Oral Formulation of Anticancer Agents for Colon Cancer

Aims/Objective: To develop and estimate enteric-coated capsules containing mucoadhesive Microspheres of Capecitabine and Oxaliplatin to treat Colon cancer. Study Design: Box Behnken. Place and Duration of Study: Department of Pharmaceutics, Parul Institute of Pharmacy and Research, Parul University, Vadodara, between 2017 to 2021. Methodology: Capecitabine and Oxaliplatin are used as antineoplastic agents and can be delivered via the oral route of administration. For the estimation of drugs Analytical method has been developed by HPLC. Box Behnken design has been used to optimize Drug: polymer ratio (1:2), Inlet temperature 170oC, and crosslinking agent with a 0.5 ml 1% Gluteraldehyde solution. The microspheres were successfully prepared by using the spray drying technique and evaluated. Results: The results of optimized Capecitabine microspheres were obtained as Particle size 87.91 μm ± 0.274,% yield 57.21± 1.5,% Mucoadhesion 57.21± 1.5,% entrapment efficiency 82.16± 0.725. Original Research Article Patel and Patel; JPRI, 33(46B): 402-418, 2021; Article no.JPRI.75792 403 The results of optimized Oxaliplatin microspheres were obtained as Particle size 99.88μm±0.034,% yield 56.0± 0.088,% Mucoadhesion 87.0± 0.80,% entrapment efficiency 82.61±0.085. The drug content of Capecitabine and Oxaliplatin in the filled capsule was 94.67% ±0.32 and 93.45%±0.712, respectively. % Drug release of Capecitabine and Oxaliplatin in Phosphate buffer pH 7.4 was found to be 94.83±0.22 and 96.94±0.11 respectively after 8 hrs. Stability study at 40 0 C±2 0 C / 75 ± 5 % RH revealed that there was no significant change in disintegration time, drug content and % CDR during 6 months. So, prepared formulation was stable during stability study. MTT assay has been performed on the formulation of Capecitabine and Oxaliplatin microspheres for assessing the % viability of both the drugs on the Caco-2 cell line. Conclusion: The present study confirmed that prepared mucoadhesive microspheres filled with enteric-coated capsules have an antitumor effect on colon cancer cells. The formulation induced high cell death within 48 hours, and less cell viability was obtained compared to API. Six months accelerated Stability study indicates that formulation is fairly stable at storage conditions.


INTRODUCTION
Cancer is a group of diseases involving abnormal cell growth to invade or spread to other parts of the body. Benign tumours do not spread to other parts of the body [1]. Colon cancer is the cancer of the epithelial cells lining the colon. Colorectal cancer is mainly divided into different stages according to the invasiveness and metastatic ability of the tumour. Diagnosis of colorectal cancer can be made by sigmoidoscopy or by colonoscopy with biopsy confirmation of cancer tissue. Treatment of colorectal cancer range from surgery in the early stages to palliative care in the most advanced stages [2]. Capecitabine is currently used as first-line therapy in patients with metastatic colorectal cancer [3]. Oxaliplatin is an anticancer ("antineoplastic" or "cytotoxic") chemotherapy drug. Oxaliplatin is used to treat colon or rectal cancer that has spread (metastasized); it is often given in combination with other anticancer drugs (fluorouracil and leucovorin) [4,5].
Capecitabine was developed as a prodrug of Florourosil, with the goal of improving tolerability and intratumor drug concentrations through tumor-specific conversion to its only active metabolite, FU, by thymidine phosphorylase. Higher levels of this enzyme are found in several tumors and the liver, compared with normal healthy tissue [3]. Capecitabine is metabolized to 5-FU which in turn is a Thymidylate synthase inhibitor, hence inhibiting the synthesis of thymidine monophosphate (ThMP), the active form of Thymidine which is required for the synthesis of DNA [1].
Capecitabine is currently use as first-line therapy in patients with metastatic colorectal cancer when single-agent fluoropyrimidine therapy is preferred. The drug is also approved for use as a single agent in metastatic breast cancer patients who are resistant to both anthracycline-and paclitaxel-based regimens or in whom further anthracycline treatment is contra indicated Improved tolerability and comparable efficacy compared with IV FU/LV in addition to oral administration make Capecitabine an attractive option for the treatment of several types of cancers as well as the focus of future trials [3].
Oxaliplatin is an anti-cancer ("antineoplastic" or "cytotoxic") chemotherapy drug. Oxaliplatin is classified as an "alkylating agent." Alkylating agents are most active in the resting phase of the cell. These drugs are cell-cycle non-specific. There are several types of alkylating agents [4].
Microspheres are small spherical particles with diameters in the micrometer range (typically 1 μm to 1000 μm). Microspheres are sometimes referred to as microparticles. Microspheres can be manufactured from various natural and synthetic materials [6,7] . Mucoadhesive microspheres enhance the intimate contact with the mucus layer and drug targeting to the absorption site by anchoring bacterial adhesions, plant lectins, antibodies etc [7,8]. Mucoadhesive Microspheres prolong the residence time of the dosage form at the site of absorption. Due to an increased residence time, it enhances absorption and hence the therapeutic efficacy of the drug. Delivery via the oral route, increase in drug bioavailability due to first-pass metabolism avoidance. The drug is protected from degradation in the acidic environment in the gastrointestinal tract: improved patient compliance -ease of drug administration. Faster onset of action is achieved due to the mucosal surface having an enormous blood supply and good blood flow rates. Therefore, site-specific activity, reduced systemic side effects (systemic cytotoxicity), reduced dose and toxicity, Increased stability, and provided constant and longer therapeutic effect. Reduces the frequency of daily administration and thereby improves patient compliance. Improve the absorption of drug hence improve the bioavailability of drug and reduce the chances of adverse effects. The morphology of Microspheres permits a controllable variability in degradation and drug release [7,8]. Oral administration of drugs is one of the most convenient and patient-accepted methods of drug delivery. However, the gastrointestinal microenvironment presents many delivery challenges, including the acidic conditions of the stomach, the proteolytic activity of the gastrointestinal tract due to the presence of digestive enzymes, and the high density of bacterial species. While intravenous administration of chemotherapeutics is common practice, the oral route provides an anatomical advantage for delivering such agents. It permits direct access to the luminal tissue affected by many diseases. One promising method for oral drug delivery involves mucoadhesive biomaterials such as chitosan [9].
Enteric-coated HPMC Capsule Shell has been commercially available to the dietary supplement industry as a vegetarian alternative to gelatin. As HPMC is often used as a pre-coating material for enteric-coated tablets, it may be expected that the application of enteric-type polymers to a capsule made from HPMC would result in suitable Polymer to Polymer adhesion and compatibility [10].
The main objectives of the study are focused on decreasing the Drawbacks associated with the Current Therapy of the Colon Cancer, through targeting of the Antineoplastic agents at the site of the Tumor. Reduction of systemic adverse effects: due to site specific drug release, the systemic sideeffects of both the drugs will be reduced.
Patient compliance as both the drugs are delivered in single formulation and convenient route of administration.

Differential scanning Calorimetry (DSC)
DSC analysis was conducted using a Thermal Analyzer. Samples, including Capecitabine, physical mixture of Capecitabine and polymer, Oxaliplatin, physical mixture of Oxaliplatin, and polymer, were weighed into an aluminium pan, which was sealed with a pinhole-perforated cover. The samples were purged with dry nitrogen at a flow rate of 20 mL/min. Heating curves were record at a scan rate of 10°C/min from 30 to 400. Heating Curves of Drugs and the Physical mixture of Polymers were recorded [11,12].

Method of preparation for Capecitabine microspheres
Dissolve chitosan in 1% v/v glacial acetic solution. Then add 150mg Capecitabine and dissolve in 10 ml water. Sonicate the solution for 5 min, then mix both the solutions and add 1% 0.5ml glutaraldehyde solution. Stir at 500 rpm/min for 30 min, then spray dried at inlet temperature 170°C, outlet temperature 120°C, and the flow rate was 5 mL/min using a spray drier. Collect dried microspheres, weigh and evaluate [13,14].

Method of preparation for Oxaliplatin microspheres
Dissolve chitosan in 1% v/v glacial acetic solution. Then add 50mg Oxaliplatin and dissolve in 10 ml water. Sonicate the solution for 5 min, then mix both the solutions and add 1% 0.5ml glutaraldehyde solution. Stir at 500 rpm/min for 30 min, then spray dried at inlet temperature 170°C, outlet temperature 120°C, and the flow rate was 5 mL/min using a spray drier. Collect dried microspheres, weigh and evaluate [13,14].

Surface Morphology
Shape and surface morphology was studied with Scanning electron microscopy (SEM) [14].

Particle size
The particle size of the microspheres was determined by using the optical microscopy method. A small amount of dry microspheres was suspended in distilled water. A small drop of the suspension was placed on a clean glass slide.
The slide containing suspended microspheres was mounted on the microscope stage, and 100 particles were measured using a calibrated ocular micrometer. The process was repeated three times for each batch prepared [14,15].

Flow Properties
The flow properties of microspheres were investigated by determining the angle of repose, bulk density, tapped density, Carr's and Hausner's ratio. Each parameter was calculated three times for each batch prepared, and results were averaged.
The angle of Repose: Angle of repose (θ) was measured according to the fixed funnel of Banker and Anderson. A funnel with the end of the stem cut perpendicular to the axis of symmetry is secured with its tip at a given height of 1cm (H) above graph paper placed on a flat horizontal surface. The microspheres were carefully poured through the funnel until the apex of the conical pile so formed just reached the tip of the funnel. Thus, the R being the radius of the base of the microspheres conical pile: θ θ Where, θ = Angle of repose, H = Height of pile, R = Radius of the pile [13,14].
Carr's Index and Hausner's Ratio: Poured density was determined by placing the exact quantity 'M ' of microsphere into a graduated cylinder and measuring the volume' V ' occupied by the microspheres. o re ensit Tapped density was determined by placing a graduated cylinder containing a known quantity (M) of the prepared microspheres on a mechanical tapping apparatus operated for a fixed number of taps until the bed volume reached a minimum. [14,15].

Percentage yield
The microspheres were evaluated for percentage yield. The % yield was calculated by the formula: [14,15].

Mucoadhesion: In-Vitro Wash-off test for Microspheres
An in-vitro wash-off test evaluates the mucoadhesive properties of the microspheres. A 2 cm x 2 cm piece of chicken intestine mucosa was tied onto a glass slide (3inch by 1inch) using thread. Fifty microspheres were spread onto the wet rinsed tissue specimen, and the prepared slide was hung onto one of the groves of a USP tablet disintegrating test apparatus. The disintegrating test apparatus is operated such that the tissue specimen was given regular up and down movements in a beaker containing the simulated intestinal fluid USP (PBS pH 7.4). At the end of 8 hours, the number of microspheres still adhering onto the tissue is counted [14,15].

% Entrapment Efficiency
150mg Capecitabine or 50 mg Oxaliplatin loaded core microspheres was weighed and washed with 10ml of phosphate buffer of pH 6.8 to remove the surface-associated drug. then microspheres were kept in a phosphate buffer of pH 7.4 for digestion for 24 h and sonicated for 1 h at room temperature, from that, 1ml of sample is withdrawn and diluted 1000 times using phosphate buffer pH 7.4 and quantified by HPLC. Entrapment efficiency is determined by using the formula [14,15]. [14,15].

In-vitro drug release of capsule
In-vitro drug release studies were conducted using a modified USP type 1 dissolution apparatus at 37°C and 75 rpm/min in 90 mL of phosphate-buffered saline (PBS), pH 7.4. At predetermined time intervals, 1 mL samples were taken, and an equivalent volume of fresh PBS was added to maintain a constant volume. Drug concentrations from collected samples were measured using an HPLC. The zero-order kinetics was carried out by plotting the square root time against percent drug release.

Stability study: [19]
Stability studies were conducted as per the International Conference on Harmonization (ICH) guidelines. The samples were stored at 40°C ± 2°C/75 ± 5% relative humidity (RH) for six months. The samples were withdrawn and evaluated for the drug content and in-vitro release at pre-determined time intervals. The variations were analyzed and compared with the freshly prepared formulations. All samples were taken in triplicates (n = 3).
The stability study has been performed and result of the stability study indicated that there was not much difference observed in disintegration time, drug content, and % drug release before and after the storage period at 40 ± 2°C/75% RH ± 5% temperature and relative humidity. This indicates that formulation is fairly stable at storage conditions.

In-vitro Cell Viability Study [20-24]
The MTT assay was performed to assess cell cytotoxic potential of Different formulations of Oxaliplatin and Capecitabine using Caco-2 cell line. MTT assays were performed to check and compare the cytotoxicity of formulation with API solution of the respective drug, in colon cancer cell line Caco-2 by measuring IC50 values.
Cancer Cells (Caco-2) (5x10 3 ) were plated in 96 well plates in 200 µL of MEM medium per well and incubated for 24 h. Cells were incubated with different concentrations of test solutions for 48 h. The medium was removed from all the wells, and wells were fe with 200 μL of fresh complete medium. 100 µL of MTT solution was added to each well plate and incubated for 4 hrs. Cell plates were centrifuged at 3000 rpm/min for 10 min &culture media was discarded. Each cell was treated with 200 µL of DMSO solution. DMSO solution was added to dissolve MTT formazan crystals. DMSO solution was measured at 540 nm with a microplate reader immediately. Capecitabine and Oxaliplatin formulations and API were diluted with serumfree DMEM to prepare various formulation concentrations at 50mg Oxaliplatin and 150 mg Capecitabine. Cells treate with 200 μL of fresh in complete medium (DMEM) were used as negative control (100% viability will be assumed from the absorbance of wells containing these cells). Cell viability was calculated, and Viability plots were plotted by plotting % viable cells against the treatment.

Differential scanning Calorimetry (DSC)
Differential scanning calorimetry is used to define the melting point of drug substances by endothermic peak in the curve to find DSC curve, the sample run at 30 to 400 °C temperature. The Endothermic peak of both drugs was observed in the DSC curve of the drug-polymer mixture at a specified temperature. The DSC graphs are shown in Fig. 1, Fig. 2, Fig. 3 and Fig. 4.

Scanning Electron Microscopy (SEM)
The morphology of Capecitabine and Oxaliplatin Mucoadhesive microspheres were examined using Scanning electron microscopy, which shown spherical morphology, narrow size distribution. It is shown in Fig. 5. and Fig. 6.  Table 1.
Particle size measurement, Flow property, tappe ensit , Carr's in ex, Ha sner's ratio of Capecitabine mucoadhesive microspheres are shown in Table 2.
From the results of above evaluation of batch F12 was optimized because of having good Mucoadhesion property, % entrapment efficiency, % yield, swelling Index and this batch was filled into capsule.

Statistical Analysis of Capecitabine microspheres
Graphical presentation of effect of factors on variable is shown in Fig. 7. (A),(B),and (C).

Statistical Analysis of Oxaliplatin Microspheres
Graphical presentation of effect of factors on variable is shown in Fig. 7. (A),(B),and (C).

Evaluation Parameters for Microspheres Filled Enteric-Coated Capsules
The final optimized microspheres filled enteric-coated capsules were evaluated as per pharmacopoeial tests for its appearance, average weight, disintegration (PBS pH 7.4), average weight of empty capsule shell, net content, drug content, and results are recorded in Table 5. The graphical presentation of % CDR of Capecitabine and Oxaliplatin in the capsule is shown in Fig. 9.
The highest % drug release of Capecitabine and Oxaliplatin was found as 94.83 ±0.22 and 96.94 ±0.11 respectively after 8h. The correlation coefficient (R 2 ) of Capecitabine and oxaliplatin and the zero-order model was found 0.9518 and 0.944, slightly higher when compared to the peppas plot and higuchi's plot for the final selected optimized batch of capsule. Hence drug release from the preparation followed zero-order kinetics, which indicated that drugs released from the capsule were in a controlled manner.

Stability Study
The stability study has been performed result of the stability study indicated that there was not much difference observed in disintegration time, drug content, and % drug release before and after the storage period at 40 ± 2°C/75% RH ± 5% temperature and relative humidity. This indicates that formulation is fairly stable at storage conditions.   Table 7.
The graph of % cell viability v/s treatment is shown in Fig. 10.  Oxaliplatin overcome the problem associated with side effect, parenteral route of administration, give cytotoxic effect. Two drugs administered in a single formulation hence patient compliance. The stability study indicates that formulation is fairly stable at storage conditions. Apart from this, compared with available literature Goutam Kumar Jena et al. [13] that microspheres were successfully prepared and optimized with maximum drug entrapment and minimum particle size. The optimized microspheres coated with Eudragit S100, having 62.5% entrapment efficiency and 100% drug release in phosphate buffer pH 7.4 in 24 h. whilst prepared mucoadhesive Capecitabine and Oxaliplatin microsphere prepared by using spray drying technology with chitosan as polymer having more entrapment efficiency and lower the particle size.
Moreover available literature Aleksandra M et.al and Rudra P. et al. [9,25] they have designed a novel "particle in a particle" form lation where oxaliplatin was first loaded into nanoparticles composed of lipid like polymeric molecules which were later encapsulated in micro-sized alginate based particles. We believe that this combinatorial approach allowed for an improved and targeted delivery of the drug to the lower gastrointestinal tract where the tumor cells reside. This study helps to conclude oral delivery of oxaliplatin can provide good therapeutic effect.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

ETHICAL APPROVAL
It is not applicable.