pharmaceutical and medical aspects of hyaluronic acid...
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2013
http://informahealthcare.com/drtISSN: 1061-186X (print), 1029-2330 (electronic)
J Drug Target, 2013; 21(6): 551–563! 2013 Informa UK Ltd. DOI: 10.3109/1061186X.2013.776054
ORIGINAL ARTICLE
Pharmaceutical and medical aspects of hyaluronic acid–ketorolaccombination therapy in osteoarthritis treatment: radiographic imagingand bone mineral density
Alia A. Badawi1, Hanan M. El-Laithy1, Demiana I. Nesseem2, and Shereen S. El-Husseney2
1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt and 2National Organization of Drug
Control and Research, Cairo, Egypt
Abstract
The objective of this study was to formulate novel painless combined hyaluronic acid (HA)–ketorolac (KT) membrane for the management of osteoarthritis with rapid analgesic onset, thusavoiding HA frequent invasive intra-articular injections and KT gastrointestinal complaintsassociated with all non-steroidal anti-inflammatory drugs. HA was chemically crosslinked withcarbodiimide/glutaraldehyde to yield membrane of low water content. Different in vitro aspects(mechanical properties, water content and in vitro release) were studied leading to anoptimized soft, flexible K8 HA membrane containing 30 mg KT that achieved the desiredbalance of excellent elasticity and low water content. Moreover, a successful retardation of KTrelease rate was achieved (82%) after 48 h with favored initial fast drug release in the first hour(32.7%) to attain rapid analgesic effect. The clinical assessments in arthritic rats revealedapparent improvement in joint space narrowing, highest increase in bone mineral density atthe proximal tibia and distal femur joints with the absence of osteophytosis only in animalgroup treated with combined HA–KT membrane. Application of K8 membrane was able topreserve KT plasma concentration above its minimum effective concentration for 48 htherefore, would able to replace six commercial tablets each of 10 mg KT.
Keywords
Bone mineral density, Freund’s completeadjuvant, hyaluronic acid, ketorolactromethamine, osteoarthritis, radiography
History
Received 30 November 2012Revised 1 February 2013Accepted 9 February 2013Published online 22 May 2013
Introduction
Osteoarthritis (OA) is a common, progressive joint disease
characterized by destruction of articular cartilage, which may
affect several joints especially weight-bearing joints such
as knee [1] leading to chronic pain and functional restrictions
[2]. The disease process of OA is characterized by progressive
erosion of articular cartilage, leading to joint space nar-
rowing, subchondral sclerosis, synovial inflammation and
marginal osteophyte formation [3]. The primary goal of
therapeutic management of OA is pain relief and prevention
of secondary functional disability and joint damage [1].
Recently, increasing interest has been given to the use
of hyaluronic acid (HA) in the treatment of OA because
of its safety and efficacy [4–6]. HA is a high molecular
weight linear polysaccharide. It is an important component
of synovial fluid and extracellular matrix of articular cartil-
age, contributing to the elasticity and viscosity of syn-
ovial fluid [7]. HA could restore the rheological and
anti-inflammatory effects of synovial fluid, which are lost
in OA by scavenging prostaglandin, metalloproteinase and
other bioactive molecules, thus reducing the level of inflam-
matory mediators [8]. However, injected HA is cleared from
joints in less than a day [9] because of its degradation in vivo
by hyaluronidase (HAase), so it does not exert a long lasting
reaction [10]. A useful approach to solve this problem could
be through the preparation of chemically crosslinked HA
membrane that shows an increased resistance towards
degradation by HAase and so, increasing biological activity
resulting in an increased half-life of 1.5–9 d [11]. Chemical
modification of HA can produce more mechanically and
chemically robust material that still retains its biocompatibil-
ity and biodegradability [10]. This biocompatible material
crosslinks and gels in minutes and swells from a flexible dry
membrane to a flexible porous hydrogel in seconds [12].
Although HA appears to be effective in improving function
and pain caused by knee OA, it is a slow-acting symptom
modifying agent, lacking rapid analgesic effects. Therefore,
the clinical use of its marketed commonly used formulation
implies the necessity of frequent administration of intra-
articular (IA) HA injections for several times [13]. This
frequent invasive dosing together with common side-effects
to injectables HA that include bruising at the injection site,
redness, slight pain, swelling and bacterial infections resulted
in inconvenient poor patient compliance [14].
Address for correspondence: Hanan M. El Laithy, Department ofPharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, CairoUniversity, 11562, Cairo, Egypt. Tel.: +20 122 312 40 34. E-mail:[email protected]; [email protected]
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It was suggested that HA and corticosteroids might act
synergistically. Although corticosteroids have been widely
used, due to their powerful and rapid effects on OA pain,
adverse systemic effects such as hyperglycemia in diabetic
patients, secondary adrenal insufficiency and Cushing’s
syndrome have been reported [15]. Moreover, adverse local
effects such as articular infection, loss of elasticity and
cartilage breakdown have been also mentioned [16].
For these reasons, non-steroidal anti-inflammatory drugs
(NSAIDs) instead of corticosteroids could be considered for
rapid analgesic onset in knee OA. Ketorolac tromethamine
(KT) has been widely used as a powerful analgesic NSAID.
KT is a non-selective cyclooxygenase (COX) inhibitor having
several mechanisms of action including inhibition of prosta-
glandin synthesis, modulator effect on opioid receptors, and
nitric oxide synthesis [17]. Clinical studies have shown that
a single dose of KT is more effective than that of morphine,
pethidine and pentazocine in severe to moderate post
operative pain [18] and has been found to be effective
in the treatment of trauma-related pain and pain associated
with cancer [19]. Thus, KT was chosen to be used rather than
corticosteroids in the present study.
Based on these considerations and to overcome all these
problems, the current work has aimed to improve HA therapy
in knee OA by developing novel combined controlled
membrane therapy containing both HA and KT. In this way,
easy, painless and continuous HA–KT delivery through
skin into the blood stream could be ensured thus producing
stable plasma concentrations over a long period avoiding
invasive frequent IA injections of HA and KT gastrointestinal
complaints associated with all NSAIDs such as bleeding,
perforation and peptic ulceration. Additional objective was
to examine the effects of the developed membranes on rat
adjuvant-induced arthritis using Freund’s complete adjuvant
(FCA), where combined treatment with HA and KT was
compared with HA treatment alone with the aid of radiog-
raphy, bone mineral density (BMD) and histopathology.
Materials and methods
Materials and animals
HA sodium salt from streptococcus equi sp.,
Polyvinylpyrrolidone (PVP) and ethyl cellulose (EC) were
purchased from Sigma-Aldrich (Steinheim, Germany).
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlor-
ide (EDC) was purchased from Fluka (Tokyo, Japan).
Glutaraldehyde (GA) and propylene glycol (PG) were obtained
from Loba Chemic (Mumbai, India). KT was a gift from
Arab Drug Company (Cairo, Egypt). Polyvinyl alcohol (PVA)
was purchased from Fluka Chemika (Buchs, Switzerland).
Octan-1-ol (n-octyl alcohol) (Synchemicals, Leicestershire;
England). Propylparaben (propyl-p-hydroxybenzoate) was
from Sigma Chemical (Dorset, UK), FCA (DIFCO
Laboratories, Detroit, MI). Formic acid (98–100%) was
obtained from Merck (Darmstadt, Germany). Acetone,
hydrochloric acid and ethanol were of analytical grade.
Male healthy white Albino rats of Sprague-Dewily strains
weighing between 180 and 220 g were obtained from Cairo
University Animal House, Cairo, Egypt. All studies per-
formed in this work were approved by the research ethics
committee for experimental and clinical studies at the Faculty
of Pharmacy, Cairo University, Egypt and the protocol was
compliant with the ‘‘Principles of Laboratory Animal Care
[NIH Publication # 85–23, revised 1985].
Preparation of crosslinked HA membrane by solutioncasting method
Chemical crosslinking is the most effective modification
of HA in retarding its hydrolytic degradation. The main
functional groups responsible for HA crosslinking are the
hydroxyl group that was crosslinked via an ether linkage
when GA was used as crosslinker or carboxyl group that was
crosslinked via an ester linkage when EDC crosslinker was
used [20]. Crosslinked membrane was prepared by dissolving
HA powder in double distilled water at room temperature to
produce HA solution of one wt% concentration. The obtained
solution was treated with different concentrations of HCl
(0.001, 0.01 and 0.1 N) followed by addition of 80% v/v
acetone in water. Different concentrations of crosslinking
agents either EDC (1.5%; 2%; 2.5%; 3%; 3.5%; 4% and 5%) or
GA (150; 200; 250; 300; 350 and 400 mM) were then added
and crosslinking reaction was allowed to proceed by slow
stirring at room temperature for 24 h. The resultant solutions
(25 mL) were casted into clean, dry 5 cm diameter glass Petri
dishes lined with aluminum foil. The solutions were allowed
to dry at room temperature under reduced pressure to yield
crosslinked transparent membranes which then peeled off and
kept in desiccators until used.
FT-IR spectroscopy
FT-IR spectra between 4000 and 400 cm�1 of HA membranes
were recorded before and after subjecting to crosslinking
with EDC and GA using FT-IR 460-plus (Jasco-Hachioji,
Tokyo, Japan). To set the thin membranes in a spectrometer
cell, they were inserted between two silicon sheets with a hole
of 10 mm diameter.
Water content of crosslinked HA membranes
Several pieces of dried, crosslinked membranes of 1� 1 cm2
were immersed in phosphate buffered saline (PBS; pH 7.4) at
room temperature for 24 h. The duration of 24 h was sufficient
to attain the equilibrated swelling [21]. The membranes were
removed and placed between two pieces of filter papers to
wipe off excess solution. The swollen membranes were
weighed, followed by drying under reduced pressure until
constant weight at room temperature then reweighed again
to determine their water content percentage according to the
following equation [22,23]
Water content %ð Þ ¼ Ws�Wdð Þ=Ws½ � � 100,
where Ws and Wd are the weights of swollen and dried HA
membranes, respectively.
Moisture uptake of crosslinked HA membranes
Membranes strips of 1� 1 cm2 were weighed accurately (Ws)
and placed in a desiccator containing saturated solution of
potassium chloride (relative humidity 80–90%) at room
temperature. After 3 d, the membranes were taken out and
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weighed (Wm). Moisture uptake was calculated according to
the following equation [24].
Moisture uptake %ð Þ ¼ Wmð Þ � WSð Þ � 100
WSð Þ
Membrane thickness and mechanical properties
Thickness of the prepared membranes was measured at three
different points using Hans Schmidt micrometer (Bayern,
Germany) and the mean values were calculated. The mech-
anical properties reflected by tensile strength (TS) and
elongation at break provide an indication of membranes
strength and elasticity respectively. It was suggested that
suitable membranes for transdermal or topical applications
should be stress resistant, flexible and elastic [25,26]. H1-KS
testing machine (Tinius Olsen, England, UK) was used to
measure the mechanical properties of crosslinked HA mem-
branes. Membranes strips of 1� 5 cm2 and free from air
bubbles or physical imperfections were held between two
clamps positioned at a distance of 5 cm. During measurement,
the strips were pulled by the top clamp at a crosshead speed of
5 mm/min. The force and elongation were measured when the
membranes were broken. Measurements were run in triplicate
for each membrane. The TS and percentage elongation (%EL)
were calculated as follows [27].
TS ¼ Breaking force Nð Þ=initial
cross sectional area of sample mm2� �
Elongation % ¼ Increase in length at breaking point
mmð Þ=original length mmð Þ � 100
Preparation of crosslinked HA–KT membranes
Medicated HA membranes containing KT were prepared by
solution casting method using PVP as a release modifier
to enhance KT permeation across epidermis [28,29], EC, a
water insoluble polymer to maintain long-term KT analgesic
effect [30] and PG as a penetration enhancer [31]. Various
ratios of PVP:EC:PG were prepared and evaluated. HA
aqueous solution of 1 wt% containing optimum concentration
of crosslinking agent was prepared as before. Appropriate
amount of KT (250 mg) was dissolved in chloroform followed
by addition of PVP and EC in requisite ratios with constant
stirring. PG was then added to the organic phase which further
added to the aqueous HA phase. The mixture was stirred at
room temperature using magnetic stirrer (MSH 420, BOECO,
Hamburg, Germany) till a uniform dispersion was produced.
The membrane was obtained by casting the resultant disper-
sion into clean, dry 5 cm diameter glass Petri dishes lined with
aluminum foil containing 4% w/v PVA backing layer casted
earlier and dried at 40 �C for 6 h. The medicated membranes
were allowed to dry at room temperature under reduced
pressure, then removed and kept in desiccators until used. The
composition of different formulations is shown in Table 1.
In vitro release study
The release of KT from different prepared HA membranes
was carried out using USP dissolution tester (Hanson
SR6, Chatsworth, CA). The paddle over disk method was
performed according to USP 29 apparatus 5. 1000 mL of PBS
(pH 7.4) was used as dissolution medium at 32� 0.5 �Cand the stirring shafts were rotated at a speed of 50 rpm.
Aliquots of 5 mL were withdrawn at predetermined time
intervals (0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 18, 24, 30, 36 and
48 h), suitably diluted and analyzed spectrophotometrically
(Shimadzu UV-1605 PC, Kyoto, Japan) at 323 nm against the
sample withdrawn at respective time interval from non-
medicated KT-free membrane treated in a similar manner.
Every withdrawal was immediately replaced with fresh media
to maintain constant volume and the dilution was taken into
account in the calculation of the amount of KT released
from the membrane. The method was validated, the accuracy,
repeatability (intraday) and intermediate precision (interday)
and reliability were ensured. The recovery% was498%. The
experiment was run in triplicate for each formula and the
mean drug percent was calculated and plotted versus time for
different formulae. The obtained release data were subjected
to kinetic treatment according to zero, first and Higuchi
diffusion models [32]. The correlation coefficient (r), the
order of release pattern and t50% value were determined in
each case. The release data were further analyzed according
to Korsmeyer–Peppas model using the following exponential
equation that is often used to describe the drug release
behavior from polymeric matrices [33].
Log Q ¼ Log K þ n Log t,
where Q is the fraction of drug released at time t, K is the
release constant characteristic for the drug polymer
Table 1. Composition and physical properties of medicated HA membrane containing KT tromethamine.
Permeation enhancer
Formula HA Wt% Crosslinker PVP:EC PG %wt/wt %Water content %Moisture uptake TS (g/mm2) EL%
K1 1% 350 mM GA – – 28.14� 0.94 14.18� 1.24 16.73� 1.62 26.79� 0.81K2 1% 350 mM GA 1:1 5% 32.59� 1.12 18.18� 1.19 18.54� 1.02 27.66� 0.99K3 1% 350 mM GA 1:1 10% 36.93� 1.09 19.26� 1.61 17.93� 1.80 29.26� 1.19K4 1% 3.5% EDC 1:1 5% 39.45� 0.78 13.11� 0.87 12.17� 0.93 19.26� 1.45K5 1% 3.5% EDC 1:1 10% 44.12� 1.33 17.12� 1.07 11.85� 1.20 22.10� 1.48K6 1% 3.5% EDC 1:3 10% 36.12� 1.78 13.46� 2.65 9.54� 0.11 13.79� 1.77K7 1% 3.5% EDC 1:5 10% 35.59� 1.13 11.26� 2.77 6.01� 0.11 6.66� 0.99K8 1% 3.5% EDC 3:1 10% 49.23� 1.49 24.06� 1.58 20.31� 1.20 27.06� 0.99K9 1% 350 mM GA 5:1 10% 54.54� 2.24 26.15� 1.33 22.23� 0.43 33.79� 1.77
DOI: 10.3109/1061186X.2013.776054 HA–KT combination therapy in osteoarthritis treatment 553
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interaction and n is an empirical parameter (diffusion
exponent) characterizing the release mechanism. The value
of n gives an indication about the release mechanism: when
n¼ 1, the release rate is independent on time (zero-order).
When n� 0.5, this indicates case I or simple Fickian diffusion
(Higuchi model). If n value falls between 0.5 and 1, this
indicates non-Fickian or anomalous release. Lastly, when
n41, indicating case II transport apparent [34,35].
Clinical animal investigations
OA induction and measurement of knee joint diameter
(Gross evaluation)
Arthritis could be induced experimentally by administration of
several reagents like FCA [36–38] Type II collagen and
streptococcal wall [39]. FCA is the most common model used
in rats for the evaluation of anti-arthritic drugs [40,41]. Studies
were carried out using 30 male albino rats randomly divided
into five groups each containing six animals. Group I was kept
as a control group and received 0.1 mL injection of normal
saline. Arthritis was induced in rats of the remaining four
groups by injecting single dose of 0.1 mL of FCA containing
heat killed Mycobacterium tuberculosis suspended in heavy
paraffin oil into the paw of the right hind limb of each rat [42].
Treatment was started on the 21st day of arthritis induction by
topical membranes application on the back of the rats after
removal of hair with hair clipper. Treatments were continued
for 28 d where, group III was treated with plain HA membrane
(KT free) and animals of group IV, and V were treated with K8
and K9 respectively. Animals of group II remained untreated
and served as diseased control. During the 28 d of treatment,
the knee volume of the animals was recorded at regular
intervals (every 3 d) by measuring the diameters of both left
(control) and right (induced) knee joints using a digital vernier
caliper (Demm, Nolan, Italy). The mean changes in the volume
of injected knee edema with respect to non-injected knee were
calculated and the percent edema inhibition produced by each
membrane-treated group was calculated using the following
formula [43].
%EI ¼ %Edema diseased controlð Þ �%Edema treatedð Þ � 100
%Edema diseased controlð Þ
Radiography
Before OA induction, knee joints of randomly selected rats
at antro-posterior and lateral projections were radiographed
under anesthesia using Shimadzu X-ray apparatus (Shimadzu
Corporation, Kyoto, Japan) set at 40 KV and 0.5 mA/s and
developed with an industrial X-ray film (Fuji photo film,
Tokyo, Japan) to rule out any abnormality. The film to source
distance was 60 cm with an exposure time of 0.5 s for anterior
posterior projection. At 20th day post FCA injection, and
after 28 d post treatment with HA membranes, joints were
re-X-rayed to monitor joint space impairment, changes in
bone morphology and response to treatment.
Bone mineral density
BMD is an estimate of bone strength measuring the amount
of mineral per square centimeter of bones (g/cm2). BMD was
used in clinical medicine as an indirect indicator of OA using
dual-energy X-ray absorptiometry (DEXA) [44]. Rats from
each group were anesthetized using diethyl ether and placed
lying flat on their back with the ankle in neutral position and
the knee was extended. BMD was determined for each rat
using Norland XR-46 bone densitometer (Norland Corp. Fort
Atkinson, WI) with a scan speed of 60 mm/s at distal end of
right femur and proximal right tibia. DEXA measurements
were performed on zero day (control, group I), 20 d after
induction to ensure OA occurrence (group II) and on day
49 after 28 d post treatment with optimized prepared mem-
branes to evaluate the effectiveness of different formulations
on BMD. The percent change in BMD was determined by
inserting the values (V) collected for each time point into the
calculation [45]:
% changein BMD
¼ V post-treatment� V pre-treatmentð Þ � 100
V post-treatment:
Histopathological examination
Rats were sacrificed by cervical dislocation after 28 d of
treatment. Their whole knees were dissected free from the
surrounding soft tissues and fixed in 5% neutral buffered
formalin for at least 3 days then decalcified with 10% formic
acid for 7 d and dehydrated through descending series of
ethanol [46]. The specimens were then embedded in paraffin
blocks and sections of 6 mm thickness from femoro-tibial
joints were cut and stained with hematoxylin and eosin for
histopathological examination using light microscope with
digital camera (Olympus microscope CX31, Tokyo, Japan).
Changes of OA occurred in the articular cartilage and
subchondral bones were evaluated.
In vivo absorption study
Study design
Based on previous experimentations, one HA–KT membrane
formulation (K8) was chosen to be evaluated in vivo. The
in vivo study was carried out to determine the pharmacokin-
etics of KT from K8 membrane containing 30 mg KT. The
study was done using six white Albino rats of Sprague-Dewily
strains weighing between 180 and 220 g. The animals were
housed for 7 d under constant environmental and nutritional
conditions according to ‘‘the principles of laboratory animals
care’’, (NIH publication 85–23, revised 1985). Before the
commencement of the experiment, the skin of every animal
was thoroughly examined for any abnormality and only those
having no structural abnormality of the skin were selected for
the study. The hair of the back areas (2� 2 cm2) was removed
with the help of electric hair clipper 1 d before starting of the
experiment and care was taken to avoid any damage of the
skin during shaving. A membrane piece of 2� 2 cm2
containing 30 mg KT was applied topically to the back of
each rat. Blood samples were withdrawn from eye vein at time
intervals of 0 (predose); 1; 2; 4; 8; 24; 28; 32 and 48 h
following drug application. All samples were collected in
heparinized glass centrifuge tubes to prevent coagulation of
blood, and immediately centrifuged at 3000 rpm for 10 min
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(Centurion SCI, West Sussex, UK). The clear plasma were
collected in capped tubes and deep frozen at �20 �C until
analysis using HPLC method.
Chromatographic conditions
The concentration of KT was determined using HPLC assay
with Agilent 1100 series HPLC system (San Diego, CA).
Thermo Hypersil BDS C18 reverse-phase column (5 mm
particle diameter, 4.6� 250 mm i.d., Thermo Hypersil-
Keystone, Bellefonte, CA) was used for separation and
quantification of KT. The mobile phase was composed of a
mixture of purified water:acetonitrile:dibutylamine phosphate
buffer (59:39:2, v/v/v) adjusted to pH 2.5� 0.05 using
phosphoric acid. The mobile phase was filtered through
0.45mm membrane filter, degassed by sonicator degasser for
15 min and delivered at flow rate of 1.8 mL/min. Effluents
were monitored using UV absorbance at 323 nm (Agilent
VWD G1314A, San Diego, CA).
Standard solutions
In a 10 mL glass centrifuge tube, 1 mL of blank plasma
sample was spiked with 1 mL of KT stock solution
(10mg/mL) to contain serial dilutions of 0.1, 0.25, 0.5, 0.75,
0.9 and 1.0 mg/mL. Propylparaben 0.1 mL of 10 mg/mL was
then added to each sample as an internal standard. Plasma
samples were then mixed with 1 mL acetonitrile, vortex-
mixed for 30 s and centrifuged for 10 min at 3000 rpm. The
upper layer was decanted into another clean centrifuge tubes,
evaporated to dryness using vacuum concentrator (Eppendorf
Concentrator 5301, Hamburg, Germany) at room temperature.
The residue was reconstituted with 250 mL of mobile phase,
and 20 mL of the resulting solution was injected into the
HPLC column. A plasma sample without the addition of KT
was also treated in the same manner. A standard calibration
curve was obtained by plotting the ratio of the peak area
of KT to that of internal standard versus KT concentration.
All the assays were done in triplicate and the intra precision
and accuracy of the method were determined after replicate
analysis (n¼ 3) of control samples spiked at three concen-
tration levels: 0.5, 0.75 and 1 mg/mL. The lower limit of
quantification was 0.10 mg/mL with a linear response
across the full range of concentrations from 0.1 to 1 mg/mL
(R2¼ 0.997). The analysis of quality control samples showed
acceptable precision of relative standard deviation below 10%
and accuracy below� 5% for intra-membrane analysis.
Pharmacokinetic analysis
Plasma level-time course following K8 treatment was plotted
and the pharmacokinetic parameters, peak concentration Cmax
(mg/mL) and the necessary time tmax (h) to attain Cmax, were
obtained. The apparent terminal elimination half-life t1/2 (h)
was calculated as t1/2¼ 0.693/k.
Statistical analysis
The data obtained from different formulae were compared
for statistical significance by one-way analysis of variance
adopting SPSS statistics program (version 16, SPSS Inc.,
Chicago, IL) followed by post hoc multiple comparisons
using the least square difference. Differences between series
were considered to be significant at p� 0.05.
Results and discussion
FT-IR of HA membranes
Since chemical crosslinking of HA using EDC or GA is
favored in acidic condition [20], different concentrations
of HCl were tried. HCl was used as a catalyst necessary for
acetalization between hydroxyl groups of HA and aldehyde
group of GA [21]. While upon using EDC crosslinker, HCl
allows ion exchange of carboxyl group of HA from COONa to
COOH, which then reacts with EDC to form an ester bond
after a series of reactions [20,47]. Figure 1(a) revealed ion
exchange effect on IR spectra of uncrosslinked HA upon
addition of different concentration of HCl. It was clear that,
the concentration of 0.01 N HCl led to sharp new peaks
at 1650 and 1740 cm�1 that correspond to the absorbance
of carbonyl group of carboxylic acid (COOH) with a
concomitant decrease in the absorbance of carboxylate salt
(COO�Naþ) at 1620 cm�1. Addition of 80% v/v acetone,
a water miscible nonsolvent for HA was substantiated by the
previous reports of the ability of acetone to prevent dissol-
ution of HA membranes into reaction aqueous solution,
prevent EDC activity loss in aqueous medium [47–49] and
allows higher diffusion of GA into HA membranes resulting
in maximum crosslinking [21]. To support crosslinking
mechanisms, FT-IR spectra of crosslinked HA membranes
was displayed in Figures 1(b) and (c). As seen in Figure 1(b),
the difference in the spectrum between virgin HA and EDC-
crosslinked membrane was noticeable at a wave number of
1714 cm�1, which was assigned to ester bond that function as
a crosslink of HA. In this way, EDC seemed to mediate acid
anhydride formation between two carboxyl groups belonging
to the same or different HA molecules. The resultant acid
anhydride might react with hydroxyl group at 3433 cm�1
(peak not shown) of HA molecule to yield an ester bond [48].
This confirms the previous suggestion that EDC was a zero-
length crosslinking agent as it did not chemically bind to HA
molecules during crosslinking [50]. On the other hand; no
new peaks were observed for crosslinked HA with GA in
Figure 1(c) except the peak at 1740 cm�1 for ion exchange.
This was in accordance with Tomihata & Ikada [21] who
reported a similarity in the chemical structure of acid treated
HA to that of acetal structure formed between hydroxyl
groups of HA and aldehyde group of GA during crosslinking.
Water content of crosslinked HA membranes
Since the non-crosslinked HA membranes were completely
dissolved within 2–3 h when immersed in PBS, the extent of
crosslinking was verified by the percentage of water content
of crosslinked membranes. The lower the water content,
the higher the extent of crosslinking. It was apparent from
Figure 2 that lowest water content of 36.7%� 1.67% and
36.1%� 1.29% are obtained for crosslinked HA membrane
when EDC and GA were used in a concentration of 3.5% and
350 mM, respectively. Interestingly, HA membranes prepared
with GA concentration below 250 mM shrinked and dissolved
within 24 h and upon increasing GA concentration, the
percentage of water content decreased. These results indicated
DOI: 10.3109/1061186X.2013.776054 HA–KT combination therapy in osteoarthritis treatment 555
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that GA5250 mM was not sufficient to ensure complete
crosslinking and by increasing its concentration, more
hydrophilic hydroxyl groups of HA were consumed in the
crosslinking reactions and membrane became less capable for
hydrogen bonding, thus leading to water content decrease
[51]. On the contrary, increasing EDC concentration to 3.5%
was accompanied by a significant decrease in the water
content (p50.05). Further EDC increase resulted in water
content% increase. This pattern was suggested to take
place due to the increased EDC steric hindrance resulting
in less crosslinking efficiency and decreased accessibility of
the EDC to HA chain [52].
Mechanical properties of crosslinked non-medicatedHA membrane
Desirable characteristics of suitable biomedical membranes
for topical or transdermal applications addressed by Ammar
et al. [53] relied not only on enough flexibility to follow the
movements of the skin without breaking but also increased
strength is required to survive handling and prevent mem-
brane abrasion and cracking during clothing contact. The
prepared HA membranes were transparent, smooth and
uniform with average thickness range of 0.13� 0.34–
0.18� 0.83 mm. Low standard deviation values of the mem-
brane thickness measurements ensured uniformity of the
prepared membranes. Experimental data in Table 2 revealed
that, the mechanical properties of all prepared crosslinked
membranes were higher than that of plain ones. Moreover,
direct proportionality did exist between the measured mech-
anical properties and concentration of crosslinking agent
used. It is rather important to mention that, the TS and EL%
scaled positively with increasing GA and EDC concentrations
up to 350 mM and 3.5%, respectively, beyond which the
elasticity decreased. This could be understood as the TS of a
polymer is closely correlated to the density of crosslinking
[54]. A possible explanation for the different behavior of EDC
and GA beyond their optimum concentrations on TS was
related to the difference in the involved mechanisms during
crosslinking reaction. Increasing GA concentration would
increase mechanical properties of HA membranes due to the
covalent inter and intramolecular crosslinking of HA [55].
However, high concentrations of EDC above 3.5% allowed
initial rapid crosslinking of HA surface thus slowed down
further diffusion of EDC and limited its efficacy [56–58].
Therefore, HA membranes crosslinked with 3.5% EDC or
GA crosslink
uncrosslink (acid form)
1640cm-1
1740cm-1
1650cm-1
1725cm-1virgin HA
EDC crosslink
1624cm-1
1714cm-1
0.1N HCl
0.01N HCl
0.001N HCl
1740cm-1
1650cm-1
1620cm-1
1700cm-1
1720cm-1 1670cm-1(a)
(b) (c)
Figure 1. FT-IR of HA (a) using different concentrations of HCl (0.001, 0.01, 0.1 N), (b) crosslinked with EDC, (c) crosslinked with GA.
556 A. A. Badawi et al. J Drug Target, 2013; 21(6): 551–563
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350 mM GA which achieved the desired balance of excellent
elasticity with high TS and low water content were chosen
for the preparation of medicated HA–KT membranes.
Physicochemical properties of medicated HA–KTmembranes
All the prepared medicated HA membranes loaded with
KT were found to be flexible, smooth and not adhesive in
their dry form, with average thickness of 0.145� 0.93–
0.19� 0.71 mm. Table 1 showed the results of medicated HA-
KT membranes prepared using different ratios of PVP:EC:PG
on water content and moisture uptake. It was clear that,
the incorporation of hydrophilic polymer PVP has led to an
increase in both water content and moisture uptake as seen in
K8 and K9 (PVP: EC, 3:1 and 5:1, respectively). Increasing
PG from 5% to 10% (K3 and K5) had the same effect as
PVP because of its humectant ability. On the other hand, the
least water content and moisture uptake were significantly
(p50.05) observed with increasing the concentration of
hydrophobic EC from 1:3 (K6) to 1:5 PVP: EC (K7)
compared to (K5). These parameters are considered important
as low water content and moisture uptake are required to
maintain the membrane stability, suppleness, reduce its
brittleness during storage and protect membrane from micro-
bial contamination [53]. Regarding the mechanical properties,
a soft and weak membrane is characterized by low TS and low
EL% as well. A hard and brittle membrane is characterized by
high TS and low EL%, while a suitable membrane for topical
application should be soft and flexible, i.e. of high TS and
high EL%. Therefore, on the basis of mechanical properties,
results in Table 1 could be explained as follows:
� Plain medicated HA–KT membrane (K1) was of low TS
(16.73� 1.62 g/mm2) and higher EL% (26.79%� 0.81%)
than non-medicated HA ones (18.69� 1.2, 21.52�1.80 g/mm2 and 16.97� 0.97%, 24.58� 1.09% for
EDC6 and GA6, respectively). This increased flexibility
might be attributed to the inclusion of KT which
impart plasticizing role as previously reported by
Alanazi et al. [59].
� Increasing concentration of PG from 5% in K2 and K4
to 10% in K3 and K5 resulted in higher membrane
flexibility and reduced brittleness. This might be
attributed to penetration of PG between polymer chains,
weakening their intermolecular binding and allowing the
polymer molecules to move more freely thus, increasing
membrane flexibility [59,60].
� Increasing hydrophobic EC concentration from 1:1 in K5
to 1:5 in K7 resulted in a decrease in the mechanical
properties. In contrast, increasing the concentration
of PVP from 1:1 in K3 to 5:1 in K9 improved the
mechanical properties and produced soft and elastic
membranes. This pattern was reported previously by
Jachowicz et al. [61] and was suggested to take place due
to the elastic and flexible nature of PVP polymer.
Therefore, based on the previous results, membrane
formulae K6, K7 (of low water content), K8 and K9
(of maximum TS and maximum EL%) were better fit the
requirements for an acceptable topical membrane
delivery.
In vitro release study
Results of KT in vitro release from different HA based
membranes are illustrated in Figure 3. It was apparent that
the incorporation of PVP and EC in K3-K9 modified the
drug liberation profile. Significant increases (p50.01) in the
percentage KT released were achieved compared to its
release modifier free membrane K1 where only 0.5% was
released after 48 h. The general features of the release
profile obtained for all prepared HA membranes exhibited
an initial fast release phase within the first hour followed by
a slow release one that was maintained till 48 h. The initial
quick drug release could be explained by the formation of
hydrophilic PVP layer that requires a very little lag time to
establish a concentration profile [43] while the drug release
in the slower phase was regulated by controlled diffusion
of entrapped drug through out pores and channels created
in the membrane as a result of hydrophilic PVP water
absorption and swelling [62–64] This profile could be
advantageous if we considered the importance of stratum
corneum saturation with initial fast drug released to attain
rapid therapeutic effect and to achieve high-concentration
gradient required for successful drug delivery to the blood,
then the drug release followed a well-defined kinetic
behavior to maintain the effect of drug for a longer time
[65]. This pattern was confirmed by proportional significant
increase in KT release with increasing PVP concentration
in K8 and K9 where 32.68%� 2.02%, 44.39%� 1.33% (after
1 h) and 81.69%� 4.25%, 88.26%�5.07 % (after 48 h) were
released, respectively, compared with 27.21%� 1.46%,
23.22%� 2.82% and 51.31%� 7.14%, 53.70%� 2.72%
from K5 and K3 having lower percentage of PVP after the
same time, respectively.
0
10
20
30
40
50
60
70
80
1.5 2 2.5 3 3.5 4 5
EDC concentration (%)
% W
ater
con
tent
0
10
20
30
40
50
60
0 250 300 350 400
GA concentration (mM)
% W
ater
con
tent
Figure 2. Water content of crosslinked HA membranes.
DOI: 10.3109/1061186X.2013.776054 HA–KT combination therapy in osteoarthritis treatment 557
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Figure 3. In vitro drug release of KT fromdifferent HA membranes in phosphate bufferpH 7.4 at 32� 0.5 �C (mean� SD, n¼ 3).
0
20
40
60
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100
0 4 8 12 16 20 24 28 32 36 40 44 48Time (h)
% K
T R
elea
sed
K1 K3 K5 K6 K7 K8 K9
Figure 4. Digital photomicrographs repre-senting right hind limbs of (a) control,(b) FCA induced, (c) treated with K8,(d) treated with plain HA.
Table 2. Mechanical parameters of crosslinked non-medicated HA membranes.
Formula code EDC conc. (%) TS (gm/mm2) EL% Formula code GA conc. (mM) TS (gm/mm2) EL%
EDC1 0 3.52� 1.32 5.21� 0.98 GA1 0 3.52� 1.32 5.21� 0.98EDC2 1.5 7.93� 0.4 9.89� 0.90 GA2 150 5.99� 0.19 9.69� 0.62EDC3 2.0 10.86� 1.1 12.79� 1.80 GA3 200 9.61� 0.85 14.76� 0.66EDC4 2.5 12.58� 1.5 13.23� 1.02 GA4 250 13.84� 0.98 16.94� 1.28EDC5 3.0 13.0� 2.5 15.22� 0.57 GA5 300 15.42� 1.22 21.18� 0.26EDC6 3.5 18.69� 1.2 16.97� 0.97 GA6 350 21.52� 1.80 24.58� 1.09EDC7 4.0 15.36� 1.3 11.68� 1.54 GA7 400 25.05� 0.48 21.94� 1.68EDC8 5.0 14.82� 0.59 8.9� 1.52
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It is important to mention the inverse relation between EC
concentration and KT release due to the hydrophobic nature
of EC. Thus, increasing its concentration will help to retain
the drug in the matrix system and slow down its diffusion by
reducing solvent penetration to the membrane [66]. This was
evident by the slow release pattern in K6 and K7 containing
high percentage of EC where only 45.54%� 3.73% and
38.36%� 6.19% KT were released after 48 h, respectively.
The kinetic analysis of the drug release data showed a linear
relationship between the amount of drug released from
different membranes and square root of time indicating
Higuchi diffusion model. Korsmeyer–Peppas equation
revealed values of n50.5 which assures Fickian diffusion
combined mechanism of KT release partially through a
swollen membrane and partially through water-filled pores
[34,67].
Therefore, based on the good mechanical properties and
higher KT released after 48 h, two optimized formulae K8 and
K9 were further progressed to clinical animal study.
Animal experiments
OA is a chronic inflammatory disease involving the release of
several mediators like cytokines and prostaglandin that induce
inflammation due to infiltration of the injured tissues by
immune cells [42]. FCA-induced arthritis in rats is commonly
Figure 6. X-ray radiograph of right knee joint showing radiographic changes in rats’ femur and tibia. (a) Control rat showing normal femoro-tibial jointspace with no marginal osteophytosis. (b) FCA-induced rat showing: (1) minute marginal osteophytosis, (2) femoro-tibial joint space narrowingat femoro-tibial articulation, (3) subchondral bone sclerosis. (c) Animal group treated with plain HA showing rather normalized joint space andno definite evidence of osteophytosis. (d) Animal group treated with K8 showing: (4) smoothness in femoral condyle, (5) restoration of joint space.(e) Animal group treated with K9 showing less improved subchondral cartilage than K8 with no definite evidence of osteophytosis.
Figure 7. (a) BMD level of femoral and tibial bone of different rat groups. (b) DEXA scan of rat knee (tibia and femur) after treatment with K8.
0
20
40
60
80
100
3 6 9 12 15 18 21 24 27
Time (day)
%E
dem
a in
hibi
tion
in r
atkn
ee
K8 K9 Plain
Figure 5. %Edema inhibition of rat knee joint.
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used to study the clinical and pathological changes character-
izing OA including tenderness, edema, joint swelling, cartil-
age destruction and erosion of the underlying bone due to
decreased concentration of endogenous HA, a major compo-
nent of synovial fluid [41,68,69]. Figure 4(b) reveals that,
inflammation signs including paw redness as well as
progressive swelling around the right knee joints and the
plantar paw region started to appear on the right hind paw
3 d after FCA injection in all induced groups. The anti-
inflammatory response was significantly higher and quick in
groups IV and V treated with KT containing membranes K8
and K9 compared to group III treated with KT free plain HA
membrane. After 27 d of treatment completion, the inflam-
mation signs were relatively disappeared in HA–KT mem-
brane treated animals (Figure 4c) while knee joint and paw
plantar side of animals treated with plain HA membranes,
although partially ameliorated, still showed evident swelling
(Figure 4d).
In order to evaluate the anti-inflammatory effect of KT
during OA treatment, the %edema reduction of right knee
joint swelling was measured. It was clear from Figure 5 that,
%edema reduction 12 d post treatment was 51% and 49% for
K8 and K9, respectively, compared to 29% reduction for plain
HA group. This significant reduction in joint swelling was
attributable mainly to addition of KT, a powerful non-
selective COX inhibitor, to HA membrane and its unique
advantage in inhibition of prostaglandin synthesis at sites of
inflammation thus, initiating rapid relief of pain associated
with this inflammatory stimulus [70]. Therefore, it should be
pointed out that, the developed KT-based HA membrane
combination was thought to be an effective treatment of
arthritis as it is intended to minimize the associated inflam-
mation, tenderness, swelling and to decelerate the progress
of arthritic symptoms as well. Previous reports [71–73]
suggested that, HA alone lacks rapid anti-inflammatory effect
and has weaker activity against edema in inflammatory
animal model while addition of KT might synergize HA
therapeutic effectiveness in OA by enhancing HA accumula-
tion in the joints and controlling inflammation [13].
� Although radiography in OA relies on destruction of
articular cartilage, yet, signs of cartilage damage in the
arthritic rats’ knee joints could not be unequivocally
Figure 8. (a) Histopathology photomicrograph of knee joint of control rat showing normal intact cartilage with normal synovium containing noinflammatory cells and normal chondrocytes (arrow). Hematoxylin and eosin, original magnification¼ 40X. (b) Histopathology photomicrograph ofknee joint of FCA-induced rat showing synovial membrane with mild edema and irregular surface (arrow). Hematoxylin and eosin, originalmagnification¼ 40X. (c) Histopathology photomicrograph of knee joint of FCA-induced rat showing subchondral bone with fragmented trabaculae(arrow) with predominance of bone marrow elements (arrowhead) and osteoids (double arrow). Hematoxylin and eosin, original magnification¼ 40X.(d). Histopathology photomicrograph of articular cartilage from femoro-tibial knee joint of plain HA group showing synovial membrane (arrow) withmild edema (double arrowhead), many intact chondrocytes (arrowhead). Hematoxylin and eosin, original magnification¼ 40X. (e) Histopathologyphotomicrograph of knee joint of K8 group showing femoro-tibial joint with intact chondrocytes (arrowhead), intact synovial membrane (arrow).Hematoxylin and eosin, original magnification¼ 40X. (f) Histopathology photomicrograph of knee joint of K8 group showing intact chondrocytesin subchondral cartilage (arrowhead), well-preserve bony trabeculae (arrow). Hematoxylin and eosin, original magnification¼ 40X.
560 A. A. Badawi et al. J Drug Target, 2013; 21(6): 551–563
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detected due to the small size of rat joints [74]. Only
early effects of OA pathogenesis showed mild cartilage
alterations detected by the presence of bone erosions,
femoro-tibial joint space narrowing and increased sub-
patellar opacity with minute marginal osteophytosis
(Figure 6b) relative to normal rats (Figure 6a). At the
end of the treatment period, X-rays of all treated animals
with and without KT were normalized to a great extent
with apparent improvement of joint space narrowing and
absence of osteophytosis evidence (Figure 6c and d).
These detected improvements supported and confirmed
the potent anti-arthritic effect of HA in the presence or
absence of KT. For better assessment of subtle differ-
ences between formulations, BMD was measured using
DEXA scan. Figure 7 revealed that the mean BMD at the
proximal tibia and distal femur of normal control rats was
0.12� 0.005 and 0.13� 0.003 g/cm2, respectively. After
20 d of FCA induction, a significant decrease (p50.05)
of 26.9% and 43.5% was observed (0.097� 0.017 and
0.091� 0.03 g/cm2). This decrease in BMD suggesting
a successful establishment of OA that was associated
with thinning and loss of bone trabeculae induced by
cytokines or chemical mediators released from inflamed
joints into the adjacent bone [75,76]. Although HA
reported to decrease bone turnover and increased BMD
by increasing osteoblasts formation, therefore regulating
bone mineralization contributing to the overall bone
restoration progression. Nevertheless, application of
KT-containing HA membranes K8, K9 demonstrated
highest increase in BMD of rat tibia and femur (19.3%
and 28.7%, respectively for K8 and 16.5% and 25.75%
for K9) compared to (11.9% and 21.2%) KT-free plain
HA membranes. This might confirm the role of KT in
enhancing accumulation of more HA in the joints [13].
� Histopathological studies showed that the animals of
group I was apparently normal with well-organized intact
cartilage and bone microstructure in femero-tibial joints.
No infiltration of inflammatory cell nor edema were seen
(Figure 8a). However, tissues of FCA-injected animals
(arthritic group II) showed many inflammatory signs
including infiltration of inflammatory cells around bone
and bone marrow along with many osteoclasts and
osteoids, disturbed cartilage integrity (irregular surface
and erosion), subchondral bone with fragmented trabe-
culae, moderate chondrocytes degeneration in the
femoro-tibial joint with focal area of complete destruc-
tion were also seen (Figure 8b and c). Comparing animals
of group III treated with KT free plain HA membrane
with group IV animals treated with KT containing HA
membrane (K8), it was shown that in both groups the
femoro-tibial joint, synovial membrane and subchondral
cartilage together with the trabeculae and bone marrow
were similar to normal largely. Moreover, Subchondral
chondrocytes and cartilage were almost intact and well
organized. These results are explained on the basis of the
presence of HA and its role in the inhibition of osteoclast
formation and induction of matrix synthesis. On the other
hand, group III still revealed slight edema in synovial
membrane (Figure 8d) that could not be seen in group IV
due to the presence of KT which decreased inflammation
and swelling in OA joints, therefore managing pain and
enhancing joint mobility (Figure 8e and f). Therefore,
these results are in good agreement with the results of
DEXA in which HA membrane containing KT was able
to produce better improvement in comparison with plain
HA membrane. Therefore, Formula K8 was selected for
further in vivo study because of its superior in vitro and
animal results.
In vivo absorption study
The mean plasma concentration–time curve following the
application of K8 membrane containing 30 mg KT on six
white rats was shown in Figure 9. The membrane delivered
large fraction of its KT content during the first two hrs of the
application period followed by a slower release phase that
extended for about 8 h. The average Cmax was calculated to be
4.73� 1.37 mg/mL with an elimination half-life (t1/2) of 22.3 h
after membrane application. Interestingly, as the minimum
effective concentration (MEC) of KT was reported to be
0.37 mg/mL [77] therefore, K8 membrane was able to
maintain effective therapeutic concentration for about 48 h.
Conclusion
The present study demonstrated a novel coupling between
HA and KT in knee OA therapy that have better therapeutic
Figure 9. Plasma concentration–time curvefollowing application of 30 mg KT fromKT–HA membrane (K8).
DOI: 10.3109/1061186X.2013.776054 HA–KT combination therapy in osteoarthritis treatment 561
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efficacy than conventional treatment with HA alone. The
coupling serves to protect joints from cartilage erosion
with apparent improvement of joint space narrowing, and
significant rapid analgesic onset due to KT powerful anti-
inflammatory role. The superior performance of the devel-
oped cross linked HA membrane containing 30 mg KT was
able to preserve KT plasma concentration over the MEC for
48 h and would be able to replace six commercial tablets
(three tablets per day) each of 10 mg KT.
Acknowledgements
The authors wish to acknowledge the superb efforts of
Dr Mohammed Shaker, Dr Rokia Elbanna, National Research
Center, biological anthropology department, as well as to
Dr Elias Makkar, an orthopedic surgeon for their valuable
support in performing radiographic and DEXA analysis.
Great appreciation to Dr Sahar Drwish, National Organization
of Drug Control and Research, histology department for her
technical assistance in histological analysis.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
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DOI: 10.3109/1061186X.2013.776054 HA–KT combination therapy in osteoarthritis treatment 563
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