200
RS and RL polymers are commonly used for the enteric A7 33 200
A8 50 200
coating of tablets and the preparation of controlled-release A9 10 500
drug forms. They are co-polymers of poly(ethylacrylate, B1 10 100
methyl–methacrylate and chlorotrimethyl–ammonioethyl B2 20 100
methacrylate), containing an amount of quaternary am- B3 33 100
monium groups between 4.5–6.8% and 8.8–12% for RS B4 50 100
B5 33 200
and RL, respectively. Both are insoluble at physiological C1 10 100
pH values and capable of swelling (Bodmeier and Chen, C2 33 200
1989), thus representing good material for the dispersion
of drugs (Kawashima et al., 1993; Perumal et al., 1999;
Pignatello et al., 2001).
injection, the mixture was highly mixed by an Ultra-Turrax
In the present paper, with the aim of improving the T25 (IKA Labortechnik, Staufen, Germany) at the agitaavailability
of IBU at the intraocular level, sodium ibu- tion speed reported in Table 1.
profen-loaded polymeric nanoparticle suspensions were The solution immediately turned into a pseudo-emulsion
made using the RS polymer. One formulation was tested in of the drug and polymer–ethanol solution in the external
vivo in the rabbit to evaluate the inhibition of miosis aqueous phase. The counter-diffusion of ethanol and water
induced by a mechanical trauma (paracentesis), in com- out of and into the emulsion micro-droplets, respectively,
and the gradual evaporation of the organic solvent de-
termined the in situ precipitation of the polymer and the
drug, with the formation of matrix-type nanoparticles
(Kawashima et al., 1989; Pignatello et al., 1997). Ethanol
parison with an aqueous eye-drop formulation containing a
soluble IBU lysine salt (solprofen, IBL).
2. Materials and methods residues were left to evaporate off under a slow magnetic
stirring of the nanosuspensions at room temperature for
2.1. Materials 8–12 h.
Two ml aliquots of the freshly prepared suspensions
Eudragit RS 100 was a kind gift from Rofarma Italia
S.r.l. (Gaggiano, Milan, Italy). IBU sodium salt (Sigma)
and Tween 80 (Fluka) were purchased from Sigma-Aldrich
Chimica S.r.l. (Milan, Italy); benzalkonium chloride (50%,
w/v, FUI X Ed. grade) is a product from Galeno (Italy).
All other chemicals were of reagent grade or higher.
were centrifuged at 11 000 rpm, 10 8C for 15 min (IEC/
Centra MP 4R centrifuge equipped with an 851-type rotor),
and the amount of unincorporated drug was measured by
UV analysis in the supernatant. Some supernatant samples
were submitted to a second centrifugation cycle, but no
further solid material could be collected.
2.2. Preparation of nanoparticles 2.3. Size analysis and z-potential
RS nanosuspensions were obtained in the presence or The mean particle size of the formulations was deabsence
of IBU, at different drug/polymer weight ratios termined by photo-correlation spectroscopy with a
and using different rates of agitation (Table 1), using an Zetamaster (Malvern Instruments Ltd., Worcs, UK)
adaptation of the quasi-emulsion solvent diffusion tech- equipped with the Malvern PCS software (v. 1.27). Every
nique (Kawashima et al., 1989).
sample was appropriately diluted with pro-injection water
The drug and polymer were co-dissolved at room and the reading was carried out at a 908 angle in respect to
temperature in ethanol (2 ml). The solution was slowly the incident beam.
21
injected (0.5 ml min ), with a syringe connected to a thin Electrophoretic mobility was obtained by a laser Dop-
Teflon tube, into 50 ml water containing Tween 80 pler anemometer using the same instrument. A suitable
(0.02%, w/v) and benzalkonium chloride (0.1%, w/v), and amount of the sample (50–100 ml) was diluted with 5 ml
kept at a low temperature in an iced-water bath. During of pro-injection water and placed into the electrophoretic

R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61 55
cell of the instrument where a potential of 6150 mV was
created. The z-potential value was calculated by the
software using Smoluchowski’s equation.
2.4. Freeze-drying and redispersibility of
nanosuspensions
Aliquots of different batches were freeze-dried to verify
the physical stability and the following redispersibility.
Ten ml aliquots of samples were frozen in liquid nitrogen
and lyophilized by an Edward Freeze-Dryer Modulyo for
24 h at 230 8C, and at a pressure of 0.05 mm Hg. Samples
were then stored at room temperature in glass containers.
At 2 month intervals the freeze-dried samples were
re-hydrated with the original volume of double distilled
water to restore the drug and polymer concentrations. Size
and z-potential changes were assessed as described above.
Reconstituted samples were also centrifuged as described
above, and the drug leakage was determined in the (cf. Table 1).
supernatant by UV analysis.
Fig. 1. In vitro dissolution profile of IBU from RS nanosuspensions
prepared at 20 500 rpm from a 100 mg total drug and polymer amount
2.5. Stability studies of the formulations (v/v) mixture of NaH2PO 4
(0.2 M, pH 6.5) and acetoni-
21
trile; the flow rate was 1 ml min . The UV detector was
The physical stability of the nanosuspensions was set at 236 nm. Chromatography was performed at 25 8C;
evaluated after storage for 24 months under different the injection volume was 20 ml. Under these conditions,
temperature conditions. Exact volumes of each nanosus- the retention time for IBU was 6.58 min. An external
pension were stored in screw-capped amber-glass bottles calibration graph was constructed by plotting the IBU
and placed at either room temperature or 4–6 8C (re- peak-areas versus the known concentrations of the drug.
frigerator) away from direct light. Aliquots of 500 ml were
withdrawn every 2 months to determine particle size, 2.7. In vivo biological assay
z-potential value and drug leakage, as described above.
2.7.1. Treatment schedule and sample collection
2.6. In vitro drug release Pharmacological studies were performed on groups of
six male New Zealand albino rabbits (Charles River,
IBU release from nanosuspensions was evaluated in Calco, Italy), weighing 1.8–2.2 kg, and with no signs of
triplicate over 3 h by a dialysis system consisting of a ocular inflammation or gross abnormalities. Animal pro-
Spectrapor membrane (cut-off: 3500 Da), loaded with 5 ml cedures conformed to the Association for Research in
of nanosuspension and soaked in a 0.14 M phosphate Vision and Ophthalmology (ARVO) resolution on the use
buffer solution (pH 7.4), at room temperature and under of animals in research.
slow magnetic stirring. At regular time intervals 1 ml The A4 formulation (Table 1) (at a 0.1%, w/v IBU
aliquots of the external medium were withdrawn and concentration), a blank RS nanosuspension batch (prepared
immediately replaced with the same volume of fresh under the same conditions as the A4 formulation but
buffer. without the drug), or an IBL solution in saline (0.1%,
To check the eventual limiting effects of the dialysis w/v), were instilled (50 ml) into the conjunctival sac
membrane on drug dissolution, separate experiments were (left-eye) 180, 120, 90 and 30 min before, and then 5, 15,
run in duplicate with a solution in the same phosphate 75 and 90 min after the first paracentesis (multiple
buffer of pure IBU at two of the drug concentrations treatment schedule). Ninety minutes before the first
present in the nanosuspensions. Similarly, an aqueous paracentesis, rabbits were treated with 1% atropine sulfate
suspension of the powdered drug (250 mm sieve) was (Allergan, Rome, Italy) instilled into both eyes (50 ml/
loaded into the dialysis bag and the test was carried out (in eye). The right eye of each animal was used as a control
duplicate) as usual. In both cases, only a limited delay in and was treated only with atropine.
drug dissolution into the receiving medium was observed To perform the paracentesis, animals were lightly anaes-
(Fig. 1).
thetized with an i.v. injection of ketamine hydrochloride
The amount of drug released was determined by RP-
® 21
(Ketalar , Parke-Davis, Milan, Italy) (20 mg kg ). One
HPLC analysis, using a Hypersil ODS C18
column (5 mm, drop of a local anaesthetic (0.4% oxybuprocaine) was
25034.6 mm I.D.). The mobile phase consisted of a 66:34 instilled into the conjunctival sac. Aqueous humour sam-

56 R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61
ples for each animal were collected with a 26 gauge needle After the analysis of variance, Student’s t-test was used to
attached to a tuberculin syringe. The needle was introduced establish the significance of the differences between mean
into the anterior chamber through the cornea, taking care values. A value of P,0.05 was considered statistically
not to damage the iris, the lens and the anterior uvea; eye significant.
conditions were carefully examined using a slit lamp every The limit of quantitation in aqueous humor was dehour
after the paracentesis. Fifty microlitres of aqueous
21
termined to be 10 ng ml for IBU, with a relative
humour were collected and analyzed by HPLC for drug standard deviation (RSD) of 3.6% and accuracy of 97%.
concentration (see below).
Validation of our assay method consisted of intra- and
In preliminary experiments, all the formulations, ad- inter-day reproducibility studies at three drug concenministered
according to the treatment schedule above trations. The results obtained (data not shown) did not
mentioned, had not altered the pupillary diameter for up to exceed 5% (RSD) for both intra- and inter-day assays.
6 h after the last instillation (data not shown).
The pupil diameters of the treated and the control eyes
were measured with a Castroviejo caliper (Stark et al., 3. Results and discussion
1986) 180 min and 5 min before the first paracentesis and
then 5 min before the second one (Fig. 4). At the end of 3.1. Preparation and characterization of IBU-loaded
the treatment schedule, the animals were killed by i.v. nanosuspensions
21
injection of 0.3 ml kg Tanax (Hoechst AG, Frankfurt am
Main, Germany).
The main advantages of the QESD technique are the
In a separate set of animals, the drug administration was avoidance of toxic organic solvents, commonly used in
carried out only before the first paracentesis, in order to micro- and nanoparticle solvent evaporation techniques,
compare the drug activity associated with a prolonged drug which increases the potential ophthalmic application of the
release from the nanosuspension to the eye surface. system, and the possibility of modifying particle morphol-
Formulations were applied 2 h before the first paracentesis ogy by choosing the agitation speed, the polymer conand
then 2 min thereafter (twice-treatment schedule). Two centration in the initial ethanol solution as well as the
hours later the second paracentesis was performed and 50 volume and injection rate of the solvent (Kopade and Jain,
ml of aqueous humour were withdrawn. 1995).
In a separate set of rabbits the potential ocular irritancy IBU-loaded nanoparticles using different preparative
and/or damaging effects of the nanosuspension formula- variables were obtained (Table 1): the drug-to-polymer
tion were evaluated according to a modified Draize test weight ratio (10, 20, 33 or 50%), the total amount of drug
(McDonald and Shadduck, 1977), using a slit lamp (Sbisa, ` and polymer in the initial ethanol solution (100, 200 or 500
Florence, Italy). The congestion, swelling and discharge of mg), and the agitation speed (from 9500 to 20 500 rpm)
the conjunctiva were graded on a scale from 0 to 3, 0 to 4 during the formation of the nanosuspension. Such variables
and 0 to 3, respectively. Iris hyperemia and corneal opacity could influence the particle size and the drug release from
were graded on a scale from 0 to 4. Nanosuspension them. However, no effect on the amount of drug associated
formulation (50 ml) was topically administered in the right with the polymer matrices was observed, drug loading
eye every 30 min for 6 h (twelve treatments). At the end of being always higher than 90% of the initial added amount
the treatment, three observations at 10 min, 6 h and 24 h (data not reported).
were carried out to evaluate the ocular tissues.
In Table 2, the average particle size and z-potential
values measured after preparation of the nanosuspensions
2.7.2. Determination of IBU concentration in the or after 24 months of storage are reported. Intermediate
aqueous humor
values (collected every 2 months of storage) have not been
Drug levels in rabbit aqueous humour were measured by reported for clarity. The nanoparticles obtained at a speed
HPLC under the conditions above described. Fifty ml of of 20 500 rpm (batches A, see Table 1) and 13 500 rpm
6% perchloric acid in methanol were added to 50 ml (batches B, Table 1) showed mean sizes between 35 and
aliquots of aqueous humour samples to precipitate pro- 125 nm. During storage all the nanosuspensions formed a
teins. The sample was vortex-mixed for 1 min and sediment, which could be easily re-dispersed by manual
centrifuged at 10 000 rpm for 5 min. The supernatant was agitation. However, significant changes were not observed
filtered through a 0.45 mm nylon membrane filter and after 24 months in the refrigerator. Storage at room
injected into the HPLC system. The recovery efficiency temperature, conversely, shows an increase of particle size
from blank rabbit aqueous humor to which 20 and 5 mg that can be partly attributed to the growth of micro-
21
ml of an IBU standard had been added were 97.2 and organisms in the preparation (the presence of microorga-
93.8%, respectively. nisms alters the results of the dimensional analysis).
IBU drug levels found are reported in Fig. 5 for the two This is confirmed by the data from two suspensions (A5
treatment protocols, respectively, as the mean drug and A6), prepared also without benzalkonium chloride as
concentration6S.D. from 5 samples made in triplicate. preservative. For them we were not able to determine the

R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61 57
Table 2
Effects of storage time and conditions on the mean size and z-potential values of IBU-loaded RS nanosuspenions
Batch Mean size z-potential
(nm)6S.D.
(mV)6S.D.
Initial After 24 After 24 Initial After 24 After 24
values months months values months months
at 462 8C
a
at r.t. at 462 8C
a
at r.t.
A1 58.565.6 74.661.1 102.964.3 110.861.1 137.061.4 153.361.2
A2 39.662.3 27.161.9 130.2620.1 132.661.4 137.260.7 152.060.3
A3 46.0610.1 41.466.7 26.867.6 131.061.4 139.060.8 151.861.2
A4 47.565.8
b
58.563.3
b
93.2622.1 117.766.2 129.9611.4
c
–
A5 16169.9 93.8611.1 – 131.261.5 145.361.4 –
A6 65.364.9 59.369.0 – 129.662.8 122.160.7 –
A7 68.669.8 95.967.7 – 126.862.9 141.861.3 –
A8 39.165.6 100.868.7 – 129.661.9 140.761.5 –
A9 100.2612.2
b
1282623.2
b
1032620.1 136.461.2
b
144.361.3
b
146.462.8
B1 18.763.3 65.0610.2 268.8634.3 129.660.1 125.760.7 117.561.8
B2 28.463.3 115611.1
b
10596100.2 136.861.3 130.260.2
b
146.461.5
B3 66.0632.1 87.469.9
b
1862634.4 128.861.0 129.165.5 –
B4 102.668.9 99.6616.6 – 132.962.5 121.166.5 –
B5 110.068.9 – – 133.562.1
b
134.160.7 –
C1 64.662.7 – – 121.661.2 – –
C2 55.4617.6 – – 128.661.2 – –
a
Room temperature 2065 8C.
b
Measured after 6 months from preparation.
c
Not determined.
dimensional parameters and z-potential even after 2 this drug, the particles maintained a positive z-potential
months after preparation. The latter two batches were also suggests that the active compound be mainly dispersed
used to verify the influence of this preservative agent of within the polymer matrix, besides of adsorbed onto their
the z-potential of the nanosuspensions. For its cationic surface. On the other hand, it is know that acidic comnature,
in fact, benzalkonium chloride could adsorb onto pounds, like IBU, interact with RS and RL polymers by
particle surface and alter their charge. Our preliminary means of electrostatic bindings between their carboxyl
findings suggested that this additive had a very limited moiety and the quaternary ammonium groups of the
effect on z-potential value and none in terms of particle polymer (Jenquin et al., 1990; Pignatello et al., 2001).
size. However, a detailed study would be necessary for a Furthermore, the relative constancy of the z-potential
better understanding of the role of this and other preserva- upon storage indicated that no significant drug leakage
tives in increasing the stability and physical properties of from the nanoparticles to the aqueous environment
nanoparticle suspensions.
occurred. In that case, in fact, the free drug molecules in
The preparations obtained at 9500 rpm (batches C1 and solution could have altered the overall electrophoretic
C2) displayed higher mean sizes (Table 2) and after 4 mobility of the suspensions. However, previous studies
months of storage, aggregation phenomena (caking) were with similar systems showed that the drug remained
observed with the formation of an unredispersible residue, absorbed onto particle surface also after several months of
so that size and surface charge data could not be de- storage at low temperatures (Pignatello et al., 2002).
termined. The formulation obtained with the higher To evaluate the possible effect of Tween 80 on the
amount of drug and polymer (500 mg total) (batch A9, z-potential values, one formulation, corresponding to batch
Table 2) gave the worst results in terms of stability, A3 (Table 1), was prepared without the drug and the
displaying a remarkable size growth during storage in both z-potential was measured immediately before the preparaconditions.
tion and after 1 h, 3 h and 24 h of dialysis against water
All the formulations exhibited positive z-potential val- p.i. No change in the electrophoretic mobility was obues
(Table 2), these were not very different from the served with respect to the initial value, thus indicating that
values obtained for plain RS nanoparticles (135 mV), the presence of the surfactant and its possible release from
whereas the drug powder suspension gave a negative particle surface did not give any interference.
z-potential value (212 mV). Such a positive charge is The pH value of the prepared nanosuspensions was
supposed to be important, since it can facilitate an effective always close to that of pure water (5.5–6.3), therefore
adhesion to the cornea surface. Up to 24 months, also the compatible with ocular administration. In separate experielectrophoretic
behavior did not change significantly dur- ments (not shown) some nanosuspensions were prepared
ing storage. The observation that, even in the presence of using phosphate buffered saline (PBS, pH 7.4) instead of

58 R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61
water as the dispersing medium; no significant changes in
the overall properties of the polymeric nanoparticles were,
however, registered.
The described RS nanosuspensions could be freezedried,
both in the absence or in the presence of a
cryoprotectant (1% p/v mannitol), forming a solid residue
which can be easily redispersed by hand agitation, without
evident size and z-potential changes of the resulting
suspension.
A preliminary evaluation in the solid state (IR spectrophotometry,
DSC analysis, and powder X-ray diffractometry)
of the prepared nanosuspension systems showed
that the drug is dispersed in the polymeric matrices in a
microcrystalline form, without polymorph change or transition
into an amorphous form. Detailed data of such study
will be reported in a separate note.
Fig. 3. Effect of the agitation speed on the dissolution rate of the drug
3.2. In vitro drug release tests from nanosuspensions with the same composition (cf. Table 1).
Figs. 1–3 display the dissolution profiles of the drug
from the nanosuspensions. IBU release was evaluated by ing the final dissolution pattern. However, previous studies
dialysis, using a pH 7.4 phosphate buffer as the dissolution on RS and RL microparticles showed that drug release is
medium.
more complex, resulting from co-existing dissolutive and
The applied formulative variables were compared for diffusional phenomena (Pignatello et al., 2000, 2001).
their influence on drug dissolution rate. Increasing the The dispersion of IBU in the polymer matrices led to a
IBU/RS weight ratio in the nanoparticles reduced the drug gradual dissolution and release of the drug, which became
release rate (Fig. 1), with the A4 formulation, containing complete within 24 h; however, over 75% of the drug was
an IBU/RS 1:1 weight ratio, showing the slowest drug already generally released after 2–3 h. The total amount of
release profile. Such behavior would suggest that the more polymer and drug in the initial ethanol solution (100 or
homogeneous and finer dispersion of drug molecules in the 200 mg) did not seem to influence the release of IBU,
polymer matrix, at lower drug concentrations, enhanced its since the batches prepared with the same drug-to-polymer
dissolution profile, allowing a better penetration of the ratio but different amounts of the ingredients in ethanol
dissolution medium through nanoparticles. Such behavior showed very similar dissolution patterns (Fig. 2).
would indicate that the drug solubility is more important Finally, by comparing the formulations obtained with
than its diffusion through the polymer network in delineat- different agitation speeds (20 500 or 13 500 rpm, batches
A and B respectively) (Table 1), nanoparticles obtained at
the higher speed gave a slower and more gradual drug
release curve (Fig. 3), although they did not show significant
differences in size compared to the other batches
(Table 2). This could be ascribed to a higher structural
homogeneity of this polymeric matrix, and also to a more
uniform distribution of the drug.
3.3. Biological evaluation of nanosuspensions: in vivo
inhibition of miosis
Rabbits treated with atropine (control), atropine plus A4,
atropine plus IBL solution, or atropine plus the empty RS
nanosuspension all showed a superimposable mydriasis 85
min after the instillation of atropine (Fig. 4). Mydriasis
induced by atropine alone (control) was not maintained
120 min after the first paracentesis. In fact, the pupil size
was significantly smaller than at 5 min (P,0.01) and even
Fig. 2. Effect of the initial drug and polymer concentration on the in vitro smaller than the basal value. Mydriasis produced by
dissolution profile of IBU from nanosuspensions (cf. Table 1 for atropine, however, was maintained in rabbits treated with
nanoparticle composition). the A4 nanosuspension, in spite of paracentesis (Fig. 4).

R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61 59
Fig. 4. Paracentesis-induced miosis antagonizing activity of IBU administered as a lysine salt (IBL) solution or in the A4 nanosuspension formulation
(both at 0.1%, w/v drug concentration), compared with a blank RS100 nanosuspension. Each bar represents the mean6S.D. (n56).
Fig. 5 shows IBU concentrations measured in the
aqueous humour samples collected from the rabbits treated
with the free drug salt (IBL) or IBU-loaded RS nanoparti-
cles, according to the multiple or the double treatment
schedule, respectively.
Similar results were obtained in rabbits treated with the
reference formulation containing IBL at the same 0.1%
concentration (Fig. 4), even though the pupil diameter
obtained from A4 nanosuspension was higher but it was
not statistically significant compared with the IBL group.
Fig. 5. IBU levels in rabbit aqueous humor samples, collected by the two paracentesis, after the multiple-treatment schedule (mean6S.D.; n55).

60 R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61
IBU levels in the aqueous humour were significantly Acknowledgements
higher (P,0.01) in the rabbits treated with IBU-RS
nanoparticles (multiple treatment schedule), compared to This work was supported by the University of Catania
the group treated with the IBL solution (Fig. 5). Similar (Ricerca di Ateneo, 2000).
findings were found in the second set of experiments,
where the rabbits were treated twice with the formulations:
after the application of the A4 formulation, IBU con-
21 21
References
centrations of 1.5460.06 mg ml and 1.4560.10 mg ml
were found, after the first and the second paracentesis, Bodmeier, R., Chen, H., 1989. Preparation and characterization of
respectively; conversely, the corresponding 0.1% IBL microspheres containing the anti-inflammatory agents, indomethacin,
solution gave a first drug concentration of 0.9360.08 mg ibuprofen, and ketoprofen. J. Contr. Rel. 10, 167–175.
21
ml , whereas no detectable amount of drug was found in Brown, R.M., Roberts, C.W., 1996. Preoperative and postoperative use of
the aqueous humor after the second paracentesis.
nonsteroidal antiinflammatory drugs in cataract. Insight 21, 13–16.
Bucolo, C., Spadaro, A., 1995. Effect of sodium naproxen on inflamma-
These results can be ascribed to an increased corneal tory response induced by anterior chamber paracentesis in the rabbit. J.
residence time of the polymeric matrix. In fact, while after Pharm. Pharmacol. 47, 708–712.
the application of a conventional eye-drop formulation the Calvo, P., Vila-Jato, J.L., Alonso, M.J., 1997. Evaluation of cationic
rapid rug leakage from corneal surface reduces the actual polymer-coated nanocapsules as ocular drug carriers. Int. J. Pharm.
available drug dosage, the gradual drug release from the 153, 41–50.
Gimbel, H.V., 1989. The effect of treatment with topical nonsteroidal
nanoparticles allowed IBU to better penetrate into the anti-inflammatory drugs with and without intraoperative epinephrine
anterior chamber of the eye.
on the maintenance of mydriasis during cataract surgery. Ophthal-
Topical application of the nanosuspension formulation mology 96, 585–588.
to rabbit eyes showed no sign of toxicity or irritation to Harmia, T., Speiser, P., Kreuter, J., Boye, T., Gurny, R., Kubis, A., 1986.
ocular tissues (data not shown).
Enhancement of the myotic response of rabbits with pilocarpine-
loaded poly-butylcyanoacrylate nanoparticles. Int. J. Pharm. 33, 187–
193.
Jenquin, M.R., Liebowitz, S.M., Sarabia, R.E., McGinity, J.W., 1990.
Physical and chemical factors influencing the release of drugs from
4. Conclusions
acrylic resin films. J. Pharm. Sci. 79, 811–816.
Kawashima, Y., Iwamoto, T., Niwa, T., Takeuchi, H., Hino, T., 1993. Size
control of ibuprofen microspheres with an acrylic polymer by chang-
A series of polymeric nanoparticle systems have been ing the pH in an aqueous dispersion medium and its mechanism.
described, obtained by co-dispersion of IBU sodium salt Chem. Pharm. Bull. 41, 191–195.
and Eudragit RS100 polymer in water. The resulting Kawashima, Y., Niwa, T., Handa, T., Takeuchi, H., Iwamoto, T., Itoh, K.,
nanosuspensions showed interesting mean sizes for ophacrylic
polymers by a novel quasi-emulsion solvent diffusion method.
1989. Preparation of controlled-release microspheres of ibuprofen with
thalmic applications, a positive surface charge, which can
J. Pharm. Sci. 78, 68–72.
help corneal adhesion and good stability upon storage, Kawashima, Y., 2001. Nanoparticulate systems for improved drug delivparticularly
at low temperatures. The formulations could ery. Adv. Drug Deliv. Rev. 47, 1–2, and the articles cited therein.
also been successfully freeze-dried to allow a long term Kopade, A.J., Jain, N.K., 1995. Self assembling nanostructures for
conservation.
sustained ophthalmic drug delivery. Pharmazie 50, 812–814.
In the in vivo test on rabbit eye, one of the nanosuspenintraocular
inflammation. J. Ocul. Pharmacol. 7, 227–241.
Kulkarni, P.S., 1991. The role of endogenous eicosanoids in rabbitsions
(containing an IBU concentration of 0.1%, so that no Le Bourlais, C., Acar, L., Zia, H., Sado, P.A., Needham, T., Leverge, R.,
further dilution of the formulation was required), showed a 1998. Ophthalmic drug delivery systems — recent advances. Progr.
very good ocular tolerability. The comparison with an Retinal Eye Res. 17, 33–58.
aqueous solution of IBU lysine salt showed that the Li, V.H.K., Wood, R.W., Kreuter, J., Harmia, T., Robinson, J.R., 1986.
nanoparticle system is able to give a gradual and prolonged Ocular drug delivery of progesterone using nanoparticles. J. Mi-
croencapsulation 3, 213–218.
release of the drug which, associated with an increased Losa, C., Calvo, P., Castro, E., Villa-Jato, J.L., Alonso, M.J., 1991.
retention to the corneal surface, resulted in higher drug Improvement of ocular penetration of amikacin sulphate by association
levels in the aqueous humour. Ultimately, this led to a to poly(butylcyanoacrylate) nanoparticles. J. Pharm. Pharmacol. 43,
higher, even though it was not significant, capacity to 548–552.
inhibit the miosis induced by a surgical trauma than the Marchal-Heussler, L., Sirbat, D., Hoffman, M., Maincent, P., 1993.
Poly(e-caprolactone) nanocapsules in carteolol ophthalmic delivery.
reference eye-drops. Pharm. Res. 10, 386–390.
By a further optimization of formulative parameters, in McDonald, T.O., Shadduck, J.A., 1977. Eye irritation. In: Marzulli, F.M.,
particular the sterility and isotonicity of the preparations, Maibach, H.I. (Eds.). Advances in Modern Toxicology, Vol. 4. Wiley
the described systems are good candidates for a valid and Sons, New York, pp. 139–191.
therapeutic approach for the topical treatment of inflammanoncorticosteroidal
antiinflammatory agent. Metabol. Pediatric Sys-
Murray, D.L., Leopold, I.H., 1982. Preclinical ocular evaluation of
tory conditions of the eye, as well as in maintaining temic Ophthalmol. 6, 201–208.
mydriasis during surgery (for instance after surgery or Perumal, D., Dangor, C.M., Alcock, R.S., Hurbans, N., Moopanar, K.R.,
traumatic events).
1999. Effect of formulation variables on in vitro drug release and

R. Pignatello et al. / European Journal of Pharmaceutical Sciences 16 (2002) 53 –61 61
micromeritic properties of modified release ibuprofen microspheres. J. Sabiston, D., Tessler, H., Sumers, K. et al., 1987. Reduction of inflamma-
Microencapsulation 16, 475–487.
tion following cataract surgery by the nonsteroidal anti-inflammatory
Pignatello, R., Vandelli, M.A., Giunchedi, P., Puglisi, G., 1997. Properties drug, ibuprofen. Ophthalmic Surg. 18, 873–877.
of tolmetin-loaded Eudragit RL100 and RS100 microparticles prepared Samiy, N.M.D., Foster, S.C., 1996. The role of nonsteroidal antiinflamby
different techniques. S.T.P. Pharma Sci. 7, 148–157. matory drug in ocular inflammation. Int. Ophtalmol. Clin. 36, 195–
Pignatello, R., Consoli, P., Puglisi, G., 2000. In vitro release kinetics of 206.
Tolmetin from tabletted Eudragit microparticles. J. Microencapsulation Stark, W.J., Fagadau, W.R., Stewart, R.H. et al., 1986. Reduction of
17, 373–383. pupillary constriction during cataract surgery using suprofen. Arch.
Pignatello, R., Amico, D., Chiechio, S., Giunchedi, P., Spadaro, C., Ophthalmol. 104, 364–366.
Puglisi, G., 2001. Preparation and analgesic activity of Eudragit Zimmer, A.K., Serbe, H., Kreuter, J., 1994. Evaluation of pilocarpine-
RS100 microparticles containing diflunisal. Drug Delivery 8, 35–45. loaded albumin particles as drug delivery systems for controlled
Pignatello, R., Bucolo, C., Spedalieri, G., Maltese, A., Puglisi, G., 2002. delivery in the eye. I. In vitro and in vivo characterization. J. Contr.
Flurbiprofen-loaded acrylate polymer nanosuspensions for ophthalmic Rel. 32, 57–70.
application. Biomaterials 23, 3247–3255.