Online - 2455-3891

Vol 9, Issue 3, 2016 Print - 0974-2441

Research Article

FORMULATION AND EVALUATION OF METRONIDAZOLE TABLETED MICROSPHERES FOR

COLON DRUG DELIVERY

SHIVANGI SINGH1*, NITHYA SHANTHI C2, ARUN K MAHATO2

1
Department of Pharmacy, IFTM University, Moradabad - 244 001, Uttar Pradesh, India. 2Department of Pharmaceutics,
SBSPGI, Balawala - 248 001, Dehradun, India. Email: [email protected]
Received: 11 March 2016, Revised and Accepted: 22 March 2016

ABSTRACT

Objective: The need of this study was to develop tableted microspheres that can be targeted to colon because metronidazole (MNZ) has good solubility
at pH 1.2; hence, coating of the drug with the suitable pH dependent is done to prevent its release in the gastric region.

Methods: Colon targeted tablets of MNZ were prepared with enteric coated microspheres using pH dependent polymers such as cellulose acetate
phthalate, hydroxypropyl methylcellulose phthalate, and Eudragit S 100 by solvent evaporation method. All the formulations were prepared by
changing drug-polymer ratio from 1:1 to 1:5 and the interactions of the drug with polymers were studied by Fourier transform infrared and thermal
analysis.

Results: Formulations F5, F8, and F14 were found to best optimized in percentage yield, drug entrapment efficiency, mean particle size and in vitro
drug release. The result obtained were found in the desired ranges where % yield ranging from 52.56% to 98.253%, drug entrapment efficiency from
42.17% to 99.017%, and mean particle size from 36.774 to 229.961 m. Then, tablet of optimized formulations was prepared by direct compression
method and in vitro drug release was performed. All the parameters of tablets were found acceptable as per IP guideline. Around 4-10% drug release
was in 0.1 N HCl after 2 hrs, 50% release at pH 7.4 phosphate buffer within 5 hrs, maximum retardation was found in the formulation of Eudragit S
100. Scanning electron microscopy permitted a surface topographical analysis.

Conclusion: The MNZ tableted microspheres showed their release at pH 7.4 thus this experimental work can be used to improve absorption of drug
in colon for successful treatment of the disease.

Keywords: Metronidazole, Tableted microspheres, Solvent evaporation method, Direct compression method.

INTRODUCTION Identification of drug

The drug was identified by melting point determination (capillary
Metronidazole (MNZ) is an amoebicidal drug, well known to kill the
tube method) on a silicone oil bath [8], Fourier transform infrared
trophozoites mainly present in the colon [1]. Thus, the development
(FTIR) spectroscopy analysis on Shimadzu and differential scanning
of tableted microspheres is necessary by coating of the drug with the
calorimeter (DSC) on precalibrated EXSTAR TG/DTA 6300.
suitable pH dependent and delayed release polymer by which the drug
can be targeted to colon as MNZ has a good solubility at pH 1.2 and the
Preparation of microspheres
pH of the terminal ileum and colon is higher (pH - 6.8 and 8). Thus,
The enteric microspheres were prepared by the solvent evaporation
coating of the drug with pH dependent polymer retards the release of
method. The formulation is given in Table 1. The polymer solution
drug from microsphere at low pH. Another problem associated with the
was prepared by dissolving it in acetone using a magnetic stirrer.
MNZ is its bitter taste, which mainly lead to patient non-compliance.
The powdered drug was then dispersed in the polymer solution. The
By entrapping the drug molecule into the polymeric layer, patient
resultant solution was then poured into a vessel of 250 ml containing
acceptance toward the drug can be increased. Microspheres are the
of liquid paraffin while stirring at the rate of minimum 1000 rpm.
carrier linked drug delivery system, in which particle size ranges from
Stirring was continued at room temperature until acetone evaporated
1 to 1000 m range in diameter having a core of drug and entirely outer
completely. After evaporation of acetone, the microspheres formed
layers of the polymer as coating material [2], constitute efficient carrier
were filtered and washed 4-5 times with n-hexane. Finally, the washed
capacity by virtue of their small size [3,4]. With regards to the final
microspheres were dried at room temperature and collected [9].
dosage form and easy administration, the microspheres are usually
formulated into single-unit dosage forms such as filling them into hard
Characterization of microspheres
gelatin capsules or compressing them into tablets [5,6]. The tableted
microspheres are preferred as a new approach in solid dosage form for Percentage yield
oral drug delivery. Microspheres can be compressed into a tablet to vary The percentage yield of microspheres was calculated by dividing the
the release properties of drugs [7]. weight of microspheres by the total weight of the added ingredients [10].

METHODS Percentage yield = The amount of microspheres obtained (g)/The

theoretical amount (g) 100
Materials
MNZ was taken as gift sample from Sunpharma New Delhi, cellulose
acetate phthalate (CAP) and other chemicals used in the study were Drug entrapment efficiency
procured from CDH, Delhi. HPMC phthalate and Eudragit S 100 were A weighed amount of drug loaded microspheres (equivalent to 25 mg
taken from Yarrow Chem Product, Mumbai. Liquid paraffin was of drug) was extracted using 10 ml of ethanol [10]. The solution was
purchased from LOBA, India. suitably diluted, and the absorbance was taken at max. The experiment
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Table 1: Formulation table for microspheres

Formulation code F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15

Drug (mg) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
CAP 100 200 300 400 500
HPMCP 100 200 300 400 500
Eudragit S 100 100 200 300 400 500
CAP: Cellulose acetate phthalate, HPMCP: Hydroxypropyl methylcellulose phthalate

was done in the triplicate. The drug entrapment efficiency was Table 2: Formulation table for tableted microspheres
calculated using the following formula:
Ingredients % composition
DEE = Actual drug content/Theoretical drug content 100
T1 T2 T3
Particle size analysis Microspheres 57 35.2 49
The diameter of microspheres from each formulation was determined Crospovidone 4.56 2.816 3.92
using an optical microscope. The samples were suspended in Magnesium stearate 1.14 0.704 0.98
Micro crystalline cellulose 37.3 61.28 46.1
dispersion, and individual microspheres diameter were measured using
micrometers. About diameter of 500 microspheres was measured, and
the mean particle diameter was calculated [10].
In vitro drug release studies
In vitro release studies The dissolution studies of marketed tablets and tableted microspheres
The drug release from the microspheres was carried out using the were performed. Initially, dissolution was carried out in pH 1.2 for
USP Type II dissolution paddle assembly. A weighed amount of enteric 2 hrs, and then, in pH 7.4 phosphate buffer using USP Type II Paddle
microspheres equivalent to 10 mg drug were dispersed in 250 ml of method at 50 rpm until the drug completely released from the tablet
0.1 N HCl (pH 1.2) maintained at 370.5C and stirred at 100 rpm. 5 ml under sink condition at 370.5C. At specific time intervals, aliquots
solution was withdrawn and replaced with fresh media after every were withdrawn and replaced by an equal volume of fresh dissolution
15 minutes up to 2 hrs. Then, dissolution medium was changed by medium to maintain a constant volume. After suitable dilution, the
pH 7.4 phosphate buffer. 5 ml solution was collected until a constant samples were analyzed max 317.6 nm [15].
release was found and analyzed at max [11].
Accelerated stability testing
Preparation of tableted microspheres The accelerated stability testing of the optimized tablet was done
The optimized MNZ loaded microspheres were compressed to form tablet according to ICH guidelines. Three set of formulation was packed in
of 250 mg using microcrystalline cellulose as diluents, crospovidone as high-density polyethylene bottle as study would be done for 3 months.
binder while magnesium stearate as a lubricant. Each tablet contains In an oven 40,C temperature and 75% humidity was maintained, and
10 mg drug, and the tablets were coded T1, T2, and T3 for each batch. these sets of formulations were kept. After every 1 month, one set of
The amount of microspheres equivalent to 10 mg drug and amount of each formulation was removed from the oven, and the analysis of drug
excipients used to prepare 250 mg tablet is given in Table 2 [12,13]. content and in vitro drug release were performed and compared with
the control sets [15].
Evaluation of tablets
Thickness RESULTS AND DISCUSSION

Thickness of the tablets was measured by vernier calipers. Three The melting point of MNZ was found to be 161.33C0.577. The reported
tablets were selected randomly from all the batches. valve of melting point is 159-163C [8]. From the result of melting point
determination of drug, the drug was identified as MNZ. The reported
Hardness test peaks (cm1) of MNZ by FTIR spectroscopy of -OH, -C-CH, -N-O, -C-O,
Hardness of tableted microspheres was determined using hardness and -C-N assignments were 3230, 3105, 1538 and 1375, 1078, and
tester. Three tablets were randomly picked from each batch and 830, respectively, whereas the observed peaks (cm1) were found to
analyzed for hardness. be 3228.09, 3096.03, 1538.76 and 1372.41, 1074.87, and 818.59,
respectively (Fig. 1). The DSC of the drug showed a sharp endothermic
peak at 160C for a pure MNZ as the melting point of drug (Fig. 2).
Weight variation
From each batch 20 tablets were selected at random and weight was After comparing the FTIR spectra of given drug and physical mixture
determined. Then, the tablets were weighed individually, and each of drug-polymer (Fig. 3), it was found that there were prominent peaks
weight was compared with an average weight. of functional group in physical mixture those can be identified in the
pure drug spectra. This revealed that there was no interaction between
Friability test drug and polymers used to prepare the microspheres. The DSC of
The friability of six tablets was determined using Roche Friabilator. The polymer, thermal transition occurs at 397C (Fig. 4), which is attributed
initial weight of these was noted. Then, all the tablets were weighed after to the melting point of the Eudragit polymer. In the physical mixture
friabilation. Friability can be determined by the following equation: of drug and polymer, the endothermic peak was observed at 160C as
the melting point of the drug. In case of the blank microspheres, an
% Friability= WtinitialWtfinal/Wtinitial 100 endothermic peak at 80C was observed due to dehydration of water
at the surface. The drug might have been dispersed in crystalline and
In vitro disintegration test amorphous form or dissolved in the polymeric matrix during formation
The tablet disintegration was carried out by placing one tablet in each of microspheres. After 250C where the polymer is in the form of liquid,
tube (6 tablets) of the basket, and the assembly was suspended in a a mild interaction between polymer or drug or degradation may be
beaker containing 0.1 N HCl (gastric pH 1.2) and operated without occurred. The evaluation of thermograms revealed that there was no
the disc for 120 minutes by maintaining temperature at 372C. The physical or chemical interaction found between drug and polymer.
experiment was carried out in triplicate [14]. For a pure polymer, thermal transition occurred at 222C and 397C

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attributed to the glass transition temperature (Tg) of polymer (Fig. 5). yield and particle size distribution of microspheres. Above 1000 rpm
The thermogram of the physical mixture showed almost the same stressing speed caused breaking of microspheres. Evaporation rate also
melting peaks at 160C and 281C with some lowering in enthalpy affected percentage yield and drug content. Below 2 hrs of evaporating
valves might be due to reduction in purity of the drug. rate resulted in incomplete emulsification of microspheres. Use of small
volume beaker for dispersion of microspheres resulted in higher yield
The microspheres were optimized with different polymers by changing and entrapment efficiency.
the volume of external phase, stirring rate, evaporation rate, and
evaporating surface area. The change in volume of external phase varied Characterization of microspheres
percentage yield, drug content, and particle size. It might be attributed Percentage yield of CAP microspheres ranges from 74 to 96,
to the fact that larger amount of external phase than 30 ml solubilized hydroxypropyl methylcellulose phthalate (HPMCP) microspheres
the microspheres formed. The stirring speed affects the percentage range 52-90%, and microspheres range 63-98%. The drug entrapment
efficiency of CAP microspheres varied from 42 to 94%, from 37 to
80% for HPMCP microspheres, and from 71 to 86 for Eudragit S 100
microspheres, respectively (Fig. 6).

Fig. 1: Fourier transform infrared spectra of drug

Fig. 4: Differential scanning calorimeter spectra of drug, Eudragit

S 100: Physical mixture of drug-eudragit S 100, blank formulation
F14, formulation F14

Fig. 2: Differential scanning calorimetry spectra of drug

Fig. 5: Tg spectra of drug, Eudragit S 100: Physical mixture of

drug-eudragit S 100, Blank formulation F14, Formulation F14

Fig. 3: Fourier transform infrared spectra of drug, cellulose

acetate phthalate (CAP): Physical mixture of drug - CAP,
hydroxypropyl methylcellulose phthalate (HPMCP): Physical
mixture of drug - HPMCP, Eudragit S 100: Physical mixture of Fig. 6: % Yield and drug entrapment efficiency of different
drug-eudragit S 100, blank formulation F14, formulation F14 formulations of microspheres

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The particle size analysis was performed on the 500 microspheres Around, 12% of drug was found to be released in 0.1 N HCl (pH 1.2) in
(Fig. 7). The mean particle size of microspheres ranged from 65 to CAP microspheres. Once the media changed to buffer of higher pH, 40%
524 for CAP, 36 to 166 for HPMCP, and 63 to 256 for Eudragit S 100, of drug was released in initial hours. At pH 7.4 more than 50% of drug
respectively. It is found that by increasing the drug-polymer ratio, there released within 3 hrs and more than 90% of drug was released within
is a shift toward the higher particles. Higher concentration of polymer 4.5 hrs (Fig. 9). The HPMCP microspheres showed about 7-9% of drug
produced a more viscous dispersion which formed larger droplets and release within 2 hrs at 0.1 N HCl, which is not significant. More than
consequently larger microspheres. The drug entrapment efficiency was 40% of released in pH 7.4 phosphate buffer within 3 hrs and 70-90% of
found to be higher for Eudragit S 100 microspheres than the CAP and drug released within 4 hrs (Fig. 10). Since the acrylic polymer used is
HPMCP microspheres because of its electric charge reduction [16]. The not soluble in acidic pH and starts to dissolve above pH 7, no significant
entrapment efficiency increased with increase in polymer concentration. amount of drug was released in 0.1 N HCl after 2 hrs, around 4% of drug
An increase in polymer concentration resulted in the formation of larger was released in 0.1 N HCl after 2 hrs. 50% of drug released within 5 hrs
microspheres entrapping greater amounts of the drug [17]. In case of at pH 7.4 phosphate buffer (Fig. 12).
particle size, it was found that mean size increased with increase in
polymer concentration. Increasing polymer concentration produced a Evaluation of tablets
significant increase in the viscosity [16], thus leading to an increase of The optimized microspheres formulation among above were
emulsion droplet size and finally a larger microsphere size [16], who compressed into the tablet form, and they were evaluated for various
suggested that the higher concentration of polymer in the sample had led parameters such as thickness, hardness, weight variation, friability, in
to an increased frequency of collision, resulting in fusion of semi-formed vitro disintegration test, and in vitro dissolution testing. The various
evaluation parameters of tableted microspheres are given in Table 3.
particles and producing an overall increase in size of microspheres. In
The scanning electron microscopy of microspheres of tableted
additional, the high viscosity of organic phase tends to restrict migration
microspheres (T3) containing individual microspheres scattered. There
of internal oil phase to external oil phase.
was no visible damage to microspheres. The microspheres inside the
tablet maintained their shape with no significant changes in their
The surface morphology and structure of microspheres were
surface properties (Fig. 13).
investigated using SEM. The surface of CAP microspheres was smooth,
spherical, and exhibited pores on its surface (Fig. 8). Such pores were
The tableted microspheres showed release of about 10% in CAP
due to the interconnectivity of internal phase droplets during the final
microspheres, 5% in HPMCP tableted microspheres, and about 2% in
stage of solvent evaporation [16, 18]. Whereas the surface of HPMCP
case of Eudragit S 100 within 2 hrs in 0.1 N HCl. The following are the
and Eudragit S 100 microspheres were rough and exhibited large pores
rank order of the drug release T1 (CAP) > T2 (HPMCP) > T3 (Eudragit
and cracks within crystalline drug on the surface of the microspheres
S 100) (Fig. 12). Eudragit S 100 tableted microspheres showed slower
(Figs. 9 and 10).
release than CAP and HPMCP in pH 6.8 maximum of about 7%, whereas
CAP showed release of 61% and HPMCP around 53%. This might be
The polymer solidified at the same time as the hardcore was formed,
due to the difference in solubility of polymer and interaction of the
resulting in the creation of coarse microspheres as reported [19]. The
drug with polymer. The in vitro drug release of MNZ loaded tableted
pores might form passages to help the drug release from inner pores of
microspheres was affected strongly by the pH of media.
microspheres (Fig. 11). The crystals of MNZ adsorbed on the surface of
microspheres contribute to a burst release and help to achieve effective One of the most important properties of a delayed release system is
concentration quickly after oral administration [20]. its resistance against the gastric condition. It is required that no more
than 10% drug degradation would occur after 2 hrs in 0.1 N HCl [21].
In vitro release profile of microspheres showed that increase in polymer When these tableted microspheres were kept in 0.1 N HCl for 2 hrs, the
concentration; decreased rate of drug release from microspheres. structural integrity of microspheres was almost maintained. However,
when these microspheres were kept in pH 7.4 phosphate buffer for
3 hrs, the microspheres progressively developed pores and tortuous

Fig. 9: Scanning electron micrograph of hydroxypropyl

Fig. 7: Particle size range of different formulation of microspheres methylcellulose phthalate microspheres

Fig. 8: Scanning electron micrograph of cellulose acetate Fig. 10: Scanning electron micrograph of Eudragit S 100
phthalate microspheres microspheres

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Table 3: Evaluation parameters of tableted microspheres

Evaluation Tableted microsphere

parameters
T1 T2 T3
Thickness (mm) 3.850.035 4.340.03 4.020.015
Hardness (kg/cm2) 5.690.02 5.600.015 5.740.03
Weight variation (mg) 252.331.15 248.330.57 252.660.57
Friability (%) 0.8 0.78 0.8
Disintegration 561 57.662.30 58.330.57
Fig. 11: Microspheres after drug release time (min)
Drug content (%) 93.8930.01 95.3450.02 98.8170.02

higher polymer concentration [16]. Further, smaller microspheres are

formed at a lower polymer concentration and have a larger surface area
exposed to dissolution medium giving rise to faster drug release.

Accelerated stability of T3 formulation was performed at 40C75% RH

for 3 months. The percentage drug content and in vitro drug release
studies were performed after every month for 3 months. The result
of percentage drug for control was 98%, whereas after 3 months was
97.56% (Fig. 14). The result was interoperated after similarity factor.
The similarity factor was found 99.76%, which is in the range 50-100%.
Thus, the formulation was considered to be stable.

CONCLUSION

The MNZ tableted microspheres showed their release at pH 7.4 thus this
experimental work can be used to improve absorption of drug in colon
Fig. 12: In vitro release studies of different formulation of for successful treatment of the disease. As MNZ has good solubility at
microspheres and tableted microspheres gastric pH and coating of drug with pH dependent polymer retards, its
release in pH 1.2. Among all the formulations tablet of F14 formulation
(T3) gave the good release. Its 3% part released in 0.1 N HCl in 2 hrs,
about 7% in pH 6.8 phosphate buffers, and rest of drug was released
in pH 7.4 phosphate buffer. There was 50% release in 4.5 hrs and 98%
release in 6.5 hrs.

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