Preparation and Evaluation of Nateglinide-Cyclodextrin Inclusion Complex

 

Venkatesh¹, Anand Kumar Y²*, C. Mallikarjuna Setty³

¹Department of Pharmaceutics, Sarada Vilas College of Pharmacy, Mysore, India.

²Department of Pharmaceutics, V.L. College of Pharmacy, Raichur, India.

³Oxford College of Pharmacy, Bengaluru, India.

 

ABSTRACT:

A physicochemical characterization of nateglinide (NT) - hydroxy propyl β-cyclodextrin (HPβCD) inclusion complex both in solution state and solid state were studied to improve the solubility and dissolution properties of nateglinide. Inclusion complex were prepared by micro wave irradiation and kneading method at 1:1M, 1:2M ratios. The prepared inclusion complex were investigated in solution state by drug content, phase solubility studies and solid state by differential scanning calorimetry (DSC), Fourier transformation-infrared spectroscopy (FTIR), X-ray diffractometry (XRD) and in vitro dissolution studies. Phase solubility studies revealed 1:1M stoichiometry inclusion complex, a true inclusion complex was observed at 1:1 and 1:2M ratios it was confirmed by FTIR, DSC, XRD studies. In vitro dissolution data suggests the dissolution properties of nateglinide was dependent on method and ratio, the inclusion complex of both methods shows superior dissolution properties when compare to corresponding physical mixture and pure nateglinide.

 

 

INTRODUCTION:

An inclusion complex is formed when a macrocyclic compound possessing an intermolecular cavity of molecular dimensions interacts with a small molecule that can enter the cavity^1,2. The macrocyclic molecule is called the host and the small included molecule is called the guest. Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides of α-D-glucopyranose containing a relatively hydrophobic central cavity and hydrophilic outer surface. For complexation, the cavity size of cyclodextrin should be suitable to accommodate a drug molecule of particular size³. Cyclodextrins enhance the bioavailability of insoluble drugs by increasing the drug solubility, dissolution and absorption/or drug permeability. They increase the permeability of insoluble hydrophobic drugs by making the drug available at the surface of the biological barrier, from where it partitions into the membrane without disrupting the lipid layers of the barrier^4-7.

 

Nateglinide is an oral antidiabetic agent used in the management of Type 2 diabetes mellitus also known as non-insulin dependent diabetes mellitus (NIDDM) or adult onset diabetes. It promotes a more rapid but less sustained secretion of insulin than do other available oral antidiabetic agent^8,9. Nateglinide is practically insoluble in water and having aqueous solubility about 0.03mg/ml and its log P value is 3.824 indicates class II drugs of BCS (Biopharmaceutical classification systems i.e. high permeability and low solubility). The poor aqueous solubility and wettability of nateglinide leads to difficulties in formulating oral solutions and variations in bioavailability. Thus, increasing the aqueous solubility of nateglinide is of therapeutic importance. Cyclodextrins are carriers able to form inclusion complex with poorly water soluble drugs. These inclusion complex have been shown to improve stability, solubility, dissolution rate and bioavailability. Hydrophilic derivatives such as hydroxy propyl-β-cyclodextrin (HPβCD) or sulfobutyl ether-β-cyclodextrin are useful for improving solubility and dissolution rate of poorly soluble drugs^10-13. In the present study, an attempt has been made to prepare inclusion complex of model drug nateglinide with HPβCD by conventional methods and characterize the inclusion complexes in solid state and liquid state.

MATERIALS AND METHODS:

Materials:

The pure Nateglinide (NAT) gift sample was procured from Divis, laboratories Hyderabad, India, Hydroxy propyl-β-cyclodextrin (HPβCD) was obtained from Glenmark, Pharma Ltd., Nasik, India and all other chemicals and solvents were of analytical grade.

 

Methods:

Physical mixture (PM):

The physical mixture of NT and HPβCD in 1:1 and 1:2M were obtained by mixing individual components that had previously been sieved (100-150μm) together with a spatula.

 

Kneading (KNE):

Accurately weighed quantities of NT and HPβCD (1:1, 1:2M) were collected in a mortar and kneaded with a small volume of dried dichloromethane. The thick slurry was kneaded for 60min and then dried at 45^oC for 48 h. The dried mass was pulverized and sieved through a 100-150μm granulometric sieve and stored in dessicator until further evaluation.

 

Microwave oven irradiation (MC):

The aqueous solution of cyclodextrin was added slowly into a solution of nateglinide dissolved in dried dichloromethane with constant stirring. These solvents containing glass containers are subjected for irradiation in microwave oven for 90sec at 60ºC. After reaction was complete, adequate amount of dried dichloromethane was added to remove the residuals. The resulting mixture was stirred for 1hr and evaporated under vacuum until dry. The dried mass was pulverized and sieved through a 100-150μm granulometric sieve and stored in dessicator until further evaluation. The different formulae were given in table 1.

 

Table 1: Different formulae of nateglinide physical mixture and inclusion complex.

 

EVALUATION:

Drug Content:

In each case physical mixture and inclusion complex equivalent to 20mg of nateglinide was accurately weighed and transferred into 25ml volumetric flask. Dried ethanol was added and mixed to dissolve the nateglinide. The volume was made up to 25ml with 0.5%w/v sodium lauryl sulphate in 0.01NHCl. From this 1ml is subsequently diluted with 0.5%w/v sodium lauryl sulphate in 0.01NHCl and measure the absorbance at 209nm by using double beam UV spectrophotometer, the nateglinide content was calculated using the calibration curve.

 

Phase Solubility Studies:

Phase solubility studies were carried out, according to the method described by Higuchi and Connors¹⁴. Excess amounts of nateglinide (50mg) were added to 25ml of CD aqueous solutions (ranging in concentration from 0.01 to 0.1M) in a series of 25ml stoppered conical flasks. The mixtures were shaken for 72h at room temperature (28^oC) on a rotary flask shaker. After 72h shaking to achieve equilibrium. 2ml aliquots were withdrawn at 12h intervals and filtered immediately using a 0.45μm nylon disc filter. The filtered samples were diluted and assayed for nateglinide by measuring absorbance at 209nm. Shaking was continued until three consecutive estimations were the same. The solubility experiments were conducted in triplicate (coefficient of variation, CV<2%). The blanks were performed in the same concentrations of HPβCD in water in order to cancel any absorbance that may be exhibited by the CD molecules. The apparent stability constants were calculated from the solubility diagrams, with the assumption of 1:1 stoichiometry, according to the equation,

 

       

 

Where S_o is NT solubility in the absence of CD.

 

Fourier transmitted infrared spectroscopy (FTIR:

The FTIR spectra were recorded for nateglinide, HPβCD, physical mixture and inclusion complexes on Shimadzu FTIR-281-spectrophotometer. Samples were prepared in KBr disks prepared with a hydrostatic press at a force of 5.2Tcm^-2 for 3 min. The scanning range was 450-4000cm^-1 and the resolution was 1cm^-1.

 

Differential scanning calorimetry (DSC):

Thermograms of nateglinide, HPβCD and inclusion complexes were recorded on a Seiko, DSC 220C model Differential scanning calorimeter (Tokyo, Japan). About 10mg of samples were sealed in aluminum pans and heated at a rate of 10ºC/min from 30ºC-300ºC.

 

Powder X-ray diffractometry (XRD):

The powder X-ray diffraction patterns of nateglinide, HPβCD, physical mixture and inclusion complex were recorded by using Philips X-ray powder diffractometer (model PW 1710) employing Cu-K_α-radiation. The diffractometer were run at 2.4⁰/min interms of 2θ angle.

 

 

Dissolution Studies:

In vitro dissolution studies of pure nateglinide, physical mixture and inclusion complex were carried out in 900ml of 0.5% w/v sodium lauryl sulphate in 0.01NHCl using a USP type 2-dissolution test apparatus by powder dispersed amount method (powder samples were spread over the dissolution medium). In each case sample equivalent to 50mg of nateglinide were used at optimum conditions viz., 900ml dissolution medium, 50rpm and a temperature of 37ºC. A 5ml aliquot was withdrawn at predetermined intervals of time, filtered using a 0.45µm nylon disc filter and replaced with 5ml of fresh dissolution medium. The filtered samples were suitably diluted, assayed for nateglinide by measuring the absorbance at 209nm using double beam UV spectrophotometer. The dissolution experiments were conducted in triplicate and the results were computed by using dissolution software PCP DISSO V3.0.

 

RESULTS AND DISCUSSION:

Drug Content:

The drug content was found to be in the range of 99.12% to 99.85% with low coefficient of variation and standard deviation i.e., less than 0.15% in all the batches prepared. Small SD and CV values indicate the method employed gave inclusion complex with uniform drug content.

 

Phase Solubility Studies:

The solubility of nateglinide increases linearly with an increase in the concentration of HPβCD, giving A_L type solubility diagram, the increase in solubility in the systems is due to one or more molecular interaction between NT and HPβCD to form complex. The HPβCD seems to optimal for entrapment of NT molecules and consequently provides the greatest solubilization effect. Solubility of nateglinide without HPβCD was 0.678±0.0012 M×10^-3 and the apparent stability constant obtained was 8.114M ±0.1778 for HPβCD. The larger constant value indicates that nateglinide interacts strongly with HPβCD produces (K_1:1) stichometric constant. Phase solubility diagram was given in figure 1.

 

Figure 1: Phase solubility profile of nateglinide in 0.5%w/v sodium lauryl sulphate in 0.01N HCl.

 

FTIR Study:

More evidence of complex formation was obtained from FTIR study, which investigated the functional groups of NT involved in the complexation. The FTIR spectra of NT shows the characteristic bands at 1244-1382cm^-1 for carboxyl, carboxylate groups; 1650cm^-1 for carbonyl stretching;  2857-3034 cm^-1 for C-H stretching; 1723cm^-1 for C=O vibration; 3369 cm^-1  for NH stretching.

 

Figure 2: Comparative FTIR spectras of nateglinide, physical mixture and its inclusion complex.

The FTIR spectra of HPβCD showed intense band at 3600-3200cm^-1 corresponding to absorption by hydrogen bonded OH groups and at 3000-2800cm^-1 stretching vibrations of–CH and –CH₂ groups. However, the spectra of inclusion complex showed rightward shifts of the band corresponding to hydrogen bonded group suggest existing bonds formed between the OH groups on the narrow side of cyclodextrin molecules which might be distributed after the formation of inclusion complex. In case of physical mixture the FTIR spectra was superimposed to pure nateglinide spectra where as in inclusion complex the aromatic carbonyl stretching band of drug appeared shifted to lower wave number 1635-1649cm-¹ and 1649-1653cm-¹ respectively for kneading and microwave irradiation inclusion complex. The FTIR results suggest the formation of hydrogen bonds between the carbonyl groups of nateglinide and the hydroxyl groups of the host cavities, during inclusion complexation^15,16 with cyclodextrin. These findings are in full agreement with other authors¹⁷ who previously reported that the carbonyl group is joined to a hydroxylic compound by hydrogen bonds; the stretching band is displaced to lower frequency due to a weakening of the carbonyl radical double bond. Comparative FTIR spectra were presented in figure 2.

 

DSC study:

The DSC curves of the NT, HPβCD and its inclusion complex are given in figure 3. The method confirms not only an interaction between the drug and HPβCD, but also a real inclusion. The DSC thermogram of NT exhibited an endothermic peak at 132.18^oC corresponding to its melting point and DSC thermograms HPβCD showed broad endothermic peaks at 68.58^oC. Lowering of the endothermic peaks in HPβCD is mainly due to their dehydration process during analysis. The DSC curves of NAT-HPβCD 1:1 and 1:2M ratios shows progressive reduction in NT endothermic peak intensity and shifted to lower temperatures, indicating the formation of an amorphous solid dispersion i.e., molecular encapsulation of nateglinide inside the HPβCD cavity suggest a true inclusion complexation.

 

X-Ray Diffractometry Studies:

Powder X-ray diffractometry is a useful tool for the detection of cyclodextrin complexation in powder or microcrystalline states. The diffraction pattern of the complex should be distinct from the superimposition of each of the components if a true inclusion complex were to form. X-ray diffraction pattern of nateglinide and its inclusion complex was presented in figure 4. Crystallinity was determined by comparing some representative peak heights in the diffraction patterns of the inclusion complexes with those of a reference. The relationship used for the calculation of crystallinity was relative degree of crystallinity (RDC) =I_sam/I_ref, where I_sam is the peak height of the sample under investigation and I_ref is the peak height at the same angle for the reference with the highest intensity. NT peak at 25.31º (2θ) values was used for calculating RDC of kneaded and microwave irradiation inclusion complex. The RDC values were found to be 0.555; 0.407; 0.450; 0.297 for kneading and microwave irradiation method inclusion complex at 1:1 and 1:2M ratios. From the RDC values it is seen that when nateglinide was considered as reference sample, there is lot of decrement in the crystalline structure of NT in 1:1 and 1:2M NT-HPβCD inclusion complex prepared by kneading and microwave irradiation method.  In these systems all the characteristic peaks were suppressed and a nearly smooth peaks were observed indicate the formation of amorphous state of the complex. These results suggest that nateglinide undergoes a strong interaction with HPβCD at both the molar ratios prepared by kneading and microwave irradiation method resulting formation of true inclusion complexes. Further these results were justified by DSC and FTIR studies.

 

Figure 3: Comparative DSC spectras of nateglinide and its inclusion complex.

 

Dissolution studies:

When an assumed drug- HPβCD inclusion complex is dispersed in a dissolution medium, a very rapid dissolution is often observed. Dissolution rate tests are based on this observation in order to characterize the inclusion complexation between drug and cyclodextrin. In the present investigation, dispersed amount method is used to investigate the various dissolution parameters of nateglinide and its inclusion complex.

Figure 4: Comparative XRD spectras of nateglinide, physical mixture and its inclusion complexes.

 

The usual method of evaluation of in vitro dissolution testing is the comparison of time taken for given proportions of active drug to be released into solution and parameters such as T₅₀ values are often used. Alternatively, the fraction of drug in solution after given time is used for comparison such as percent released in 30 minutes i.e.DP₃₀ and also relative dissolution rate at 30minutes i.e. RDR₃₀ are calculated to assess improvement in extent of dissolution rate enhancement. Another parameter suitable for the evaluation of in vitro dissolution has been suggested by Khan^18,19 who introduced the idea of ‘Dissolution Efficiency’ (D.E.). Dissolution Efficiency is defined as the area under the dissolution curve upto a certain time‘t’, expressed as a percentage of the area of the rectangle described by 100% dissolution in the same time.

Dissolution efficiency (DE) =

 

The dissolution efficiency can have a range of values depending on the time interval chosen. In any case, constant time intervals should be chosen for comparison. In the present investigation DE₃₀ and DE₆₀ values were calculated from the dissolution data of each product and used for comparison. The dissolution data of nateglinide and its inclusion complex were studied by using dissolution software PCP DISS0 V.3.0 and data are computed in table 2 and the comparative dissolution profiles are shown in figure 5. The dissolution data obtained were subjected to model fitting and the model which fits the observed dissolution data was evaluated by correlation coefficient (r) between the variables involved. The dissolution of standard nateglinide and from various inclusion complexes obeyed both Hixson-Crowell’s cube root dissolution rate law and first-order dissolution models. T_50, DP₃₀, RDR₃₀, DE_10, DE₃₀ and DE₆₀ values were calculated from the dissolution software and are given in table 2. The results of the dissolution rate studies indicated higher dissolution rate of nateglinide from inclusion complex when compared to nateglinide itself and the corresponding physical mixtures.

 

Figure 5: Comparative dissolution profiles of pure drug, physical mixture and inclusion complex.

Table 2: Dissolution parameters for pure drug, physical mixtures and NAT-HPβCD inclusion complex.

 

One-way ANOVA was used to test the statistical significance of difference between pure and treated samples. Significant differences in the means were tested at 95% confidence. The DE₃₀ and DE₆₀ values were significantly higher (P<0.05) in inclusion complexes prepared by microwave irradiation method when compared to other inclusion complex and standard nateglinide. The slight increase in dissolution rate and efficiency values recorded for the physical mixture may be explained on the basis of the solubility of the drug in aqueous HPβCD solutions. Since the HPβCD dissolve more rapidly in the dissolution medium than the drug alone, it can be assumed that, in early stages of the dissolution process, the HPβCD molecule will operate locally on the hydrodynamic layer surrounding the particles of the drug^{20, 21, 22}. This action results in an in situ inclusion process, which produces a rapid increase of the amount of the dissolved drug. Overall the rank order of improvement in dissolution properties of nateglinide with HPβCD concentration 1:2>1:1M and with methods MC > KNE > PM.

 

CONCLUSIONS:

Physicochemical characterization of NT- HPβCD inclusion complex in solution state by phase solubility revealed 1:1M complexation of nateglinide with HPβCD. A true inclusion of NT with HPβCD at 1:1 and 1:2 M was observed in both kneaded and microwave irradiated inclusion complex and were confirmed by FTIR, DSC, powder XRD studies. Dissolution properties of NT-HPβCD inclusion complexes were superior when compared to pure NT. Overall microwave irradiation method showed superior dissolution properties when compared to kneaded system, physical mixtures and pure drug nateglinide. Thus, from the research work it was concluded that aqueous solubility and dissolution rate of NT can be improved by complexation with cyclodextrin.

 

ACKNOWLEDGMENT:

The authors are thankful to Divis Laboratories Hyderabad, India, for providing gift sample of nateglinide. The authors are also grateful to the Principal, Staff and Management of V. L. College of Pharmacy, Raichur for providing all necessary facilities to carry out the research work.

 

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Received on 18.09.2017         Modified on 24.10.2017

Accepted on 01.11.2017      © RJPT All right reserved