1. INTRODUCTION:

The solubility behavior of a drug is a key determinant of its oral bioavailability. With the recent advent of high throughput screening of potential therapeutic agents, the number of poorly soluble drug candidates has risen sharply and the formulation of poorly soluble compounds for oral delivery now presents one of the most frequent and greatest challenges to formulation scientists in the pharmaceutical industry. The dissolution rate is directly proportional to saturation solubility of drug. Therefore aqueous solubility of a drug can be used as first approximation of its dissolution rate. Drugs with low aqueous solubility have low dissolution rates and hence suffer from oral bioavailability problems. So if the solubility of the drug is less than desirable, steps are to be taken to improve its solubility. Therefore, an attempt to improve the solubility and therapeutic efficacy of an oral anti-diabetic (Nateglinide) was chosen for the present work.

Enzymatic hydrolysis of starch usually results in formation glucose, maltose and a long range of linear and branched dextrins. However, a number of different microorganisms and plants produce certain enzymes called cyclodextrin glucosyltrnasferases (CGTs), which degrade starch to cyclic products called cyclodextrins. These are cyclic oligosaccharides consisting of a lipophilic central cavity and a hydrophilic outer surface.

Because of such characteristics, cyclodextrins form inclusion complexes both in solution and in solid state, in which each guest molecule is surrounded by the hydrophobic environment of Cyclodextrin cavity; this can lead to alteration of the physical, chemical and biological properties of the guest molecules and can eventually have considerable pharmaceutical potential. Cyclodexstrins have been found to be of extensive application in many fields including pharmaceutical technology to improve the aqueous solubility, dissolution rate, bioavailability and stability of the drugs. Many poorly soluble drugs have been complexed by cyclodextrins to enhance solubility, chemical stability and bioavailability. The binding forces within the inclusion complex may involve hydrophobic, Van der Waals, hydrogen bonding, or dipole interactions.

Most of the antidiabetic agents including Metaglitinide analogues belong to class II of BCS in view of the poor aqueous solubility. The major drawback in their therapeutic application and efficacy as oral dosage form in their very low aqueous solubility because of their hydrophobic nature (1). This causes poor dissolution rate and significant inter-individual variation in their bioavailability. This may lead to irreproducible in response or therapeutic failure in some cases due to sub-therapeutic plasma drug levels (2). In view of above mentioned reasons, an attempt was made in the present investigation to formulate and evaluate inclusion complexes of Nateglinide with β-Cyclodextrin by using Kneading methods with the aim to improve their solubility, dissolution rate and hypoglycemic activity.

2. MATERIALS AND MATERIALS:

2. 1) MATERIALS:

β-cyclodextrin-Signet chemical Pvt. Ltd, Mumbai, Phosphate buffer pH 7.4, Nateglinide-Bal Pharm, Banglaore, FT-IR spectrophotometer (model 460 Plus,Jasco,Japan) and DSC by using (SDTQ 600 V 20.9 BUILD 20, Universal V4.5ATA Instrument)

2.2) METHODS:

A. Drug Excipient Interaction:

Fourier Transform Infra Red Spectrometry

Infra red spectrometry is a useful analytical technique utilized to check the chemical interaction between the drug and other excipients used in the formulations. The samples were powdered and intimately mixed with dry powdered potassium bromide. The powdered mixture was taken in a diffuse reflectance sampler of FT-IR spectrophotometer. The mixtures were taken in a diffuse reflectance sampler and infrared spectra of the Nateglinide, β-CD and the inclusion complexes were recorded scanning the 400-4000 cm^-1wavelength region in an FT-IR spectrophotometer (Model 460 Plus, Jasco, Japan).The IR spectrum of formulation was compared with that of Nateglinide to check for any possible drug excipient interaction and chemical integrity of drug in formulation (3-4).

B. Phase-Solubility Studies:

Phase solubility was performed in triplicate at room temperature (250 C) according to the method reported in the literature (5). Excess amounts of Nateglinide were added to distilled water containing various proportions of β-CD (0.3-1.5mM) in a series of stoppered volumetric flasks and shaken for 72hrs on a rotary shaker (Secor Laboratory Instruments, New Delhi, India), the saturated solution was sonicated for 20 minutes and then centrifuged; the supernatant were filtered through a Whatman filter papar No.1. The filtrate was suitably diluted and analyzed spectrophotometrically at the wavelength of 210nm using a UV-VIS Spectophotometer (Model UV 1700 PC Schimadzu Corp., Kyoto, Japan) at 210nm using reagent blanks prepared with same concentrations of β-CD in the distilled water (6). The apparent stability constant (Ks) of the complexes was calculated from the slope and the intercept of the phase solubility diagram.

C. Preparation of Inclusion Complexes:

The Nateglinde with β-CD inclusion complexes were prepared by using Kneading method. Nateglinide was mixed with β-CD in different drug and polymer ratios like 1:1, 1:2 and 1:3 in a mortar with a small amount of water (10ml) and kneaded for 45 minutes to obtain a homogeneous paste. The resulting paste was dried in an oven at 450C for 48 hr, and the solid obtained was grounded and sieved through a 100µm sieve. All formulation drug and polymer ratio is 1:1, 1:2 and 1:3, formulation code is 1:1 is C1, 1:2 is C2 and 1:3 is C3. After preparing the inclusion complexes, the characterization of the Kneaded complexes were done.

1.1. EVALUATION OF PREPARED INCLUSION COMPLEXES:

i. In-vitro Dissolution Studies:

Dissolution study was carried out using USP XXII dissolution test apparatus type II. The dissolution medium used was 900ml of pH 7.4, which was maintained at 37degreeC±10⁰C. The stirrer (basket type) speed was kept at 50 rpm throughout the study. 10ml of samples were withdrawn at 5min, 10min, 15min, 30min, 45min and 60 minutes of time intervals and 10ml of fresh dissolution media maintained at the same temperature was replaced. Pure drug release pattern also done. The samples were analyzed spectrophotometrically at 210nm using pH7.4 as a blank. The dissolution data was analyzed for calculating the amount of drug released at different time intervals.

ii. LC-MS Study:

Prepared inclusion complexes and Nateglinide were subjected to LC-MS by using Shimdzu.

iii. NMR Study:

All prepared inclusion complexes and Nateglinide, NMR study was done by using BRUKER (Spect). MeOD was used as a solvent system.

Sample details in NMR spectra are: 1 in1 = 1:1(C1), 1 in 2 = 1:2(C2), 1 in 3 = 1:3(C3)

4 = Pure drug. All the NMR spectras were reported in the Graph 8, 9, 10 and 11.

iv. Characterization Studies of Nateglinide and Nateglinide- β-CD complex:

X-Ray Diffraction Study:

Every compound that is crystalline will give characteristic X-ray diffraction pattern⁸. These patterns are very useful for characterization of complexes. The diffraction studies were carried out in powder X-ray diffractometer(Philips PW 3710 BASED) with vertical goniometer. Using Cr anode tube, Chromium filter, radiation (λ=2.29A⁰), at a voltage of 40 Kv and a current of 25 mA. Powder XRD patterns of Nateglinide, β-CD and Nateglinide- β-CD complex were obtained by scanning the samples from 10 to 70⁰2 θ values at steps of 10 at room temperature.

v. Differential Scanning Calorimetry and Thermogravimetric Analysis (DSC-TGA):

DSC has been one of the most widely used calorimetric techniques employed to characterize the solubility and solid state of drug in the complex (1). DSC by using (SDTQ 600 V 20.9 BUILD 20, Universal V4.5ATA Instrument). Thermograms of Nateglinide, β-CD complex and Nateglinide- β-CD complex were recorded using DSC and were compared. The samples (5mg) were hermetically sealed in flat bottomed aluminium pans and heated over a temperature range of 50-300⁰C at rest if 10⁰C/min with purging of Nitrogen (100ml min^-1) using alumina as a reference standard.

vi Scanning Electron Microscopy (SEM) Study:

Equipment used SEM-JEOL, JSM-840 A, Japan. The samples to be examined were mounted on the SEM sample stab using a double-sided adhesive tape. The samples mounted were coated with gold (200 A⁰) under pressure (0.001 torr) for 5 min to improve the conductivity using an Ions Sputtering Device (JEOL, JFC-1100E, Japan). The gold coated samples were observed under the SEM and photomicrographs of suitable magnifications were obtained.

3. RESULT AND DISCUSSION:

Phase-solubility studies:

Solubility experiments showed that the concentration of Nateglinide pH 7.4 buffers was notably affected by the presence of β-cyclodextrin. Phase-solubility diagram investigated in pH 7.4 buffer was linear in a wide range of β-cyclodextrin concentrations and correspond to A_p-type profiles (Higuchi and Connors, 1965). The apparent stability constant (K_1:1) was estimated from the slope of the straight line of the phase-solubility diagram using equation.

The values of apparent stability constant Ks, between each drug–carrier combination were computed from the phase-solubility profiles, as described below:

Ks = Slope / Intercept (1- slope).

The stability constant was found to be 981.6 M^-1. These results are in accordance with the well established formation of soluble complexes between water soluble polymeric carriers and poor water soluble drugs. Increased solubility may be due to the improved dissolution of Nateglinide particles in aqueous solution by β-cyclodextrin..

Graph 1 (Phase Solubility):

Nateglinide standard graph was taken in pH 7.4 by UV-spectrometer at 210nm. Inclusion complexes of Nateglinide with β-CD in various ratios of Nateglinide and β-CD (C1- 1:1, C2-1:2 and C3-1:3) were prepared by Kneading method.

FT-IR Study:

The mixtures were taken in a diffuse reflectance sampler and infrared spectra of the Nateglinide, β-CD and the prepared inclusion complexes (C1, C2 and C3) were recorded scanning the 400-4000 cm-I wavelength region in an FT-IR spectrophotometer. All ratios of prepared inclusion complexes FT-IR were done. FT-IR spectra are in the Graph 2. FT-IR of Nateglindie and prepared inclusion complexes of Nateglinide were showed no interactions between the Nateglinide and β-CD.

Graph 2 (FT-IR Spectra of Prepared Nateglinide Inclusion complexes):

FT-IR Characteristics peaks observed:

Characteristic peaks of FT-IR	Wave number cm^-1
Characteristic peaks of FT-IR	Nateglinide	C1	C2	C3
-NH and-OH (Stretching)	3305.39	3305.39	3311.18	3307.32
-CH aromatic (Stretching)	3074	3079	3034.76	--
-CH aliphatic (Stretching)	2933	2930.31	2929.38	2928.38
-CH aliphatic (Stretching)	2860.88	2860.88	2863.77	--
-C=O (Stretching)	1706	1708.62	1710.55	1709.59
C=O of –COOH group	1644	1643.05	1642.09	1644.02

No much changes in the C1 and C2 characteristic peaks of prepared formulations and as compared with Nateglinide. C3 peaks due to –CH aromatic and –CH aliphatic merging, it indicates that formation of inclusion complex with β-CD.

In-vitro Dissolution study:

In-vitro dissolution study was subjected to all prepared inclusion complexes (C1, C2 and C3) and Pure Drug (PD). Dissolution study data was recorded in the Graph 3.

From the release study it can be observed that among all prepared Nateglinide inclusion complexes C3 formulation showed better drug release pattern compared with pure drug release.

Graph 3 (In-vitro release pattern):

LC-MS Study:

LC-MS was performed on all prepared inclusion complexes (C1, C2 and C3) and on pure drug (Nateglinide), the results were recorded in the Graph 4, Graph 5, Graph 6 and Graph 7, this showed no much change in the Nateglinide with that of the C3 prepared inclusion complex.

Graph 4 (Prepared inclusion complex C1) :

Graph 5 (Prepared inclusion complex C2) :

Graph 6 (Prepared inclusion complex C3) :

Graph 7 (Nateglinide) :

The Retention time is 3.76.

Quantation was performed using a selected reaction monitoring(SRM) of the transition m/z 320-166 for Nateglinide.

Nateglinide would form [M-H]^- ion under Electron Spray Ionization (ESI) conditions. However, it was found that Nateglinide favorably produced M+2. Several fragment ions were observed in the product ion spectra. The major fragment ions at m/z 166 for Nateglinide.

In the LC-MS spectra of Nateglinide and all prepared Nateglinide inclusion complexes but in the formulation C3 showed almost same fragmented molecule at m/z 166 and 320 as that of the Nateglinide.

NMR Study:

Further NMR study was done on all prepared inclusion complexes (C1, C2 and C3) and on pure drug (Nateglinide), the results were recorded in the Graph 8, this also showed no much change in the NMR spectra of Nateglinide with that of the C3 prepared inclusion complex.

NMR Spectra: Graph 8 (Prepared inclusion complexes):

Table 1: NMR peaks observed

mHz	Groups
7.08-7.17 (m, 5H)	Aromatic protons
4.5-4.53 (t, 1H)	Benzylic proton
3.09-3.20 (m, 1H)	Benzylic proton
2.82-2.88 (m, 1H)	Proton adjacent to acid carbonyl
1.98 (t, 1H)	Proton adjacent to amide carbonyl
1.64-1.72 (m, 4H)	Cyclohexyl proton + 1 methylene proton
1.2-1.29 (m, 3H)	Cylcohexyl proton
0.90 (brs, 3H)	Cylcohexyl proton
0.75-0.77 (d, 6H)	Both methyl proton

brs-broad singlet, t-triplet, d-doublet, m-multiplet.

Minute detectable shifts in the spectra of C1, C2 and C3 complexes.

DSC-TGA Study:

The endothermic peak at 124.48 ⁰C of the drug represented the melting point transition of the crystalline drug.

The broad band that appeared between 140- 180 ⁰C in the thermogram of β-CD can be attributed to a loss of water or molecular dehydration process.

The reduction in the band area and reduction in the energy required for the melting transition. The β-CD dehydration band in the DSC scan of the Kneaded product shifted to lower temperature.

Graph 9 (DSC Spectra of Prepared Nateglinide Inclusion complexes):

Scanning Electron Microscopy (SEM) Study:

a) Nateglinide

b) β-Cyclodextrin

c) C1 (1:1 Nateglinide: β-CD)

d) C2 (1:2 Nateglinide: β-CD)

e) C3 (1:3 Nateglinide: β-CD)

Figure: SEM Photographs.

Figure ‘a’ and ‘b’ in irregular crystalline shapes. The photomicrograph of the formulations (Figure ‘c’, ‘d’ and ‘e’) showed the topological changes produced in the β-CD particles. The β-CD surface seems to be more porous in nature.

In the prepared Nateglinide inclusion complexes displayed a number of irregular shaped microcrystals, in C3 formulation (figure ‘e’) showed formation of complex.

4. CONCLUSION:

Among all prepared inclusion complexes, prepared inclusion complex C3 (1:3 Nateglinide:β-CD) showed very good drug release pattern, this was confirmed by the evaluation tests like FT-IR, DCS, XRD, NMR and SEM. Therefore, among all prepared inclusion complexes (C1, C2 and C3), C3 formulation was showed the best drug release pattern.

5. REFERENCES:

1. Yalcon Ozan, Tamer Atay, Necti Dikmen, Askon, Hassan and Aboul Enein., Pharmaceutical Acta Helvetiae 365-370, Vol.74, Issue 4, April 2000.

2. Ammar HO, Salama HA, Ghorab H, Mohmoud AA., International Journal of Pharmaceutics 129-138, Vol 309, Issues 1-2, Feb 2006.

3. Shivakumar HN, Sarsija S, Venkataram S, Design and evaluation of multiparticulate system for chronotherapeutic delivery of diclofenac sodium, Indian J Pharm Sci. 133-137, 64(2), 2002.

4. Shivakumar HN, Sarsija S, Desai BG, Design and evaluation of controlled onset extended release multoparticulate system for chronotherapeutic delivery form Ketoprofen, Indian J Pharm Sci. 76-82, 68(1), 2006.

5. Zingone G and Rubessa I, “Pre-formulation studies of Inclusion complex Warfarin-Beta-Cyclodextrub,” Int. J. Pharm. 291, 3-10 (2005).

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Received on 28.04.2011 Modified on 27.05.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1159-1164