INTRODUCTION:
Systemic
therapy with variety of macrolides has been the most common method to treat
skin infections for many years. However, antibiotics given systemically may
cause severe allergic reactions and side effects1. The stratum
corneum of the skin acts as a barrier to the permeation of topically applied
drugs. Therefore, currently available topical preparations are not effective in
delivery of antibiotics deep in to the skin.
Erythromycin
[EM] is a lipophlic macrolide antibiotic with MW 734 Da. It has been widely
used for the dermatological treatment of acne which is characterized by
persistent, recurring reddish blemishes leading to serious painful inflammatory
conditions, if neglected. EM is highly unstable in aqueous system and
undergoes rapid degradation in alkaline as well as acidic conditions. The
alkaline hydrolysis of EM produces pseudo erythromycin enol ether. The major
degradation product of acidic hydrolysis is enol ether.2-4 The
stability problem of EM necessitates use of extempore preparations. Attempts
have been made to improve the stability of EM by formulating hydrogels5
and lotions6.
These
dermal vehicles require the use of penetration enhancers and have limited
stability against temperature, moisture and microbial contamination. Therefore,
it is desirable to develop topical vehicle system having better stability that
does not require the use of chemical enhancers to facilitate drug permeation
through the skin. In recent years, interest in organogels has been increased
dramatically with the discovery and synthesis of very large numbers of diverse
molecules that gel a range of organic solvents at low concentrations.7
Lecithin, the natural bio-friendly molecules are ubiquitous phospholipids that accounts for more than 50 %
of the lipid matrix of biological membranes.
Soybean lecithin in an apolar organic solvent, on addition of water, forms an
entangled dynamic network of long and flexible worm-like multi-molecular
aggregates termed as ‘organogels.’ 8 These are characterized
by high viscosity and complete optical transparency. Lecithin organogels are emerging as carriers for drug molecules with
diverse physicochemical properties including macromolecules9. Transdermal transport
rates of scopolamine and broxaterol from lecithin organogels were faster than
commercial patches. 9 Similarly,
improved skin penetration of indomethacin and diclofenac has been observed with
lecithin-based organogels in isopropyl palmitate.10 Piroxicam has
been successfully incorporated in lecithin organogels.11 Recently
results have shown that ketorolac tromethamine could be incorporated at high
concentrations into lecithin organogels. 12
Thus,
with the inflow of several research reports on the varied fundamental aspects
of lecithin organogels, along with some promising results on the front of drug
delivery, these systems can be seen as potential tools in the field of topical
drug delivery applications. However, lecithin organogels has not been evaluated
for improving the drug stability till date.
The
present research work was undertaken to enhance EM hydrolytic resistance and to
improve its penetration through the skin. To achieve this objective we
formulated and evaluated lecithin based organogel containing EM. Lecithin
organogel was chosen as a matrices for delivering EM topically because (a) its
ability to solublilize guest molecules of different chemico- physical
properties; (b) its low acute and cumulative skin irritation potential; (c) its
chemical composition and thermo reversible nature; (d) its isotropicity
allowing the use of spectroscopic methods to detect possible structural changes
of the guest molecules and (e) its long term stability.13
MATERIAL
AND METHODS:
Soy
lecithin (Epikuron-200) was a generous gift from Degussa Bioactive (Germany). Erythromycin base was kindly supplied as gift sample by IPCA Lab. Ltd. (Mumbai, India). Carbopol 940, triethanolamine, propylene glycol, isopropyl alcohol, IPM
were purchased from S.D.Fine chemicals. (Mumbai, India) and all were of AR
grade. Micrococus luteus (ATCC 9341) culture was obtained from Indoco
Pvt. Ltd. (Mumbai, India). Double distilled water was used through out the
experiment.
HPLC Analysis of EM
The
standard solutions of EM were prepared in the range of 0.5 -2.5 mg/ml in the
mobile phase [Acetonitrile-0.2M Ammonium acetate- Methanol-Water (35: 15: 5: 45)]. HPLC analysis of EM was performed using a Jasco SERIES 2000 pump set, 1 ml/min
flow rate and a Spectra Jasco SERIES UV 2075 detector. The column used was
Hypersil C-18 (250 mm X 4.6 mm, 5.0μm particle size). Jasco Borwin
version 1.5, LC-Net II/ADC software was used for data analysis. The correlation
coefficient of standard curve was found to be 0.9941.
Preparation of EM Gels
Lecithin
organogel were prepared according to a technique reported by Angela et al.14
Accurately weighed quantity of lecithin (15.2%, 22.8%, and 30.4% w/v)
was dissolved in IPM. The solubilization was facilitated by
sonication at a frequency of 15000 KHz for 10 min. The EM was incorporated in
lecithin solution. The organogelation was induced by adding distilled water
[Wo=3; molar ratio of water to lecithin for IPM]. The drug containing
organogels with different concentration of lecithin were evaluated in a similar
fashion.
Hydrogel containing EM was
prepared with carbopol 940, triethanolamine, and isopropyl alcohol.15
Carbopol 940® was dissolved in propylene glycol and mixed with
impeller at 100 rpm for 2h. The contents were transferred to the planetary
mixer. Triethanolamine dissolved in propylene glycol was admixed in thin stream
with polymer solution. The pH was adjusted (7.5 to 8). The EM dissolved in
mixture of isopropyl alcohol and propylene glycol was admixed with above
contents in planetary mixer. The speed of the planetary mixer was controlled to
prevent air entrapment.
In vitro skin permeation
studies
Ethical
clearance was obtained from the institutional animal experimental committee
before the study.Full thickness abdominal skin of albino rats [125-150g] was
used. The dermal surface was carefully cleaned to remove subcutaneous tissues
and fats without damaging the epidermal surface. The diffusion of EM from the different
gel samples was investigated across animal skin using Keshary-Chien type
diffusion cells. The capacity of diffusion cell was 20ml and effective surface
area was 3.14 cm2. The receptor compartment was filled with saline
phosphate buffer [pH=7.4].The cells were thermostated at 37+1 0C
and the receptor solution was stirred with a magnetic bar at 200 rpm. One gram
of various gel samples (organogel, hydrogel and marketed cream) was loaded on
the membrane. The aliquots of 2 ml samples were withdrawn every hour for 4 h
from the receptor compartment and replaced with fresh medium. The samples were
diluted with mobile phase solvent and then analyzed by HPLC for EM content. The
mean cumulative amount of drug permeated per unit surface area of the skin was
plotted versus time.
Evaluation of Gels
Content
Uniformity
Tubes
containing organogel, hydrogel and marketed preparations were cut into three
different portions (top, middle, and bottom). The EM content in each portion
was analyzed using HPLC method. The analysis of each sample was performed in
triplicate.
RhIeological Studies
a.
Spreadability
The
spreadability of the formulations was determined using an apparatus suggested
by Goud et al 16with slight
modification. It consisted of a wooden block provided by a pully at one end. A
rectangular ground glass plate was fixed at this end. An excess of the gel /
cream (3g), under study, was placed on ground glass plate. The gel/ cream
sample was sandwiched between ground glass and additional glass plate. The
second glass was attached to a hook. The top plate was subjected to a pull of
50 g with the help of a sting attached to the hook. The time (in sec) required
by the top plate to cover a distance of 10 cm was noted. A shorter time
interval indicates better spreadability.
b. Viscosity
Viscosity of both plain and medicated organogel sample
was determined. For the viscosity measurements, a Cone and Plate viscometer
CAPΠ H+ was used. All measurements were made on freshly
prepared samples.
Table 1: Comparative in vitro skin permeation
data of 3 different formulations containing erythromycin 3 %( w/w)
|
Sr. No.
|
Formulations
|
Cumulative amount released
(µg)
|
Percent drug released
|
|
1
|
Organogel
|
7940.49 µg
|
26.46%
|
|
2
|
Hydrogel
|
6220.40 µg
|
20.73%
|
|
3
|
Cream [marketed]
|
5534.41 µg
|
18.44%
|
Table
2: Content uniformity of different formulations containing erythromycin 3 %(
w/w)
|
Portion
of tube analyzed
|
Organogel
(μg/ml)
|
Hydrogel
(μg/ml)
|
Cream
(μg/ml)
|
|
Top
|
1404.51
|
1361.46
|
1035.40
|
|
Middle
|
1403.23
|
1398.02
|
1404.4
|
|
Bottom
|
1399.4
|
1404.51
|
1302.96
|
Table
3: Stability data compilation for an erythromycin organogel
|
Tests
|
Initial
Time
|
400C/75%
R.H
|
500C/75%
R.H
|
|
1st
Month
|
2nd
Month
|
3rd
Month
|
1st
Month
|
2nd
Month
|
3rd
Month
|
|
Microbiological
assay
(%)
|
101.98
|
99.15
|
96.15
|
94.65
|
97.62
|
93.22
|
88.63
|
|
Chemical
assay (%)
|
100
|
97.5
|
96.11
|
94
|
95.1
|
90.4
|
86
|
The 0.5 milliliters of sample was used for
measurements. The measurements were carried out at different speeds ranging
from 100 rpm to 900rpm at 37 ± 0.5 0C.
Histopathological Investigation of Organogel
The rat abdominal skin region measuring approximately
4cm2 was mounted on two different modified Keshary-Chien diffusion
cells. A 3.0 g of organogel was placed on the skin membrane of one diffusion
cell, whereas 3.0 g of water [control] was placed on the skin membrane of the
other diffusion cell. The skin was fixed in 10% neutral formalin for 24 hours
and then cut vertically against the surface at the central region (4mm width).
Each section was dehydrated using graded solutions of ethanol and then embedded
in paraffin wax. Tissues were divided into small pieces and stained with
haematoxylin and eosin .The sections were observed under 100x magnification and
photographed.
Stability Studies
Chemical Assay
The structural integrity and chemical stability of EM
under accelerated storage conditions were estimated for developed organogel,
hydrogel and marketed cream. The organogel and hydrogel containing 3% w/w of EM
was placed in collapsible aluminum tubes. Samples were stored at 40+2oC/75%RH and 50+2oC/75%RH for periods of up to 3 months and observed for
physical changes. The drug content was estimated (HPLC method) each at 0, 1, 2,
and 3 months interval in accordance with ICH guidelines. Shelf life of the gel
formulations was predicated using Arrhenius plots. 17
Estimation of Stability
of Erythromycin Organogels using Microbiological Assay
Standard EM solutions were prepared by dissolving known
concentration of EM in 90% ethanol and then diluted to 50 ml in a phosphate
buffer of pH 8 (0.523 g KH2PO4 and 16.73 g K2HPO4,
in distilled water 1 L.)5 Working solutions of 0.25 and 1mg/ml
were prepared in a phosphate buffer pH 8 as standard low and standard high,
respectively. The susceptible organism used was Micrococus luteus (ATCC
9341). Extracted drug from an organogel was placed in each well with a control
(vehicle free drug). The analysis was carried out by two level factorial assays.
Mean zone of inhibition (the antibacterial activity) was calculated by using
the following equations.
. (1)
The
value “a”may be positive or negative & should be used algebraically
Where,
I is ratio of dilution (1:4) log I = 0.6021 and
Where,
U1 and U2: unknown sample at
lower and higher concentration,
S1 and S2: standard sample at
lower and higher concentration.
RESULTS
AND DISCUSSION:
Effect of Lecithin Concentration on Drug Release
The drug release from organogels is affected by the
strength of gels. The gel strength is based on existence of three-dimensional
network in the oils. The gelator concentration is expected to enhance the gel
strength.18 The effect of lecithin concentration on EM release was
studied. Three different concentrations of
lecithin organogels were prepared (15.2%, 22.8%, and 30.4% w/v), all containing
3%w/w EM. All the three formulations were subjected to permeation studies. The
cumulative release data was obtained from three different lecithin organogels
at the end of 4hr. The viscosities of these three gels were of the increasing
order of 15.2% < 22.8% < 30.4% w/v and the cumulative amount of drug
released were 8350.25 µg, 7940.49µg, and 6323.89µg
respectively. The viscosity ranged from 375- 500 centipoises
(Cps) at 100rpm for 30.4% w/v lecithin organogel. The release of drug from
these organogel was low. The result reveals that the drug release decreased
with increasing concentration of lecithin. The effect is attributed to
establishment of more lecithin micellar networks causing increase in viscosity.
The established connected channels are expected to promote the drug diffusion. However, the drug being less water-soluble
is more portioned in micellar phase. Thus increasing the distance for diffusion
controlled release. Among all the three formulations 15.2%w/v organogel gave
high release rates but its viscosity was very low, 30.4%w/v organogel showed
lowest release rate and it was highly viscous. The consistency, viscosity and
release pattern of 22.8% w/v organogel was satisfactory. Hence it was finalized
for further studies.
In vitro skin permeation study
The
cumulative amount of drug release was plotted versus time .After 4 hr of
release study the amount of drug release was found to be 5534.41 µg, 6220.40
µg, and 7940.49 µg for marketed cream, hydrogel and organogel respectively
(Table 1). Comparative in vitro skin permeation data of 3 different
formulations containing EM 3 %( w/w) shown in Figure 1.Higher permeation
profiles of organogel compared to that of hydrogel and marketed cream may be
attributed to the presence of lecithin in the organogel. Lecithin enhances skin
permeation by affecting the lipids of stratum corneum, altering their
arrangement and disordering them transiently The trans-skin permeability of
propranolol hydrochloride, a poorly permeable and water-soluble drug
incorporated in lecithin organogel, across human cadaver skin has been
investigated and significantly enhanced (approximately 10 times higher)
permeability of micellar-borne drug across the human skin was observed
employing drug in 200 mM lecithin/iso-octane/water organogel system in
comparison to that of
pure
drug in solution form or emulsified in the petroleum jelly 19 .
Content Uniformity
Organogels are prepared as isotropic solutions and then converted to gels 20. These gels are basically interconnected network of two continuous phase and exhibits complete optical transparency. Uniformity will be a problem in biphasic system but organogels are extension of single-phase system and hence there is no problem of drug homogeneity in organogels. Concentration from all the three sites i.e. top, middle and bottom was found to be almost similar in organogel, where as it was not same in case of hydrogel and marketed cream (Table 2).
Figure 2. Histopathology
(a) test (b) control
sample
Rheological Studies
a. Spreadability
The
rheological properties of topical preparation influence the performance of drug
delivery systems. The spredability is important for uniform and ease of
application of topical preparations. The spredability of the preparations was
in the decreasing order of organogel> hydrogel > marketed cream. It is
based on the composition and principle of formulation. The spreadability of organogel,
hydrogel and marketed cream was found to be 18 ± 0.98, 23 ± 1.03 and 29 ± 1.35 seconds to travel the distance of 10 cm
respectively. Each value represents mean ± S.D. (n =
3). The marketed cream being an emulsion has less spredability. The
organogels are micellar aggregates and hence show much better spredability.
b. Determination of Viscosity
For
any vehicle to be used for topical drug delivery applications, it is essential
to study its rheological behavior. The latter is important for its efficacy in
delivering the molecules onto or across the skin site. Lecithin
organogel, prior to gelling, i.e., before the addition of polar phase, exhibit
Newtonian behavior but follow Maxwell’s rheological (viscoelastic) behavior on
addition of the polar phase.21 As soon as critical amount of water is added to
lecithin-oil mixture there is drastic increase in viscosity.
Figure 3: First order plot of
erythromycin organogel at (a) 40 0C (b) 50 0C
The
physical properties of the drugs alter micellar aggregation of lecithin. The
interaction results in weakening the micellar aggregation. Plain organogel has
higher viscosity than organogel containing EM. This was observed because EM is
hydrophobic in nature; the drug will be partitioned predominantly in
hydrophobic region of micellar aggregate. This results in decreased hydrophobic
interaction among lecithin molecules present in oil. This is expected to
produce weak network of lecithin and hence low viscosity. The viscosity of
plain IPM organogel and of medicated IPM organogel observed was 392.4 and 223 centipoises at 100 rpm respectively.
At 900 rpm, viscosity observed was 88 and 68.3
centipoises respectively. The structure of gel is altered in presence of
stress. As the stress increases the viscosity decreases. The organogel
structure is gradually lost at higher stress.
Histopathological Investigation of Organogel formulation
The
histology of excised rat skin in control and treated with organogel formulation
after 24 hours is shown in Figure 2. The
microscopic observations indicate that the organogel has no significant effect
on the microscopic structure of the skin. The surface epithelium lining and the
granular cellular structure of the skin were totally intact. No major changes
in the ultra structure of skin morphology could be seen and the epithelial
cells appeared mostly unchanged. Hence, it can be concluded that IPM organogels
containing EM are safe for dermatological purpose. Willimann and Luisi have
also reported that IPM lecithin organogels does not shows any toxic effect on
the skin. 9 IPM is widely used in
cosmetics and topical formulations and is generally regarded as nontoxic. Lecithin used in the formulation has GRAS status
and it has been included in the FDA Inactive Ingredients Guide for inhalations;
IM and IV injections; otic preparations; oral capsules, suspensions and
tablets; rectal, topical, and vaginal preparations.Hence,it can be safely
concluded that the IPM based lecithin organogels are biocompatible and safe for
topical applications.
Stability Studies
a.
Study of Degradation Kinetics
To determine the degradation kinetics of EM in
formulations at 25 0 C , k value for 40 0C and 50 0C
was found and the values obtained from first order plots at 40 0C
and 50 0C are shown in Figure 3,4,and 5 for EM organogel, hydrogel
and marketed cream respectively. As derived from Arrhenius plots (Figure 6),
the degradation rate constant (Kdeg) x 10-3 was found to
be 19.5, 24.7, 30.2 per month at 40 0C and 48.7, 60.8, 74.2 per
month at 50 0C for organogel, hydrogel and marketed cream
respectively.
Figure 4: First order plot
of erythromycin hydrogel at (a) 40 0C (b) 50 0C
b.
Arrhenius Plot
From
Arrhenius plot given for different formulations, it is concluded that the
calculated kinetic parameters proved that EM is more stable in organogel as
compared to hydrogel and marketed cream. Arrhenius plots for erythromycin in
organogel, hydrogel, and marketed cream at 40 0C and 50 0C
are represented in Figure 6.
The
stability of EM was of the decreasing order of organogel > hydrogel>
marketed cream. This has reflected in EM degradation rate constants. The
temperature and humidity of storage has significant effect on stability of EM
in these three formulations. The higher temperature and humidity increased
hydrolytic degradation of EM. The shelf life of EM was predicted from the
accelerated stability data. The EM in organogels, hydrogel and cream has a
shelf life of 24, 18 and 15 months respectively. These organogels have provided
effective barrier for hydrolytic degradation of EM. The organogels are not
sensitive to moisture at low temperature. The higher temperature must have
dissolved the lecithin thus weakening the gel structure. This has caused some
degree of degradation at high temperature and humidity. The EM stability in the
organogel formulation at room temperature was found to be well above 95% up to
six months. The degradation was evident in hydrogels and creams (7-9%).
Negligible drug loss was observed in organogels. The stability of EM in
organogels was further confirmed by microbiological assay.
Figure 5: First-order plot of
erythromycin marketed cream at (a) 40 0C (b) 50 0C
Figure 6: Arrhenius plots for erythromycin
in (a) organogel, (b) hydrogel, and (c) marketed cream at 40 0C and
50 0C.
Stability of Erythromycin Organogels using Microbiological Assay
To
determine the antibacterial activity of EM in organogel, microbiological assay
was carried out. Organogel of EM is a novel formulation and it is necessary to
determine its stability by using chemical as well as microbiological assay
according to ICH guidelines. It was observed that temperature and humidity
affects the activity of EM in organogel that reduces to around 6% to 7% for 400C/75%
R.H and around 11% for 500C/75% R.H after 3 months. From this it
can be concluded that shelf life of EM organogel would be approximately around
2 years. 22 Comparing chemical assay with microbiological assay it
is observed that organogel formulation of EM is stable and it will remain
stable physically, chemically and microbiologically for about 2 years. (Table
3).Variation is not observed in both assays due to homegenicity in organogel
formulation.
CONCLUSION:
The
chemical stability of EM was significantly enhanced by incorporating the drug
in lecithin based organogel containing isopropyl myristate (IPM) as oil phase.
The stability of EM was in the decreasing order of organogel > hydrogel>
marketed cream. This has reflected in EM degradation rate constants. EM release
was found to be highest for organogels as compared to other two formulations.
The physical instability commonly observed in hydrogels and creams was
successfully overcome by an organogels. The stability of EM in organogel was
also confirmed by microbiological evaluation and it was found to be within the
specifications. Organogels are less sensitive to water and provided desired
shelf life for erythromycin. Histopathology study revealed that the formulation
has no toxic effect on skin even after a longer contact and all the surface
epithelium lining and the granular structure remain intact. This ensures the
safety of lecithin organogel formulation for topical delivery of EM.
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