Introduction

Rheumatoid arthritis is a chronic inflammatory syndrome which mainly causes the destruction of joints integrity. The patients with this disease have symptoms such as joint pain and functional disability which mainly persist in the early morning (1). These symptoms are mainly characterized due to the diurnal variations in the levels of circulating pro-inflammatory cytokines, tumor necrosis factor-α and/or interleukin-6 (2). Rheumatoid arthritis can be well treated by the concept of chronotherapy to maintain the highest concentration of drug in the bloodstream in the early morning, so that peak of the pain and stiffness of the disease can be overcome (3, 4). In this case, intentionally delayed absorption or colon targeting of drug can be advantageous in order to have a uniform therapeutic effect as the release of drug occurs after a lag time and can be delivered in higher concentration during the time of its greatest need. Thus, good therapeutic effectiveness and patient compliance can be achieved (5, 6).

A number of approaches can be used for targeting the drugs at the colonic junction which some of them are performed by using the enzyme and pH dependent approaches (7). In the former approach, polymers or carriers which are degraded by the enzymes produced by colonic bacteria were used. Because the colonic region is rich in micro flora and their energy is fulfilled by fermenting various types of substrates that have been left undigested in the small intestine such as disaccharide, trisaccharides, and polysaccharides. This fermentation takes place,

Mini-tablets are very small tablets having a diameter equal to or smaller than 3 mm which can be either filled into a capsule shell or placed in sachets for easy administration (10-13). Mini-tablets are having several advantages over single unit larger tablets, such as consistent drug release, uniform clinical performance, more flexibility during the formulation development and maximum stability on storage (12, 13). Moreover, mini-tablets are very easy to prepare using direct compression method, involving fewer steps using simpler equipments, saving the time and cost. Other benefits of mini-tablets include regular shapes and excellent size uniformity (10-15). In this research, the purpose of the designing matrix-mini-tablets-filled capsule dosage form is to develop a more reliable formulation which has all the advantages of a single unit larger tablet and yet devoid of the problems such as danger of dose dumping and alteration in drug release profile and formulation behavior due to unit to unit variation.

Lornoxicam possess potent analgesic and anti-inflammatory properties and comes under the class of NSAIDs (16). It is widely used for the symptomatic treatment of inflammation and pain in rheumatoid arthritis (17, 18). Lornoxicam mechanism of action is based on decreasing prostaglandin synthesis by inhibition of cyclo-oxygenase enzymes as similar to other NSAIDs (19).

The previous published works requires the usage of solvents, additional number of steps and highly specialized equipments which increases the time and the costs of the pharmaceutical industry (20-22).

By considering the above facts about treating the early morning peak symptoms of rheumatoid arthritis, colon targeted matrix-mini-tablets of lornoxicam filled in a capsule were developed.

 Materials and Methods

Materials

Lornoxicam pure drug was received as a gift samples from Spectrum Pharma training and IPS institute, Hyderabad, A.P, India. Guar gum, sodium alginate, microcrystalline cellulose, Avicel PH 102and Aerosil^® were purchased from SD Fine Chemicals, Mumbai. Magnesium stearate was purchased from Himedia Chem Lab, Mumbai. pH sensitive methacrylic acid co-polymers (Eudragit® L-100 and S-100) were supplied as gifts samples by Degussa India Pvt. Ltd., Mumbai, India. Empty HPMC capsules (almost all sizes) were obtained as a gift sample from ACG Associated capsules Pvt. Ltd. Mumbai. All the remaining used materials were of analytical grade.

 Pre-formulation studies

Identification of Absorption Maxima (λmax)

Lornoxicam pure drug was mixed in different pH range solutions (pH 1.2, 6.5, 6.8, 7.2 and 7.4) to the concentration of 10µg/ml. These prepared solutions were scanned between 200-400 nm regions on UV-Spectrophotometer in order to identify the absorption Maxima (λmax).

 Fourier Transform Infrared (FTIR) spectral analysis

The pure drug lornoxicam, polymers and physical mixtures of drug and polymers used in this experimental condition were evaluated for compatibility by recording the spectra using FT-IR Spectrophotometer (Perkin Elmer, spectrum-100, Japan). The evaluation was performed by taking 5% of sample in potassium bromide (KBr) and the mixture was ground into a fine powder and then compressed into KBr pellets at a compaction pressure of 4000 Psi for 2 min. The range of scanning was 400 to 4000 cm^-1 and the resolution was 1 cm^-1. 

 Diffraction scanning calorimetric (DSC) studies

DSC thermograms of pure drug lornoxicam and physical mixtures were recorded using Diffraction scanning calorimeter (DSC 60, Shimadzu, Japan). The measurement was performed between 30 and 350⁰C at heating rate 10⁰ C/min.

Formulation methods

Preparation of matrix-mini-tablets of lornoxicam

The matrix-mini-tablets of lornoxicam were prepared by using a direct compression technique as shown in Table 1. At first, lornoxicam, polymer and microcrystalline cellulose Avicel PH 102 were passed through 60 mesh sieve, weighed as per the formulation tablet and mixed. Then magnesium stearate and aerosil were separately passed through the same sieve and after weighing were added to the above mixture and blended thoroughly. Then the prepared blend was compressed into matrix-mini-tablets by using 3mm flat round concave punches in a rotary tablet press (Model RSB-4, Rimek mini-press, Karnavathi engineering, Ahmadabad).

Table 1. Composition of matrix-mini-tablets formulations

 Preparation of matrix-mini-tablets-filled capsule formulations

For preparing the capsule formulation, 4 matrix-mini-tablets equivalent to 8 mg of lornoxicam were filled into size 4 HPMC capsule (as shown in            Figure 1).

 Evaluation methods

Pre-compression parameters

The prepared powder blends ready for compression containing drug, polymers and various excipients were evaluated for pre-compression parameters to study their flow properties, and maintain uniformity of matrix-mini-tablets weight.

 Angle of repose (q)

The angle of repose for the prepared powder blend was determined by taking accurately weighed powder blend into the funnel. The height of the funnel was adjusted as the tip of the funnel touched the apex of the blend. Then the blend was allowed to flow through the funnel freely on to the surface. From the formed powder cone, height and radius was measured and the angle of the repose was calculated using the following equation, where, h and r are the height and radius of powder cone respectively (23, 24).

                 tan θ = h/r

 Bulk density and tapped density

For the powder blends both loose bulk density (LBD) and tapped bulk density (TBD) were determined. Two gram of powder blend from each batch, which previously was shaked to break any formed agglomerates, was introduced into 10 ml of measuring cylinder. Noting the initial volume, the cylinder was allowed to fall under its own weight over a hard surface from the height of 2.5 cm at 2 sec intervals. Tapping was continued until no further change in volume was noted. LBD and TBD were calculated using the following equations (23, 24).

LBD= Weight of the Granules/Untapped Volume of the packing

TBD= Weight of the Granules/ Tapped Volume of the packing

 Hausner’s ratio: Hausner’s ratio is considered as an indirect index of the ease of the powder flow.  It is calculated by using the following equation (23, 24).

Hausner ratio =

 Where, r_t is tapped density and r_d is bulk density. 

 Carr’s compressibility index

 The compressibility index of the powder blend was determined by Carr’s compressibility index. Carr’s Index (%) can be calculated by using the following equation (23, 24).

Carr’s Index (%) = x 100

Post-compression parameters

The prepared matrix-mini-tablets were evaluated for post-compression parameters to study their physicochemical properties.

 Hardness test

The hardness of matrix-mini-tablets was determined using Pfizer hardness tester. From each formulation batch three matrix-mini-tablets were randomly taken and the values were calculated             (23, 24).

 Friability test

The test was performed by initially weighing (W_initial) twenty matrix-mini-tablets and then transferring into a Veego friabilator. The friabilator was operated at 25 rpm and run up to 100 revolutions. Then the mini-tablets were weighed again (W_final) (23, 24).

The % friability was then calculated by using the following equation.

F =                          

Weight variation test

From each formulation batch twenty mini-tablets were randomly taken and weighed individually to check the weight variation (23, 24).

 Uniformity of thickness

From each formulation batch six matrix-mini-tablets were randomly taken and were measured for thickness using screw gauge (23, 24).

 Drug content uniformity

Ten matrix-mini-tablets were chosen and crushed in a mortar then weighed powder containing equivalent to 8 mg of lornoicam was extracted in phosphate buffer of pH 6.8. The solution was filtered through a Millipore filter of 0.45 μm pore size and the drug content was determined spectrophotometrically at λ max of 375 nm after suitable dilution.

 In vitro dissolution testing for matrix-mini-tablets-filled capsule formulations F1 to F15

Dissolution studies were carried out by using USP XXIII dissolution test apparatus using basket method. One HPMC capsule filled with 4 matrix mini-tablets were immersed completely at a time. To maintain the conditions of GI tract, three dissolution media with pH 1.2, 7.4 and 6.8 were used sequentially. These three media represents the stomach, the small intestine and the colon respectively. When performing studies, 750 ml of medium with pH 1.2 was first used for 2 hr, then continued with 900 ml of pH 7.4 phosphate buffers for 3 hr and was further continued with 900 ml of pH 6.8 dissolution medium containing 0.05 mg/ml of betagalactomannase enzyme for subsequent hours. Rotation speed was 100 rpm and temperature was maintained at 37±0.5°C. At predetermined time intervals (0, 1, 2, 3, 4, 5, 6, 7, 8, 10 and 12 hr), 5 ml of dissolution media were taken and replaced with fresh dissolution media. The withdrawn samples were analyzed at 376 nm, 377 nm and 375 nm respectively for pH 1.2, 7.4 and 6.8 buffers, respectively by UV absorption spectroscopy and the mean cumulative percentage drug release was calculated over the sampling times (25-27).  

 In vitro dissolution testing for matrix-mini-tablets-filled capsule formulations F16 to F36

Dissolution studies were carried out by using USP XXIII dissolution test apparatus using basket method. One HPMC capsule filled with 4 matrix mini-tablets were immersed completely at a time. To match the increased pH changes along the GI tract, four dissolution media with pH 1.2, 6.5, 6.8 and 7.2 were used sequentially. These four media represents the stomach, proximal and distal parts of the small intestine, and terminal ileum respectively. When performing studies, 750 ml of pH 1.2 medium was first used for 2 hr, followed by 900 ml of pH 6.5, 6.8 and 7.2 phosphate buffers for 1, 2 and subsequent hours respectively. Rotation speed was 100 rpm and temperature was maintained at 37±0.5°C. At predetermined time intervals (0, 1, 2, 3, 4, 5, 6, 7, 8, 10 and 12 hr), 5 ml of dissolution media were taken and replaced with fresh dissolution media. The withdrawn samples were analyzed at 376 nm for pH 1.2 buffer and 375 nm for pH 6.5 and 6.8 buffers, whereas 377 nm for 7.2 buffer by UV absorption spectroscopy and the mean cumulative percentage drug release was calculated over the sampling times (28, 29).

 Drug release kinetics

The In vitro dissolution data were fitted to mathematical models representing zero-order kinetics (Mean % cumulative drug release versus time), First-order kinetics (Log Mean % drug unreleased versus time), Higuchi’s (Mean % cumulative drug release versus square root of time) and Korsemeyer-Peppas equation (Log mean % cumulative drug release versus Log time) in order to know the mechanism of release. The data was processed for regression coefficient analysis using Microsoft office Excel 2003 software (30).

 Stability studies

These studies were performed both at room temperature and accelerated stability conditions.

The conditions used for storing in room temperature were kept at 30±2°C and 65±5 % relative humidity and for accelerated stability were stored at 40±2°C and 75±5 % RH in a stability chamber. At regular intervals of 2, 3 and 6 months samples were withdrawn and tested for physical stability parameters such as Appearance, Weight variation, hardness, thickness, friability, drug content and In vitro release profile (31, 32).

 Results

Evaluation of the prepared powder blend

The prepared powder blends of all the formulation batches were found to exhibit good flow properties as evident from the results.

 Evaluation of prepared matrix-mini-tablets

The physicochemical properties of all the matrix- mini-tablets formulations were found to be within limits as evident from the results.

 Table 2. Results of physical evaluation of Pre-compression Blend

Table 3. Evaluation results of matrix-mini-tablets

In vitro dissolution testing of matrix mini-tablets-filled capsule systems

From the dissolution testing of all these matrix mini-tablets-filled capsule formulations, it was found that formulation F30 prepared with 40% combined concentrations of Eudragit L100 and Eudragit S100 polymers in 1:3 ratio was able to release lornoxicam after a lag time of 5.02±0.92 hr. It was also found that this formulation rlease the drug more immediately at the ileo-colonic junction. Our aim in this dissolution testing was also to identify a suitable formulation which immediately releases lornoxicam after a lag time (less than 10 %) of minimum 5 hr or at colonic junction. Thus, it was considered to be the best formulation.

 Drug Release kinetics

The mechanism of drug release kinetics was determined by fitting the in vitro dissolution data to Zero order, First order, Higuchi’s plot and Korsemeyer-peppa’s plot. The results suggest that none of the formulation have followed any of the mathematical models significantly because of the obtained low ‘R²’ values from all of the four plots.

 Stability studies

The results of physical stability revealed that the optimized formulation F30was found to be stable for a period of 6 months based on ICH guidelines.

 FT-IR studies

In order to check the compatibility between drug and polymers the spectra of pure drug, polymers and their physical mixtures were recorded. The spectra showed that all the peaks of pure drug and polymers were also visible in their physical mixtures. Absence of any peak was not noted, as they were found to be intact. Thus, it confirmed that combination of pure drug lornoxicam and the used polymers in matrix-mini-tablets can be suitable for designing a formulation intended for its desired therapeutic purpose.

 DSC studies

The above observation of FT-IR studies was further confirmed by DSC studies. The DSC thermograms showed that the physical mixtures of drug and polymers did not show any significant shift in their endothermic peaks. Thus, the DSC thermograms results also confirmed that there is no chemical interaction between the physical mixtures of drug and polymers used in matrix mini-tablets.

Table 4. Results of In vitro drug release studies

In vitro release profile of matrix-mini-tablets-filled capsule formulations

From the dissolution testing release profile results, it was found that as the concentrations of polymer or a combined polymer was increasing, the release rate of lornoxicam frommatrix-mini-tablets formulations was decreasing. When guar gum in combination with sodium alginate or Eudragit S100 in combination with Eudragit L100 polymers was used, it was found that the release rate of lornoxicam was increased comparing with formulations prepared with individual polymers. It was also found that 10-50 % concentrations of guar gum polymer, all the concentrations of Eudragit L100 polymer and 10-30 % of Eudragit S100 polymer in matrix-mini-tablets were not suitable for targeting lornoxicam at the colonic junction. Whereas 60% concentration of guar gum polymer and 40-60 % of Eudragit S100 polymer were able to target lornoxicam at the colonic junction.  When sodium alginate concentration was increased from 10 to 20 % along with 10 % concentration of guar gum polymer, the release rate of lornoxicam was decreased, but when it was further increased to 30 %, the release rate of lornoxicam was decreased. This similar effect was not seen in formulations when guar gum was also used in high concentration along with sodium alginate. In all the formulations, only the release from formulation F30 prepared with   40 % combine concentrations of Eudragit L100 and Eudragit S100 polymers in 1:3 ratio was found to be more immediate at the ileo-colonic junction and was also found to release lornoxicam after a lag time of 5 hr. Thus, it was considered as the best formulation.

 Discussion

As mentioned above, the objective in this research was to develop matrix-mini-tablets of lornoxicam for treating early morning peak symptoms of rheumatoid arthritis. So, a pre-determined target release profile was set. Optimized formulation should release lornoxicam after a lag time (i.e less than 10 %) of minimum 5 hr and maximum till the end of 8 hr. This target release profile is based upon the assumption that if the patient takes our optimized formulation at night

 Table 5.  Curve fitting analysis results for different formulations

10:00 AM (before going to bed) the drug starts releasing after 3:00 AM and be available in maximum concentration between 4:00 to 6:00 AM which is the time when the rheumatoid arthritis symptoms are worsened.

In this research, the designed formulations are based on microsomal enzyme and pH-sensitive
dependent delivery system. The device was formulated into two steps: First, lornoxicam was prepared as matrix mini-tablets by using microsomal enzyme dependent or pH sensitive polymers with different combinations and concentrations. Second, these matrix-mini-tablets were filled into an empty HPMC capsule (Size ‘4’).

 Table 6. Results of physical stability studies for optimized formulation F30

Figure 2. FTIR spectra of a) Pure drug lornoxicam b) Guar gum c) Sodium alginate d) Eudragit L100 e) Eudragit S100 f) Physical mixture of lornoxicam+ guar gum+ sodium alginate g) Physical mixture of lornoxicam+eudragit L100+eudragit S100

 Identification of Absorption Maxima (λmax)

 When lornoxicam pure drug was subjected to scanning in 200-400 nm regions on UV-Spectrophotometer, the λmax was found as 376 nm for pH 1.2 buffer and 375 nm for pH 6.5 and 6.8 buffers, whereas 377 nm for pH 7.2 and 7.4 buffers. Thus, these obtained results of λmax were used for calculating the In vitro release profile of lornoxicam.

 FT-IR studies

In order to evaluate the compatibility between drug and polymers, the FT-IR spectra was recorded between 400-4000 cm^-1 for pure drug lornoxicam, polymers and their physical mixtures in the formulations (as shown in Figure 2). In present research, lornoxicam was taken as the model drug. It has shown -OH, -Ar-CH, -NH, -C=O, –CONH, SO₂N and CCl stretchings by the presence of characteristic peaks at 3512 cm^-1, 3100 cm^-1, 3066 cm^-1 , 1647 cm^-1, 1594 cm^-1,1327 cm^-1 and 790 cm^-1,  respectively. These are all the characteristic peaks of lornoxicam. The spectra of guar gum polymer shows -OH, -CH and -CO stretchings by the presence of characteristic peaks at 3382 cm^-1, 2938 cm^-1 and1241 cm^-1,respectively. Whereas, the spectra of sodium alginate polymer also shows similar -OH, -CH and -CO stretchings by the presence of characteristic peaks at 3567 cm^-1, 2943 cm^-1 and1302 cm^-1,   respectively. The spectra of Eudragit L100 polymer shows –OH, -OCH _3, -CH ₃ and _-CO stretchings by the presence of characteristic peaks at 3258 cm ^-1,2997 cm ^-1,2952 cm ^-1 and1731 cm ^-1,  respectively. Whereas, spectra of Eudragit S100 polymer also shows similar –OH, -OCH _3, -CH ₃ and-CO stretchings by the presence of characteristic peaks at 3225 cm ^-1,2998 cm ^-1,  2953 cm ^-1 and  1727 cm ^-1,  respectively.

Figure 3. DSC spectra of a) Pure drug lornoxicam b) Guar gum c) Sodium alginate d) Eudragit L100 e) Eudragit S100 f) Physical

Figure 4. In vitro release profile of matrix-mini-tablets-filled capsule formulations prepared with a) Guar gum b) Combination of guar gum and sodium alginate c) Eudragit L100 d) Eudragit S100 e) Combination of Eudragit L100 and Eudragit S100