glycosaminoglycan in osteoarthritic cartilage. The duration of morning stiffness and pain
intensity are reduced and spinal mobility improved, by ACF in patients with
ankylosing spondylitis (Laurent et al., 2000). ACF is metabolized to a major
metabolite, 4'-hydroxy ACF and to a number of other metabolites including
5-hydroxy ACF, 4'-hydroxydiclofenac, diclofenac and 5-hydroxydiclofenac (Hinz et al., 2003)
There are reports describing the use
of hydroxyl propyl cellulose (HPC) in transdermal
patches and ophthalmic preparations (Cohen et al., 1979; Harwood et al., 1982; Dumortier et al., 1990) and
ethyl cellulose (EC) transdermal delivery systems as
well as other dosage forms for controlled release of drugs (Kusum
et al., 2003; Limpongsa and Umprayn,
2008; Sakellariou et al., 1986). HPC is freely water
soluble, whereas EC is hydrophobic. So the transdermal
delivery systems were prepared using HPC and EC to study the effect of
hydrophilic and hydrophobic nature of polymer on release of ACF. A large number
of fatty acids and their esters have been used as permeation enhancers. Oleic
acid has been shown to be effective as a permeation enhancer for many drugs,
for example increasing the flux of salicylic acid 28-fold and 5-fluorouracil
flux 56-fold, through human skin membranes in-vitro (Cooper, 1984;
Goodman and Barry, 1989). It has also been used for ketoprofen
(Hu, J.H., and Zhu, 1996), flurbiprofen
(Chi et al., 1995), 5-FU, estradiol (Goodman and
Barry, 1988), zalcitabine, didanosine,
zidovudine (Kim and Chien,
1996), etc.
The aims of the present study were to
(1) prepare transdermal patches of ACF using hydrophilic and
hydrophobic polymer; (2) optimization of transdermal
patch formulation using 3 2 full factorial design;
and (3) study the in-vitro diffusion behavior of prepared transdermal patch formulations in the presence and absence of penetration enhancer. The
purpose was to provide the delivery of the drug at a controlled rate across
intact skin.
2. MATERIALS AND METHODS
2.1 Materials
ACF was received as a gift samples
from Lincoln Pharmaceticals,
Ahmedabad, India. Hydroxyl propyl
cellulose (HPC) and ethyl cellulose (EC) were generous gift from Colorcon Asia Pvt. Ltd (Mumbai, India) and Maan Pharmaceuticals Ltd. (Ahmedabad,
India), respectively. Oleic acid (OA) and di-n-butyl-phthalate
(DBP) were procured from Sigma Chemicals Ltd. (Ahmedabad,
India). Other materials used in the study (chloroform, methanol,
dichloromethane, glycerol, potassium dihydrogen
phosphate, etc.) were of analytical grade. Double-distilled water was used
throughout the study.
2.2 Methods
a) Investigation of Physicochemical
Compatibility of Drug and Polymer
The physicochemical compatibility
between ACF and polymers used in the films was studied by using differential
scanning calorimetry (DSC- Shimadzu 60 with TDA trend
line software, Shimadzu Co., Kyoto, Japan) and fourier
transform infrared (FTIR- 8300, Shimadzu Co., Kyoto, Japan) spectroscopy.
In DSC analysis, the samples were
weighed (5 mg), hermetically sealed in flat bottom aluminum pans, and heated
over a temperature range of 50 to 300°C at a constant increasing rate of
10°C/min in an atmosphere of nitrogen (50 mL/min).
The thermograms obtained for ACF, polymers, and
physical mixtures of ACF with polymers were compared. The infrared (IR) spectra
were recorded using an FTIR by the KBr pellet method
and spectra were recorded in the wavelength region between 4000 and 400 cm1.
The spectra obtained for ACF, polymers, and physical mixtures of ACF with
polymers were compared.
b) Preparation of Transdermal Films
Transdermal patches containing ACF were prepared by the
solvent evaporation technique in cylindrical glass molds with both sides opens
(Arora et al., 2002). The backing membrane was cast
by pouring a 2 % (m/V) polyvinyl alcohol
(PVA) solution followed by drying at 60 °C for 6 h. The drug reservoir was
prepared by dissolving HPC or EC in Chloroform: Methanol (1:1) mixture. Dibutyl phthalate 15 % (w/w of dry polymer
composition) was used as a plasticizer. The drug 50 mg (in 5 mL solvent mixture Chloroform: Methanol) was added into the
homogeneous dispersion under slow stirring with a magnetic stirrer. The uniform
dispersion was cast on a PVA backing membrane and dried at room temperature.
(Table 1) The films were stored between sheets of wax paper in a desiccator.
c)
Physicochemical characterization of films
Thickness
The thickness of
patches was measured at three different places using a micrometer (Mitutoyo Co., Japan) and mean values were calculated (Amnuaikit 2005).
Weight Variation
The patches were
subjected to mass variation by individually weighing randomly selected patches.
Such determinations were carried out for each formulation (Verma
2000).
Drug Content
Patches of specified area (1 cm2) were
dissolved in 5 mL of dichloromethane and the volume
was made up to 10 mL with phosphate buffer pH 7.4;
dichloromethane was evaporated using a rotary vacuum evaporator at 45 °C. A
blank was prepared using a drug-free patch treated similarly. The solutions
were filtered through a 0.45 μm membrane, diluted
suitably and absorbance was read at 274 nm in a double beam UV-Vis
spectrophotometer.
Flatness
Three longitudinal strips were cut out from each film:
1 from the center, 1 from the left side, and 1 from the right side. The length
of each strip was measured and the variation in length because of
non-uniformity in flatness was measured by determining percent constriction,
with 0% constriction equivalent to 100% flatness (Arora 2002).
Folding Endurance
This was determined
by repeatedly folding one film at the same place till it broke. The number of
times the film could be folded at the same place without breaking/cracking gave
the value of folding endurance (Devi 2003).
Tensile
strength
In order to determine
the elongation as a tensile strength, the polymeric patch was pulled by means
of a pulley system; weights were gradually added to the pan to increase the
pulling force till the patch was broken. The elongation i.e. the distance
traveled by the pointer before break of the patch was noted with the help of
magnifying glass on the graph paper, the tensile strength was calculated as kg
cm-2.
Percentage of Moisture Content
The films were weighed individually and kept in a
desiccators containing activated silica at room temperature for 24 hours.
Individual films were weighed repeatedly until they showed a constant weight.
The percentage of moisture content was calculated as the difference between
initial and final weight with respect to final weight (Gupta, 2003).
Water vapour transmission
rate (WVTR)
WVTR is defined as
the quantity of moisture transmitted through unit area of film in unit time
(Krishna, 1994). Glass cells were filled with 2 g of anhydrous calcium chloride
and a film of specified area was affixed onto the cell rim. The assembly was
accurately weighed and placed in a humidity chamber (80 ± 5% RH) at 27 ± 2 °C
for 24 hours.
d) In-vitro skin permeation studies
In-vitro skin
permeation studies were performed by using a Franz diffusion cell with a
receptor compartment capacity of 22.5 mL. The excised
rat abdominal skin (Wistar
albino) was mounted between
the donor and receptor compartment of the diffusion cell. The formulated
patches were placed over the skin and covered with paraffin film. The receptor
compartment of the diffusion cell was filled with phosphate buffer pH 7.4. The
whole assembly was fixed on a magnetic stirrer, and the solution in the
receptor compartment was constantly and continuously stirred using magnetic
beads at 50 rpm; the temperature was maintained at 32 ± 0.5 °C. The samples were withdrawn at different time
intervals and analyzed for drug content spectrophotometrically. The receptor
phase was replenished with an equal volume of phosphate buffer pH 7.4 at each
sample withdrawal. The cumulative percentages of drug permeated per square
centimeter of patches were plotted against time.
e) Full factorial
design
A 32
randomized full factorial design was used in the present study. In this design
two factors were evaluated, each at 3 levels, and experimental trials were
performed at all 9 possible combination. The amount of Oleic acid (X1)
and the amount of Isopropyl myristate (X2)
were selected as independent variables. The drug release at 8 hrs was selected
as dependent variable. The design lay out is depicted in Table 4.
f) Permeation Data
Analysis
The flux (΅g cm-2 hr-1) of ACF
was calculated from the slope of the plot of the cumulative amount of ACF
permeated per cm2 of skin at steady state against the time using
linear regression analysis (Julraht et al., 1995; Ho et
al., 1998). The steady
state permeability coefficient (Kp) of the
drug through rat epidermis was calculated by using the following equation (Yamune
et al., 1995):
(1)
Where, J is the flux and C is the concentration of ACF
in the patch. The penetration enhancing effect of penetration enhancer was
calculated in terms of enhancement ratio (ER), and was calculated by using the
following equation (Williams et al., 1991):
(2)
g) Kinetic modeling of drug release
To analyze the mechanism of drug release from the
patches, the release data were fitted to the following equations:
Zero-order equation:
(3)
Where Q is the amount of drug released at time t, and
k0 is the release rate.
First-order equation:
(4)
Where Q is the percent of drug release at time t, and
k1 is the release rate constant.
Higuchis equation:
(5)
Where Q is the percent of drug release at time t, and
k2 is the diffusion rate
constant.
h) Stability study of
optimized formulation
Stability
study was carried out for optimized patch formulation at 40 oC
temperature in a humidity chamber having 75 % RH for 3 months. After 3 months
samples were withdrawn and evaluated for physicochemical properties and in-vitro diffusion study.
3.
RESULTS AND DISCUSSION
3.1
Investigation of Physicochemical Compatibility of Drug and Polymer
Differential scanning calorimetry enables the quantitative detection of all
processes in which energy is required or produced (i.e., endothermic or
exothermic phase transformations). The thermograms of ACF (A), HPC (B), physical mixture of ACF with excipient of HPC patch formulation (C), EC (D) and
physical mixture of ACF with excipient of EC patch
formulation (E) are presented in Figure 1. The ACF showed a melting peak
at 158.22 °C. Peak of ACF at 158.22 °C was present at the same
position i.e. near to 160 oC in the
physical mixture of drug with both HPC and EC patch formulation excipients. This confirmed the physicochemical stability of
drug with the formulation excipient used in the
study.
Drug - excipient
interactions play a vital role with respect to release of drug from the
formulation amongst others. FTIR techniques have been used here to study the
physical and chemical interaction between drug and excipients
used. Infrared (IR) spectra of ACF (A), physical
mixture of ACF
Figure 1: DSC study of ACF
(A), HPC (B), physical mixture of HPC formulation excipient
with ACF (C), EC (D), physical mixture of EC formulation excipients
with ACF (E)
they are uniform in
thickness. The weights ranged between 10.11 ± 0.30 mg and 15.13 ± 0.45 mg, which indicates that
different batches patch weights, were relatively similar. Good uniformity of
drug content among the batches was observed with all formulations and ranged
from 97.9 ± 2.93 % to 99.2 ± 2.97 %. The results indicate that the process
employed to prepare patches in this study was capable of producing patches with
uniform drug content and minimal patch variability. The flatness study showed
that all the formulations had the same strip length before and after their cuts,
indicating 100% flatness. Thus, no amount of constriction was observed; all
patches had a smooth, flat surface; and that smooth surface could be maintained
when the patch was applied to the skin. Folding endurance test results
indicated that the patches would not break and
Figure 2: FTIR spectra of
ACF (A), physical mixture of HPC with ACF (B), physical mixture of EC with ACF
(C)
would maintain their
integrity with general skin folding when applied. Moisture content and
moisture uptake studies indicated that the increase in the concentration of
hydrophilic polymer was directly proportional to the increase in moisture
content and moisture uptake of the patches. The moisture content of the
prepared formulations was low, which could help the formulations remain stable
and reduce brittleness during long term storage. The moisture uptake of the
formulations was also low, which could protect the formulations from microbial
contamination and reduce bulkiness (Mutalik et al., 2004).
Figure 3: Release profile of ACF from patches
containing different concentration of HPC and EC, mean ± SD (n = 3)
3.3 In-vitro skin permeation
The in-vitro release profile is an important tool that predicts in
advance how a drug will behave in vivo (Katayose et al.,
1997). The results of in-vitro skin
permeation studies of ACF from transdermal patches
are shown in Figures 3. In the present study hydrophilic (HPC) and hydrophobic
(EC) polymers are used to prepared patches. Formulation A1 exhibited greatest
84.8 ± 3.39 % of drug release value, while formulation A6 exhibit lowest 44.73
± 1.789 % of drug release value.
The cumulative amount of drug released from formulations containing
hydrophilic polymer release drug at faster rate than hydrophobic polymer. The
cumulative amount of drug released from formulations A1, A2 and A3 is much
higher than formulation A4, A5 and A6. In addition to nature of polymer
concentration of polymer also affect the drug release. As the concentration of
polymer increased drug release decreased. The drug release from the patch is
ordered as A1 > A2 > A3 > A4 > A5 > A6. Unlike the formulations
A2, A3, A4, A5 and A6, the formulations A1 achieved a high cumulative amount of
drug permeation at the end of 10 hours. Based on physiochemical and in-vitro release experiments, A1 was
chosen for further studies.
3.4
Full factorial design
3.4.1 Physicochemical properties of factorial design batches
The results of the physicochemical characterization of the patches are
shown in Table 3.
3.4.2
In-vitro drug release study of
factorial design batches
The cumulative
percentage of drug permeated through the rat epidermis from the patch
containing different concentration of penetration enhancer is shown in Figure
4.
Table
5 shows the results of analysis of variance (ANOVA), which was performed to
identify independent factors. The high value of correlation coefficient for Q8hr
indicates a good fit. The equation can be used to obtained estimates of
the response as a small error of variance was noticed in the replicates. The
significant level of coefficients b11 was found to be greater than P
= 0.05. Hence it was omitted from the full model to generate the reduced model.
The results of statistical analysis are shown in Table 5. The coefficients b1,
b2, b22 and b12 were found to be significant
at P < 0.05. Hence they were retained in the reduced model. An increase in
concentration of Oleic acid leads to an increase in Q8hr because the
coefficient b1 bears positive sign. An increasing the concentration
of oleic acid from 5 to 10 % the Q8hr value increased from 81.13 %
to 88.14 %.An increase in concentration of Isopropyl Myristate
leads to increase in Q8hr because the coefficient b2
bears positive sign. As increasing concentration of Isopropyl myristate from 5 to 10 % the Q8hr value
increased from 84.36 % to 90.31 %.
Here the coefficient
of interaction terms showed negative value. The interaction term indicate that Q8hr was not significantly
affected by interaction of two penetration enhancer. This indicates that by
changing two factors at a time there no effect on Q8hr.
The maximum amount (Q8hr) of ACF that permeated
during the 8 hr of the study was 99.64 % from formulation F9. The flux was
calculated by dividing the cumulative amount of drug permeated per cm2
of the skin with time. Thus the corresponding flux of ACF was 216.54 ΅g cm-2 hr-1 from
formulation F1 (transdermal patch without penetration
enhancer). A marked effect of penetration enhancer on ACF permeation was
observed when they were incorporated in patch in varying concentration. The
cumulative percentage of ACF that permeated over 8 hr was found to increase
ranging from 81.13 to 99.64 % for HPC patches. The corresponding flux values
were ranging from 242.56 to 256.57 ΅g cm-2 hr-1.
Formulation F9 shows highest flux among all the formulation. Formulation F9
shows 1.369 fold enhancements in drug permeation. This result indicate that the formulation containing
15 % of oleic acid with 10 % Isopropyl myristate give
better penetration of ACF through rat skin.
3.5 Kinetic modeling of drug release
The cumulative amount of
drug permeated per square centimeter of patches through rat skin was plotted
against time was fitted to zero, first and higuchi
kinetic model. As indicated in Table 7, the release profile of ACF followed
mixed zero-order and first-order kinetics in different formulation. However, the
release profile of the optimized formulation F9 (r2 = 0.9935 for
Higuchi) indicated that the permeation of the drug from the patches was
governed by a diffusion mechanism.
3.6 Stability study
In order to determine the change in physicochemical
parameter and in-vitro release profile on storage, stability study was
carried out. The physicochemical parameter of the optimized formulation was not
significantly changed on storage. The in-vitro release profile before
and after storage is shown in Figure 5. The result indicates that the
formulation was stable on the required storage condition.
4.
CONCLUSION
The method of preparation of transdermal
patches of Aceclofenac
presented in this research work is simple. All formulation also showed good physicochemical properties
like thickness, weight variation, drug content, flatness, folding endurance,
moisture content and moisture uptake. The in-vitro release data showed
that drug release from the patch formulation have been affected by types of
polymer and concentration of polymer. Effect of penetration enhancer like oleic
acid and isopropyl myristate have been checked on in-vitro
permeation of drug. These studies indicated that as the concentration of
penetration enhancer increased drug permeation was increased. The finding of
this result revealed that the problems of Aceclofenac on oral administration like dissolution rate limited
absorption and gastric side effects can be overcome by applying Aceclofenac topically in the form of transdermal
patch.
Figure 4: Release profile of ACF from
formulation F1 - F9, men ± SD (n = 3)
Figure 5: Drug
release profile of ACF before and after stability study for formulation F9
5.
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