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Drug Dev Ind Pharm, Early Online: 110


! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.788011

ORIGINAL ARTICLE
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Sildenafil vaginal suppositories: preparation, characterization, in vitro


and in vivo evaluation
Srinivasan Shanmugam1, Young-Hun Kim1, Jeong-Hee Park1, Ho Taek Im1, Young Taek Sohn2, Kyeong Soo Kim1,
Yong-Il Kim1, Chul Soon Yong3, Jong Oh Kim3, Han-Gon Choi4, and Jong Soo Woo1
1
Pharm. R&D Institute, Hanmi Pharm. Co., Ltd., Hwasung, Gyeonggi, South Korea, 2College of Pharmacy, Duksung Womens University, Dobong-Gu,
Seoul, South Korea, 3College of Pharmacy, Yeungnam University, Gyongsan, South Korea, and 4College of Pharmacy, Hanyang University, Ansan,
Republic of Korea

Abstract Keywords
Aim: The main objective was to investigate the in vitro release profile/kinetics, and in vivo Intravaginal delivery, PK/biodistribution,
plasma pharmacokinetics (PK) and organ biodistribution (BD) of the prepared sildenafil vaginal release kinetics, sildenafil, vaginal
suppositories (SVS). suppositories
Methods: Suppositories containing 25 mg of sildenafil were prepared by the cream melting
technique using Witepsol H-15 as a suppository base. The suppositories were characterized History
for weight variation, content uniformity, hardness, disintegration time and crystallinity change.
Received 6 December 2012
For personal use only.

The in vitro dissolution in pH 4.5, and in vivo plasma PK and organ BD of sildenafil from SVS in
female Sprague Dawley rats, were also investigated. Revised 8 March 2013
Results: The mean weight variation, content uniformity, hardness and disintegration time of the Accepted 16 March 2013
prepared SVS were 1.127  0.020 g, 98.25  2.50%, 2.5  0.08 kg and 9  1.0 min, respectively. Published online 19 June 2013
The release of sildenafil from the SVS was more than 90% at 30 min, with a release kinetic of
HixsonCrowell model and non-Fickian diffusion (n 0.464). The plasma PK study demon-
strated a significantly lower Cmax (10 times) and AUC024 h (13 times) of sildenafil in plasma
following intravaginal (IVG) administration of suppositories compared to oral (PO) administra-
tion of sildenafil solution. Nevertheless, the organ BD study showed a phenomenally higher
Cmax (40 times) and AUC024 h (20 times) of sildenafil in uterus following IVG administration
of suppositories than PO administration of sildenafil solution.
Conclusion: This study demonstrated enhanced sildenafil exposure in the uterus following IVG
administration of SVS, which could be used to target the uterus for therapeutic benefits.

Introduction estrogens and low dose aspirin with varying degree of success8,9.
Besides, much interest has now been focused on improving
Recurrent spontaneous abortion (RSA) is the most common
uterine blood flow through vasodilation of endometrial vascula-
complication of pregnancy. RSA is defined as three or more
ture for the proliferation of the endometrial lining in women10,11.
clinically recognized pregnancy losses. i.e. expulsion or extraction
Recently, sildenafil, 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-
of the embryo or fetus weighing less than 500 g which is
oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenylsulfonyl]-
13
equivalent to approximately 2022 weeks of gestation1,2. It has
20
4-methylpiperazine, a type 5-specific phosphodiesterase (PDE)
remained a significant health problem affecting 25% of
inhibitor, has been reported to improve uterine blood flow in
reproducing couples worldwide3. In vitro fertilization (IVF) is
women following intravaginal (IVG) administration1,4,1215. This
one of the useful treatment methods used for the treatment of this
increased blood flow has lead to estrogen-induced proliferation of
type of infertility with variable outcomes4,5.
the endometrial lining in patients with poor endometrial devel-
A trilaminar endometrial thickness of 8 mm as determined by
opment resulting in successful pregnancy4,13. Besides, sildenafil
ultrasonographic pattern of the endometrium on the day of human
is considered as a potential candidate for treating pregnant women
chorionic gonadotrophin has been shown to be correlated with a
with intrauterine growth retardation (IUGR) or as a tocolytic
high chance of pregnancy in patients being treated with IVF6,7.
agent (anti-contraction agent)4,1315.
Eventually, a myriad of treatment options have been proposed to
However, oral sildenafil therapy is not considered effective
improve endometrial proliferation, including treatment with
in pregnant women because of its possible low target (uterus)
concentration and related side effects such as hypotension,
flushing and headaches in patients4,13. Thus, targeting drug
Address for correspondence: Jong Soo Woo, PhD, Pharm. R&D Institute, delivery through vagina is of particularly appealing for substances
Hanmi Pharm. Co., Ltd., Hwasung, Gyeonggi, 445-913, South Korea. Tel:
+82-31-356-3311. Fax: +82-31-356-7139. E-mail: jswoo@hanmi.co.kr
such as sildenafil that are destined to exert their primary action
Han-Gon Choi, PhD, College of Pharmacy, Hanyang University, 1271, on the uterus itself. For this reason, the development of
Sa-3-Dong, Ansan 426-791, Republic of Korea. Tel: +82-31-400-5802. appropriate IVG delivery system for effective delivery of
Fax: +82-31-400-5958. E-mail: hangon@hanyang.ac.kr sildenafil to its target organ (uterus) for the treatment of RSA,
2 S. Shanmugam et al. Drug Dev Ind Pharm, Early Online: 110

IUGR and other pregnancy-related complications, has been individually and the average weights were determined.
gaining momentum. No suppositories should deviate from mean weight by more
In recent years, scientists have demonstrated the effectiveness than 5% except two, which may deviate by not more than 7.5%.
of the vaginal route for delivering drugs such as progesterone and
danazol, preferentially to the uterus while minimizing systemic Content uniformity
levels and side effects1618. Among various kinds of vaginal drug
Five suppositories were randomly selected during preparation and
delivery systems, suppositories are semi-solid preparations widely
assayed individually for sildenafil content. First, the suppository
used for vaginal applications due to their low cost, safety and easy
was dissolved in 100 ml volumetric flask containing about 80 ml
administration19,20. Unfortunately, there have been no reports
of methanol using sonication for about 5 min. Then, the volume
on the in vivo plasma pharmacokinetics (PK) and/or organ
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was made up to 100 ml using methanol. Four ml of the above


biodistribution (BD) of sildenafil following IVG application.
solution was taken into 50 ml volumetric flask and was diluted
Thus, the main objective of this study was to investigate the
with mobile phase. The final solution was then syringe filtered
in vivo PK and BD of sildenafil vaginal suppositories (SVS)
and analyzed by HPLC to determine the content uniformity of
following IVG application in female rats. Suppositories contain-
prepared SVS. The standard sildenafil powder was simultaneously
ing 25 mg of sildenafil were prepared and characterized for
analyzed using 25 mg of sildenafil by the above-mentioned
weight variation, content uniformity, hardness, disintegration
procedure.
time and crystallinity change. The in vitro dissolution profile
and release kinetics of sildenafil in pH 4.5 dissolution medium
Hardness
was also investigated.
The hardness of the prepared SVS was tested using hardness
Materials and methods tester (Erweka hardness tester, Heusentamm, Germany) at
ambient temperature. The weight required for suppository to
Materials
collapse was taken as measure of hardness of the suppository.
Sildenafil was purchased from Dongwoo Syntech Co., Ltd., Hardness test or fracture point test was carried to determine
(Seoul, Korea). Witepsol H-15 Pastillen was purchased from the tensile strength of the suppositories to access whether
Cremer Oleo GmBH & Co., (Hamburg, Germany). Telmisartan they will be able to withstand the hazards of packing and
was purchased from Alembic Ltd., (Vadodara, India). transporting. The measurement was performed in triplicates.
Triethylamine, phosphoric acid, ammonium acetate, and formic
acid were purchased from Sigma Chemicals Co. Ltd., (St. Louis, Disintegration time
MO). Bovine serum albumin (BSA) was purchased from Lonza
For personal use only.

(Walkersville, MD). Methanol, acetonitrile and all other chem- The disintegration time of SVS was determined using tablet
icals were of HPLC grade and used without further purification. disintegrator by the method suggested by Kaewnopparat et al.23
Briefly, the suppository to be tested was placed in a cylindrical
Preparation of SVS glass container with perforated ends and immersed in 1 l of buffer
solution (citric acid/phosphate buffer pH 4.5) maintained at
The composition used for the preparation of the SVS is shown 37  0.5  C temperature. The cylindrical glass container was
in Table 1. Suppositories containing 25 mg of sildenafil were moved up and down in the buffer. The time for disintegration was
prepared by a cream melting technique using Witepsol H-15 as a the time taken for the suppository to completely melt in the
suppository base21. Primarily, an appropriate amount of Witepsol medium. The mean values of six different suppositories were
H-15 was melted in a water bath maintained at a temperature determined.
of 65  2  C and was mixed with 25 mg of sildenafil powder. The
mixture was then allowed to cool down to 55  C until it becomes
sticky. This was then heated again and poured into the suppository Differential scanning calorimetry
molds. The suppositories were then inserted into suppository The thermal property of Witepsol H-15, sildenafil, and supposi-
package and stored at 4  2  C until used. tory containing both was assessed by thermograms obtained using
differential scanning calorimetry (DSC Q200 v24.2 build 107,
Characterization of SVS TA Instruments, New Castle, DE). The samples of about 2.00 mg
Weight variation were placed in standard aluminum pans and dry nitrogen was used
as effluent gas. All samples were scanned at a temperature ramp
The weight variation test was determined according to the speed of 5  C/min and the heat flow was set from 25 to 60  C.
British Pharmacopeia22. Briefly, 20 suppositories were weighed Before the experiment, the DSC was calibrated using pure Indium
and heat of fusion (Hfusion).
Table 1. Composition of SVS and properties of suppository base,
Witepsol H15.
In vitro release of sildenafil from SVS
Ingredients Weight (mg)
The release of sildenafil from the SVS was determined using
Sildenafil API 25 USP XXIII, dissolution apparatus II with 900 ml of pH 4.5
Witepsol, grade H15 Suppository base 1102 phosphate buffer solution as dissolution medium to mimic the
Total 1127
Witepsol H15 properties
pH of the vaginal environment at physiologic temperature
Composition Glycerides, coco mono-, di- and tri-, of 37  0.5  C with paddle speed of 50 rpm. Vaginal suppository
hydrogenated containing 25 mg of sildenafil was introduced into the dissolution
Additives None medium using sinker. At predetermined time intervals, an aliquot
Function Lipophilic base of 5 ml was collected, filtered and analyzed for the content
Hydroxyl value 515 of sildenafil by HPLC. An equivalent volume (5 ml) of fresh
Melting point 34.5  1  C
dissolution medium was replaced to compensate the loss due to
Solidification point 33.5  1  C
sampling.
DOI: 10.3109/03639045.2013.788011 Sildenafil vaginal suppositories 3
HPLC analyses of sildenafil standard (1 mg/ml of telmisartan in 1:1 methanol:acetonitrile
mixture) was added and vortexed for 5 s. To this, 1 ml of
The sildenafil in the prepared samples of content uniformity study
dichloromethane was added, and vortexed again for 10 min
and release study was measured by Hitachi HPLC system
followed by centrifugation at 12 000  g for 3 min. The super-
equipped with L-2130 pump, L-2200 Autosampler and L-2400
natant organic layer was separated and evaporated under nitrogen
UVVIS detector at 290 nm was used. The column used was
gas at 40  C. The residue was then reconstituted with 100 ml
Inertsil ODS-3 (4.6  150 mm, GLScience Inc., Tokyo, Japan)
of mobile phase using vortex mixer for 30 s followed by
with a column temperature of 30  C. The mobile phase was
centrifugation at 12 000  g for 3 min, and 5 ml of this solution
prepared by adding 20.3 ml of triethylamine into 2.88 l of water,
was injected into HPLC for sildenafil measurement. The same
and the pH was adjusted to 3.0 using ortho-phosphoric acid.
procedure was used for the extraction of sildenafil from the organ
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To this, 1250 ml of methanol and 850 ml of acetonitrile was


samples except for 100 ml of initial homogenized organ sample
added. The mobile phase was then filtered using 0.45 mm filter and
and 200 ml of mobile phase used for reconstitution.
sonicated to remove any dissolved gas before use. The injection
volume of the sample was 20 ml and flow rate was 1.0 ml/min.
LC-MS/MS method for analyses of sildenafil in plasma
Linearity of sildenafil was investigated before analyses and
and organ samples
the calibration curves showed excellent linearity over the range
of 150 mg/ml with satisfactory coefficients of determination LC analysis was performed using a Waters Alliance 2795 HPLC
with R240.9997. The method was precise and accurate with system (Waters Corp., Milford, MA). Compounds were separated
coefficient of variations of less than 10%. on an Atlantis dC18 Column (2.1  50 mm, 3 mm, Waters Corp.,
Milford, MA). The isocratic mobile phase was mixture of
Plasma PK and organ BD evaluation acetonitrile and 10 mM ammonium acetate solution (90:10 v/v)
containing 0.1% formic acid. The flow rate of the mobile phase
Animals. All animal treatment protocols were in accordance and the column oven temperature were set at 0.3 ml/min and
with National Institute of Health guidelines, Korea. Healthy, 30  C, respectively. The injection volume of the sample was 5 ml.
female Sprague Dawley (SD) rats (46 weeks old, about 250 g) The LC system was coupled to an API 4000 Qtrap mass
were supplied by Samtacho (Kyeonggi, Korea) and quarantined spectrometer equipped with turbo ion spray ionization source (AB
for one week prior to use. Animals were maintained on sawdust MDS Sciex, Toronto, Canada). The turbo ion spray ionization
bedding free of any known chemical contaminants in a 12 h source was operated in a positive mode. The curtain gas, nebulizer
photoperiod (light on at 08:00 and off at 20:00) in our animal gas and the turbo gas (nitrogen) pressures were set at 30, 40 and
facility at 23  2  C and 5080% relative humidity. The animals 40 psi, respectively. The turbo gas temperature was set at 350  C,
For personal use only.

were provided with Purina Certified Rodent Chow No. 5002 meal and the ion spray needle voltage was adjusted to 5500 V. The mass
(Ralston Purina, St. Louis., MO) and had free access to water. spectrometer was operated at a unit resolution for both Q1 and Q3
in the multiple reaction monitoring (MRM) mode with a dwell time
Plasma PK study. For the plasma PK study, female SD rats were of 300 ms in each transition. The transition of the precursors to the
randomly divided into two groups (n 6). The femoral vein was product ion was monitored at 474.9 ! 100.2 for sildenafil and
cannulated with 23-gauge polyethylene cannula under anesthesia 515.0! 276.3 for telmisartan (IS). The collision energy was set at
with diethyl ether. Either oral solution or vaginal suppository medium. Data acquisition was preformed with the Analyst 1.4
was administered with a dose of 16.6 mg/kg of body weight of software (AB MSD Sciex, Toronto, Canada). Calibration curves
rats. The oral solution was intubated through mouth into the were prepared by spiking the blank plasma (200 ml) or tissue sample
stomach using lab animal syringe feeder, while the suppository (100 ml) with standard solution at various concentration ranges.
was carefully inserted into the vagina without damaging the
surrounding tissue. About 0.5 ml of blood samples were collected PK parameter and statistical data analyses
into heparinized tubes at 0, 0.25, 0.50, 1, 1.5, 2, 4, 6, 8 and 24 h
after administration. The collected blood samples were centri- The plasma concentration of sildenafil versus time profile was
fuged at 12 000  g for 3 min and the plasma was stored at 70 C analyzed by a non-compartmental method using WinNonlin
until analysis. Professional Edition, Version 2.1 for windows (Pharsight, Cary,
NC). The relative bioavailability % of sildenafil from vaginal
Organ BD study. For BD study, female SD rats were randomly suppository and oral solution was calculated using the following
divided into two groups containing 18 rats each. Three animals equation;
from each group were sacrificed at 0.5, 1, 2, 4, 8 and 24 h after AUCtest Dosereference
administration of either oral solution or vaginal suppository at a Relative bioavailability %   100
AUCreference Dosetest
dose of 16.6 mg/kg of body weight of rats. The organs such
as liver, lung, kidney and uterus were collected immediately, and where AUC is the area under plasma drug concentration curve
cleansed with saline solution for few times to remove blood and from time zero to the last sampling time. The PK parameters were
adhered drug and/or suppository. The weights of tissues were analyzed for statistical significance by unpaired Students t-test
measured after removing of lipid or any extra tissues. A 4% BSA with significance level of p50.05.
solution was added to the tissues, and homogenized by Ultra-
Turrax (Janke and Kunkel, IKA Labortechnik, Staufen, Results and discussion
Germany) homogenizer at 20 000 rpm under ice bath. The diluted
homogenates were transferred into respective tubes and stored in a The placenta plays a central role in fetal programming by directly
deep-freezer (70  C) until further analyses. regulating blood flow, transporter activity, fetal nutrient supply
and fetal growth24. Placental vascular development is a crucial
process required for adequate fetal development. It has been
Extraction of sildenafil from plasma and organ samples
suggested that therapeutic agents, which target placental blood
Prior to extraction, frozen plasma samples were thawed at flow and vascular development, ameliorate fetal growth restric-
ambient temperature, and an aliquot of 200 ml of plasma was tion25,26. Nitric oxide (NO), the main vasodilator in the placenta,
used for extraction of sildenafil. To the plasma, 50 ml of internal is involved in the regulation of feto-placental vascular reactivity,
4 S. Shanmugam et al. Drug Dev Ind Pharm, Early Online: 110

placental bed vascular resistance, trophoblast invasion and Table 2. Physical properties and characterization of prepared SVS.
apoptosis and platelet adhesion and aggregation in the intervillous
space25,27,28. NO is synthesized in the vasculature from L-arginine Properties Values
by the endothelial nitric oxide synthase isoforms. NO produced Physical properties
by the endothelium stimulates guanylate cyclase in vascular Shape Conical
smooth muscle, resulting in the production of cGMP and Color/Gloss White/Dull
thus vasodilation. Conversely, PDE is a family of isoenzymes Size 2.7  0.8 cm (L  W)
that hydrolyzes cyclic nucleotides, such as cGMP preventing Characterization
Weight variation (g) 1.127  0.020
NO production leading to reduced blood flow to the uterus29,30. Content uniformity (%) 98.25  2.50
Sildenafil is a PDE inhibitor that prevents the breakdown Hardness (kg) 2.5  0.08
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of cGMP and potentiates the effects of NO3134. However, oral Disintegration time (min) 9  1.0
use of sildenafil has been reported to produce various side effects
including hypotension, flushing, and headaches in patients, which Each suppository contains 25 mg of sildenafil.
is of unpleasant nature especially during pregnancy4,15,3537.
Besides, the clinical PK data suggest that a 100 mg oral dose
The physical properties and characterization of the prepared
of sildenafil citrate produces a plasma concentration in excess
SVS containing sildenafil were shown in Table 2. The prepared
of 100 nmol/l for 45 h after oral dosage38,39. This concentration
suppositories were conical shaped with a dimension of
is still 100-fold less than that required for nonspecific inhib-
2.7  0.8 cm (L  W). The mean weight of the prepared
ition of other PDE isoforms by sildenafil citrate (which is
suppositories was 1.127  0.020 g with a content uniformity of
suggested to be in the 110 mmol/l range) and the concentration
98.25  2.50%. Drug contents were all homogeneous for all
of sildenafil in uterus following oral administration would thus
suppositories according to the content uniformity test. The weight
be insufficient to produce the required vasodilatory effect in
variation study for all the suppositories were found to be within
uterus39,40. For these reasons, delivery of sildenafil targeting
the acceptable range of 55%, which indicated perfect calibration
uterus has been considered beneficial in terms of safety and
of mold. The hardness and disintegration time of the suppositories
efficacy. The vagina provides a promising site for local effect as
were 2.5  0.08 kg and 9  1.0 min, respectively. The disintegra-
well as systemic drug delivery because of its large surface area,
tion time of the SVS was performed to understand the time taken
rich blood supply, avoidance of the first-pass effect, relatively
by the suppositories to soften or disintegrate when placed in an
high permeability to many drugs and self-insertion4143.
immersion liquid. According to BP, the disintegration should
Numerous reports have reported the vaginal use of sildenafil
occur in not more than 60 min. The prepared SVS disintegrated in
as a novel and safe method for the treatment of RSA, IUGR
For personal use only.

less than 10 min, suggesting conformation of the prepared


and other pregnancy-related complications4,1315,37,44. Abbott
suppositories to the BP requirement for disintegration22.
et al., reported that the assisted pregnancy was successful
Besides, the drug content of the five suppositories did not deviate
in female rats with sildenafil dose in the range of 360 mg/kg35.
by more than 10% from the labeled amounts, which were found to
According to Sher and Fisch, IVG administration of sildenafil
be in accordance with BP requirements for content uniformity22.
at a dose of 25 mg q.i.d. produced successful IVF in female
patients13. So, we prepared and evaluated SVS containing 25 mg
of sildenafil in this study. Although myriad of studies reported Differential scanning calorimetry
the beneficial effects of IVG sildenafil, unfortunately there The DSC thermograms of Witepsol H-15, sildenafil, and
have been no reports on its PK/BD in vivo, especially no data the vaginal suppository formulation containing the both were
available on the amount of exposure of sildenafil in uterus shown in Figure 1. The thermogram of Witepsol H-15 showed
following IVG application. Even, earlier research by Degim et al., two endothermic peaks at 33.52 and 37.54  C, corresponding
failed to report on the in vivo PK of the prepared controlled to the melting point of the base49,50. The thermogram of
release sildenafil suppositories and focused only on the in vitro sildenafil showed no endothermic peak due to its amorphous
release studies20. Eventually, we have investigated the PK and BD nature. The sildenafil vaginal suppository showed endothermic
of sildenafil in vivo following IVG application of suppositories peak that is corresponding to the melting points of Witepsol H-15
containing 25 mg of sildenafil. The suppositories were prepared with no extra peaks, suggesting no crystalline change of the
and characterized before the in vitro release and in vivo PK and ingredients during the preparation process of the suppository
organ BD studies. formulation5153.

Preparation and characterization of SVS Release study of SVS


The composition of SVS was shown in Table 1. The prepared Sildenafil release from the prepared SVS was determined at pH
suppositories were well formed with a smooth white surface 4.5 to mimic the vaginal environment and the release profile of
without any fissures, cracks or contraction holes. Witepsol, sildenafil from the SVS is shown in Figure 254,55. The dissolution
hydrogenated cocoa mono-, di- and tri-glycerides, is one of the of sildenafil was a little slower up to 15 min with a release of
commonly used suppository bases because of its unaffected 55.6%. However, the release of sildenafil was more than 90% at
solidification point even at high temperatures, good resistance to 30 min, and was complete at 60 min. The initial phase drug
oxidation due to their high amount of saturated fatty acid content, release is limited by the available surface area of the suppository
and availability of various grades with different melting points45. and is believed to be controlled by the spreading or dissolution of
In our study, we chose Witepsol H-15 because it has the least the base. The second phase of the dissolution profile then
difference between its solidification point (33.5  1  C) and corresponds to the dissolution of the drug from the base23,45,48.
melting point (34.5  1  C), and thus sets quickly without the Witepsol H-15 has a melting point of 34.5  1  C and therefore
risk of sedimentation of the suspended drug molecules. Besides, melts rapidly and spreads on dissolution, producing a very rapid
it does not contain any additives that are commonly available dispersion process. The complete release of sildenafil from the
with other suppository bases4648. The composition and properties vaginal suppository is probably due to the very low partition of
of Witepsol H-15 were shown in Table 1. sildenafil in the lipophilic base23,45,48.
DOI: 10.3109/03639045.2013.788011 Sildenafil vaginal suppositories 5
Figure 1. DSC thermograms of (A) Witepsol
H-15, (B) suppository containing sildenafil
and Witepsol H-15 and (C) sildenafil.
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Figure 2. Release profile of sildenafil from 120


the IVG suppository formulations. All sam-
ples were performed in triplicates and the
For personal use only.

values are shown as mean  SD (n 3).


100

80
Sildenafil release %

60

40

20

0
0 15 30 45 60 75 90
Time (min)

The release kinetics of the prepared SVS containing 25 mg of in case of systems, which dissolute or erode over time60. The drug
sildenafil was also determined. Table 3 shows the release kinetic release data of sildenafil were fitted to these kinetic models
models, and their respective correlation coefficients, and other to explain the drug release kinetics and mechanism from the
related release kinetic parameters of sildenafil from the SVS. The suppository prepared. Criterion of selecting the most appropriate
zero order model describes the systems, where the drug release model was based on the best goodness of fit61,62. The values
is independent of its concentration56. The first order model of the release exponent (n) and correlation coefficient (r2) were
describes the release from systems, where release rate is calculated and presented in Table 3.
concentration dependent57. According to Higuchi model, the The suppository formulation did not seem to follow a zero
drug release from matrix is directly proportional to a square root order profile of drug release based on the lower correlation
of time and is based on the Fickian diffusion58. A more coefficient (r2) values obtained compared to the other three
comprehensive, but still very simple, semi-empirical equation to kinetic models examined. On the other hand, the r2 values
describe drug release mechanism from polymeric systems more obtained from examining the Higuchi and the KorsmeyerPeppas
precisely is the so-called KorsmeyerPeppas power law59. The models were found to be very close to each other with a value of
HixsonCrowell cube root law describes the release from the around 0.7947 and 0.7982, respectively. The r2 value for Hixson
systems, where it depends on the change in surface area and Crowell model was slightly higher than the other models
diameter of the particles or tablets with time and mainly applies (0.9534), showing a better conformance to this model (Table 3).
6 S. Shanmugam et al. Drug Dev Ind Pharm, Early Online: 110

Applicability of the release curves to HixsonCrowell model IVG administration of 16.6 mg/kg of oral solution and SVS,
indicated a change in surface area and diameter of the respectively, was shown in Figure 3. The PK parameters of
suppositories, with a progressive dissolution of the suppository sildenafil after PO and IVG administration of 16.6 mg/kg of
base as a function of time61,62. The value of release exponent (n) sildenafil were shown in Table 4. The administration of both
determined for SVS was 0.464 and confirmed that the formula- formulations through PO as well as IVG was well tolerated by the
tions followed non-Fickian diffusion kinetics (anomalous trans- rats.
port), i.e. the release is ruled by both diffusion of the drug and The AUC024 h and Cmax of sildenafil in plasma was
dissolution of the suppository base, Witepsol H-1559,60,63. 11 907.76  8499.20 ng h/ml and 1684.86  589.40 ng/ml, and
1497.77  745.06 ng h/ml and 155.55  98.84 ng/ml, following
Plasma PK study oral and IVG administration, respectively. The AUC024 h (about
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eight times) and Cmax (about 11 times) was significantly higher


To assess the PK behavior of sildenafil from the SVS, the plasma
(p50.05) following the oral administration compared to IVG.
profile of sildenafil was obtained after IVG administration of
Earlier PK studies of sildenafil demonstrated similarities between
suppository in female SD rats. The PK profile of sildenafil
the rat and human in metabolite formation in vivo64. Both species
following oral administration in female SD rats was also assessed
produce five principal metabolites and form UK-103,320 as the
for comparison purpose. The sildenafil dose for both administra-
primary circulating metabolite. Another in vitro study using
tions was fixed at 16.6 mg/kg of body weight of animals. The
human liver microsomes demonstrates that 79% of sildenafil
mean plasma concentration-time profile of sildenafil after PO and
biotransformation to UK-103,320 is attributable to CYP3A65.
A small percentage of metabolite formation is due to CYP2C9
Table 3. Release kinetic models, correlation coefficients (r2 value) and (20%), CYP2D6 and CYP2C19 (collectively less than 2%)
kinetic parameters of sildenafil from the SVS.
activity. These data are consistent with the findings of Hyland
et al.66. However, plasma clearance in the male rat is eight times
Model Equation R2 value
greater than that observed in male volunteers. Plasma clearance
Zero order F k0 t 0.3171
First order F 1001  ek1 t 0.9217
Higuchi F kH t0:5 0.7947 Table 4. Pharmacokinetic parameters of sildenafil following oral solution
KorsmeyerPeppas F kKP tn 0.7982 and IVG suppository application in female SD rats.
HixsonCrowell F 100[1  (1  kHCt)3] 0.9534
Release kinetic parameters Ratio
Release extent at 90 min (%) 100.60  5.90 PK parameters PO IVG IVG/PO
Release exponent (n) 0.464
For personal use only.

Release kinetic model HixsonCrowell AUC024 h (ng  h/ml) 11 907.76  8499.20 1497.77  745.06* 0.13
Release mechanism Non-Fickian (Anomalous)* Cmax (ng/ml) 1684.86  589.40 155.55  98.84* 0.09
Tmax (h) 0.65  0.14 10.00  11.01 15.39
F is the fraction (%) of drug released in time t; k0 is the zero-order release t1/2 (h) 2.95  1.08 5.24  0.83* 1.78
constant; k1 is the first-order release constant; kH is the Higuchi release Vd (ml) 0.009  0.006 0.079  0.007* 8.95
constant; kKP is the release constant incorporating structural and z (h1) 0.26  0.09 0.13  0.02 0.50
geometric characteristics of the drug-dosage form; kHC is the release
constant in HixsonCrowell model. All data are expressed as mean value (n 6).
*For the case of cylindrical geometries such as suppositories, the release *Statistically significant (p value50.05) in comparison to control.
constant (n) obtained using KorsemeyerPeppas power law could be PO per oral.
categorized into, n 0.45 corresponds to a Fickian diffusion release AUC024h area under the concentrationtime curve from the time of
(Case I), 0.455n50.89 to a non-Fickian (Anomalous) transport, dosing (0 h) to last observation (24 h); Cmax maximum measured plasma
n 0.89 to a zero order (Case II) release kinetics and n40.89 to a super concentration; Tmax time of maximum plasma concentration. t1/2 half
Case II transport. life; Vd volume of distribution; z elimination rate constant.

Figure 3. Mean plasma concentration-time 2500


PO
profile of sildenafil following p.o. or IVG
IVG
administration of suppository formulations in
SD rats. Mean  SD (n 6).
2000
Plasma sildenafil level (ng/ml)

1500

1000

500

0
0 5 10 15 20
Time (hr)
DOI: 10.3109/03639045.2013.788011 Sildenafil vaginal suppositories 7
in the female rat is comparable to that found in the human and, (z) of sildenafil appears to be different following various routes
hence, is significantly lower than clearance in the male rat64. The of application, it was statistically insignificant (p40.05). The
differences in clearance between species may be attributable, relative bioavailability % of sildenafil from the IVG in compari-
in part, to differences in the affinities of the enzymes controlling son to oral was only about 12.57%. The low systemic exposure
the metabolic pathways. While UK-103,320 formation is of sildenafil from the suppositories is highly desirable considering
largely mediated by CYP3A in humans, it appears to be largely the intended use as IVG local application, as higher concentration
dependent on CYP2C11 in the male rat65. Also, a preliminary PK in plasma was reported to produce various side effects including
study performed by authors demonstrated a female to male hypotension, flushing and headaches in patients, which is of
ratio of Cmax and AUC024 h of sildenafil was about 23 and nine unpleasant nature4,15,3537.
times following 16.6 mg/kg of sildenafil PO, respectively (data Drugs are transported across the vaginal membrane by
Drug Development and Industrial Pharmacy Downloaded from informahealthcare.com by Gachon Univ of Medicine and Science (Gil Hospital) on 11/04/13

not shown). The higher AUC024 h and Cmax of plasma sildenafil the transcellular route, intracellular route, or vesicular and
following PO in female rats could be due to this significant gender receptor-mediated transport mechanisms54. The physiological
difference (metabolism-related slow clearance rate). factors (e.g. cyclic changes in the thickness and porosity of the
The Tmax was 0.65  0.14 h for oral administration, which was epithelium, volume, viscosity and pH of the vaginal fluid) and
about 15 times shorter than the Tmax of 10.00  11.01 for IVG physicochemical properties of drugs (e.g. molecular weight,
administration. However, there were huge variations between lipophilicity and ionization) affect absorption across the vaginal
the Tmax values of sildenafil in rats following IVG administration. epithelium19,20,54,67. One or combination of these factors could
The concentration of sildenafil in plasma was 52.6 ng/ml even have contributed to the reduced absorption of sildenafil from the
after 24 h following IVG application of the suppositories, while vagina leading to a reduced plasma concentration. However,
it was less than 10 ng/ml for oral administration at the same considering the formulation of sildenafil suppositories is intended
time period. Besides, the mean plasma concentration of sildenafil for targeting uterus through IVG administration, these factors
between 0.5 and 24 h was 79.9  17.89 ng/ml following IVG would have least or no effects.
application of the suppositories suggesting sustained slow
absorption of sildenafil through the vaginal wall45. The t1/2 of
BD study
oral and IVG application were 2.95  1.08 h and 5.24  0.83 h,
respectively. This study was performed to investigate the sildenafil exposure
The volume of distribution (Vd) of sildenafil following oral in the organs in terms of AUC following IVG administration of
and IVG was 0.009  0.006 and 0.079  0.007 ml, respectively. suppositories to female rats. The mean concentration-time profile
The higher Vd of sildenafil following IVG suggests the higher of sildenafil in liver, lung, kidney and uterus after oral and IVG
concentration of sildenafil in extracellular fluids and/or tissues administration of 16.6 mg/kg of sildenafil is shown in Figure 4.
For personal use only.

rather than in plasma18. Although the elimination rate constant Sildenafil was found to be widely distributed into most organs and

(A) (B) 16000


10000
14000 PO
PO IVG
IVG
Sildenafil concentration (ng/ml)
Sildenafil concentration (ng/ml)

8000 12000

10000
6000
8000

4000 6000

4000
2000
2000

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (hr) Time (hr)

(C) (D) 250000


25000
PO
PO IVG
200000
Sildenafil concentration (ng/ml)

IVG
Sildenafil concentration (ng/ml)

20000

150000
15000

100000
10000

5000 50000

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (hr) Time (hr)

Figure 4. Mean concentration-time profile of sildenafil in organs (BD), (A) liver, (B) lung, (C) kidney and (D) uterus/vagina, following p.o. or IVG
administration of suppository formulations in SD rats. Mean  SD (n 3).
8 S. Shanmugam et al. Drug Dev Ind Pharm, Early Online: 110

Table 5. BD of sildenafil following oral and IVG application in female SD rats.

Organs PK parameters PO IVG Ratio IVG/PO


Liver AUC024h (ng  h/ml) 5528.84  1290.00 2742.21  1208.72 0.50
Cmax (ng/ml) 7032.39  1926.52 3833.62  1346.93 0.55
Lung AUC024h (ng  h/ml) 9749.07  1586.47 4398.00  378.93 0.45
Cmax (ng/ml) 10 002.42  3428.93 5494.78  618.05 0.55
Kidney AUC024h (ng  h/ml) 7133.92  1058.75 2252.58  675.44 0.32
Cmax (ng/ml) 9867.56  1694.76 3268.24  1654.41 0.33
Uterus AUC024h (ng  h/ml) 5470.85  470.36 106 684.87  33 954.46* 19.50
Cmax (ng/ml) 4490.13  1563.48 177 718.87  35 537.57* 39.58
Drug Development and Industrial Pharmacy Downloaded from informahealthcare.com by Gachon Univ of Medicine and Science (Gil Hospital) on 11/04/13

All data are expressed as mean value (n 3).


*Statistically significant (p value 50.05) in comparison to control.
PO per oral.
AUC024 h area under the concentration-time curve from the time of dosing (0 h) to last observation (24 h); Cmax maximum
measured plasma concentration.

the AUC and Cmax of sildenafil in different organs could be seen complex25,43,73. The tubal corner must receive its blood supply
in Table 5. Sildenafil is a weakly basic compound (pKa 6.5) from a different source, probably the ovarian artery, since the
which is therefore only partially ionized at physiological pH. The arterial blood was not cooled41,43,7173. The findings of this study
compound is moderately lipophilic (log D7.4 2.7) resulting in indicated that sildenafil was highly available in uterus, which is
good solubility68. While these desirable physicochemical param- the target site of its action following IVG delivery of suppositories
eters confer the excellent absorption properties, the moderate containing 25 mg of sildenafil. The increased exposure of
lipophilic and weakly basic nature result in extensive tissue sildenafil in the uterus coupled with decreased exposure in the
distribution69. Highest concentration of sildenafil was found in plasma would be a classical approach for local targeted delivery,
lungs following PO administration, while it was highest for the and sildenafil vaginal suppository could thus be a promising
uterus following IVG administration of SVS in female SD rats. approach.
As expected the sildenafil exposure was significantly higher
in uterus following IVG administration of SVS with AUC024 h Conclusion
and Cmax of 106 684.87  33 954.46 ng  h/ml and 177 718.87 
For personal use only.

35 537.57 ng  h/ml, respectively. This AUC024 h and Cmax of In summary, SVS containing 25 mg of sildenafil were prepared,
sildenafil in uterus from SVS was about 20 and 40 times characterized and investigated for its in vitro dissolution and
higher than the sildenafil from oral administration, respectively. in vivo PK and organ BD in female SD rats after IVG
The distribution pattern of sildenafil in organs in terms administration. The results demonstrated that the prepared vaginal
of AUC024 h following oral administration was in the order sildenafil suppositories containing Witepsol H-15 suppository
of lung4kidney4liver4uterus, while it was in the order of base followed HixsonCrowell model of dissolution kinetics,
uterus44lung4liver4kidney for SVS following IVG adminis- exhibited non-Fickian diffusion release mechanism in dissolution
tration. These data demonstrated that a first uterine pass effect medium mimicking vaginal pH of 4.5. The in vivo PK study
occurs when sildenafil is delivered vaginally, thereby providing an performed in female SD rats showed significantly lower Cmax and
explanation for the unexpectedly high uterine concentrations AUC024 h of sildenafil in plasma following IVG administration of
relative to the low plasma concentration observed after vaginal suppositories compared to PO administration of sildenafil solu-
administration19,41,43,70. tion. Nevertheless, the in vivo BD study performed in female SD
The vaginal route has been reported to have the potential for rats demonstrated a phenomenally higher Cmax and AUC024 h of
the uterine targeting of active agents such as progesterone and sildenafil in uterus following IVG administration of suppositories
danazol16,71. The plasma concentrations of vaginally administered than oral administration of sildenafil solution. Our study results
progesterone were found to be higher in the uterine artery than in suggested the possible involvement of first uterine pass effect of
the radial artery, indicating a preferential distribution of proges- sildenafil following vaginal administration of SVS. Thus, SVS
terone to the uterus16,71. This confirmed the existence of direct could be utilized to enhance the effectiveness of sildenafil by
local transport from the vagina to the uterus, termed the first delivering it preferentially to the uterus while minimizing
uterine pass effect or direct preferential vagina-to-uterus systemic levels and possible side effects.
transport19,42,43,70,71. Our study results with SVS comply with
these earlier studies and suggested the possible involvement of Declaration of interest
first uterine pass effect of sildenafil, higher than expected uterine The authors report no declarations of interest.
tissue concentrations of sildenafil in uterus, after vaginal admin-
istration of suppositories. References
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