Pharmaceutical and medical aspects of hyaluronic acid...

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


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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.


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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.


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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

80

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.


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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).


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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.

References

1. Ozturk C, Atamaz F, Hepguler S, et al. The safety and efficacyof intraarticular hyaluronan wit/without corticosteroid in kneeosteoarthritis: 1 year, single-blind, randomized study. RheumatolInt 2006;26:314–19.

2. Lorenz H, Richter W. Osteoarthritis: cellular and molecularchanges in degenerating cartilage. Prog Histochem Cytochem2006;40:135–63.

3. Sun SF, Chou YJ, Hsu CW, et al. Efficacy of intra-articularhyaluronic acid in patients with osteoarthritis of the ankle: aprospective study. Osteoarthritis Cartilage 2006;14:867–74.

4. Bannuru RR Natov NS, Obadan IE, et al. Therapeutic trajectory ofhyaluronic acid versus corticosteroids in the treatment of kneeosteoarthritis: a systematic review and meta-analysis. ArthritisRheum 2009;61:1704–11.

5. Kim SB, Kwon DR, Kwak H, et al. Additive effects of intra-articular injection of growth hormone and hyaluronic acid in rabbitmodel of collagenase-induced osteoarthritis. J Korean Med Sci2010;2:776–80.

6. Foti C, Cisari C, Carda S, et al. A prospective observational studyof the clinical efficacy and safety of intra-articular sodiumhyaluronate in synovial joints with osteoarthritis. Eur J PhysRehabil Med 2011;47:407–15.

7. Sun SF, Chou YJ, Hsu CW, Chen WL. Hyaluronic acid as atreatment for ankle osteoarthritis. Curr Rev Musculoskelet Med2009;2:78–82.

8. Hakshur K, Benhar I, Bar-Ziv Y, et al. The effect of hyaluronaninjections into human knees on the number of bone and cartilagewear particles captured by bio-ferrography. Acta Biomater 2011;7:848–57.

9. Juni P, Reichenbach S, Trelle S, et al. Efficacy and safety of intra-articular hylan or hyaluronic acids for osteoarthritis of the knee: aRandomized Controlled Trial. Arthritis Rheum 2007;56:3610–19.

10. Pitarresi G, Palumbo FS, Tripodo G, et al. Preparation andcharacterization of new hydrogels based on hyaluronic acid and a,b-polyaspartyl hydrazide. Eur Poly 2007;43:3953–62.

11. Brown MB, Jones SA. Hyaluronic acid: a unique topical vehicle forthe localized delivery of drugs to the skin. J Eur Acad DermatolVenereol 2005;19:308–18.

12. Luo Y, Kirker KR, Prestwich GD. Cross-linked hyaluronic acidhydrogel films: new biomaterials for drug delivery. J ControlRelease 2000;69:169–84.

13. Lee SC, Rha DW, Chang WH. Rapid analgesic onset ofintraarticular hyaluronic acid with ketorolac in osteoarthritisof the knee. J Back Musculoskelet Rehabil 2011;24:31–8.

14. Van Dyke S, Hays GP, Caglia AE, Caglia M. Severe acute localreactions to a hyaluronic acid-derived dermal filler. J Clin AesthetDermatol 2010;3:32–35.

15. Shapiro PS, Rohde RS, Froimson MI, et al. The effect oflocal corticosteroid or ketorolac exposure on histologic andbiomechanical properties of rabbit tendon and cartilage. Hand(NY) 2007;2:165–72.

16. Desai A, Ramankutty S, Board T, Raut V. Does intraarticularsteroid infiltration increase the rate of infection in subsequent totalknee replacements? Knee 2009;16:262–4.

17. Galan-Herrera JF, Poo JL, Maya-Barrios JA, et al. Bioavailabilityof two sublingual formulations of ketorolac tromethamine 30 mg:a randomized, open-label, single-dose, two-period crossover com-parison in healthy Mexican adult volunteers. Clin Ther 2008;30:1667–74.

18. El-Setouhy DA, El-Ashmony SM. Ketorolac trometamol topicalformulations: release behavior, physical characterization, skinpermeation, efficacy and gastric safety. J Pharm Pharmacol 2010;62:25–34.

19. Angeles Gonzalez-Fernandez M. Appropriateness of ketorolac usein a trauma hospital. Rev Calid Asist 2009;24:115–23.

20. Collins MN, Birkinshaw C. Comparison of the effectiveness of fourdifferent crosslinking agents with hyaluronic acid hydrogel filmsfor tissue-culture applications. J Appl Polymer Sci 2007;104:3183–91.

21. Tomihata K, Ikada Y. Crosslinking of hyaluronic acid withglutaraldehyde. J Polym Sci A Polym Chem 1997;35:3553–9.

22. Jinghua C, Jingtao C, Zheng X, Qisheng G. Characteristic ofhyaluronic acid derivative films crosslinked by polyethylene glycolof low water content. J Med Coll PLA 2008;23:15–19.

23. Merrett K, Liu W, Mitra D, et al. Synthetic neoglycopolymer-recombinant human collagen hybrids as biomimetic crosslinkingagents in corneal tissue engineering. Biomaterials 2009;30:5403–8.

24. Shivaraj A, Panner Selvam R, Tamiz Mani T, Sivakumar T. Designand evaluation of transdermal drug delivery of ketotifen fumarate.Int J Pharm Biomed Res 2010;1:42–7.

25. Peh K, Khan T, Ch’ng H. Mechanical, bioadhesive strengthand biological evaluations of chitosan films for wound dressing.J Pharm Pharm Sci 2000;3:303–11.

26. Claudia Cristiano MZ, Fayad SJ, Porto LC, Soldi V. Protein-basedfilms cross-linked with 1-ethyl-3-(3-dimethylamino-propyl) carbo-diimide hydrochloride (EDC): effects of the cross-linker and filmcomposition on the permeation rate of p-hydroxyacetanilide as amodel drug. J Braz Chem Soc 2010;21:340–48.

27. Kerch G, Korkhov V. Effect of storage time and temperature onstructure, mechanical and barrier properties of chitosan-basedfilms. Eur Food Res Techno 2011;232:17–22.

28. Tiwary AK, Sapra B, Jain S. Innovations in transdermal drugdelivery: formulations and techniques. Recent Pat Drug DelivFormul 2007;1:23–36.

29. Saei K, Pongpaibul Y, Viernstein H, Okonogi S. Factorsinfluencing drug dissolution characteristics from hydrophilicpolymer matrix tablet. Sci Pharm 2007;75:147–63.

30. Tanabe M, Watanabe M, Yanagi M, et al. Controlled indomethacinrelease from mucoadhesive film: in vitro and clinical evaluations.Yakugaku Zasshi 2008;128:1673–9.

31. Zhang J, Liu M, Jin H, et al. In vitro enhancement of lactate esterson the percutaneous penetration of drugs with different lipophili-city. AAPS PharmSciTech 2010;11:894–903.

32. Higuchi T. Mechanism of sustained-action medication. Theoreticalanalysis of rate of release of solid drugs dispersed in solid matrices.J Pharm Sci 1963;52:1145–9.

33. Ritger PL, Peppas NA. A simple equation for description of soluterelease II. Fickian and anomalous release from swellable devices.J Control Release 1987;5:37–42.

34. Peppas NA. Analysis of Fickian and non-Fickian drug release frompolymers. Pharm Acta Helve 1985;60:110–11.

35. Lara MG, Bentley MV, Collet JH. In vitro drug release mechanismand drug loading studies of cubic phase gels. Int J Pharm 2005;293:241–50.

36. Dhaneshwar S, Patel V, Patil D, Meena G. Studies on synthe-sis, stability, release and pharmacodynamic profile of a


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rsity

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novel diacerein-thymol prodrug. Bioorg Med Chem Lett 2013;23:55–61.

37. Włodarski KH, Dickson GR. Evaluation of locally inducedosteoarthritis by the complete and incomplete Freund’s adjuvantin mice. The application of DEXA measurements. Folia Biol(Praha) 2002;48:192–9.

38. Richard EF, Marilyn JB, Peggy JD, Alicia ZK. Anesthesia andanalgesia in laboratory animals. Copyright Elsevier Inc; June 2008.

39. Brahn E. Animal models of rheumatoid arthritis. Clues to etiologyand treatment. Clin Orthop Relat Res 1991;265:42–53.

40. Kumar VL, Roy S, Sehgal R, Padhy BM. A comparative study onthe efficacy of rofecoxib in monoarticular arthritis induced by latexof Calotropis procera and Freund’s Complete Adjuvant.Inflammopharmacology 2006;14:17–21.

41. Patil KR, Patil CR, Jadhav RB, et al. Anti-arthritic activity ofBartogenic acid isolated from fruits of Barringtonia racemosaRoxb. (Lecythidaceae). Evid Based Complement Alternat Med2011;2:1–7.

42. Kilimozhi D, Parthasarathy V, Amuthavalli N. Effect ofClerodendrum phlomidis on adjuvant induced arthritis in rats – aradiographic densitometric analysis. Int J PharmTech Res 2009;1:1434–41.

43. Arora P, Mukherjee B. Design, development, physicochemical,in vitro and in vivo evaluation of transdermal patches containingdiclofenac diethyl ammonium salts. J Pharm Sci 2002;91:2076–89.

44. Multani NK, Kaur H, Chahal A. Impact of sporting activities onbone mineral density. J Exercise Sci Physiotherapy 2011;7:103–9.

45. Rzonca SO, Suva LG, Gaddy D, et al. Bone is a target for theantidiabetic compound rosiglitazone. Endocrinology 2004;145:401–6.

46. Al-saffar FJ, Ganabadi S, Fakurazi S, et al. Chondroprotectiveeffect of Zerumbone on monosodium iodoacetate induced osteo-arthritis in rats. J Applied Sci 2010;10:248–60.

47. Young JJ, Cheng KM, Tsou TL, et al. Preparation of cross-linkedhyaluronic acid film using 2-chloro-1-methylpyridinium iodide orwater-soluble 1-ethyl-(3,3-dimethylaminopropyl) carbodiimide.J Biomater Sci Polym Ed 2004;15:767–80.

48. Park SN, Park JC, Kim HO, et al. Characterization of porouscollagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking. Biomaterials2002;23:1205–12.

49. Collins MN, Birkinshaw C. Physical properties of crosslinkedhyaluronic acid hydrogels. J Mater Sci Mater Med 2008;19:3335–43.

50. Everaerts F, Torrianni M, Hendriks M, Feijen J. Quantificationof carboxyl groups in carbodiimide cross-linked collagen sponge.J Biomed Mater Res 2007;83:1176–83.

51. Parida UK, Nayak AK, Binhani BK, Nayak PL. Synthesis andcharacterization of chitosan-polyvinyl alcohol blended with cloisite30B for controlled release of the anticancer drug curcumin.J Biomater Nanobiotech 2011;2:414–25.

52. Jeon O, Song SJ, Lee KJ, et al. Mechanical properties anddegradation behaviors of hyaluronic acid hydrogels cross-linked atvarious cross-linking densities. Carbohydr Polym 2007;70:251–7.

53. Ammar HO, Ghorab M, El-Nahhas SA, Kamel R. Polymeric matrixsystem for prolonged delivery of tramadol hydrochloride, Part I:physicochemical evaluation. AAPS Pharm Sci Tech 2009;10:1065–70.

54. Lee MW, Chen HJ, Tsao SW. Preparation, characterization andbiological properties of Gellan gum films with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linker. CarbohydrPolym 2010;82:920–6.

55. Park SK, Bae DH, Rhee KC. Soy protein biopolymers cross-linkedwith glutaraldehyde. J Am Oil Chem Soc 2000;77:879–84.

56. Powell HM, Boyce ST. EDC cross-linking improves skin substitutestrength and stability. Biomaterials 2006;27:5821–7.

57. Olde Damink LH, Dijkstra PJ, Van Luyn MJ, et al. Cross-linking ofdermal sheep collagen using a water-soluble carbodiimide.Biomaterials 1996;17:765–73.

58. Angele P, Abke J, Kujat R, et al. Influence of different collagenspecies on physico-chemical properties of crosslinked collagenmatrices. Biomaterials 2004;25:2831–41.

59. Alanazi FK, Abdel Rahman AA, Mahrous GM, Alsarra IA.Formulation and physicochemical characterization of buccoadhe-sive films containing ketorolac. J Drug Del Sci 2007;17:183–92.

60. Lemmouchi Y, Murariu M, Margarida Dos Santos A, et al.Plasticization of poly (lactide) with blends of tributyl citrate andlow molecular weight poly (d, l-lactide)-b-poly (ethylene glycol)copolymers. Eur Polymer J 2009;45:2839–48.

61. Jachowicz J, McMullen R. Mechanical analysis of elasticity andflexibility of virgin and polymer-treated hair fiber assemblies.J Cosmet Sci 2002;53:345–61.

62. Patel VM, Prajapati BG, Patel MM. Design and characterization ofchitosan-containing mucoadhesive buccal patches of propranololhydrochloride. Acta Pharm 2007;57:61–72.

63. Shidhaye SS, Thakkar PV, Dand NM, Kadam VJ. Buccal drugdelivery of pravastatin sodium. AAPS Pharm Sci Tech 2010;11:416–24.

64. Antesh KJ, Bhattacharya A, Pankaj VA. Formulation and in vitroevaluation of sustained release matrix tablets of metoprololsuccinate using hydrophilic polymers. Int J PharmTech Res 2009;1:972–77.

65. Dash S, Murthy PN, Nath L, Chowdhury P. Kinetic modeling ondrug release from controlled drug delivery systems. Acta Pol Pharm2010;67:217–23.

66. Ganesh S, Radhakrishnan M, Ravi M, et al. In vitro evaluation ofthe effect of combination of hydrophilic and hydrophobic polymerson controlled release zidovudine matrix tablets. Indian J Pharm Sci2008;70:461–65.

67. Yassin AB, Alsarra IA, Al-Mohizea AM. Chitosan beads as a newgastroretentive system of verapamil. Sci Pharm 2006;74:175–88.

68. Khaled KA, Sarhan HA, Ibrahim MA, et al. Prednisolone-loadedPLGA microspheres, in vitro characterization and in vivo applica-tion in adjuvant-induced arthritis in mice. AAPS PharmSci Tech2010;11:859–69.

69. Ando A, Hagiwara Y, Chimoto E, et al. Intra-articular injection ofhyaluronan diminishes loss of chondrocytes in a rat immobilized-knee model. Tohoku J Exp Med 2008;215:321–31.

70. Zhang Y, Shaffer A, Portanova J, et al. Inhibition ofcyclooxygenase-2 rapidly reverses inflammatory hyperalgesiaand prostaglandin E2 production. J Pharmacol Exp Ther 1997;283:1069–75.

71. Campo GM, Avenoso A, Campo S, et al. Efficacy of treatment withglycosaminoglycans on experimental collagen-induced arthritis inrats. Arthritis Res Ther 2003;5:R122–31.

72. Petrella RJ. Hyaluronic acid for the treatment of knee osteoarthritis:long-term outcomes from a naturalistic primary care experience.Am J Phys Med Rehabil 2005;84:278–83.

73. Chou LW, Wang J, Chang PL, Hsieh YL. Hyaluronan modulatesaccumulation of hypoxia-inducible factor-1 alpha, inducible nitricoxide synthase, and matrix metalloproteinase-3 in the synovium ofrat adjuvant-induced arthritis model. Arthritis Res Ther 2011;13:R90–102.

74. Kinne RW, Meyer P, Grunder W, et al. Rat antigen-inducedarthritis: cartilage alterations assessed with iodine-123-antileuko-proteinase. J Nucl Med 1998;39:1638–45.

75. Goldring SRMB. Bone and cartilage in osteoarthritis: is what’s bestfor one good or bad for the other? Arthritis Res Ther 2010;12:143–4.

76. Okazaki A, Koshino T, Saito T, Takagi T. Osseous tissue reactionaround hydroxyapatite block implanted into proximal metaphysis oftibia of rat with collagen-induced arthritis. Biomaterials 2000;21:483–7.

77. Cohen MN, Christians U, Henthorn T, et al. Pharmacokinetics ofsingle-dose intravenous ketorolac in infants aged 2–11 months.Anesth Analg 2011;112:655–60.


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Pharmaceutical and medical aspects of hyaluronic acid...

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