ORIGINAL ARTICLE

Characterisation of DM-b-cyclodextrin:prednisolone complexesand their formulation as eye drops

Andre Sa Couto • Joana Vieira • Helena F. Florindo •

Mafalda A. Videira • Helena M. Cabral-Marques

Received: 20 December 2013 / Accepted: 13 May 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Several ocular treatment options have been

developed to overcome a broad range of ocular infections

and corneal pathologies. Even though, commonly used

ophthalmic formulations are only able to promote a short

therapeutic effect, demanding a frequent dosing regimen.

This study took advantage of dimethyl-b-cyclodextrin to

overcome prednisolone low water solubility through com-

plexes formation. These complexes were characterized by

phase-solubility studies (Ks = 732; CE = 0.864), 1H-

NMR, Differential Scanning Calorimetry and Fourier

Transform Infrared Spectroscopy. Particle size distribution,

prednisolone assay, rheology and osmolality were assessed

to evaluate dimethyl-b-cyclodextrin and HPMC influence

on the eye formulation main physicochemical properties.1H-NMR studies showed a 1:1 molar ratio complexes’

stoichiometry; and the other physical characterisation

methods (FTIR spectra and DSC thermograms) proved a

successful interaction between prednisolone and dimethyl-

b-cyclodextrin. Dimethyl-b-cyclodextrin promoted a sta-

tistical significative water solubility increase of drug and

the particle size of all suspensions prepared presented a d90

lower than 90 lm. The presence of dimethyl-b-cyclodex-

trin did not change the pseudoplastic behaviour of this

HPMC-based suspension, but a lower viscosity was

obtained in the presence of the complexes. As the final

formulation was hypotonic its osmolality was adjusted with

NaCl. Overall, dimethyl-b-cyclodextrin:prednisolone

complexation in the presence of hydrophilic polymer

HPMC appears to be an advantageous approach for the

ocular administration of this drug.

Keywords Complex formation � Ocular drug delivery �Dimethyl-b-cyclodextrin � Prednisolone � HPMC

Introduction

Several ocular preparations have been developed to address

different eye conditions, disorders or pathologies, such as

external infections and intraocular diseases, namely corneal

pathologies like glaucoma and uveitis [1, 2].

Most of the commercial ophthalmic formulations are for

topical application being a well-accepted route of admin-

istration for the treatment of various eye disorders. These

formulations are mostly presented as eye drop solutions/

suspensions and ointments [3, 4].

However, the efficient and complex structure of the eye

promotes the maintenance of eye physiological integrity by

preventing eye desiccation and the adherence/invasion of

the ocular surfaces by external agents. In addition, blink-

ing, baseline and reflex lachrymation, and drainage ensure

the rapid elimination of foreign substances (drugs inclu-

ded), preventing not only its therapeutic effect by low

bioavailability, but also leading to possible adverse side

effects in the gastrointestinal tract, due to its systemic

absorption via the conjunctiva or the nasolacrimal duct [4].

Even in the case of aqueous-based systems, the viscosity

inherent to those formulations causes loss of vision for a

reasonable period of time [4, 5]. Therefore, the design of

alternative ocular viscous therapeutic systems must take

into consideration those challenging barriers resultant from

the particular ocular anatomy and physiology, in order to

increase drug residence time and to control drug

A. S. Couto � J. Vieira � H. F. Florindo �M. A. Videira � H. M. Cabral-Marques (&)

Faculdade de Farmacia, Instituto de Investigacao do

Medicamento (iMed.ULisboa), Universidade de Lisboa, Av.

Prof. Gama Pinto, 1649 - 003 Lisbon, Portugal

e-mail: hcmarques@ff.ul.pt

123

J Incl Phenom Macrocycl Chem

DOI 10.1007/s10847-014-0420-8

elimination rate. These major disadvantages may be over-

came using newer drug delivery systems, for example,

liposomes, in situ gel, microemulsions, nanoparticles,

nanosuspensions, complex formation, etc. that will increase

drugs bioavailability in a sustained and controlled manner

[4, 6]. The use of polymers for ophthalmic instillation is

intended to be administered as liquid (either suspensions or

solutions) and then undergo to a gel after getting in contact

with the eye. In fact, those polymers have led to better

release profiles due to their ability to increase the formu-

lations viscosity [6].

Corticosteroids, namely the synthetic glucocorticos-

teroid prednisolone (PRD), constitute one of the major

molecules used for ocular treatment. PRD (Fig. 1) has

been used for several decades and is still studied using

different formulation approaches and for different ocular

diseases [7–11]. Even though, the development of simple

and safe aqueous ocular formulations containing this

drug is impaired by its low solubility in water. Cyclo-

dextrins (CyDs) can be used to overcome those major

limitations, since they improve drug solubility, chemical

stability, dissolution rate and prevent drug interactions

that may cause eye irritation and discomfort [12–18].

Due to their lypophilic cavity and hydrophilic external

surface, these cyclic oligosaccharides are able to solubi-

lise poorly water-soluble drugs via non-covalent inter-

actions. Being hydrophilic molecules, CyDs are not

likely to overcome biological membranes and thus a

considerable reduction in adverse side effects can be

expected [12, 13].

Thus, this study took advantage of CyDs, namely

dimethyl-b-cyclodextrin (DM-b-CyD) for the preparation

of an eye drop dosage form. The structural features of DM-

b-CyD, namely cavity size and aqueous solubility (57 g/

100 mL at 25 �C for DM-b-CyD and 1.85 g/100 mL at

25 �C for b-CyD) are expected to allow higher complex-

ation efficiencies in comparison to b-CyD [19], and thus

DM-b-CyD was chosen to investigate the formation of

PRD complexes and further characterisation.

Materials

DM-b-CyD was a generous gift from Wacker Chemie AG

(Burghausen, Germany). Micronized PRD and hydroxy-

propyl methycellulose (HPMC) were purchased from

Fluka, UK. Tween 20 was purchased from Vaz Pereira,

Portugal. Purified water used for the experiments met the

Eur. Ph. requirements for purified water [20]. All other

reagents were of analytical grade.

Methods

Preparation of PRD and DM-b-CyD binary systems

Binary systems of PRD and DM-b-CyD (Table 1) were

prepared i) as physical mixtures of both entities at different

DM-b-CyD ? PRD molar ratios (1:1, 2:1, 4:1 and 6:1) for

10 min using a glass mortar and pestle and ii) by kneading

[21] at the same ratios (DM-b-CyD:PRD), which were

further dried at 40 �C for 12 h.

Phase-solubility studies

Phase-solubility studies were carried out to characterise

DM-b-CyD:PRD complexes according to Higuchi and

Connors [22].

An excess of PRD was added to DM-b-CyD (0, 0.0125,

0.025, 0.0375 and 0.05 M) water solutions. The suspen-

sions were kept in a water bath at 37 �C for 6 days under

constant agitation. Samples were collected at predeter-

mined periods of time (1, 2, 5 and 6 days), and PRD

concentration was assessed, after filtration, by UV spec-

trophotometric method (Hitachi U-200 UV–visible spec-

trophotometer) at 254 nm.

Thus, complexes apparent stability constant (Ks),

complexation efficiency (CE) and drug:cyclodextrin

molar ratio (D:CD molar ratio) values were determined

in order to evaluate the solubilising ability of DM-b-CyD

[23].

Fig. 1 Prednisolone (PRD) chemical structure

Table 1 Weights of DM-b-CyD and PRD needed to prepare the

binary systems (physical mixtures and complexes)

Molar ratio (DM-b-CyD:PRD) DM-b-CyD (g) PRD (g)

1:1 0.7845 0.2155

2:1 0.8792 0.1208

4:1 0.9357 0.0643

6:1 0.9562 0.0438

All the presented weights were calculated to 1 g of complex

J Incl Phenom Macrocycl Chem

123

Proton nuclear magnetic resonance (1H-NMR)

Proton Nuclear Magnetic Resonance (1H-NMR) spectros-

copy has been employed to examine the interaction mode

of the DM-b-CyD with PRD. This technique is based on

the observation of the CyD proton chemical shifts as a

result of the PRD influence in different molar ratios (1:1,

2:1, 4:1, 6:1). Spectra were performed on a Bruker AMX

using residual solvents as internal reference: H = 7.26

(CHCl3) at 300 MHz.

Differential scanning calorimetry (DSC)

The thermal behaviour was investigated on a TA instru-

ments DSC Q200 calorimeter, using 4 mg samples of each

sample (DM-b-CyD:PRD physical mixture, DM-b-

CyD:PRD complex, PRD and DM-b-CyD) in open alu-

minium pans at a heating rate of 10 �C/min from 30 to

270 �C.

Fourier transform infrared spectroscopy (FTIR)

Infrared spectra (IR) were performed on a spectropho-

tometer (Shimadzu IRAffinity-1) using the KBr disk

method and scanned from 4,000 to 400 cm-1.

Effect of HPMC on drug solubility

The effect of a water-soluble polymer (HPMC) on the

solubilising ability of PRD either pure or as DM-b-CyD

complex (1:1 molar ratio) was further evaluated. Thus, 4

systems were prepared with equal amounts of each corre-

sponding excipient (PRD ? H2O; PRD ? 0.25 % (w/v)

HPMC; DM-b-CyD:PRD complex ? H2O; DM-b-

CyD:PRD complex ? 0.25 % (w/v) HPMC) and kept for

48 h in a water bath at 24 �C under constant agitation. PRD

concentration in all suspensions was then analysed by UV

spectroscopy as described above.

Particle size distribution

Particle size analysis of the different suspensions

(PRD ? H2O; PRD ? HPMC; DM-b-CyD:PRD ? H2O;

DM-b-CyD:PRD ? HPMC) were performed using puri-

fied water [20] by laser diffraction spectroscopy (LDS;

Mastersizer Hydro 2000S, Malvern Instruments, UK). The

influence of Tween 20 in the particle size distribution of the

latter suspensions was also assessed.

Rheological analysis

The rheological properties of both PRD ? HPMC and

DM-b-CyD:PRD ? HPMC suspensions were studied

using a controlled speed Brookfield DVII? rotational vis-

cometer/rheometer (Brookfield engineering laboratories,

INC; USA).

Osmolality

The osmolality of different ocular suspensions

(PRD ? HPMC; DM-b-CyD ? HPMC; DM-b-

CyD:PRD ? HPMC) was determined by its direct mea-

surement in a Knaur automatic osmometer using NaCl (400

mOsM/kg) as a reference.

Results and discussion

Phase-solubility studies

The main goal of the phase-solubility studies was to assess

the ability of DM-b-CyD to promote the solubilisation of

an active substance slightly soluble in water.

The phase-solubility curves (Fig. 2) were classified as

AL-type according to Higuchi and Connors [22], which

0.000

0.005

0.010

0.015

0.020

0.025

0 0.01 0.02 0.03 0.04 0.05 0.06

[Pre

dnis

olon

e] (

M)

[DM-β-CyD] (M)

24h48h120h144h

Fig. 2 Phase solubility diagrams of DM-b-CyD:PRD (Mean ± SD;

n = 3)

Table 2 Phase-solubility studies of PRD complexation with DM-b-

CyD according Higuchi and Connors [22]

Time

(h)

R2 Slope Ks (M-1)

[23]

CE

[23]

D:CD ratio

[23]

24 0.9827 0.353 461 0.544 2.84

48 0.9556 0.464 732 0.864 2.16

120 0.9892 0.455 707 0.834 2.20

144 0.9857 0.402 570 0.673 2.49

J Incl Phenom Macrocycl Chem

123

shows a linear increase in solubility of the drug with

increasing DM-b-CyD concentration. Considering Ks and

the CE values (Table 2), 48 h seem to be the optimal time

for complex formation, under the used conditions.

Based on curve trend-lines slope values (\1) it could be

assumed that only a 1:1 complex is formed, however,

having in consideration the obtained Ks and the CE, D:CD

molar ratio [23] was calculated (Table 2) pointing to higher

ratios than 1:1. In order to make sure of the most probable

cyclodextrin:drug ratio it was decided to perform 1H-NMR

studies covering a wider range, i.e., between 1:1 and 6:1

ratios.

Proton nuclear magnetic resonance (1H-NMR)

DM-b-CyD has primary and secondary OH groups

crowning opposite ends of its torus: H-3 and H-5 directed

towards the interior, H-6 on the rim and H-1, H-2 and H-4

located to the exterior. It is expected that if inclusion does

occur, protons located within or near the cavity (e.g. H-3,

H-5 and H-6) should be strongly shielded whereas protons

located on the exterior of the torus should be relatively

unaffected [24]. In the present case it seems that the

association takes place in both surfaces, the inner cavity

and the exterior of the CyD. This assumption is shown by

the more significant shifts on the protons (H-3, H-2 and

H-4) compared to the other protons, probably the com-

plexes formed are a mixture of inclusion and non-inclusion

types. Figure 3 presents the most relevant DM-b-CyD

proton shifts plotted against the different molar ratios,

showing the highest shifts for the CyD:PRD molar ratio of

1:1. For this reason further studies were performed with 1:1

molar ratio binary systems.

Differential scanning calorimetry (DSC)

The DSC thermogram of PRD (Fig. 4a) shows a sharp

endothermic peak at 247.43 �C, which is not in accordance

with its melting point described in literature (237 �C). This

difference can be attributed to the presence of impurities or

to high heat rate used for this analysis. DM-b-CyD DSC

thermogram (Fig. 4b) presents a peak at 66.12 �C due to

the expected water release from the inner cavity of this

molecule. In addition, DM-b-CyD did not melt until

270 �C and does not show any noticeable thermal event.

The DSC thermogram of DM-b-CyD ? PRD (1:1)

physical mixture (Fig. 4c) presents a similar peak at

65.31 �C that can be also attributed to the loss of water

from this sample. Additionally, it would be expected to

observe a peak at 247.43 �C, corresponding to PRD.

However, this was not observed, which may predict the

formation of CyD complex by simple physical mixing of

the drug with DM-b-CyD may be due to the heat generated.

The absence of PRD characteristic peak can also be due to

the low mass proportion of DM-b-CyD ? PRD (75 %/

25 %, w/w) used.

Regarding the DSC thermogram of DM-b-CyD:PRD

complex (Fig. 4d), prepared by kneading method, it is

possible to observe the presence of an endothermic water-

related peak at 63.92 �C. This endothermic peak is smaller

than the one obtained by physical mixture (Fig. 4c) or DM-

b-CyD (Fig. 4b), as expected due to its incubation at 40 �C

for 12 h during complex preparation procedures. Despite

this small difference, the PRD endothermic peak was not

present, similarly to what have been observed for the

physical mixture thermogram (Fig. 4c). Thus, it is possible

to predict the successful formation of the DM-b-CyD:PRD

complex.

Nevertheless, to confirm the formation of DM-b-

CyD:PRD complexes, FTIR was used to corroborate and

support the above data.

Fourier transform infrared spectroscopy (FTIR)

The FTIR spectrum of PRD shows three characteristic

bands at 1,710.87 and 1,654.46 (C=O bond) and

1,612.99 cm-1 which relates to the conjugated double

bond and those were used to analyse the interaction

between this drug molecule and DM-b-CyD (Table 3). But,

DM-b-CyD characteristic bands (Fig. 5) obtained at its IR

spectrum were 3,411.62 (O–H bonds), 2,927.48 (C–H

bonds), 1,086.90, 1,045.43 and 1,157.78 cm-1. Besides

being present at both the physical mixture and the complex

FTIR spectra, 888.71 cm-1 band does not seem to repre-

sent PRD characteristic stretching vibrations.

Fig. 3 Representation of the H-NMR chemical shifts for the DM-b-

CyD:PRD complexes in several molar ratios

J Incl Phenom Macrocycl Chem

123

None of the CyD-based formulations presented addi-

tional bands to those observed on PRD and DM-b-CyD

FTIR spectra and therefore there was no formation of

covalent bonds neither in DM-b-CyD ? PRD physical

mixture nor in DM-b-CyD:PRD complexes (Fig. 5). Even

though, some of the drug and CyD characteristic bands

were shifted towards higher frequencies (Table 3). How-

ever, besides being more prominent in complexes IR

spectra, none of those shifts are significant, in opposition to

peak intensities. Interestingly, physical mixture IR spec-

trum evidenced higher peak intensities for both PRD and

CyD-associated bands, while the opposite trend was shown

in the IR analysis of the complex. The presence of those

characteristic bands at nearly the same frequencies indi-

cates that most probably there was no interaction between

CyD internal cavity and groups responsible for IR

absorption. Even though, the lower intensity referred above

suggests that the PRD complexation by DM-b-CyD prob-

ably occurred.

Effect of HPMC on drug solubility

Regarding the pure PRD it can be seen an increase of 30 %

of its solubility in HPMC compared to water. In Fig. 6 it

can also be seen that HPMC also increases the PRD sol-

ubility as complex, but in less extent (19 %). This increase

is less noticeable due to the huge increase in the water

solubility caused by complex formation in comparison to

the pure drug (more than 700 fold). All 4 systems

(PRD ? H2O; PRD ? 0.25 % (w/v) HPMC; com-

plex ? H2O; complex ? 0.25 % (w/v) HPMC) PRD sol-

ubility is significant and statistically different from each

other.

Fig. 4 DSC thermogram of

(a) PRD, (b) DM-b-CyD,

(c) DM-b-CyD ? PRD (1:1)

physical mixture and (d) DM-b-

CyD:PRD (1:1) complex. Heat

flow endothermic down (mW)

versus temperature

Table 3 PRD, DM-b-CyD, DM-b-CyD ? PRD physical mixture and DM-b-CyD:PRD complex IR data

PRD DM-b-CyD Physical mixture DM-b-CyD ? PRD DM-b-CyD:PRD complex

Wavenumber (cm-1) Intensity Wavenumber (cm-1) Intensity Wavenumber (cm-1) Intensity Wavenumber (cm-1) Intensity

1,710.87 43.743 1,710 nd (1) 1,711.84 59.258

1,612.99 28.932 1,612.99 69.165 1,613.47 64.075

1,654.46 10.381 1,651.56 60.151 1,653.01 19.515

888.71 44.157 888.71 80.589 888.71 66.919

3,411.62 26.695 3,415.00 44.124 3,415.96 18.788

2,927.48 47.887 2,928.83 59.699 2,929.41 31.701

1,045.43 12.126 1,042.05 31.671 1,040.06 6.256

1,086.90 16.466 1,087.38 36.792 1,087.38 9.18

1,157.78 28.646 1,158.26 47.519 1,158.75 16.636

The intensity of the band t 1,710 cm-1 could not be displayed since this area in the spectrum suffered interferences that prevented the

identification of value intensity

J Incl Phenom Macrocycl Chem

123

These findings suggest the benefit of this polymer to

potentiate the complexation of this drug by using DM-

b-CyD. Similar trends were reported by other studies,

which evidenced that hydrophilic polymers as HPMC,

polyvinylpirrolidone (PVP) and polyethileneglycol

(PEG) promote complexation and higher solubilising

efficiencies of different drugs as hydrocortisone [25],

piroxicam [26], finasteride [27] and pioglitazone [28],

using CyDs. In addition, in the study herein under

discussion, HPMC seems to allow the use of DM-b-

CyD lower concentrations to deliver equivalent thera-

peutic amounts of PRD.

Particle size distribution

Particle size analyses were carried out in order to study the

influence of HPMC polymer in the particle size distribution

(d10/d50/d90, corresponding to percentiles 10, 50, 90 %;

for example d10 means that 10 % of the measured particles

are below the given value) of free drug and complex, in

comparison with water (Table 4). Particle size distribution

(d10, d50, d90) was also assessed in the presence of Tween

20 to predict the existence of aggregates in the suspension.

In fact, there were not significant changes in particle size

distribution of PRD in the presence of this surfactant.

Fig. 5 IR spectra

correspondent to PRD (a) and

DM-b-CyD:PRD 1:1 molar

ratio complex (b)

J Incl Phenom Macrocycl Chem

123

The complexes formed between PRD and DM-b-CyD

present significantly lower diameters in water than in

HPMC, but in the presence of Tween 20 no significant

difference was noticed neither in water nor in HPMC

(Fig. 7).

However, for pure PRD in both suspensions (water and

HPMC) the Tween 20 promoted a statistical significant

decrease in the particle size (Fig. 8).

It can be assumed that PRD alone in suspensions forms

aggregates being the latter dispersed by the addition of the

surfactant leading to smaller particles. Probably the com-

plexes are physically stable entities (at least in terms of

size) as the Tween 20 did not promote any significant

effect on the particles’size (Fig. 8). Despite of the com-

plexes particle sizes in water are slightly smaller than in

HPMC, the formulation is prepared with this polymer

because size is still acceptable and it has the advantage to

increase the viscosity of the system (as it will be discussed

below).

Anyway in all suspensions the d90 was lower than

90 lm (higher Pharmacopoeial limit for ocular dosage

forms) [29] which is an important finding for ocular ther-

apy, namely to increase the contact time of drug with

ocular surface due to higher surface area. These are

important features for topical applications and/or to allow

drug amounts at therapeutic levels for prolonged periods of

time in the case of systemic applications [30].

Rheological analysis

HPMC, a bioadhesive and viscosity-enhancer polymer was

used to address the desired mechanical and pharmaceutical

properties of the suspensions developed to be used for

ocular delivery of PRD. The dispersion of this type of

polymers confers certain rheological properties to these

suspensions that may promote or improve drug contact

and/or permeation through ocular surfaces, mainly due to

their adhesive properties. Thus, the HPMC rheological

effect on several suspensions was assessed.

Regarding these analysis, both PRD ? HPMC (Fig. 9)

and DM-b-CyD:PRD ? HPMC (Fig. 10) suspensions

presented a pseudoplastic rheological profile, as presented

lower viscosity at higher shear rates.

Even though, the formulation containing DM-b-

CyD:PRD complex presented lower viscosity than that

obtained when the free drug was suspended in exactly the

same viscous phase (Table 5).

In fact, once within aqueous polymeric solution, the

internal cavity of CyD is expected to complex with the

hydrophobic portion of polymer chains, which will not be

then available to be dispersed in the aqueous phase. These

observations are in accordance with previous studies that

showed that CyD has the ability to decrease the viscosity of

aqueous polymeric solutions. In addition, those findings

were corroborated by the addition of surfactants to the

polymeric solution, as those compete with hydrophobic

portion of water-soluble polymers for their inclusion in

CyD hydrophobic cavity [31, 32].

Note: PRD corresponds to pure prednisolone; Complex corresponds to 1:1 molar ratio DM-β-CyD : PRD.

0.30 0.39

2.51

2.99

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

H2O+PRD HPMC+PRD H2O+Complex

HPMC+Complex

PR

D w

ater

sol

ubili

ty (

mg/

mL

)

Fig. 6 Influence of HPMC on the PRD water solubility when pure

and as CyD complex (Mean ± SD; n = 3)

Table 4 Particle size

distribution for the studied

systems (mean ± S.D.; n = 3)

d10/d50/d90 corresponds to

percentiles 10, 50, 90 %: for

example, d10 means that 10 %

of the measured particles are

below the given value. Particle

size distribution expressed as

mean ± S.D. (n = 3)

Formulation Particle size distribution (lm)

d (10) d (50) d (90)

PRD ? H2O 1.60 ± 0.08 20.15 ± 0.54 49.90 ± 3.54

PRD ? HPMC 2.40 ± 0.36 20.22 ± 0.75 54.24 ± 2.39

DM-b-CyD:PRD ? H2O 4.07 ± 0.35 24.34 ± 0.15 50.15 ± 1.61

DM-b-CyD:PRD ? HPMC 3.94 ± 0.30 24.69 ± 0.43 69.79 ± 2.65

PRD ? H2O ? Tween 20 1.19 ± 0.07 17.46 ± 0.81 44.06 ± 1.71

PRD ? HPMC ? Tween 20 2.58 ± 0.55 20.56 ± 1.04 48.19 ± 1.13

DM-b-CyD:PRD ? H2O ? Tween 20 3.71 ± 0.33 23.58 ± 0.59 49.72 ± 2.82

DM-b-CyD:PRD ? HPMC ? Tween 20 6.33 ± 0.32 25.65 ± 0.15 72.40 ± 2.92

J Incl Phenom Macrocycl Chem

123

Osmolality

Osmolality is a physical property usually associated to

ocular irritation resultant upon instillation. The lacrimal

fluid presents an osmolality of about 308 mOsmol/kg

(value attributed to the tonicity of a 0.9 % (w/v) NaCl

solution). The desired osmolality values for ophthalmic

suspensions range from 255 to 315 mOsmol/kg [26].

The influence of free PRD, DM-b-CyD, HPMC and

DM-b-CyD:PRD complex in the osmolality of the for-

mulation under study must be evaluated as only isoto-

nicity or hypertonicity are allowed for ophthalmic

preparations.

Nevertheless, none of the developed formulations pre-

sented values within the accepted osmolality range, and the

following equation has been used to determine the exact

Fig. 7 Comparison of the effect

of HPMC/water on the particle

size distribution of DM-b-

CyD:PRD complex (a) and

effect of Tween 20 on the latter

complexes (b)

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

Par

ticl

e si

ze (

μm)

Fig. 8 Comparison between the

d90 values of the different

systems

J Incl Phenom Macrocycl Chem

123

amount of NaCl that should be added to increase the sus-

pension osmolality, and thus address the required proper-

ties for ocular administration of drugs:

Excipient osmolalityðmOsmol=kgÞ¼Weightðg/Lþmolecular weight � number of ions

� 1; 000Þ

According to the above equation, the sum of the

osmolality value of all excipients (49.73 mOsmol/kg) was

subtracted from 308 mOsmol/kg, yielding 258.27 mO-

smol/kg. Given the molecular weight of NaCl (58.44 g/

mol), it is required to add 15.09 g/L of NaCl to the

formulation.

This amount of NaCl was added to a suspension of the

PRD:DM-b-CyD complex in HPMC and the osmolality

obtained was 490 mOsmol/kg (Table 6). This value is

within the accepted limits of tonicity for ophthalmic

preparations intended to avoid marked discomfort for the

eye [33].

Conclusion

Eye drops of PRD complexed with DM-b-CyD were for-

mulated as viscous suspension with particle size and

osmolality within accepted ophthalmic criterion.1H-NMR studies proved that the most probable stoi-

chiometry for the complex is a 1:1 molar ratio. IR spectra

and DSC thermograms have revealed the successful inter-

action of PRD with DM-b-CyD, showing the existence of

molecular bounds between both molecules.

DM-b-CyD improved PRD solubilisation in both aque-

ous and polymer solutions although was not possible to

solubilise the total PRD amount. The best size profile was

obtained for the complex in water; however HPMC was

chosen in order to increase the system viscosity. As this

formulation was hypotonic, osmolality was adjusted with

NaCl.

The therapeutic outcome of ocular preparations is

expected to be considerably improved by the development

of alternative viscous systems (e.g. HPMC) that would not

only prolong the residence time at the ocular surface, but

also decrease drug elimination rate and in addition would

stabilise the complex and physically stabilise the

suspension.

The dispersion of DM-b-CyD:PRD complex in HPMC

solution constitutes a promising approach for the applica-

tion of water-insoluble molecules in ocular delivery.

IncreasingDecreasing

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.00

0 2 4 6 8 10 12

η (P

a.s)

(Pa

.s)

D (s-1)

Fig. 9 Rheological profile/viscosity behaviour (viscosity (g) vs shear

rate (D)) of PRD dispersed in a HPMC-based suspension

Increasing

Decreasing

0.00

5.00

10.00

15.00

20.00

25.00

0 2 4 6 8 10 12

D (s-1)

η (P

a.s)

(Pa

.s)

Fig. 10 Rheological profile/viscosity behaviour (g vs D) of DM-b-

CyD:PRD complex dispersed in a HPMC-based suspension

Table 5 PRD effect on viscosity (Pa s) of HPMC-based suspensions

Shear rate (s-1) Viscosity (Pa s)

PRD ? HPMC DM-b-CyD:PRD ? HPMC

0.13 39.991 19.996

0.22 21.115 14.877

0.66 11.518 9.278

1.32 10.478 8.238

2.64 9.558 7.118

5.50 9.156 6.546

8.80 8.818 6.515

11.0 8.753 6.354

Table 6 Osmolality values for HPMC-based formulations

Formulation Osmolality (mOsmol/kg)

HPMC 3

PRD ? HPMC 50

DM-b-CyD ? HPMC 27

DM-b-CyD:PRD ? HPMC 29

DM-b-CyD:PRD ? HPMC ? NaCl 490

J Incl Phenom Macrocycl Chem

123

Acknowledgments The financial support provided by the FCT,

Fundacao para a Ciencia e Tecnologia (PTDC/SAU-FCF/098733/

2008) and the Portuguese NMR Network (IST-UTL Center) for

providing access to the NMR facility are gratefully acknowledged.

The authors also thank Stephanie Monod for technical assistance.

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