FORMULATION AND EVALUATION OF TETRACYCLINE …
Transcript of FORMULATION AND EVALUATION OF TETRACYCLINE …
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FORMULATION AND EVALUATION OF TETRACYCLINE
NIOSOMAL TOPICAL GEL DRUG DELIVERY SYSTEM
Sorlin Selva Joice P.*1, Rubina Reichal C.
2, Thirumoorthy N.
2 and Sangeetha M.
2
1Assistant Professor, Department of Pharmaceutics, Cherraan’s College of Pharmacy,
Coimbatore, Tamil Nadu, India.
2Department of Pharmaceutics, Cherraan’s College of Pharmacy, Coimbatore, Tamil Nadu,
India.
ABSTRACT
The core objective of the present study is to formulate Tetracycline
niosomes for topical delivery system by hand shaking method. The
prepared niosomes are to be characterized for their size, shape,
stability, entrapment efficiency, invitro drug release and retention
study. The formulation 1(F1) was more stable when compared to other
formulations with smaller size vesicles and showed higher entrapment
efficiency. The best formulation was prepared as gel with suitable gel
base. The Tetracycline Niosomal gel delivery may reduce the
frequency of dosing intervals and improve patient compliance.
KEY WORDS: Tetracycline, Niosomal Gel, Topical Drug Delivery,
Acne Vulgaris.
INTRODUCTION
Even the oral route is the convenient route for delivery of drugs, it has some limitation in the
treatment of skin diseases. The topical delivery system has better percutaneous absorption
than the semi solid preparations.[1-4]
Recently niosomes are becoming popular in the field of
topical drug delivery due to its outstanding characteristics like enhancing the penetration of
drugs, providing a sustained pattern of drug release and ability to carry both hydrophilic and
lipophilic drugs. Topically applied Niosomes can increase residence time of drug in the
stratum corneum and epidermis. Topical applicability of niosomes was further enhanced by
developing niosomal gel formulation using carbopals.[5,6]
The release from the niosomal gel
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6.647
Volume 6, Issue 8, 2644-2657 Research Article ISSN 2278 – 4357
Article Received on
20 June 2017,
Revised on 11 July 2017, Accepted on 1 August 2017,
DOI: 10.20959/wjpps20178-9963
*Corresponding Author
Sorlin Selva Joice P.
Assistant Professor,
Department of
Pharmaceutics, Cherraan’s
College of Pharmacy,
Coimbatore, Tamil Nadu,
India.
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was highly prolonged when compare to conventional gel formulation. As well as the presence
of other ingredients that act as skin permeation co-enhancers.[7,8]
Chemically Tetracycline is (4S,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12,12a-
pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
(Fig No.1 ) inhibits protein synthesis by binding to 30S ribosomes the attachment of
aminoacyl- t-RNA to the acceptor A site of mRNA ribosome. As a result, the peptide chain
fails to grow10,11
. The action is usually inhibitory and reversible upon withdrawal of the drug.
Mammalian cells are less vulnerable to the effect of tetracyclines, despite the fact that
tetracycline binds to the small ribosomal subunit of both prokaryotes and eukaryotes (30S
and 40S respectively). This is because bacteria actively pump tetracycline into their
cytoplasm, even against a concentration gradient, whereas mammalian cells do not. This
accounts for the relatively small off-site effect of tetracycline on human cells.
Fig. 1: Chemical Structure of Tetracycline.
It is a broad spectrum antibiotic, used to treat Acne Vulgaris, Syphilis, Chlamydia infection,
and Bronchitis.
In the novel drug delivery system, there is number of permeation enhancers has been
introduced to effective plasma drug level, in this niosomes play a vital role in topical drug
delivery. The presence of nonionic surfactants increases the permeability of Tetracycline
through biological membrane and also reduces the systemic toxicity. The niosomal gel of
tetracycline may increase efficacy for sustained activity.
MATERIALS AND METHODS
Tetracycline was obtained as a gift sample from Emil Pharma, Maharastra.All other
chemicals used were of analytical grade.
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Preformulation Studies
Compatibility Studies
Drug and excipients interaction study is carried out by FTIR technique.
Preparation of Niosomes
Cholesterol and span 20 were taken in specified ratios of (1:1, 1:1.5,1:2,1:2.5,1:3 & 1:3.5)
and transferred in to a clean round bottom flask. Then the lipid mixture was dissolved in 10
ml of diethyl ether. The flask was continuously vortexed to form a thin film along the sides of
the flask. An appropriate amount of tetracycline was dissolved in phosphate buffer saline
(PBS) pH.7.4. This was poured into the thin film and vortexed continuously for a period of
30 min at room temperature.[4,6]
Table 1: Composition of the Tetracycline Niosomal formulations.
Evaluation of Prepared Tetracycline Niosomes
By optical microscopy
Take one drop of prepared niosomes (diluted with water if needed) in a clean glass slide; it is
then placed on the stage of the optical microscope. First it was viewed under low power then
changed to high power and the vesicles were counted up to 100 numbers.
By scanning Electron microscopy (SEM)
The size of the vesicles was measured by scanning electron microscopy (HITACHI S – 150).
Percentage Encapsulation of drug
Tetracycline encapsulated niosomes were separated from unentrapped drug by dialysis
method for 24 hrs. The formulation were transferred into a standard flask, then lysed with1ml
of 2.5% SLS solution, then incubated at 370 1
0 C for 2 hrs. Then it is filtered through
whatman filter paper. Filtrate of 1 ml was diluted to 10 ml with PBS and absorbance of the
resulting solution was measured spectrophotometrically at 255 nm.
Formulation Code Drug Span 20 Cholesterol Diethyl ether
F1 100 mg 10 ml 10 mg 10 ml
F2 100 mg 15 ml 10 mg 10 ml
F3 100 mg 20 ml 10 mg 10 ml
F4 100mg 25ml 10mg 10ml
F5 100mg 30ml 10mg 10ml
F6 100mg 35ml 10mg 10ml
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Determination of percentage of drug encapsulated in the niosomes
This was carried out to find out the percentage of drugs encapsulated in the niosomes by
using the following formula:
Drug leakage studies from vesicles
Vesicle stability with respect to drug leakage and drug degradation upon storage was studied
at refrigeration (40c), room temperature (25
0c) and high temperature (37
0c) for a period of
one month on niosomal suspension and gel samples containing a known amount of
Tetracycline, contained in light resistant containers. Samples were withdrawn at weekly
intervals, and entrapment efficiency was determined.
In Vitro Release Pattern of Tetracycline Niosomal Formulations
The niosomal gel preparation was taken in a dialysis tube, which acts as a donor
compartment. Dialysis tube was placed in a beaker containing 250 ml of phosphate buffer
saline of pH 7.4, which acts as a receptor compartment diffusion medium was replaced after
every withdrawal so that the volume of the diffusion medium was maintained. The collected
samples were analysed at 255nm.[11,12]
Formulation of Gel
Carbopol 940 was soaked in water for overnight, then required quantity of niosomal
suspension was dispersed in solution with stirring and required amount of tri ethanolamine
and glycerine was slowly added with stirring to obtain a clear gel.[16,17]
Table 2: Composition of the Tetracycline Niosomal gel Formulation.
Ingredients For 25 gm
Tetracycline niosomal suspension 0.25 ml
Carbopol 0.25 gm
Tri ethanolamine 0.125 ml
Glycerol 2.5 ml
Purified water 21.8 ml
Evaluation of Tetracycline Niosomal gel formulation
The formulated gel was examined for following tests.
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Physical appearance
The prepared gel was examined for clarity, color, homogenicity and the presence of
aggregates.
pH
2.5 g of gel were accurately weighed and dispersed in 25 ml of distilled water. The pH of the
dispersion was measured by using a digital pH meter.
Rheological study
Viscosity was determined by Brookfield viscometer.
Drug content
Tetracycline encapsulated niosomal gel were transferred into a standard flask, then lysed
with1ml of 2.5% SLS solution, then incubated at 370 1
0 C for 2 hrs. Then it is filtered by
using whatman filter paper. Filtrate of 1 ml was diluted to 10 ml with PBS and absorbance of
the resulting solution was measured spectrophotometrically at 255 nm.
In vitro drug diffusion study
The niosomal gel preparation was taken in a dialysis tube, which acts as a donor
compartment. Dialysis tube was placed in a beaker containing 250 ml of phosphate buffer
saline of pH 7.4, which acts as a receptor compartment diffusion medium was replaced after
every withdrawal so that the volume of the diffusion medium was maintained. The collected
samples were analysed at 255nm.[13]
Study of Release Kinetics
The optimized formulation F1 was subjected to graphical treatment of assesses the kinetics of
drug release. The data obtained from the best formulation were fitted to various kinetics
equations (first order, second order, higuchi’s and koresmeyer’s –peppas model) to determine
the mechanism of drug release and release rate as indicated by higher correlation co efficient
(r2).
Stability Studies
Vesicle stability with respect to drug leakage and drug degradation upon storage was studied
at refrigeration (40c), room temperature (25
0c) and high temperature (37
0c) for a period of
one month on niosomal suspension and gel samples containing a known amount of
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Tetracycline, contained in light resistant containers. Samples were withdrawn at weekly
intervals, and entrapment efficiency was determined.
RESULTS AND DISCUSSION
FT-IR Studies
The IR Spectrum of pure Tetracycline drug was compared with the IR spectrum of physical
mixture of drug and excipients.
Fig. 2: FTIR Spectrum of Tetracycline.
Fig. 3: FTIR Spectrum of Carbopol.
Fig. 4: FTIR Spectrum of Cholesterol.
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Fig. 5: FTIR Spectrum of Tetracycline Carbapol.
Fig. 6: FTIR spectrrum of Tetracycline Cholesterol.
From the spectra, (Fig 2-6) there is no appearance or disappearance of any characteristics
peaks. This shows that there is no interaction between the drug and excipients used in the
vesicles preparation.
Table 3: Size Distribution of Niosomes by Hand Shaking Method.
Size range
(m)
Number of Niosomes
F 1 F 2 F 3 F 4 F 5 F 6
Below 0.1 18-20 16-18 8-9 18-20 13-15 6-8
0.1-5 70-72 72-75 85-87 65-70 68-74 82- 86
Above 5 13-17 6-9 5-9 3-6 4-6 4-5
Optical Microscopic view of Tetracycline loaded Niosomes prepared by Hand Shaking
Method
The optical microscope of the prepared niosomes reveals that they are discrete and spherical
in shape. The vesicles are slightly black in color. There is a thickening around the inner
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compartment of the vesicles prepared by hand shaking method. Individual vesicles could be
seen under high power as shown in Fig. 7.
Fig. 7: Optical Microscopic view of Tetracycline Loaded Niosome.
By scanning electron microscopy (SEM)
The prepared niosomes vesicles sizes were performed by SEM. The size range was observed
to be 0.5-5m. This is shown in Fig.8.
Fig. 8: Scanning Electron Microscopic view of Tetracycline Loaded Niosomes.
Fig. 9: Scanning Electron Microscopic view of Tetracycline Loaded Niosomal Gel.
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Drug Retention Studies
Table 4: Percentage of Drug Retention in Tetracycline Niosomal formulation.
Formulation
Code
Percentage of drug retention in niosomes
Refrigeration
Temp. (40 1 C)
Room Temp
(250 1 C)
High Temp.
(370 1 C)
Days Days Days
7 14 21 28 7 14 21 28 7 14 21 28
F1 100 95 90 86 99 88 84 77 94 85 73 69
F2 100 96 95 87 99 90 84 79 95 84 75 70
F3 100 97 95 90 100 94 87 80 95 85 76 70
F4 100 95 93 85 98 93 84 77 93 84 73 68
F5 100 97 94 86 99 95 85 78 94 85 74 69
F6 100 98 95 90 100 95 88 80 96 86 75 72
From the results, Niosomal formulation F1 showed good retention when comparing with
other formulations.
Table 5: Percentage of Drug Retention in Tetracycline Niosomal Gel formulation.
Formulation
Code
Percentage of drug retention in Niosomal Gel
Refrigeration
Temp. (40 1 C)
Room Temp
(250 1 C)
High Temp.
(370 1 C)
Days Days Days
7 14 21 28 7 14 21 28 7 14 21 28
Tetracycline
Niosomal Gel 100 99 98 96 98 90 86 81 96 86 79 70
The results of the prepared niosomal gel (F1) had showed good retention.
Fig. 10: Drug Retention Plot for Tetracycline Niosomal Gel.
Percentage of Drug Encapsulation
The percentages of drug encapsulated in niosomes are given in the Table 6. The Percentage
of drug encapsulation varies when the Span 20 proportion was varied. This explained that
encapsulation was increased with decrease in Span 20 content while cholesterol content was
maintained at constant value.
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Table 6: Encapsulation Efficiency for Tetracycline Niosomal Formulations.
Formulation Code Amount of drug used in (mgs) Percentage of drug encapsulated
F 1 100 mg 88.63
F 2 100 mg 81.34
F 3 100 mg 71.56
F 4 100 mg 70.23
F 5 100 mg 79.67
F 6 100 mg 86.29
Invitro Drug Release Study
In vitro drug release was studied for all the batches of niosomes. The studies were performed
up to 12 h for all the batches. The results show that the percentage of drug release at 12hrs of
F1 was found to be 61.40. It confirmed that F1 showed the sustained action.
Table 7: In vitro Dissolution Study of Trial Formulations of Tetracycline Niosomes.
Time F1 F2 F3 F4 F5 F6
0 0 0 0 0 0 0
2 16.48 18.55 19.62 20.3 17.69 17.29
4 26.34 28.37 32.64 36.94 29.8 30.47
6 29.56 43.78 47.56 48.78 34.38 53.49
8 42.24 55.04 65.76 64.87 47.8 72.88
10 56.74 68.8 72.78 73.28 62.48 84.93
12 61.40 74.89 84.23 87.18 70.08 93.73
Fig. 11: Invitro % Drug release of Trial formulations of Tetracycline Niosomes.
Study of Release Kinetics
Studies revealed that the release of drug from niosomes F1 formulation were found to be
Zero order kinetic indicating that the concentration was independent of drug release .More
over Korameyer – Peppas model indicated a good linearity(r2=0.989) and Peppas model was
0.5< n which implies that the drug follows Non-Fickian transport. From this study it was
found that the niosomal F1 formulation was diffusion controlled.
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Fig. 12: Zero Order Release . Fig. 13: First Order Release.
Fig. 14: Higuchi Model. Fig. 15: Korsmeyer’s Model.
Stability Studies
Vesicle stability with respect to drug leakage and drug degradation upon storage was studied
at refrigeration (40c), room temperature (25
0c) and high temperature (37
0c) for a period of
one month on niosomal suspension and gel samples containing a known amount of
Tetracycline, contained in light resistant containers. Samples were withdrawn at weekly
intervals, and entrapment efficiency was determined.
Table 8: Stability Studies.
Temperature
Amount of Drug Retained (%) ± S.D
Initial After 1
month
After 2
months
After 3
months
After 4
months
After 5
months
After 6
months
Refrigeration
(4°±1°C) 99.68±0.44 98.24±0.16 96.2±0.36 95.74±0.41 94.83±0.67 94.06±0.28 94.02±0.62
Room
Temperature
(25°C±2°C,
60%±5% RH)
99.68±0.44 97.17±0.34 95.36±0.23 93.74±0.72 93.24±0.36 93.18±0.81 92.17±0.73
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Fig. 16: Amount of Drug Retained.
Table 9: Invitro Release Profile of Optimized Formulation Before and After Storage at
Room Temperature (25°±2°C, 60%±5% Rh) for 6 Months.
Time in Hours Cumulative Percentage of Drug Released ±S.D
Before Storage After Storage
0.25 4.88±0.28 4.25±0.24
0.50 6.01±0.31 6.08±0.81
0.75 11.06±0.51 9.45±0.13
1.00 15.03±0.42 14.24±0.31
1.50 19.41±0.72 17.36±0.42
2.00 25.95±0.83 24.23±0.62
2.50 29.2±0.62 28.24±0.16
3.00 32.5±0.54 33.90±0.12
4.00 37.24±0.43 36.51±0.71
5.00 41.09±0.47 39.03±0.59
6.00 46.37±0.32 45.64±0.68
7.00 51.02±0.67 50.82±0.37
8.00 55.95±0.14 54.42±0.58
9.00 61.05±0.26 59.08±0.53
10.00 66.15±0.19 65.47±0.41
11.00 71.61±0.54 69.22±0.52
12.00 74.64±0.32 70.50±0.81
13.00 76.51±0.41 72.74±0.39
14.00 76.98±0.58 73.23±0.73
15.00 78.00±0.61 76.78±0.37
16.00 78.04±0.32 76.19±0.86
17.00 77.25±0.59 76.05±0.31
From these results, it was observed that there was no change in the drug release before and
after storage.
CONCLUSION
Niosomal gel drug delivery has been applied for topical application to increase the skin
penetration and skin retention. The prepared tetracycline topical gel can used for the
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treatment of Acne Vulgaris and showed good entrapment efficacy with smaller vesicle size.
From this it was concluded that the prepared tetracycline niosomal gel drug delivery can
improve the patient compliance and showed sustained activity.
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