ORIGINAL PAPER

In vitro–in vivo evaluation of in situ gelling and thermosensitiveketoprofen liquid suppositories

Isık Ozguney • Anita Kardhiqi • Gulbeyaz Yıldız •

Gokhan Ertan

Received: 3 March 2013 / Accepted: 19 September 2013

� Springer-Verlag France 2013

Abstract The main objective of this study was to

investigate the release and pharmacokinetic profiles of

ketoprofen (KP) from developed thermosensitive and mu-

coadhesive liquid suppositories. Thermosensitive liquid

suppositories were prepared using KP, poloxamer 407 (P

407), poloxamer 188 (P 188) and various amounts of dif-

ferent mucoadhesive polymers. In vitro release studies was

monitored by the USP XXVI paddle method. The results

thus obtained were evaluated kinetically and mechanism of

release was analyzed. Identification of poloxamer gel

localization in vivo was conducted using white male rab-

bits by adding 1 % methylene blue. For in vivo studies,

twenty-four white male rabbits were randomly divided into

three groups. The rabbits in each group were administered

with liquid suppository F1 [P407/P188/KP (4/20/2.5 %)],

F5 [P407/P188/KP/C (4/20/2.5/0.8 %)] or conventional

suppository (F–C) into the rectum. The plasma concen-

tration of KP was analyzed by high performance liquid

chromatography (HPLC). Cmax, AUC, MRT and Tmax were

evaluated. The release of KP was variously affected by the

mucoadhesive polymers. In vitro release studies showed

that Carbopol 934 P(C) has significant effect on release rate

among the mucoadhesive polymers. When the formulations

were evaluated kinetically, different kinetic models were

obtained. Formulation F6 [P407/P188/KP/C (4/20/2.5/

1.6 %)] which contains the highest C concentration and

very high viscosity, shows a significantly better fit with

Higuchi kinetic model. n value of this formulation was also

found approximately 0.5. n exponent results of the other

formulations showed that KP might be released from the

suppositories by non-Fickian diffusion. Identification of

poloxamer gel localization in vivo showed that the sup-

positories remain in the rectum without leakage after

administration. With regard to the results of in vivo studies,

the AUC6?14 values of KP in liquid suppository containing

C are significantly higher than those in liquid suppository

without C. MRT0?24 and MRT0?? values of liquid sup-

pository containing C are significantly higher than those in

liquid suppository without C and conventional suppository.

Conventional suppository and liquid suppository without C

significantly gave faster time to reach the maximum plasma

concentrations of KP. With regard to the in vitro and

in vivo experiments, liquid suppository formulation F5

might be a promising formulation for the development of

an effective rectal dosage form.

Keywords Ketoprofen � Thermosensitive gel �In situ gelling � Liquid suppository �Sustained release � In vivo

1 Introduction

The potential of thermally reversible gels as vehicles for the

delivery of drugs has been widely studied. These systems

have been investigated for use as drug delivery systems for

ophthalmic, rectal, nasal, subcutaneous, dermatological and

intraperitonael administration (Miyazaki et al. 1998).

I. Ozguney (&) � A. Kardhiqi � G. Ertan

Department of Pharmaceutical Technology, Faculty of

Pharmacy, Ege University, 35100 Bornova, Izmir, Turkey

e-mail: isik.ozguney@ege.edu.tr

G. YıldızDepartment of Biopharmaceutics and Pharmacokinetics, Faculty

of Pharmacy, Ege University, 35100 Bornova, Izmir, Turkey

G. YıldızEge University Center For Drug Research and Development and

Pharmacokinetic Applications (ARGEFAR), 35100 Bornova,

Izmir, Turkey

123

Eur J Drug Metab Pharmacokinet

DOI 10.1007/s13318-013-0157-6

Rectal use has been favorable for infants, children and

unconscious patients. However, a conventional solid sup-

pository formulation can cause patient discomfort and lead

to patient refusal, possibly lowering patient compliance. A

solid-type suppository that might reach the end of colon

also may allow the carried drugs to undergo the first-pass

effect (Huang et al. 1987). The ideal suppository should be

easy to administer without any pain during insertion and

has a suitable mucoadhesive force so as not to reach the

end of the colon and to avoid the first-pass effect in the

liver and gastrointestinal tract (Choi et al. 1998a; Choi

et al. 1998b).

In addition, in hot climate countries, a further disad-

vantage of conventional suppository is the proximity of its

melting point to average room temperature. Especially,

some problems could be observed on transportation and

storage. Therefore, in situ gelling liquid suppositories

could have advantages because they could be liquefied in a

short time easily at ?4 �C.

KP is an analgesic and nonsteroidal anti-inflammatory

(NSAI) drug usually employed in the therapy of rheu-

matic disorder. In the usual oral administration of NSAI

drugs, the tablets and capsules have led to peptic ulcer-

ation and anorexia (Thomas and Kantor 1986). Rectal

administration of NSAI drug would be an alternative

dosage route for patients with peptic ulcers and children

(Tarımcı and Ermis 1997). KP is an appropriate model

drug for formulation of controlled release dosage forms

due to its short plasma elimination half-life and poor

solubility in unionized water, which affects its bioavail-

ability (Ozguney et al. 2007).

In this study, the sustained release and pharmacokinetic

profiles of KP from thermosensitive and mucoadhesive

liquid suppositories, which were developed in our labora-

tory, and the effect of mucoadhesive polymers on KP

release and bioavailability were investigated. The bio-

availability of KP when administered by rectal adminis-

tration to rabbits in the poloxamer gel with or without

mucoadhesive polymer is compared with that achieved

when this drug is administered in conventional PEG

suppositories.

2 Materials and methods

2.1 Materials

P 407 and P 188 were gifted from BASF (Ludwigshafen,

Germany). Hydroxypropylmethylcellulose (HPMC), poly-

vinylpyrrolidone (PVP), carbopol 934 P(C), PEG 400 and

PEG 6000 were kindly supplied by Mustafa Nevzat Co

(Istanbul, Turkey). Carboxymethylcellulose (CMC) was

purchased from Sigma (St. Louis, MO, USA). KP was

kindly supplied by Zentiva (Istanbul, Turkey). Hydro-

chloric acide, diethyl ether and acetonitrile were purchased

from E. Merck (Darmstadt, Germany). All other chemicals

were used as analytical grade.

2.2 Methods

2.2.1 Preparation of conventional suppository

F–C containing 100 mg KP was prepared by fusion method

at 48 �C using the mixture of PEG 400 and PEG 6000 in

ratio of 40:60, as described previously (Ozguney et al.

2007). The drug was mixed with the melted base. The melt

mass was poured into the steel molds and allowed to

solidify at room temperature. After solidification, the

formed suppositories were removed from the mold, wrap-

ped with aluminium foil and stored in a desiccator in the

refrigerator at ?4 �C.

2.2.2 Preparation of liquid suppositories

2.5 % KP and various amounts (0.2, 0.4, 0.6, 0.8 and

1.6 %) of different mucoadhesive polymers (PVP, CMC,

HPMC and C) were completely dispersed in distilled water

with continuous agitation at room temperature and cooled

down to 4 �C. The mixture of P 407 and P 188 was then

slowly added to the solution with continuous agitation. The

liquid suppository was left at 4 �C through the night until a

clear solution was obtained. Four gram of each formulation

contains 100 mg KP. The composition of the formulations

is listed in Table 1.

2.2.3 In vitro drug release from liquid suppositories

In vitro drug release of KP from liquid suppositories was

monitored by the USP XXVI paddle method at a rotating

speed of 100 rpm in 500 mL phosphate buffer, pH 7.2 at

37 ± 0.58C. Four grams of each formulation containing

100 mg of KP was inserted into a semipermeable mem-

brane tube (Spectra/por� 1 Dialysis Membrane, Spectum

Medical Industries Inc., Los Angeles, CA, USA). Both

sides of the tube were tied up with a thread to prevent

leakage. The semipermeable membrane tube was then

immersed in the dissolution medium. In the experiments, a

0.5 mL sample was withdrawn from dissolution medium at

selected times with the aid of an injector fitted with a

Millipore HA 0.45 lm filter paper. An equal volume of

medium was returned to the system after withdrawal. The

samples were then assayed spectrophotometrically at

261 nm. The experiments were performed in triplicate.

In vitro drug release of KP from F–C was conducted

using USP XXVI paddle method in the same conditions

with non membrane method.

Eur J Drug Metab Pharmacokinet

123

2.2.4 Kinetic evaluations

The results thus obtained were evaluated kinetically by zero-

order, first-order, Higuchi, and Hixson-Crowell equations. The

determination coefficents (r2) and the residuals were calculated

by means of a computer program (Ege et al. 2001). The

mechanism of release of KP from liquid suppositories was

analyzed using the following equations: where Mt/M is the

fraction of released drug at time t, k is a release characteristic

constant of the suppository, and n is an release exponent

indicative of the release mechanism. As the k value becomes

higher, the drug is released faster. The n value of 1 corresponds

to zero-order release kinetics, 0.5\n\1 means a non-Fickian

release model and n = 0.5 indicates Fickian diffusion (Higuchi

model) (Peppas 1985). From the plot of log(Mt/M) versus log(t),

kinetic parameters, n and k, were calculated.

Mt=M ¼ ktn ð1ÞLog Mt=Mð Þ ¼ log k þ n logðtÞ ð2Þ

2.2.5 Identification of poloxamer gel localization in vivo

The method was based on Choi et al. (1998b) and Miyazaki

et al. (1998) with some modifications. Two white male

rabbits weighing 2.5–3 kg were fasted for 24 h prior the

experiment but allowed free access to water to reduce the

fecal content in the rectal canal. The body temperatures of the

rabbits were determined before the beginning of the experi-

ment. The liquid suppository formulation P407/P188/KP/C

(4/20/2.5/0.8 %) (Gelation temperature = 35.7 �C) by add-

ing 1 % methylene blue was administered into the rectum

2 cm above the anus through a catheter onto which was fitted

a disposable syringe. At 30 min after administration, the

rectum was sectioned and the localization of the liquid sup-

pository formulations in the rectum was identified by blue

color. The investigations were performed after the approval

by ethical committee at the Faculty of Pharmacy of Ege

University (B. 30. 2. EGE. 0. 01. 00. 01/04-845-187).

2.2.6 In vivo experiments

White male rabbits were choosen for in vivo experiments

because a lot of sample points were used in pharmacoki-

netic profile analysis. They have more blood volume than

rats for adequate plasma samples and levels for analysis by

HPLC which is readily accessible and has adequate sen-

sitivity and specificity for determining plasma concentra-

tion of KP. White male rabbits weighing from 2.0 to 2.5 kg

were randomly divided into three groups. Each group

contains eight animals. They were fasted for 24 h prior to

the experiments but allowed free access to water. The

rabbits in each group were administered with liquid sup-

pository F1 [P407/P188/KP (4/20/2.5 %)], F5 [P407/P188/

KP/C (4/20/2.5/0.8 %)] or F–C into the rectum 2 cm above

the anus through a catheter onto which was fitted a dis-

posable syringe. All formulations (conventional or liquid)

contain 100 mg drug. 1 ml of blood sample was collected

from the ear vein immediately before administration and at

1, 2, 4, 6, 8, 10, 12, 14, 16 and 24 h after administration of

the formulations. Plasma was separated by centrifugation at

4 �C and 4,000 rpm for 20 min and stored at -20 �C. The

plasma concentration of KP was analyzed by HPLC using

method of Yamada et al. (2001) with slight modifications.

Individual plasma concentration–time profiles were evalu-

ated by non-compartmental analysis using WinNonlin. The

pharmacokinetic parameters were calculated using fol-

lowing equations: where MRT (mean residence time) is

average amount of time a particle remains in a compart-

ment or system, AUMC is area under the moment curve,

AUC is area under a concentration of analyte vs. time

curve. Theoretically, if C(t) denotes the concentration of

analyte at time t, then.

AUCINFðAUC10 Þ

is AUC (area under a curve) extrapolated to infinity.

MRT ¼ AUMC=AUC ð3Þ

Table 1 Composition of in situ gelling liquid suppository

formulations

Formulation P407/P188

(%/ %)

Ketoprofen

(%)

C

(%)

HPMC

(%)

CMC

(%)

PVP

(%)

F 4/20 – – – –

F1 4/20 2.5 – – – –

F2 4/20 2.5 0.2 – – –

F3 4/20 2.5 0.4 – – –

F4 4/20 2.5 0.6 – – –

F5 4/20 2.5 0.8 – – –

F6 4/20 2.5 1.6 – – –

F7 4/20 2.5 – 0.2 – –

F8 4/20 2.5 – 0.4 – –

F9 4/20 2.5 – 0.6 – –

F10 4/20 2.5 – 0.8 – –

F11 4/20 2.5 – 1.6 – –

F12 4/20 2.5 – – 0.2 –

F13 4/20 2.5 – – 0.4 –

F14 4/20 2.5 – – 0.6 –

F15 4/20 2.5 – – 0.8 –

F16 4/20 2.5 – – 1.6 –

F17 4/20 2.5 – – – 0.2

F18 4/20 2.5 – – – 0.4

F19 4/20 2.5 – – – 0.6

F20 4/20 2.5 – – – 0.8

F21 4/20 2.5 – – – 1.6

Eur J Drug Metab Pharmacokinet

123

AUCba ¼

Z b

a

cðtÞdt ð4Þ

Cmax, AUC and MRT parameters were analyzed using

SAS with Tukey LSD (t test) which is used in conjunction

with an ANOVA to find means that are significantly

different from each other. AUC and Cmax which were

not normally distributed evaluated after logharithmic

transformation. Tmax was evaluated using nonparametric

test by SAS program. It is analyzed with Kruskal–Wallis

test and then reanalyzed with Wilcoxon rank sum to find

means that are significantly different from each other.The

investigations were performed after the approval by ethical

committee at the Ege University (2011-46).

2.2.7 Blood sample analysis

Plasma (0.25 mL) was mixed with 0.1 mL of hydrochloric

acid and was shaken by vortex. 2 mL diethyl ether was

added and the mixture was shaken by vortex again. It was

then centrifuged at 2,000 rpm and ?4 �C for 10 min to

extract KP. The upper organic phase was separated and

evaporated to dryness at 40 �C under nitrogen gas. The dry

residue was dissolved in 0.25 mL of mobile phase and the

produced solution was filtered through a Millipore filter

(0.22 lm) and 20 lL of the filtrate was injected into the

HPLC column (HPLC: Agilent 1,100 series; column:

Fig. 1 Release of KP from in situ gelling liquid suppository

formulations containing different mucoadhesive polymers in different

concentrations a C (F1, 0 %; F2, 0.2 %; F3, 0.4 %; F4, 0.6 %; F5,

0.8 %; F6, 1.6 %); b HPMC (F1, 0 %; F7, 0.2 %; F8, 0.4 %; F9,

0.6 %; F10, 0.8 %; F11, 1.6 %); c CMC (F1, 0 %; F12, 0.2 %; F13,

0.4 %; F14, 0.6 %; F15, 0.8 %; F16, 1.6 %); d PVP (F1, 0 %; F17,

0.2 %; F18, 0.4 %; F19, 0.6 %; F20, 0.8 %; F21, 1.6 %) (n = 3)

Fig. 2 Release of KP from in situ gelling liquid suppositories F1;

[P407/P188/KP (4/20/2.5 %)], F5; [P407/P188/KP/C (4/20/2.5/

0.8 %)] and conventional suppository (F–C)

Eur J Drug Metab Pharmacokinet

123

Eclipse XDB-C 18, 4.6–150 mm, 5 l). The mobile phase

was a mixture of 0.05 M phosphate buffer of pH 7.0 and

acetonitrile in ratio of 80:20 v/v, respectively. The mobile

phase used was pumped at a flow rate 1 mL/min, using UV

detector at 256 nm (Yamada et al. 2001).

3 Results and discussion

3.1 In vitro drug release from liquid suppositories

After the selection of the formulation F1 (P407/P188/KP (4/

20/2.5 %)) having suitable gelation temperature (37.1 �C),

different mucoadhesive polymers were added to this for-

mulation in different ratios to test their effects on release

rate of KP. The release of KP was variously affected by the

mucoadhesive polymers. As to the obtained results of

in vitro drug release studies, C has significant effect on

release rate among the mucoadhesive polymers. C delayed

the release rates of KP from the concentration of 0.2 % and

the release rate decreased with increase in C concentration.

The decrease of release rate for the formulations containing

C in the concentrations of 0.2, 0.4, 0.6, 0.8 and 1.6 % (F2,

F3, F4, F5 and F6) was 7, 13, 25, 31 and 56 %, respectively,

during 8 h compared to the formulation without C (F1)

(Fig. 1a). For the formulations prepared with HPMC as

Table 2 Release kinetic parameters of KP from in situ gelling liquid suppositories

Formulation Zero-order First-order Higuchi Hixson-Crowell

r2 P(Resid)2/n - 2 r2 P

(Resid)2/n - 2 r2 P(Resid)2/n - 2 r2 P

(Resid)2/n - 2

F1 0.959 2,504.1 0.992 116.6 0.995 45.1 0.999 156.8

F2 0.955 2,517.2 0.997 25.8 0.990 68.5 0.990 391.6

F3 0.985 1,692.4 0.996 41.8 0.989 62.2 0.999 343.9

F4 0.997 763.5 0.986 58.0 0.969 111.8 0.994 239.4

F5 0.998 658.2 0.987 31.7 0.973 79.5 0.994 248.6

F6 0.981 1,066.5 0.991 7.6 0.994 4.9 0.988 823.3

F7 0.927 3,256.1 0.988 37.5 0.986 104.4 0.979 624.9

F8 0.931 3,380.6 0.995 24.5 0.988 80.9 0.986 662.5

F9 0.968 1,415.5 0.995 75.7 0.994 49.4 0.998 60.5

F10 0.969 1,452.9 0.997 38.1 0.996 28.6 0.998 105.2

F11 0.988 810.2 0.991 113.9 0.982 116.6 0.996 69.6

F12 0.954 1,786.9 0.993 30.7 0.988 85.8 0.986 269.7

F13 0.966 1,517.3 0.994 36.7 0.988 78.9 0.991 217.7

F14 0.955 1,065.0 0.966 24.1 0.989 79.8 0.989 98.6

F15 0.946 1,382.7 0.996 19.3 0.990 76.9 0.987 151.4

F16 0.972 1,161.8 0.989 128.5 0.984 126.7 0.992 79.5

F17 0.946 1,331.5 0.987 54.3 0.985 114.8 0.982 157.2

F18 0.939 1,474.8 0.995 37.9 0.986 111.3 0.983 182.9

F19 0.924 2,374.2 0.990 66.7 0.981 142.2 0.975 446.8

F20 0.955 1,198.7 0.996 44.2 0.987 106.0 0.989 98.2

F21 0.962 1,476.9 0.995 80.9 0.989 89.4 0.994 71.5

Table 3 n exponent assessments of release data of KP from in situ

gelling liquid suppositories

CODE n k R2

F1 0.649 25.234 0.991

F2 0.659 23.388 0.986

F3 0.662 20.417 0.996

F4 0.667 15.703 0.977

F5 0.651 14.621 0.987

F6 0.473 12.941 0.997

F7 0.609 26.303 0.970

F8 0.608 26.363 0.968

F9 0.706 21.379 0.993

F10 0.673 21.978 0.996

F11 0.783 16.557 0.995

F12 0.704 20.653 0.984

F13 0.707 19.906 0.989

F14 0.796 17.258 0.976

F15 0.751 19.230 0.974

F16 0.780 18.197 0.989

F17 0.772 18.492 0.972

F18 0.775 18.706 0.962

F19 0.698 21.978 0.957

F20 0.829 16.904 0.967

F21 0.747 20.230 0.986

Eur J Drug Metab Pharmacokinet

123

mucoadhesive polymer, the decrease of release rate was

4 % up to the concentration of 0.6 %, it was 6 and 11 for the

concentrations of 0.8 and 1.6 %, respectively, compared to

the formulation without C (F1) (Fig. 1b). The decrease of

release rate for the formulations containing CMC and PVP

in the concentration of 1.6 % was 8 and 5 %, respectively,

during 8 h compared to the formulation without C (F1).

However, the concentration increases of CMC and PVP had

almost no effect on release rate (Fig. 1c, d).

The release profiles of KP in liquid suppositories F1, F5

and F–C which were choosen for in vivo studies are

illustrated in Fig. 2. As to the obtained in vitro release

results, it was seen that 100 % of KP in conventional

suppository (F–C) was released out within 30 min, about

90 % of KP in liquid suppository formulation without

mucoadhesive polymer (F1) was released out within 8 h.

On the other hand, 60 % of KP in liquid suppository

formulation containing 0.8 % C (F5) was released out

within 8 h. It was thought that the sustained effect could be

seen using formulation F5 much better and it was choosen

for obtaining sustained effect in in vivo studies besides F1

and F–C.

3.2 Kinetic evaluations

When the formulations were evaluated kinetically as to r2,

it was seen that the kinetic models of the formulations are

different, because of having different polymer combina-

tions (Table 2). Nonetheless, the release results of most

formulations fit with first order or Hixson-Crowell kinetic

models. In the first-order model, drug activity within the

reservoir was assumed to decline exponentially and the

rate of drug release was proportional to the residual

activity. Hixson-Crowell model was developed to describe

Fig. 3 Log-log plots of released fractions of KP versus time. In situ gelling liquid suppositories were composed of 4/20/2.5/0–1.6 % P407/P188/

KP/mucoadhesive polymer a C, b HPMC, c CMC, d PVP

Table 4 Absorption parameters

of KP following rectal

administration of the

conventional and in situ gelling

liquid suppositories

Each value represents the

mean ± SD (n = 8)a Minimum and maximum

values of Tmax

F–C F5 F1

LogCmax (lg/mL) 4.931 ± 0.564 4.369 ± 0.498 4.773 ± 0.359

LogAUC0?2 (lg x h/mL) 12.160 ± 0.525 11.522 ± 0.508 11.951 ± 0.272

LogAUC2?6 (lg x h/mL) 12.652 ± 0.480 12.459 ± 0.448 12.399 ± 0.348

LogAUC6?14 (lg x h/mL) 12.309 ± 0.413 12.613 ± 0.558 11.690 ± 0.642

LogAUC0?24 (lg x h/mL) 6.609 ± 0.419 6.613 ± 0.515 6.238 ± 0.369

LogAUC0?? (lg x h/mL) 6.681 ± 0.406 6.692 ± 0.547 6.403 ± 0.318

MRT0?24 (h) 4.956 ± 0.722 7.241 ± 1.604 4.077 ± 0.996

MRT0?? (h) 6.06 ± 1.147 8.713 ± 2.47 5.97 ± 1.377

Tmax (h) 1.125 (1–2)a 2.5 (1–6)a 1.125 (1–2)a

Eur J Drug Metab Pharmacokinet

123

the release from dosage forms which show dissolution

rate limitation and which do not dramatically change

during the release process (Karasulu et al. 2003). For-

mulations F4 and F5 fit with zero order kinetic, but

constant release begins after a burst effect as from first

hour, because of this reason it could be accepted as

pseudo-zero order kinetic. Formulation F6 which contains

the highest C concentration and very high viscosity,

shows a significantly better fit with Higuchi kinetic

model. According to the Higuchi model, the data obtained

were plotted as cumulative percentage drug release versus

square root of time. It was thought that a dense mass

forms in the semipermeable membrane tube during the

dissolution and thus a Fickian diffusion occurs. This sit-

uation was supported with n value of the formulation F6

which was found approximately 0.5. When n exponent

results were evaluated to understand the release mecha-

nisms of KP from suppositories, it was seen that the

n values of the formulations are between 0.5 and 1,

suggesting that KP might be released from the supposi-

tories by non-Fickian diffusion except formulation F6

(Table 3). The relatively parallel slopes of the plots

(Fig. 3a, b, c and d) indicated that the content of muco-

adhesive polymers and their increased concentration

except formulation F6 might not affect release

mechanisms. This situation fits with n values (Table 3).

Formulation F6 has the smallest k value, indicating that

the drug was most slowly released from suppository.

k values did not change in formulations prepared with

PVP and CMC; however, in formulations prepared with

HPMC and C, k values decreased with increase in mu-

coadhesive polymer concentration from 0 to 1.6 %, indi-

cating that the drug release rate decreased. The decrease

of drug release rate is more evident in the formulations

containing C. It was thought that the reason of the slow

release is the increase of gel hardness with increase in

C concentration (data not shown). The higher gel hard-

ness means stronger viscosity and more compact structure

of poloxamer molecules in formulations. C, which

enhances gel hardness and decreases gelation temperature

(data not shown) could distort or squeeze the diffusion

channels, delaying the release process. This result fits into

the literature (Choi et al. 1998a).

3.3 Identification of poloxamer gel localization in vivo

The retention of the liquid suppository formulation in the

rectum was observed. The suppository remain in the rec-

tum without leakage after administration. At 30 min after

administration, the rectum was sectioned and the blue color

of the in situ gelled formulations was clearly shown in the

rectum.

3.4 In vivo experiments

The pharmacokinetic parameters of KP were determined

after rectal administration of liquid suppository formula-

tion containing C (F5), liquid suppository formulation

without C (F1) and conventional suppository (F–C). As to

the obtained results of in vivo studies, there was no sig-

nificant difference in extent of bioavailability among the

formulations according to AUC0?24 and AUC0?? values

Table 5 Statistical significances of differences among pharmacoki-

netic parameters of the formulations (p \ 0.05)

Results of ANOVA/

Kruskal–Wallis*

Results of Tukey LSD/

Wilcoxon rank sum**

F5/F1/F–C F5 F1

LogCmax 0.0774 F1 0.2369

F–C 0.0729 0.7913

LogAUC0–2 0.0300 F1 0.1629

F–C 0.0260 0.6286

LogAUC2–6 0.4784 F1 0.9577

F–C 0.6448 0.4761

LogAUC6–14 0.0090 F1 0.0076

F–C 0.5158 0.0826

LogAUC0–24 0.1701 F1 0.2244

F–C 0.9998 0.2320

LogAUC0–? 0.3382 F1 0.3930

F–C 0.9986 0.4201

MRT0–24 \0.0001 F1 \0.0001

F–C 0.0022 0.3086

MRT0–? 0.0074 F1 0.0139

F–C 0.0174 0.9943

Tmax 0.0183* F1 0.0430**

F–C 0.0157** 0.5874**

* Result of Kruskal–Wallis test

** Result of Wilcoxon rank sum test

Fig. 4 Plasma concentration–time profiles of KP after the rectal

administration of in situ gelling liquid and conventional suppositories

to rabbits. F1; [P407/P188/KP (4/20/2.5 %)], F5; [P407/P188/KP/C

(4/20/2.5/0.8 %)] and (F–C); conventional suppository

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(p = 0.1701 [ 0.05; p = 0.3382 [ 0.05) (Tables 4 and 5).

This result fits into the literature (Park et al. 2003). For this

reason, plasma concentration–time profiles of KP were

investigated individually between 0–2 h, 2–6 h and 6–14 h

to determine the release behaviour of sustained release

formulations much better. From 0 to 2 h, the area under the

curve values of plasma concentration–time profiles

(AUC0?2) of KP in F–C were significantly higher than

those in the liquid suppository containing C

(p = 0.026 \ 0.05) (Table 5). However, the AUC2?6 and

AUC6?14 values of KP in conventional suppositories were

not significantly different from those in the liquid sup-

positories with and without C (p = 0.6448 [ 0.05;

p = 0.4761 [ 0.05 for AUC2?6, p = 0.5158 [ 0.05;

p = 0.0826 [ 0.05 for AUC6?14, respectively) (Table 5).

On the other hand, the AUC6?14 values of KP in liquid

suppository containing C are significantly higher than those

in liquid suppository without C (p = 0.0076 \ 0.05)

(Table 5), whereas there is no significant difference

between two liquid suppositories for the AUC0?2 and

AUC2?6 values (p = 0.1629 [ 0.05; p = 0.9577 [ 0.05,

respectively) (Table 5). In addition, MRT0?24 and

MRT0?? values of liquid suppository containing C are

significantly higher than those in liquid suppository without

C and conventional suppository (for MRT0?24 p = 0.0001

\ 0.05; p = 0.0022 \ 0.05 and for MRT0?? p =

0.0139 \ 0.05; p = 0.0174 \ 0.05, respectively). Accord-

ing to MRT0?24 and MRT0?? values, there is no signif-

icant difference between liquid suppository without C and

conventional suppository (p = 0.3086; p = 0.9943,

respectively) (Table 5). The highest residence time of the

liquid suppository formulation containing C indicates that

KP was effective for a longer time when administered

using this vehicle rather than liquid suppository without

C and conventional suppository. Therefore, liquid sup-

pository containing C, which releases the KP in more

sustained rate than did liquid suppository without C, gave

more prolonged plasma levels of KP (Fig. 4, Table 4).

Same results were obtained in the literature using different

mucoadhesive polymers (Choi et al. 1998a; Miyazaki et al.

1998; Ryu et al. 1999). This phenomena might be depen-

dent on some characteristics of liquid suppository con-

taining C that it was dispersed rapidly in the rectum, gelled

and attached on the rectal mucous membranes, since it was

a fluid initially. The gel strength and mucoadhesive force

of liquid suppositories containing C have an important

effect on this phenomena too. Conventional suppository

and liquid suppository without C significantly gave faster

time to reach the maximum plasma concentrations (Tmax)

of KP (p = 0.0157 \ 0.05 and p = 0.043 \ 0.05, respec-

tively) (Table 5) indicating that in rabbits KP from con-

ventional suppository and liquid suppository without C can

be absorbed faster than that from liquid suppository con-

taining C (Fig. 4; Table 4).

4 Conclusion

From the in vitro experiments, liquid suppositories with

and without mucoadhesive polymer can be considered as

suitable suppositories for sustained release formulations.

As to the in vivo experiments, which are in limited

number in the literature, it was observed that drug release

and absorbtion of liquid suppository formulation contain-

ing C goes on in the elimination phase and its effectiveness

could be sustained in more controlled plasma level com-

pared to liquid suppository formulation without mucoad-

hesive polymer. KP could be effective for a longer time

when administered using this vehicle rather than liquid

suppository without C and conventional suppository. From

these findings, liquid suppositories containing C could be

useful to deliver KP in a sustained blood level and might be

a promising formulation for the development of an effec-

tive rectal dosage form.

Acknowledgments The authors wish to thank Research Foundation

of Ege University for financial support given to this study. Project

number: 06/ECZ/005.

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