INTRODUCTION:

Therapeutic effectiveness of a drug depends upon the bioavailability and ultimately upon the solubility of drug molecules. Solubility is one of the important parameter to achieve desired concentration of drug in systemic circulation for pharmacological response to be shown. Currently only 8% of new drug candidates have both high solubility and permeability. Up to 40% of new chemical entities discovered by the pharmaceutical industry today are hydrophobic compounds [1,2]. The solubility issues complicating the delivery of these new drugs also affect the delivery of many existing drugs.

Relative to highly soluble compounds, low drug solubility often manifests itself in a host of in-vivo consequences including decreased bioavailability, increased chance of food effect, in-complete release from the dosage form and higher inter patient variability. The bioavailability from conventional tablet formulations may be unacceptable for water insoluble drugs, which often have water-solubility of less than 1 μg/ml [3]. Poor drug solubility remains a significant and frequently encountered problem for pharmaceutical scientists. Various techniques available which are accepted worldwide to improve solubility of biopharmaceutics class II drugs, such as reduction in particle size, use of surfactants, solid dispersion techniques i.e. cyclodextrin complexation, which is widely used and accepted to improve solubility.

Cyclodextrins are cyclic oligosaccharides composed of 6 – 8 dextrose units. The interior of these molecules is relatively lipophilic and the exterior relatively hydrophilic. Cyclodextrins and their commercially available derivatives are able to incorporate lipophilic molecules or parts of molecules inside their hydrophobic cavity. This constitutes a true molecular encapsulation. The water-soluble inclusion complexes exhibit new physicochemical characteristics compared with the original guest molecules, such as better stability, high water solubility, increased bioavailability or decreased undesirable side effects. A large number of researchers have reported complex formation between cyclodextrin or their derivatives and poorly water soluble drug to improve their water solubility [4, 5]. Till today various techniques such as kneading, spray drying, freeze drying and microwave irradiation are reported to prepare inclusion complexes for poor solubility drugs.

Irbesartan (irb) used orally for treatment of hypertension, is a non peptide, specific competitive antagonist of the Angiotensin II receptor (AT1 subtype). The drug is lipophilic with a pka value of 4.90±0.09 and practically insoluble in water and in acidic buffer pH 1.2 with absolute bioavailability of 60 to 80 %. The low solubility and slow dissolution may lead to irreproducible clinical response or therapeutic failure [6]. The objective of the study is to improve the dissolution and solubility of irb utilizing the approach of inclusion complexation of irb with β-cyclodextrin (βCD) by using novel techniques as grinding, freeze drying, ball milling and microwave irradiation technique and also to observe the effect on solubility behaviour of irb-β-cyclodextrin complex in presence of water-soluble polymers [7] like PEG 400, HPMC, PVP K30, Cremophore RH 40, Soluplus etc.

MATERIAL AND METHODS:

Irbesartan was obtained as a gift sample from Que Pharam Pvt, Ltd, Surendarnagar, Gujarat, India. β-cyclodextrin, Soluplus, Cremophore RH 40 was supplied as a gift sample from signet chemical corporation, Mumbai, India and BASF Mumbai, India, Ltd, Mumbai, India.

EXPERIMENTAL:

Phase solubility studies:

Phase solubility studies [8] are carried out in water according to the method described by Higuchi and Connors. Excess amount of irb (50 mg) was added to stoppered conical flasks containing distilled water (20 ml) and acidic buffer pH 1.2 (20 ml) at various concentrations of β-CD (0.050, 0.100, 0.125, 0.150, 0.175, 0.200 g) respectively. The flasks were placed on mechanical shaker at ambient temperature. After equilibrium of 24 hrs the suspension was filtered through whatman filter paper 0.22µ and analyzed for irb concentration by UV spectrophotometer at 240 nm. Concentration of irb in mMoles was plotted against the concentration of β-CD in mMoles for distilled water and acidic buffer pH 1.2 respectively. The data was treated and the stability constant was calculated by using formula,

Slope

K_s= -------------------

S_o(1 – Slope)

S_o = Solubility of irb in water/ acidic buffer pH 1.2

Effect of water soluble polymers on solubility of Irb-β-CD systems:

This study was carried out to study the solubility behaviour of 1:1 molar ratio of irb:β-CD in presence of PEG 400, HPMC, PVP K30, Soluplus and Cremophore RH 40. These polymers were already used to improve solubility of poorly water soluble drugs by using techniques such as solid dispersion, self micro-emulsifying drug delivery system [9].

The solubility behaviour of 1:1 molar ratio of irb:β-CD was determined by adding it to conical flasks containing distilled water (40 ml). The effect of water soluble polymers; PEG 400 in different proportion 5 %, 10 %, 20 %, 30 % w/w, PVP K30 5 %, 10 %, 20 %, 30 % w/w, HPMC 5 %, 10 %, 20 %, 30 % w/w, Soluplus 5 %, 10 %, 20 %, 30 % w/w, Cremophore RH 40 5 %, 10 %, 20 %, 30 % w/w in different proportion on solubility of irb:β-CD was determined respectively for each proportion and for each polymer.

The flasks were placed on mechanical shaker at ambient temperature. After equilibrium of 24 hrs the suspensions were filtered through Whatman filter paper 0.22 µ and analyzed for IRB concentration by UV spectrophotometer at 240 nm.

Preparation of binary inclusion complexes:

Preparation of binary inclusion complex by Physical Mixture:

Irb (500 mg) and β-CD (1330 mg) was weighed accurately and mixed geometrically.

Preparation of binary inclusion complex by Grinding technique:

β-CD (1330 mg) was weighed accurately and 1 ml of distilled water was added to produce paste with the help of mortar and pestle. To the previously prepared paste irb (500 mg) was added. The resultant paste was ground in a mortar for 45 mins and dried in an oven at 40°C for 24 hrs [11].

Preparation of binary inclusion complex by Freeze drying technique:

β-CD (1330 mg) was weighed accurately and dissolved in a 30 ml of water. To it irb (500 mg) was added. The resultant solution was kept on magnetic stirrer for 6 hrs. The solution was stored in deep freezer for 24 hrs at -30°C and lyophilized using Martin Christ LD plus 1-2 models [12].

Preparation of binary inclusion complex by Microwave irradiation technique:

β-CD (1330 mg) was weighed accurately and 1 ml of distilled water was added to produce paste with the help of mortar and pestle. To the previously prepared paste irb (500 mg) was added and it is irradiated at 420 W with different energy levels of 50 %, 60 % and 70 %. Different samples were prepared and irradiated at 420 W for 15 min, 30 min, 45 min, 1 hr and 2 hr respectively.

Preparation of binary inclusion complex by Ball Mill technique:

β-CD (1330 mg) was weighed accurately and it was mixed with the irb (500 mg) in gemometric proportion and milled for 1 hr using planetary ball mill [13].

Preparation of ternary inclusion complexes:

Preparation of ternary inclusion complex by Physical Mixture:

Irb (500 mg), β-CD (1330 mg) was weighed accurately; to it soluplus/ PEG 400 was added.

Preparation of ternary inclusion complex by grinding technique:

β-CD (1330 mg) was weighed accurately and 1 ml of distilled water was added to produce paste with the help of mortar and pestle. To the previously prepared paste irb (500 mg) was added, to it soluplus/ PEG 400 (0.4 gm = 30 % w/w). The resultant paste was ground in a mortar for 45 min and dried in an oven at 40°C for 24 hrs.

Preparation of ternary inclusion complex by freeze drying technique:

β-CD was weighed accurately and dissolved in distilled water. To it irb was added, to it soluplus/ PEG 400 (0.4 gm = 30 % w/w) was added respectively. The resultant solutions were kept on magnetic stirrer for 6 hrs. The solutions were stored in deep freezer for 24 hrs at -20°C and lyophilized using Martin Christ LD plus 1-2 models.

Preparation of ternary inclusion complex by Microwave irradiation technique:

β-CD was weighed accurately and 1 ml of distilled water was added to produce paste with the help of mortar and pestle. To the previously prepared paste of irb, third component soluplus/ PEG 400 (0.4 gm = 30 % w/w) was added. It is irradiated at 420 w with energy level of 50 %, 60 % and 70 %. Different samples were prepared and irradiated at 420 W for 15 min, 30 min, 45 min, 1 hr and 2 hr respectively.

Preparation of ternary inclusion complex by Ball Mill technique:

β-CD (1330 mg) was weighed accurately and it was mixed with the irb (500 mg) in gemometric proportion. To it third component soluplus/ PEG 400 (0.4 gm = 30 % w/w) was added and resultant mass was milled for 1 hr using planetary ball mill.

Evaluation of binary and ternary inclusion complexes

Solubility of microwave irradiated samples:

50 mg of complex (16 mg equivalent) were added to 30 ml of the distilled water maintained at ambient temperature. The suspensions were allowed to equilibrate for 24 hrs. The suspension were filtered through 0.22 µ whatman filter paper and analyzed for dissolved content for irb at 240 nm.

In-vitro dissolution studies:

The dissolution studies of plain IRB, physical mixture of IRB with β-CD and binary as well as ternary inclusion complexes prepared by grinding, Microwave irradiation, freeze Dried and ball milled complexes were subjected to in-vitro dissolution study using USP type II (paddle) apparatus with distilled water 900 ml maintained at 37±0.5^oC and 50 rpm. 10 ml of aliquots was taken out and replaced with the same volume of fresh medium for predetermined time interval of 15, 30, 45, 60 and 90 min, respectively. The solution was filtered through whatman filter (0.22 µ), suitably diluted and dissolved irb was determined by spectrophotometer at 240 nm. Plot of percent cumulative release vs time were constructed.

Characterization of Binary and Ternary inclusion complexes:

Differential Scanning Calorimetry (DSC):

Plain irb, physical mixture of irb with β-CD, binary inclusion complexes and ternary inclusion complexes prepared by grinding, Microwave irradiation, freeze drying and ball milled samples were subjected to DSC studies using Perkin Elmer. 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):

Plain irb, physical mixture of irb with β-CD, binary inclusion complexes and ternary inclusion complexes prepared by grinding, Microwave irradiation freeze drying and ball milled samples to FTIR studies using model shimadzu FTIRNITY-1.

¹H Nuclear magnetic resonance spectroscopy:

Plain irb, physical mixture of irb with β-CD, freeze dried binary and ternary inclusion complexes were subjected to ¹H NMR studies using Shimazdu FT-NMR spectrometer operating at 300 MHz. Trimethyl silane was used as an external reference (solvent used: DMSO).

Powder X-ray diffractometry (XRD):

Plain irb, freeze dried and ball milled binary and ternary inclusion complexes 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).

RESULT AND DISCUSSION:

Phase solubility confirms the solubility enhancement capacity of the β-CD in distilled water and acidic buffer pH 1.2 for irb. The intrinsic solubility So of Irb in distilled water and acidic buffer pH 1.2 was found to be 0.64 mMoles and 2.86 mMoles respectively. β-CD showed improvement in the solubility of irb in distilled water and acidic buffer pH 1.2, it is in concentration range of 0 to 22 mMoles and 0-35 mMoles respectively ( Figure 1 and Figure 2 ). The stability constant (Ks) for Irb with β-CD in distilled water and acidic buffer pH 1.2 was found to be 1493 and 144 respectively. The phase solubility study confirms the formation of 1:1 stochiomety of formed inclusion complex in solution state.

Figure 1: Phase solubility of Irb with β-CD in distilled water

Figure 2: Phase solubility of Irb with β-CD in acidic buffer pH 1.2.

Effect of water soluble polymers on solubility of irb-β-CD:

The solubility of irb was found to be 0.0050 mg/ 40 ml; it is in agreement with the literature. From the phase solubility data it is confirmed that irb can form 1:1 molar complex with cyclodextrins. Based on this study was initiated to observe the solubility of 1:1 Molar irb with β-CD in presence of third component.

The solubility of 1:1 molar ratio of irb: β-CD was found to be 0.10 mg/40 ml. In case of PEG 400, solubility of irb is enhanced as compared to plain irb and 1:1 molar ratio of irb: β-CD, there with increase in concentration of PEG 400 from 5 % w/w to 30 % w/w. Three fold increases in solubility of irb was observed with 30 % w/w PEG 400. In case of Soluplus, solubility of irb is enhanced as compared to plain irb and 1:1 molar ratio of irb: β-CD, there with increase in concentration of Soluplus from 5 % w/w to 30 % w/w. Solubility of Irb increases with 30 % w/w Soluplus (Figure 3).

In case of PVP K30, HPMC and Cremophore RH 40, decrease in solubility of Irb with respect to 1:1 molar ratio of Irb: β-CD. This could be attributed to the decreased wettability of 1:1 molar ratio of Irb: β-CD, which renders the drug moiety as lipophilic.

Figure 3: Effect of Different Polymers on Solubility of IRB with β-CD

Evaluation of binary and ternary inclusion complexes

Solubility of Microwave irradiated samples:

The solubility of irb irradiated at 420 W (50 % energy) was found to be 0.708 mg/30 ml, it suggest that plain drug irradiated in microwave do not have significant difference in solubility as that of drug which is not irradiated ( Figure 4). The solubility of inclusion complexes of irb:β-CD irradiated at 15 min, 30 min, 45 min, 1 hr and 2hr showed improvement in solubility of irb over plain irb. So attempt was made to improve solubility of irb by increasing the energy level to 60 % and 70 % respectively at 420 W (Figure 5).

Figure 4: Solubility of microwave irradiated samples with different time intervals at 50 % energy.

In case of PEG 400 as a ternary component showed reduced solubility of irb as that of irb-β-CD irradiated at 60 and 70 energy for 1 hr and 2 hr respectively. This could be attributed to reduced wetability of prepared inclusion complex in presence of PEG 400. The solubility of irb is improved significantly in case of soluplus as a ternary component as that of plain drug as well as irb-β-CD. It is observed for both the time intervals of 1 hr and 2 hr respectively.

Figure 5: Solubility of microwave irradiated samples

In-vitro Dissolution Study:

The physical mixture and IRB alone show lower dissolution rates compared to inclusion complexes. The physical mixture showed a slight improvement in dissolution rate over that of pure drug. The total drug release after 90 min was found to be 5 %, 7 %, 10 %, 15 %, 10 %, 15 %, 34 %, 63 %, 20 % and 45 % for physical mixture (IRB-β-CD), grinding (IRB-β-CD), grinding (IRB-β-CD-soluplus), microwave (IRB-β-CD), microwave (IRB-β-CD-soluplus), Freeze dried (IRB-β-CD), freeze dried (IRB-β-CD-Soluplus), Ball mill (IRB-β-CD) and Ball milled (IRB-β-CD-Soluplus) inclusion complexes ( Figure 6 and Figure 7).

The dissolution rate of irb from β-CD-soluplus complex was found to be rapid as compared to that of IRB: β-CD. Among various techniques ball milling [14] and freeze drying was found to be better technique as that of grinding and microwave irradiation technique. The improvement in the dissolution of IRB as observed during the dissolution studies can be attributed to concurrence of several factors: increased particle wettability and decreased cyrstallinity of the drug. The XRD and DSC studies confirm the high energetic amorphous state and reduction in crystallinity of IRB following complexation leading to significant improvement in dissolution.

Figure 6: In-vitro dissolution study of binary and ternary inclusion complexes of Irb with βCD, PEG 400 and Soluplus in distilled water

Figure 7: In-vitro dissolution of ball milled binary and ternary inclusion complexes

Characterization and Evaluation of inclusion complexes:

Fourier-transform infra-red spectroscopy (FTIR)

The FTIR spectrum of irb showed a vibration band at 1732 cm^-1, 1024 cm^-1, 1614 cm^-1, 2341 cm^-1, 2360 cm^-1 and 3600 cm^-1for the possible functional groups of anhydride C=O, ketone, aromatic stretch, aliphatic –CH group and amine groups respectively, these are characteristics functional groups for the drug. The FTIR spectrum of physical mixtures (binary and ternary), Grinding Mixtures (binary and ternary), Microwave (binary and ternary) as well as Freeze dried binary and ternary with PEG 400 showed characteristic vibrations of irb. But one broad wide peak has been observed at 3200 cm^-1 , 3230 cm^-1, 3300 cm^-1, 3236 cm^-1, 3273 cm^-1, 3221 cm^-1, 3300 cm^-1, 3200 cm^-1, 3200 cm^-1, 3200 cm^-1, 3251 cm^-1 and 3265 cm^-1respectively, this suggest possible hydrogen bonding between –OH group of the β-CD with the amine functional group of irb ( Figure 8). In case of freeze dried ternary with soluplus showed complete disappearance of the widening of the peak as well as aliphatic –CH frequencies not observed in the spectrum ( Figure 9).

DSC

The DSC thermogram of IRB showed sharp endothermic peak at 191.2^oC. Physical mixture binary (IRB:β-CD) and ternary (IRB:β-CD) showed endothermic peak at 191.72^oC and 190.79^oC respectively (Figure 10 and Figure 11). All binary inclusion complexes showed a characteristic endothermic peak of irb but area of the peak is reduced indicates possible interaction between IRB and β-CD (Table 1). In case of ternary mixtures endothermic peak has been observed for complex prepared by grinding technique, microwave technique and ball mill technique. In case of freeze dried complex, complete absence of characteristic IRB endothermic peak was observed, indicating strong inclusion complex formation. It suggests the possible guest host interaction between irb and β-CD in presence of soluplus as ternary component.

Figure 8: FTIR Spectra of A: Irb, B: β-CD, C: Physical Mixture Irb: β-CD, D: Grinding Irb: β-CD, E: Microwave irradiated Irb:β-CD, F: Freeze Dried Irb:β-CD, G: Ball milled Irb: β-CD.

Figure 9: FTIR Spectra of H: Physical Mixture Irb:βCD:Soluplus, I: Grinding Irb: βCD: Soluplus, J: Microwave irradiated Irb: βCD:Soluplus, K: Ball milled Irb: βCD:Soluplus, L: Freeze dried Irb: βCD:Soluplus.

Figure 10: DSC Thermogram of A: Irb, B: β-CD, C: Physical Mixture Irb: β-CD, D: Grinding Irb: β-CD, E: Microwave irradiated Irb:β-CD, F: Freeze Dried Irb:β-CD, G: Ball milled Irb: β-CD

Figure 11: DSC Thermogram: H: Physical Mixture Irb:βCD:Soluplus, I: Grinding Irb: βCD: Soluplus, J: Microwave irradiated Irb: βCD:Soluplus, K: Ball milled Irb: βCD:Soluplus,

L: Freeze dried Irb: βCD: Soluplus.

CONCLUSION:

Irbesartan can form 1:1 molar ratio inclusion complex with b-cyclodextrin by grinding, microwave, freeze drying and ball mill technique. Enhancement of solubility of irb was observed under experimental conditions with the use of β-cyclodextrin and soluplus as a ternary component. Freeze drying was better than ball milled inclusion complex, grinding and microwave irradiation inclusion complex method with respect to dissolution properties of irbesartan and it is confirmed by physicochemical characterization. Thus, solubility of poorly water soluble irbesartan was successfully improved by use of the β-cyclodextrin along with the ternary component soluplus using freeze drying technique.

ACKNOWLEDGEMENT:

The author is thankful to Prof. Dr. Balvir S. Tomar, The Founder chancellor and Chairman, NIMS Univeristy, Dr. (Mrs.) Shobha Tomar, Managing Director, NIMS University for providing the platform to carry out research work. The author is also thankful to Prof. Dr. K. C. Singhal, Vice chancellor and Dr. K. P. Singh, Registrar, NIMS University for their support and encouragement. The author is also thankful to Chairman, P. E. Society, Pune for providing necessary research facilities.

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Received on 03.07.2014 Modified on 17.07.2014

Research J. Pharm. and Tech. 7(9): Sept. 2014 Page 987-994

Sr. No.	Sample	Peak ^oC	Peak height mW	Area mJ	Delta H J/g
1	Irb	191.94	16.75	660.17	66.01
2	Physical Mixture binary (irb:β-CD)	192.71	10.18	373.92	37.3.3
3	Physical mixture ternary (irb:β-CD:soluplus)	190.79	3.06	96.96	9.69
4	Ball mill Binary (irb:β-CD)	193.29	8.09	344.70	34.47
5	Ball mill Ternary (irb:β-CD:soluplus)	189.84	0.80	32.56	3.25
6	Grinding (irb:β-CD)	193.01	11.20	422.29	42.22
7	Grinding ternary (irb:β-CD:soluplus)	189.94	5.91	182.31	18.23
8	Microwave binary (irb:β-CD)	192.21	9.07	370.19	37.01
9	Microwave ternary (irb:β-CD:soluplus)	187.49	1.09	36.76	3.67
11	Freeze dried Irb binary (irb:β-CD)	188.73	4.82	159.77	15.97
12	Freeze Dried Ternary (irb:β-CD:soluplus)	-	-	-	-