INTRODUCTION:

Complexation with cyclodextrins has been widely used to improve the solubility and dissolution rate of poorly water soluble drugs. Cyclodextrins are macrocyclic oligosaccharides with six to eight d-glucose units called α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin, respectively. The most important structural feature of cyclodextrin is that they have hydrophobic central cavities capable of forming stable complexes with properly sized drug molecules. Cyclodextrin have attracted the attention of many formulation experts due to their improvement in solubility of poorly water-soluble drugs, enhancement in physical and chemical stability of drugs and elimination of the undesired properties of drugs, such as unpleasant odor and taste. Among these cyclodextrins, β-cyclodextrin and its hydrophilic derivative, such as hydroxypropyl-β-cyclodextrin are the first choices because of their suitable cavity sizes and low price¹

The efficiency of complexation is not frequently very high and consequently, relatively large amounts of cyclodextrins must be used to complex small amounts of the drug. The total solubility of a drug in the presence of CDs can be highly improved by pH adjustment or use of a proper third component. Likewise, the combined effect of inclusion complexation has been studied to improve the solubility drugs².

Aceclofenac ([2-(2, 6-dichlorophenylamino) phenyl] acetoxyacetic acid) is a phenyl acetic acid derivative that shows analgesic properties and good tolerability profile in a variety of painful conditions. It is used in the treatment of rheumatic disorders and soft tissue injuries. Aceclofenac inhibits the cyclooxygenase enzyme and thus exerts its anti-inflammatory activity by inhibition of prostaglandin synthesis. This effect seems to be correlated to the appearance of acute protocolitis associated with nonsteroidal anti-inflammatory drug therapy. However, the bioavailability of aceclofenac is very low which is mainly due to its low solubility in water³. Therefore, it is very important to introduce effective methods to enhance the solubility and dissolution rate of aceclofenac.

The aim of the work is to investigate the possibility of complex formation of aceclofenac with β-cyclodextrin in solution and in solid state and to select a suitable complex, which is a crucial step in the development of aceclofenac formulation. The type of complexation and stability constant were established according to phase solubility study. The inclusion complexes of aceclofenac with β-cyclodextrin were prepared by various methods, i.e. Kneading technique and Spray drying technique. The effect of water soluble polymer poly ethylene glycol 6000 was studied. The ternary inclusion complex was prepared by kneading technique (aceclofenac, β-cyclodextrin, PEG 6000). The prepared binary and ternary inclusion complexes were evaluated for drug content and In-vitro dissolution studies and characterized by differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD), Fourier-transform infrared spectroscopy (FT-IR) and Pharmacodyanmic studies in animals.

Materials:

Aceclofenac was supplied as gift sample from Arati drugs Ltd, Mumbai, India. β-cyclodextrin was supplied as gift sample from S. A. Pharma, Mumbai, India and Signet Chemical Corporation, Mumbai, India. All other chemicals and solvents used were of analytical grade.

Animals:

Male or females mice weighing 20-25 g were used for study. They were maintained under standard laboratory conditions. The mice were acclimated for at least 5 days and fasted overnight but supplied with ad libitum before the experiment. The permission for the use of animal was taken from the Institutional ethical review committee (1036/a/07CPCSEA).

Methods:

A phase-solubility study was carried out in water according to the method described by Higuchi and Connors (1965)⁴. Excess amount of aceclofenac was added to stoppered volumetric flask containing various concentrations of β-cyclodextrin. The suspensions were then shaken at ambient temperature for 48 hrs. After equilibrium attainment, the samples were filtered through whattman filter and the concentration of aceclofenac was determined spectrophotometrically on a JascoV-506 instrument at 275 nm.

The stability constant of the complex with aceclofenac was calculated from the phase-solubility diagram,

Slope

Kc = -----------------------------

[Intercept (1 - slope)]

Preparation of binary inclusion complex of Aceclofenac with β-cyclodextrin

Preparation of Inclusion Complexes:

The preparation of physical mixture of aceclofenac with β-cyclodextrin, binary and ternary inclusion complexes was prepared by different techniques (kneading and spray drying).

Physical mixture:⁵

Physical mixtures of aceclofenac with β-cyclodextrin 1:1 molar ratio were prepared by simple blending in a ceramic mortar.

Kneading method:

Preparation of Binary Inclusion Complexes:⁶

β-cyclodextrin was added in a mortar, wetted with a excess quantity of distilled water and then triturated to obtain paste like consistency. Subsequently, aceclofenac was added in stoichiometric proportions (1:1 molar). The mixture was then kneaded with the addition of a few drops of distilled water. This process was continued for 45 min and the product was dried at 37°C for 48 h (Selecta, model 204).

Preparation of Ternary Inclusion Complexes:⁷

β-cyclodextrin was added in a mortar, wetted with a excess quantity of distilled water and then triturated to obtain paste like consistency. Subsequently, aceclofenac was added in stoichiometric proportions (1:1 molar). Then water soluble polymer, polyethylene glycol 6000 is added in different proportions 5 % and 10 % respectively. The mixture was then kneaded with the addition of a few drops of distilled water. This process was continued for 45 min and the product was dried at 37°C for 48 h (Selecta, model 204).

Spray Drying Technique:⁸

Spray-drying was performed in Labultima LU 222 mini spray dryer. β-cyclodextrin was dissolved in 450 ml of distilled water on a magnetic stirrer, to it acelofenac was added. The solution was subjected to spray-drying. The drying conditions were as follows, inlet temperature 140^oC, feed pump 150 ml/h and aspiration speed 40.

Characterization of inclusion complexes:

Differential scanning calorimetry (DSC):

Plain acelofenac, physical mixture of aceclofenac with β-cyclodextrin and binary inclusion complexes and ternary inclusion complexes prepared by kneading technique and spray drying technique were subjected to DSC studies using model DSC 821E (Mettler Toledo). Alumina was used as a reference material and samples were scanned at the rate of 10^oC/min.

Fourier-transform infra-red spectroscopy (FT-IR):

Powder X-ray diffractometry (XRD):

Plain acelofenac, physical mixture of aceclofenac with β-cyclodextrin and binary inclusion complexes and ternary inclusion complexes prepared by kneading technique and spray drying technique were subjected to XRD studies using an x-ray generator (PW 1729) and an automatic x-ray diffractometer model PW 1710 (Philips, Eindhoven, The Netherlands).

Evaluation of inclusion complexes:⁸

In-vitro Dissolution studies:

The dissolution studies were performed using USP type II apparatus. In-vitro Dissolution studies were carried out in distilled water as a dissolution medium at temperature 37 ± 5^oC. The rotation speed was 100 rpm. Powdered samples containing 100 mg of aceclofenac or its equivalent amount of aceclofenac -β-cyclodextrin was added to the dissolution medium (900 ml). 10 ml of solution was taken out and replaced with the same volume of fresh medium in 15, 30, 45, 60 and 90 min, respectively. The solution was immediately filtered through whattman filter, suitably diluted and determined spectrophotometrically at 275 nm. Percent cumulative release was calculated.

Pharmacodynamic Activity:⁹

Analgesic potential of aceclofenac kneaded (10 % PEG) complex, aceclofenac- β-cyclodextrin spray dried complex and as compared to plain aceclofenac. An attempt was made to compare the onset of action of both the drugs after oral administration. The abdominal writhing test in mice was carried out to study the analgesic activity of aceclofenac and its complexes. 5 ml of water was taken and it was mixed with about 1.057 mg of sodium carboxymethyl cellulose (equivalent to 0.25% w/w of drug) gradually in small portion to form uniform slurry. An accurately weighed amount of aceclofenac (50 mg) or complex containing equivalent amount of aceclofenac mixed with the slurry (dose equivalent to 50 mg aceclofenac/kg). The remaining amount of water was added slowly to make 50 ml. The number of writhes those made from 0 min following the acetic acid injection was counted until 10 min.

RESULT AND DISCUSSION:

Phase Solubility Studies:

The aqueous solubility (So) of aceclofenac at 25°C was 0.21 mMole, which data is in good agreement with the reported literature. The phase solubility profile of aceclofenac with β-cyclodextrin is presented in Figure.1. The phase solubility diagram of aceclofenac with β-cyclodextrin could be classified as A_L-type according to Higuchi and Connors. The solubility of aceclofenac is increased with the increasing of concentration of β-cyclodextrin in the range of 0–15 mMole. The apparent stability constant was found to be (Kc) - 638 M^-1, which indicated that a complex with 1:1 molar ratio was formed in the solution.

Charactarization of inclusion complexes binary and ternary:

The DSC curves for all the binary and ternary are represented in Figure 2. As shown in Fig., aceclofenac exhibits a characteristic endothermic peak at 158.28°C. The Physical mixture of aceclofenac-β-cyclodextrin, kneaded complex of aceclofenac-β-cyclodextrin showed sharp endothermic peak at 154.4°C and 154.36°C respectively, which indicates presence of crystallanity. The ternary inclusion complex of aceclofenac-β-cyclodextrin-5% PEG 6000 shows weak endothermic peak at 153.53°C. However, the complete disappearance of endothermic peak corresponding to aceclofenac was observed for aceclofenac-β-cyclodextrin-10 % PEG 6000, indicating the formation of an amorphous inclusion complex, the molecular encapsulation of the aceclofenac inside β-cyclodextrin cavity.

Figure 1. Phase solubility diagram of aceclofenac with β-cyclodextrin.

Figure 2. DSC thermogram of A) aceclofenac , B) β-cyclodextrin, C) physical mixture (aceclofenac-β-cyclodextrin) , D) Kneaded (aceclofenac-β-cyclodextrin), E) Kneaded (aceclofenac-β-cyclodextrin-5% Polyethylene Glycol 6000), F) Kneaded (aceclofenac- β-cyclodextrin-10 % Polyethylene Glycol 6000), G) Spray dried inclusion complex (aceclofenac- β-cyclodextrin).

Inclusion complexes were also analyzed using FTIR spectroscopy. The FTIR spectra of wave number from 4000 to 400 cm⁻¹ are presented in Figure 3. Aceclofenac showed a very strong absorption bands between 3340 cm⁻¹. Inclusion complexes prepared by Kneading binary, Kneading ternary (10 % PEG 6000) and spray dried showed intense broad peak at 3340 cm⁻¹, this could be attributed to possible hydrogen bonding in between aceclofeanc and β-cyclodextrin.

Figure 3. FTIR spectras of A) Aceclofenac, B) β-cyclodextrin C) Physical mixture (aceclofenac- β-cyclodextrin), D) Kneaded (aceclofenac- β-cyclodextrin), E) Kneaded (aceclofenac- β-cyclodextrin- 10 % Polyethylene Glycol 6000), F) Spray dried inclusion complex (aceclofenac- β-cyclodextrin).

Powder XRD study was used to measure the crystallinity of the formed inclusion complexes. The angle of diffraction is an identification tool of a crystal structure, whereas the number of peaks is a measure of sample crystallinity in a diffractogram. The formation of an amorphous state proves that the drug was dispersed in a molecular state with cyclodextrin. All the XRD patterns of aceclofenac–cyclodextrins inclusion complexes and physical mixtures are shown in Figure 4. The powder X-ray diffraction pattern of pure aceclofenac exhibited a series of intense peaks at 2θ value which were indicative of their crystallinity. In physical mixtures of aceclofenac–cyclodextrins, most of the principal peaks of aceclofenac and cyclodextrins are present and the diffraction pattern is approximate superimposition of the pattern of the raw materials. This indicated partial interaction between the pure components in physical mixture. The diffraction patterns of kneaded binary, kneaded ternary and spray-dried products displayed a crystalline state, but in comparison with the diffractrogram of the correspondent physical mixture it was possible to observe the disappearance of some diffraction peaks of β-cyclodextrin and aceclofenac (Figure 4). Furthermore, kneaded binary, kneaded ternary and spray-dried products, the obtained diffraction patterns were diffused indicating the amorphous state reached by the kneading and spray-drying techniques.

Figure 4. Powder x-ray diffraction patterns of A) Aceclofenac, B) β-cyclodextrin, C) Physical mixture (aceclofenac- β-cyclodextrin), D) Kneaded (aceclofenac- β-cyclodextrin), E) Kneaded (aceclofenac- β-cyclodextrin- 10 % Polyethylene Glycol 6000), F) Spray dried inclusion complex (aceclofenac- β-cyclodextrin).

In-Vitro dissolution Studies:

The dissolution diagrams of aceclofenac, Physical Mixture, binary, ternary and spray dried inclusion complex in distilled water at 37°C shown in Figure 5. It is evident that physical mixture does not show significant improvement in dissolution rate with respect to aceclofenac alone. The kneaded inclusion complex shows improvement in dissolution rate over plain aceclofenac. The kneaded (Aceclofenac-β-cyclodextrin- 10 % poly ethylene glycol 6000) shows improvement in dissolution rate as compared to kneaded inclusion complex. The kneaded (Aceclofenac-β-cyclodextrin-10 % Poly ethylene glycol 6000) and spray dried inclusion complex exhibits fast dissolution as compared to plain aceclofenac.

Figure 5. In-Vitro dissolution study of binary and ternary inclusion complex of aceclofenac with β-cyclodextrin. All studies carried out in triplicate.

In kneaded (Aceclofenac-β-cyclodextrin-10 % Poly ethylene glycol 6000) case, the enhancement of dissolution rate may be attributed to the reduction in crystallinity of the product, confirmed by DSC and X-ray diffraction studies, and a greater extent of complexation by effect of water soluble polymer in kneaded technique. Spray dried inclusion complex showed fast dissolution as compared to all inclusion complexes prepared by various techniques. In-Vitro dissolution parameter of binary and ternary inclusion complex in distilled water are shown in Table 1.

Pharmacodynamic Activity:

The mean number of writhing shown by the vehicle treated control, spray dried inclusion complex (50 mg equivalent), Kneaded- Aceclofenac-β-cyclodextrin-10 % Poly ethylene glycol 6000 (50 mg equivalent) and aceclofenac (50 mg) treated mice were found to be 34 ±4.655, 1.6 ±0.6667, 12.5 ±5.078 and 21.16 ±2.81 respectively. Pretreatment to the animals with spray dried complex and kneaded complex significantly decreased the mean number of acetic acid induced writhing as compared to the vehicle treated control group animals and thus exhibited significant analgesic activity whereas plain aceclofenac was found to be insignificant (Figure 6 and Table 2). Results are expressed as mean ± SEM. (n = 6). Data was analysed by one way analysis of variance (ANOVA) followed by Dunnetts test. *P<0.05, **P<0.01.

Table 1. Dissolution parameter of binary and ternary inclusion complex in distilled water.

*Powders*	*t_50%*	*t_70%*	*Hixon-crowell constant*
Aceclofenac	275 min	315 min	0.003039 (0.794853)
Physical Mixture (Aceclofenac- β-cyclodextrin)	125	175	0.009141 (0.97579)
Kneaded (Aceclofenac- β-cyclodextrin)	98	136	0.015597 (0.95946)
Kneaded (Aceclofenac- β-cyclodextrin-5 % PEG)	16	110	0.025977 (0.86983)
Kneaded 10 % PEG (Aceclofenac-β-cyclodextrin-10 % PEG)	15	55	0.027912 (0.87545)
Spray Dried (Aceclofenac- β-cyclodextrin)	6	15	0.047703 (0.902)

Hixon-crowell constant K (coefficient of regression r)

Figure 6: Effect of aceclofenac-β-cyclodextrin spray dried complex and aceclofenac kneaded Kneaded (aceclofenac- β-cyclodextrin- 10 % Polyethylene Glycol 6000) and Plain aceclofenac on acetic acid induced writhing test in mice.

Table 2: Effect of aceclofenac-β-cyclodextrin spray dried complex and aceclofenac kneaded complex (aceclofenac- β-cyclodextrin- 10 % Polyethylene Glycol 6000) and plain aceclofenac on Acetic acid induced Writhing test in mice.

Groups	No. of writhings (Mean± SEM)
Control	34±4.655
Spray Dried (Aceclofenac- β-cyclodextrin) (50 mg)	1.6±0.6667**
Kneaded 10 % PEG (Aceclofenac-β-cyclodextrin-10 % PEG) (50 mg)	12.5±5.078**
Plain Aceclofenac (50 mg)	21.16±2.81

CONCLUSION:

Aceclofenac can form 1:1 molar inclusion complexes with β-cyclodextrin by kneading and spray drying technique. Enhancement of the solubility of aceclofenac was observed in the presence of β-cyclodextrin. Spray drying technique was better than kneaded (aceclofenac- β-cyclodextrin) in respect of dissolution properties of aceclofenac. But kneaded (Aceclofenac-β-cyclodextrin-10 % Poly ethylene glycol 6000) exihibit better dissolution rate as compared to kneaded (aceclofenac-β-cyclodextrin-5 % Poly ethylene glycol 6000) and kneaded (aceclofenac-β-cyclodextrin). This could be attributed to the effect of water soluble polymer poly ethylene glycol 6000. In phramcodynamic study, spray dried complex and kneaded complex (aceclofenac-β-cyclodextrin-10 % poly ethylene glycol 6000) significantly decreases the mean number of acetic acid induced writhing as compared to the control group animals and thus exhibited significant analgesic activity, where as plain aceclofenac was found to be insignificant. Thus fast acting inclusion complex with better bioavailability could be successfully developed by employing β-cyclodextrin as a solubility and dissolution rate enhancer and effect of water soluble polymer on inclusion complex prepared by kneading technique was studied.

ACKNOWLEDGMENT:

The authors are grateful to Dr. G. R. Ekbote, President Progressive Education society, Pune, India for providing necessary facilities. The authors are also grateful to Aarti Pharmaceutical Ltd, Mumbai, India and Signet chemical corporation Pvt. Ltd, Mumbai, India for providing gift samples of aceclofenac and β-cyclodextrin respectively. This work was funded by University of Pune, Pune, India.

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