Formulation and Development of Ethyl cellulose coated Pectin based Capecitabine Loaded Microspheres for Colorectal Cancer

 

Dilip M. Kumbhar¹*, Kailas K. Mali, Remeth J. Dias, Vijay D. Havaldar,  Vishwajeet S. Ghorpade, Nitin H. Salunkhe.

YSPM’s, YTC, Faculty of Pharmacy, Wadhe, Satara, 415011 India

 

 

ABSTRACT:

The main objective of present investigation was to design the colorectal microspheres of Capecitabine by using inexpensive natural polysaccharide based polymers. The pectin microspheres were prepared by single emulsification technique using calcium chloride as crosslinking agent. Pectin loaded microspheres were coated with ethyl cellulose by solvent evaporation method. The prepared microspheres were characterized by entrapment efficiency, particle size, in-vitro drug release, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and Differential scanning calorimetry (DSC). The in-vitro drug release behaviour of ethyl cellulose coated pectin microspheres done in varying pH conditions up to 12 hr. Optimized uncoated batch of pectin based capecitabine microspheres showed optimum particle size with good drug encapsulation efficiency and spherical in nature. All the formulations showed less than 20 % drug release in acidic environment. Ethyl cellulose coated microspheres mininised initial burst effect and showed drug release in the range of 85.33 to 95.55% at the end of 12h. As concentration of ethyl cellulose was increased drug release was found to be retarded well. From above results it can be concluded that capecitabine loaded ethyl cellulose coated pectin based microspheres may be the best alternative to conventional tablets to treat the colon cancer.

 

 

INTRODUCTION:

Among various diseases, cancer has become a big threat to human beings globally. Cancer is the second most common disease in India responsible for maximum mortality with about 0.3 million deaths per year. This is owing to the poor availability of drug for prevention, diagnosis and treatment of the disease¹. Colorectal cancer is the second leading cause of cancer death in USA and more than 66 thousand of colon cancers are reported to occur in the Indian subcontinent every year².

 

Conventional cancer therapy is not very effective for treatment of colorectal cancer. Therefore, effective treatment of colonic cancer by conventional therapy requires relatively large doses to compensate drug loss during passage through the upper GI tract. This large dose may be associated with undue side effects which can overcome by site specific delivery of the drug molecule to colon³.

 

The chemotherapeutic agents can be administered via the oral route. In comparison to injection, oral administration of anti-cancer agents is expected to improve the quality of life of patients and increase the cost-effective treatment through reducing the duration of hospitalization. The transformation of injectable to oral route is deemed to be a feasible approach in drug administration. Indeed, the orally administered capecitabine has been recommended clinically as an equivalent alternative to intravenously administered 5-fluorouracil-leucovorin⁴.

 

Microsphere delivery systems have been used for localized drug delivery to reduce side effects as well as to improve the therapeutic response at the local site⁵. Polysaccharides are bacterial enzymes that are available in sufficient quantity to be exploited in colon targeting of drugs. Based on this approach, various polysaccharides have been investigated for colon specific drug release. These polysaccharides include pectin, alginate, guar gum, amylase, inulin, dextran and chitosan⁶.

 

Capecitabine (N-[1-(5-deoxy-beta-D-ribofuranosyl)-5-fluoro-1,2-dihydro-2-oxo-4-pyridinyl]-n-Pentylcarbamate) is a crystalline substance with a molecular weight of 359.35. It is highly soluble in water and is a prodrug of flurouracil following oral administration. It is used for breast cancer and colorectal cancer. It is readily absorbed from gastrointestinal tract. It has a short half-life of 0.5 to 1h. The frequent need for capecitabine dosage adjustment due to adverse effects in both the colorectal and breast cancer populations suggests that a lower starting dose may be beneficial⁷.

 

Based on this, we planned to develop microsphere based colon targeted drug delivery which prevents drug release in upper part of gastrointestinal track and target colon. The rationale behind this is, ethyl cellulose prevents the drug release in the upper part of the GIT and gives the local action in the colon to treat the colonic cancer. Hence, attempt was made to prepare and evaluate the colon specific ethyl cellulose coated pectin alginate capecitabine loaded microspheres for the treatment of colorectal cancer and to study effect of processing variables on the drug entrapment efficiency, particle size and in-vitro drug release.

 

MATERIALS AND METHODS:

Materials

Capecitabine was obtained as gift sample from Aarti Drugs Pvt. Ltd., Mumbai, India, and pectin, Span 85, calcium chloride, n-hexane, and acetone were purchased from Loba Chemie Mumbai. Ethyl cellulose and dichloromethane were purchased from SD Lab., Mumbai. All other reagents used were of analytical grade.

 

Methods

Method of preparation of capecitabine microspheres

Capecitabine loaded pectin microspheres were prepared by single emulsification method with modification ^8,9. Detailed formulation is given in table 1. In brief, accurately weighed pectin (450mg) and sodium alginate (50mg) was transferred into 10ml distilled water with continuous stirring on magnetic stirrer for 30 min. 100mg drug was dispersed into above solution and 40ml n-hexane containing 1.25% Span 85 [1.83ml]  was added to it and stirred at 1100 rpm (RQG-126/D, Remi, Ahmadabad) for 10 min. Finally, 30ml acetone was added for hardening of microspheres and stirred for 10min. Then 10ml calcium chloride (22%) was added as a cross linking agent and again stirred for 10min at same speed. Resulting microspheres were washed with n-hexane, filtered, dried at room temperature and stored in dessicator.

 

 

 

 

Table 1. Formulation of capecitabine loaded pectin microspheres

Batch  No	Drug (mg)	n-Hexane (ml)	Acetone (ml)	Pectin: SA 9:1 %    (8ml)	CaCl2 (%) (10 ml)	Span 85 (%)	Speed (rpm)
M1	100	40	30	3%	22%	1.25%	1100
M2	100	40	30	4%	22%	1.25%	1100
M3	100	40	30	5%	22%	1.25%	1100
M4	100	40	30	6%	22%	1.25%	1100
M5	100	40	30	5%	22%	0.75%	1100
M6	100	40	30	5%	22%	1%	1100
M7	100	40	30	5%	22%	1.50%	1100
M8	100	40	30	5%	22%	1.25%	900
M9	100	40	30	5%	22%	1.25%	1000
M10	100	40	30	5%	22%	1.25%	1200

 

 

 

Preparation of ethyl cellulose coated drug loaded pectin microspheres

Ethyl cellulose coated capecitabine loaded pectin microspheres were prepared by solvent evaporation method. Details of formulation batches are given in table 2. Ten millilitre solution of ethyl cellulose was prepared in dichloromethane. Then accurately weighed 400mg capecitabine microspheres were transferred into it and solvent was evaporated to dryness. Then coated microspheres were transferred into petri dish for air drying.

Table 2. Formulation of Ethyl cellulose coated capecitabine loaded pectin microspheres

 

 

Drug-polymer interaction study

Fourier transform-infrared spectroscopy (FTIR) studies

IR spectrum of capecitabine, sodium alginate, pectin, ethyl cellulose and physical mixture were recorded on ATR Fourier Transform Infrared Spectrophotometer (MIRacle 10, Shimadzu, Japan). The spectrum was scanned in the wavelength region of 4000 to 400 cm^-1. The spectra obtained for capecitabine, polymers and physical mixture of capecitabine with polymers were compared.

 

Differential scanning Calorimetry (DSC) studies

Thermal analysis by differential scanning Calorimetry (DSC) was carried out on the plain drug, pectin polymer, physical mixture and the optimized formulation using Model-SDT Q600 V20.9 Build 20 with a computerized data station. Samples were placed in an aluminium pan and heated at a rate of 10°C/min in the temperature range of 30–300°C. The thermal analysis was performed under nitrogen atmosphere.

 

Percentage yield

The prepared microspheres were collected and weighed. Percent yield was calculated by using following  equation ¹⁰:

              

 

Particle size and shape

The particle size of microspheres was determined by using Motic microscope (BA210). Initially, all magnification lenses were calibrated using calibration slide provided by manufacturer. Microspheres were transferred to slide and slide was fixed in position on the stage. 10X magnification lens was used for determination of particle size. Then in built camera was turned on. After the coarse and fine adjustment image was captured by using Motic Images plus 2.0 software. Finally, particle size was determined.

 

 

 

 

Drug entrapment efficiency

To determine entrapment efficiency, an accurately weighed 100mg of microspheres was dispersed in100 ml of phosphate buffer pH7.4 in volumetric flask and shaken vigorously for 24 h using rotary shaker (Bio-Technics, India). Supernatant was filtered using a Whatman filter and analyzed for drug content by UV Spectrophotometer (10).  Entrapment efficiency was measured by using following formula:

 

% Drug entrapment =

[Practical content/ Theoretical content] X100

 

Surface morphology

The shape and Surface morphology of Pectin microspheres and ethyl cellulose coated pectin microspheres were observed by using scanning electron microscopy. Microspheres was sprinkled on to double side tape, sputter coated with platinum and examine in the microscope at 15 kV electron beam.

 

In vitro drug release

The in vitro release of microspheres was carried out in USP paddle Type II dissolution apparatus using 900ml of simulated gastric fluid 0.1N HCl for 2 h and, the dissolution medium was replaced with simulated colonic fluid (phosphate buffer pH 7.4) for next 10 h. Temperature of dissolution medium was maintained at 37.0±0.5°C and a stirring rate was of 50 rpm. Microspheres ware weighed accurately and filled in cellophane tube. The cellophane tubes were tied using thread to paddle and loaded into flask of dissolution apparatus containing 900ml of dissolution medium. At predetermined time intervals aliquots of samples (5ml) are collected and replenished with same volume of fresh media to maintain the sink conditions. The drug content in the samples was estimated using UV spectrophotometer at 303 nm wavelength. Release data was fitted to kinetic models.

 

RESULT AND DISCUSSION:

Drug-polymer interaction study

The overlay ATR-FTIR spectra of capecitabine, polymers and physical mixtures is shown in figure 1. IR spectrum of pure capecitabine exhibits presence of characteristic peaks at 3388 cm^-1 for N-H stretching, 2850 to 2950 cm^-1 for C-H stretching due to alkyl, 1712 cm^-1 represented (C=O) stretching due to carbonyl bond, 1064 cm^-1 for C-O-C stretching due to ether. The spectra of physical mixtures revealed presence of all capecitabine peaks at same wavelength with same intensity indicating that there were no any unwanted interaction between drug and excipients used in the study¹¹.

 

 

 

 

Figure 1. Overlay IR spectra

A-Capecitabine, B-Pectin, C-Sodium alginate D-Ethyl cellulose, E- Capecitabine+Pectin, F-Capecitabine+sodium alginate, G-Capecitabine + Ethyl cellulose, H-Capecitabine+Pectin+Sodium alginate+Ethyl cellulose

 

 

 

The overlay of DSC Thermogram is shown in figure 2. The DSC Thermogram of capecitabine showed an endothermic peak at 120ºC, which corresponds to the melting point of capecitabine. In case of pectin a small endothermic peak was observed at 79ºC corresponding to the glass transition temperature (TG) of pectin. It may also be due to the elimination of bound water in the pectin sample. The DSC of pectin also showed an endothermic peak 186ºC, which indicates the melting temperature of pectin. The physical mixture of capecitabine and pectin showed a small endothermic peak at 124ºC corresponding to the melting point of drug, which suggested lack of interaction in between capecitabine and pectin in the physical mixture. However, the DSC of formulation M3 showed disappearance of melting peak of capecitabine, which may be due to molecular dispersion of capecitabine within the polymeric matrix of pectin microspheres¹².

 

 

Figure 2 .Overlay of DSC Thermograms A- Capecitabine,

B-Pectin, C-Physical mixture, D-formulation M3

 

Percentage Yield

Percentage yield of microspheres was found to be in the range of 70 to 92 %. The loss of materials during preparation of microspheres may be due to the process parameters as well as during filtration of microspheres¹³.

 

 

Particle Size and shape

Motic images of all batches are given in figure 3. Size of M1 to M10 microspheres batches was found to be in the range of 140-260µm. Particle size of all batches are given in table 3. Shape of batch M2, M4, M5 and M6 was found to be irregular while M1, M3, M7, M8, M9 and M10 were found to be spherical.  This may be due to variation in polymer concentration, stirring speed and curing time.

 

By increasing the amount of pectin for microsphere preparation, a progressive increase in both mean size and shape was observed. This may be due to higher concentration of polymer that produced a more viscous dispersion, which formed larger droplets and consequently larger microspheres¹⁴.

 

Effect of stirring speed on mean particle size (µm), and shape of microspheres were determined. The stirring speed was varied from 900 to 1200 rpm. The mean diameter of microspheres was found to be decreased with increasing agitation speed of the mechanical stirrer. This result was expected because high stirring speed provides the shearing force needed to separate oil phase into smaller globules. The stirring speed of 1100 rpm was found to be optimum for pectin microspheres. High stirring speed (1200rpm) produced an irregular shape of microspheres¹⁵.

 

The amount of surfactant was varied from 0.75 to 1.50%. The mean diameter of microspheres was found to be varied with increase in emulsifier concentration (Span 85) from 0.75 to 1.5% w/v. Increased surfactant concentration led to the formation of particles with a lower mean diameter. Increasing Span 85 concentration from 0.75% to 1.5 % w/v led to stabilization of the emulsion droplets avoiding their coalescence, resulting in smaller microspheres¹⁶.

 

 

 

 

 

Figure 3. Motic images (10X) of pectin based uncoated microspheres of Capecitabine

 

 

 

 

 

 

 

Table 3. Particle size and % DEE of capecitabine loaded pectin microspheres

 

 

Drug entrapment efficiency

Entrapment efficiency for all batches was found to be in the range of 35.28 to 58.60%. When the polymer concentration was increased percent drug encapsulation was found to be increased. This may be due to more entrapment of drug at high polymer concentration.(14) Based on drug entrapment efficiency 5% pectin concentration was optimized which exhibited 58.6% drug entrapment efficiency. The drug loading efficiency was found to be increased with increasing emulsifier concentration from 0.75% to 1.25% during preparation of pectin microspheres ^8,17-18. When stirring speed was increased entrapment efficiency was found to be decreased.

 

Surface morphology

SEM study of batch M3 indicates that the capecitabine loaded pectin microspheres have smooth surface and are spherical in shape, which is given in figure 4.

 

 

 

Figure 4. SEM of pectin based uncoated microspheres of Capecitabine

 

 

In vitro drug release of capecitabine microspheres

Optimised batch of pectin based capecitabine microspheres (M3) showed 98.33 % drug release at the end of 12h. Initially, burst effect was observed due to sudden release of drug from the surface of microspheres. Afterwards  drug release was retarded due to swelling of the polymer up to 8h then drug release was increased due to polymer erosion.

 

Evaluation of ethyl cellulose coated pectin microspheres

Particle size and shape

Motic images of all five batches are given in figure 5. Particle size of all batches were found to be in the range of 154-329µm (table 4).   Particle shape of all batches was found to be spherical. This indicates that the coating of microspheres by ethyl cellulose was performed successfully. As concentration of polymer was increased particle size of microspheres was also found to be increased. This may be due to the increased polymer solution coating on the microspheres¹⁹.

                             

 

 

Figure 5. Motic images (10X) of pectin based coated Microspheres of Capecitabine

Surface morphology

SEM of pectin based ethyl cellulose coated microspheres of Capecitabine showed in figure 6. Scanning Electron microscopy of Ethyl cellulose Capecitabine loaded pectin microspheres showed spherical shape and smooth surface of microspheres.

 

 

Figure 6. SEM of pectin based ethyl cellulose coated microspheres of Capecitabine

 

In-vitro drug release

The in vitro drug release of ethyl cellulose coated microspheres showed drug release retardation in the colon. As the concentration of ethyl cellulose increased the rate of drug release decreased. All formulations showed release in the range of 85.33 to 95.55 % at the end of 12h (figure 6). The burst effect was avoided due to ethyl cellulose coating which has insoluble nature. All formulations retarded drug release as compared to uncoated microspheres. A sudden drug release rate was increased due to erosion of ethyl cellulose coating as well as pectin after 7h.

 

All formulations followed zero order release kinetic model (table 4). Release exponent ('n' value) was found to be in the range of 0.96 to 1.19. Anomalous drug release was observed for uncoated microspheres indicating swelling with erosion mechanism. All ethyl cellulose coated microspheres showed n value above one which indicates that drug release is controlled by more than one process²⁰. Drug release data was also fitted to model independent parameters, dissolution efficiency and mean dissolution time. It was observed that dissolution efficiency was found to be decreased and mean dissolution time was increased in case of ethyl cellulose coated pectin microspheres.

 

 

Table 4. Evaluation of Ethyl cellulose coated batches

Formulation Code	M3	M3A	M3B	M3C	M3D	M3E
Particle Size (µm)	184.25 ±25.01	196.13± 22.3	201.98±29.1	216.98±29.1	243.06±22.7	265.5± 31.62
% drug release, 1h	14.07	13.44	12.8	11.33	11.05	10.07
% drug release, 12h	98.33	95.55	92.33	90.45	88.66	85.33
Dissolution efficiency (%)	43.67	40.84	38.65	36.25	34.02	31.88
Mean dissolution time (h)	6.67	6.87	6.97	7.19	7.39	7.51
Best fit model (r value)	Zero order (0.951)	Zero order (0.937)	Zero order (0.935)	Zero order (0.927)	Zero order (0.907)	Zero order (0.894)
Release exponent, n	0.968	1.017	0.995	1.073	1.157	1.191

 

 

 

Figure7. Release profiles of Capecitabine microspheres

 

 

CONCLUSION:

The present investigation concluded that the ethyl cellulose coated pectin based microspheres of capecitabine for colorectal cancer reduces early drug release in acidic environment which may avoid unwanted side effects and absorption of capecitabine at upper GIT tract as compared to the other oral conventional formulations. Therefore, it could be concluded that ethyl cellulose coated capecitabine pectin microspheres have potential to treat the colorectal cancer effectively.

 

ACKNOWLEDGEMENT:

Authors are grateful to Arati Drugs, Mumbai for providing gift sample of drug. Also authors are thankful to Prof. D. B. Sagare, Founder President and Prof. A. D. Sagare, Vice President YSPM’s Yashoda Technical Campus, Satara

 

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Received on 05.07.2016       Modified on 31.10.2016

Accepted on 20.11.2016     ©A&V Publications All right reserved