Guar Gum Microspheres of 5-Fluorouracil for Colon Targeting

 

Amit Kumar Panigrahi*1, Mathrusri M. Annapurna2 and K. Himashankar3

1Aurobindo Pharma Ltd., Hyderabad

2GITAM Institute of Pharmacy, Visakhapatnam

3Bristol Laborotaries, Luton, United Kingdom

Corresponding author: amit.panigrahi@gmail.com

 

ABSTRACT:

The purpose of this investigation was to prepare and evaluate the colon-specific guar gum microspheres of 5-fluorouracil for the treatment of colon cancer. Guar gum microspheres were prepared by water-in-oil emulsification followed by cross-linking method using glutaraldehyde as a cross linking agent. Different ratios of drug and polymer (1:3 to 1:5), emulsifier concentrations (1%-3% w/v) and stirring speeds (1000-3000 rpm) were tried and the effects of these variables on drug delivery were studied. Particle size, shape, and surface morphology were significantly affected by drug: Polymer ratio, emulsifier concentration (Span 80) and stirring rate. Guar gum microspheres were evaluated for swellability, percentage drug entrapment, and in vitro drug release in simulated gastrointestinal fluids (SGF). The in vitro drug release studies of the formulations were also performed in simulated colonic fluid in the presence of 2% rat cecal content. The release profile of 5-FU from Guar gum microspheres was pH dependent. In acidic medium, the release rate was much slower; however, the drug was released quickly at pH 7.5. Guar gum microspheres showed adequate potential in achieving local release of drug in in-vitro release studies, It is concluded from the present investigation that Guar gum microspheres are promising controlled release carriers for colon-targeted delivery of 5-FU.

 

KEYWORDS: 5-Fluorouracil, guar gum, microspheres, colon targeting.

 


INTRODUCTION:

Colorectal cancer is the second leading cause of cancer deaths in the United States, and more than 66,000 cases of colon cancer are reported to occur in the Indian subcontinent every year. Conventional cancer chemotherapy is not very effective for treatment of colorectal cancer, as the drug molecule does not reach the target site at therapeutic concentration. Therefore effective treatment of colon cancer by conventional therapy requires relatively large doses to compensate for drug loss during passage through the upper gastrointestinal (GI) tract. These large doses may be associated with undue side effects. This can be overcome by site-specific delivery of the drug molecule to colon1. The approaches used in achieving colonic delivery of drugs include the use of prodrugs2, 3, pH-sensitive polymer coating4, 5, and time-dependent formulations6, 7, In addition, the use of biodegradable polymers such as azo-polymer and polysaccharide (e.g. gums, pectin and dextran etc.) for colon targeting are also reported in the literature8, 9

 

Several polysaccharides are being investigated as carriers for colon-specific drug delivery. The polysaccharides that are under active investigation for colon-specific drug delivery include pectin and its salts10, 11, chondroitin sulfate12, amylose13, dextran14 and chitosan15. It was reported previously that guar gum is a potential carrier for colon-specific drug delivery16. The degradation of guar gum in simulated colonic fluids by the action of bacterial enzymes is well documented. For example, Wong et al.17 demonstrated that galactomannanase (>0.1%) accelerated dissolution of dexamethasone and budesonide from guar gum matrix tablet containing 60.5% of guar gum, with the extent of drug dissolution depending on the concentration of galactomannanase. Also, in another two studies18, 19 the inclusion of galactomannanase, α-galactosidase or β-mannase (enzymes which act on guar gum) in the dissolution medium degraded guar gum or its derivatives and thereby released the drug contained in the guar-based formulations.

 

Since its introduction by Heidelberger et al in 195720, 5-fluorouracil (5-FU) has been the only agent with clinical activity against colorectal cancer. It is also used for other types of malignancies, such as those of the breast, head, and neck. Given its structural resemblance to natural pyrimidines, 5-FU interferes with nucleic acid synthesis, inhibits DNA synthesis, and eventually halts cell growth21, 22. Because of its incomplete and erratic oral bioavailability, 5-FU is commonly administered intravenously23. However, patients prefer oral rather than intravenous therapy24, with oral treatment potentially more convenient and less costly. The present regimens include an intravenous bolus or continuous infusion of 5-FU modulated with folinic acid (leucovorin)25, 26. On intravenous administration, 5-FU produces severe toxic effects of gastrointestinal, hematological, neural, cardiac and dermatological origin27. Site-specific delivery of 5-FU may reduce the systemic side effects and provide effective and safe therapy of colorectal cancer that may reduce the dose and duration of therapy when compared with the conventional treatment.

 

Guar gum is a polysaccharide derived from the seeds of Cyamopsis tetragonolobus of the Leguminosae family. It consists of linear chains of (1→4)-β-D-mannopyranosyl units with α-D-galactopyranosyl units attached by (1→6) linkages. In pharmaceutical formulations, guar gum is used as a binder, disintegrant, suspending agent, thickening Composition of the guar gum coat formulation for compression over agent and stabilising agent. The present paper describes the development and evaluation of colon targeting drug delivery systems for 5-fluorouracil using guar gum microspheres

 

MATERIALS AND METHODS:

Materials:

The 5-FU was a gift from Dabur Research Foundation (Ghaziabad, India). Guar gum (Sigma-Aldrich) was obtained from Alembic Ltd (Gujarat, India). Glutaraldehyde, castor oil and Magnesium Chloride were procured from Himedia, India. Isopropyl alcohol was obtained from Spectrochem, India. Hydrochloric Acid and Sulphuric acid were obtained from Qualigens Fine chemicals, India. All other reagents were of analytical grade or better.

 

Methods:

Preparation of Guar gum microspheres:

Guar gum microspheres wereprepared by using a combined method ivolving water-in-oil (w/o) emulsification and cross linking method following the method reported by Chourasia and Jain. Guar gum microspheres were prepared by using a combined method involving water-in-oil (w/o) emulsification and cross linking method28. The guar gum was allowed to swell in water and acidified with 5 ml dilute sulfuric acid. Drug (30 mg) was added to the guar gum solution with stirring. The emulsion was first prepared by dispersing the guar gum solution through syringe into a continuous oil phase consisting of 75 ml castor oil and 25 ml isopropanol with 2% (w/v) Span 80 in a 250 ml beaker at room temperature. The dispersion was stirred using a stainless steel stirrer (Remi, India) with half moon paddle at 1500 rpm for 10 minutes and thereafter 3 ml of glutaraldehyde was added into the beaker under stirring. The cross-linking reaction was allowed to proceed for total time of 3 hrs. Hardened microspheres were filtered and washed repeatedly with isopropanol and water to remove castor oil and unreacted glutaraldehyde, dried under vacuum at 40°C overnight and kept in a desiccator until further use. Similarly guar gum microspheres were prepared by taking polymer: drug in a ratio of 1:3, 1:4 and 1:5, stirring rate 1000 rpm, 2000 rpm and 3000 rpm and emulsifier (span 80) concentration 1%, 2% and 3%.

 

Surface morphology:

The shape and surface morphology of guar gum microspheres were investigated using scanning electron microscopy (SEM). The samples for SEM study were prepared by lightly sprinkling the formulation on a double-adhesive tape stuck to an aluminum stub. The stubs were then coated with gold to a thickness of ~300 Å under an argon atmosphere using a gold sputter module in a high-vacuum evaporator. The coated samples were then randomly scanned and photomicrographs were taken with a scanning electron microscope.

 

Particle size and particle size distribution:

The particle size and particle size distribution was measured in particle size analyzer (Malvern, USA). Microspheres were suspended in distilled water and the particle size and size distribution were determined using the software provided by the manufacturer.

 

Swellability:

A known weight (50 mg) of various FU-loaded guar gum microspheres were placed in enzyme-free simulated gastric fluid (SIF, pH 1.2) (for 2 hrs) followed by simulated intestinal fluid (SIF, KH2PO4/NaOH buffer, pH 7.4) (for 3 hrs) and allowed to swell for the required period of time at 37ºC±0.5ºC. The microspheres were periodically removed and blotted with filter paper; then their change in weight (after correcting for drug loss) was measured until attainment of equilibrium. The swelling ratio (SR) was then calculated using the following formula:

SR =

Where SR indicates swelling ratio, wo is initial weight of microspheres and wg is final weight of microspheres.

Percentage drug entrapment:

The percentage of drug entrapped in the microspheres was determined by digesting the microspheres (50 mg) in sufficient saline phosphate buffer pH 7.4 for 48 hrs. It was centrifuged at 3000 rpm for 30 min and the supernatant were analyzed spectrophotometrically at 266.6 nm. The percentage drug entrapment of guar gum microspheres was determined in the same manner.

 

Percentage drug entrapment= X 100

 

% Drug loading =  X 100

 

In-vitro drug release:

An accurately weighed amount of microspheres, equivalent to 100 mg of 5-FU, was added to 900 ml of dissolution medium and the release of 5-FU from microspheres was investigated using rotating paddle dissolution test apparatus (Electrolab, India) at 100 rpm and 37±0.5°C. The simulation of gastrointestinal transit conditions was achieved by altering the pH of dissolution medium. Initially it was kept at pH 1.2 for 2 hrs with 0.1N HCl. Then KH2PO4 (1.7 g) and Na2HPO4 .2H2O (2.225 g) were added to the dissolution medium adjusting the pH 4.5 for 3rd and 4th hr and adjusted with NaOH to 6.8 for 5th hr. After 5th hr, the pH of the dissolution medium was adjusted to 7.5 and maintained upto 8 hr. The final volume in all case was kept 900 ml. The samples were withdrawn from dissolution medium at various time intervals using a pipette fitted with micro-filter at its tips and analyzed spectrophotometrically at 266.6 nm.

 

Similarly In-vitro study was performed in simulated colonic fluid (pH 7.5 media) with 2% rat cecal matter. Rat cecal content was prepared by the method reported by Van den Mooter et al29. Four albino rats, (Sprague-Dawley strain) of uniform body weight (150-200 g) with no prior drug treatment, were used for all the present in vivo studies; they were weighed, maintained on normal diet, and administered 1 mL of 2% dispersion of guar gum in water, and this treatment was continued for 7 days for polymer induction to animals. Thirty minutes before starting the study, each rat was humanely killed and the abdomen was opened. The cecal were traced, legated at both ends, dissected, and immediately transferred into phosphate buffered saline (PBS) pH 6.8, which was previously bubbled with CO2. The cecal bag was opened; the contents were weighed, homogenized, and then suspended in PBS (pH 7.5) to give the desired concentration (2%) of cecal content, which was used as simulated colonic fluid. The suspension was filtered through cotton wool and ultrasonicated for 10 minutes in an ice bath at 40% voltage frequency using a probe sonicator at 4°C to disrupt the bacterial cells. After sonication, the mixture was centrifuged (Remi) at 2000 rpm for 20 minutes. Microspheres (100 mg) were placed in 200 mL of dissolution media (PBS, pH 7.5) containing 2% wt/vol rat cecal content. The experiment was performed with continuous CO2 supply into the dissolution medium. At different time intervals, the samples were withdrawn and replaced with fresh PBS. The experiment was continued up to 24 hours. The withdrawn samples were pipetted into a series of 10-mL volumetric flasks, and volumes were made up to the mark with PBS and centrifuged. The supernatant was filtered through 0.45-μm membrane filter (Millipore) and the filtrate analyzed for FU content at 266.6 nm using HPLC method. All the experiments were performed in triplicate.

 

Statistical Analysis:

The mean percentage of FU released in SGF (at different pH) from guar gum microspheres was prepared by using various drug: polymer ratios and compared. The Student t test was used to find the statistical significance. A value of P less than 0.05 was considered statistically significant.

 

RESULT AND DISCUSSIONS:

Guar gum microspheres of FU were successfully prepared by a combined method involving water-in-oil (w/o) emulsification and crosslinking method. Spherical microspheres were obtained as shown in scanning electron photomicrographs (Figure-1). The method was optimized using different ratios of drug and polymer, stirring speeds and emulsifier concentrations (details of the formulations given in Table-1) to produce microspheres of proper size and narrow size distribution, high drug loading efficiency and controlled drug release at the colonic pH. The details are discussed in following respective sub-headings.

 

Figure 1: SEM photomicrograph of guar gum microspheres.

 

Particle size and particle size distribution:

The particle size distributions of the microspheres of different formulations are given in the Table-2.

 

The particle size of the microspheres increased from 26.27±2.35 µm to 35.49±1.73 µm as the drug: polymer ratio was increased from 1:3 to 1:5.The increase in size of the microspheres may be attributed to an increase in viscosity of polymer solution with increasing concentration, which resulted in the formation of larger emulsion droplets and finally greater size of microspheres.

 

As the concentration of the emulsifying agent (Span 80) was increased from 1% to 3% w/v, the particle size of the microspheres was decreased from 36.38±2.51 µm to 28.65±2.27 µm. This may be due to the decrease of interfacial energy between the two droplets and the presence of emulsifying agent in the crosslinking medium, allowing the stabilization of the preformed microspheres to maintain their size until completion of the crosslinking reaction.

 

As the stirring rate was increased from 1000 rpm to 3000 rpm, the particle size of the microspheres was decreased from 35.62±1.62 µm to 27.36±2.63 µm. This may be due to formation of small size droplets on higher stirring rate.


Table-1: Different formulation approaches

Formulation code

FA1

FA2

FA3

FB1

FB2

FB3

FC1

FC2

FC3

Variables

Drug : Polymer ratio

Emulsifier (SPAN 80) concentration

Stirring rate

Values

1:3

1:4

1:5

1% w/v

2% w/v

3% w/v

1000 rpm

2000 rpm

3000 rpm

 

 


Swellability:

The swellability of different formulations performed in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.5) at 37±0.5°C are given in the Table-3.

 

Table-2: Particle size of microspheres prepared by different formulation approaches.

Sr. No.

Formulation code

Mean diameter of microspheres (in µm)

1

FA1

26.27±2.35

2

FA2

32.64±3.22

3

FA3

35.49±1.73

4

FB1

36.38±2.51

5

FB2

32.23±0.91

6

FB3

28.65±2.27

7

FC1

35.62±1.62

8

FC2

29.31±1.87

9

FC3

27.36±2.63

 

Table-3: Swelling ratio of microspheres prepared by different formulation approaches.

Sr. No.

Formulation code

% Swelling ratio

SGF

SIF

1

FA1

1.78±0.22

1.37±0.23

2

FA2

1.27±0.32

1.17±0.07

3

FA3

1.13±0.05

1.09±0.03

4

FB1

1.76±0.14

1.65±0.08

5

FB2

1.43±0.11

1.32±0.09

6

FB3

1.25±0.08

1.08±0.06

7

FC1

1.28±0.17

1.30±0.12

8

FC2

1.18±0.09

1.20±0.06

9

FC3

1.09±0.11

1.08±0.08

 

The result indicates that swelling ratio was increased with increase in drug: polymer ratio (from 1:3 to 1:5). A possible reason for this result may be due to the denser crosslink between the guar gum molecules, producing more packed structures in the formulations having more concentration of polymer (drug: polymer ratio less). Such a structure can be characterized by a lower and slower penetration of the solvent through the polymer chain.

 

Percentage drug entrapment:

The percentage drug entrapments of different formulations are given in the Table-4.

 

Table-4: Percentage drug entrapment of microspheres prepared by different formulation approaches.

Sr. No.

Formulation code

% Drug entrapment

1

FA1

76.32±1.45

2

FA2

81.29±1.17

3

FA3

86.78±2.71

4

FB1

71.14±0.81

5

FB2

74.54±2.23

6

FB3

76.14±1.17

7

FC1

85.34±0.78

8

FC2

79.55±0.26

9

FC3

75.21±1.08

The result shows that, on increasing drug: polymer ratio from 1:2 to 1:4, the entrapment efficiency was increased from 76.32±1.45 % to 86.78±2.71 %.

 

As the stirring rate was increased from 1000 rpm to 3000 rpm, the entrapment efficiency was decreased from 85.34±0.78 % to 75.21±1.08 %. This may be due to formation of small size microspheres with increased surface area. Higher stirring rate enhanced the diffusion of drug from such microspheres, resulting in the loss of drug from microspheres with a consequent lowering in the entrapment efficiency.

 

However the results showed that the change in the concentration of the emulsifying agent (span 80) had no significant effect in entrapment efficiency of the microspheres.

 

In-vitro drug release:

In-vitro drug release was carried out for guar gum microspheres in pH progression medium and simulated colonic fluid (pH 7.5 media) with 2% rat cecal matter and without 2% rat cecal matter.

Drug release from guar gum microspheres in pH progression media is represented in figure 2

 

Figure 2: Drug release from guar gum microspheres in pH progression media.

 

The cumulative percentage drug release from guar gum microspheres showed the desired rate, as there was no measurable drug release observed up to 2 hours in SGF (pH 1.2), while at pH 4.5, the drug release was quite insignificant (<2%) up to 4 hours.

 

The result indicates that, when drug:polymer ratio was increased in the preparation of guar gum microspheres, the in-vitro drug release from microspheres was decreased which may be due to increased path length for diffusion of drug molecule from microspheres. Drug release after 8 hrs was found to be 99.18±1.75% in case of microspheres prepared using 1:2 drug:polymer ratio, while it was 89.27±1.26% for microspheres prepared with 1:4 drug:polymer ratio.

 

Microspheres which were prepared using 1% w/v of emulsifying concentration, released 95.36±1.11% of drug after 8 hrs while those prepared using 2% and 3% w/v of emulsifying agent released  96.27±2.01% and 97.43±1.71% of drug after the same period. The result revealed that the concentration of emulsifying agent had no significant effect on drug release of the microspheres.

 

Microspheres which were prepared at stirring speed of 3000 rpm, released 97.28±2.15% of drug after 8hrs, while those prepared at 2000 rpm released 93.17±1.18% of drug after 8 hrs. The size of the microspheres prepared at 1000 rpm was large and hence effective surface area was less in comparison to those prepared at 2000 rpm and 3000 rpm, which could probably be the reason for the lesser amount of drug release (90.29±2.56% after 8 hr) from microspheres prepared at 1000 rpm.

 

Figure 3: drug release from guar gum microspheres in simulated colonic Fluid (pH 7.5) without rat cecal content.

 

Figure 4: drug release from guar gum microspheres in simulated colonic Fluid (pH 7.5) with rat cecal content.

 

For comparison, the in-vitro drug release study of the guar gum microspheres was performed in simulated colonic fluid (pH 7.5) with and without rat cecal contents. The drug release from guar gum microspheres in simulated colonic fluid (pH 7.5) with and without rat cecal contents is represented in figure 3 and figure 4 respectively.

 

The in vitro release of drug from guar gum microspheres in presence of 2% rat cecal content in simulated colonic fluid showed faster drug release at different time periods when compared with release study without rat cecal content. This finding could be attributed to the various anaerobic bacteria present in cecal content and responsible for digestion/degradation of guar gum in order to release drug from microspheres.

 

CONCLUSION:

The designed site-specific delivery of 5-FU from the system may reduce the side effects of the drug caused by its absorption from the upper part of the GI tract when given in conventional dosage forms such as tablets and capsules. The experimental results demonstrated that guar gum microspheres have the potential to be used as a drug carrier for an effective colon-targeted delivery system.

 

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Received on 03.12.2011          Modified on 15.12.2011

Accepted on 14.01.2012         © RJPT All right reserved

Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 267-272