Ternary Systems of HP β-Cyclodextrin Felodipine Inclusion Complexes: Preparation, Characterization and Solubility Studies

 

Minal Raghunath Narkhede1*, Bhanudas Shankar Kuchekar2 and Jitendra Yadav Nehete3

1Department of Biopharmaceutics, Govt. College of Pharmacy, Karad, Dist-Satara (M.S.) 415124 (INDIA).

2Maharashtra Institute of Pharmacy, Pune-411038 (M.S) (INDIA).

3Department of Pharmacognosy, M.G.V’s Pharmacy College, Panchavati, Nasik 03.

Corresponding author: minalnehete@rediffmail.com

 

ABSTRACT:

The aim of this study was to investigate the effect of presence of water soluble polymer, PVP K -25, PEG 6000 and HPMC, on complexation of felodipine with HP β CD. Solid complexes at mole ratio 1:1 were obtained by freeze drying method. The phase solubility studies indicated the formation of HP- β cyclodextrin complexes at a 1:1M ratio in solution, in presence and absence of water soluble polymers. All complexes  were studied by X- ray diffractometry, Differential Scanning Calorimetry, FT-IR spectroscopy and dissolution study. X-ray diffraction data revealed decrement in crystallinity of binary and ternary systems. All DSC curves of binary and ternary systems showed shifting of characteristics melting peaks of pure drug. The process of felodipine complexation with HP β CD, in presence of water soluble polymer was shown to involved aromatic ring, the carbonyl groups in ester bonds and carbon atom of DHP (dihydropyridine) linked via ester bonds. One of aim of complexation was to improve drug solubility and hence the dissolution rate of binary and ternary system tested. As result of inclusion complex formation, obtained by freeze drying method, brought dramatic 20 fold increase Felodipine solubility in presence of water soluble polymer HPMC.

In photo-stability studies it is observed that upon inclusion complexation of  felodipine with HP β CD showed dramatic decreases in degradation rate constants and increases in the values of  t0.1% .The possible reasons for  protection of complexed felodipine against photo degradation could be due to inclusion of dihydropyridine ring into CD cavity where, dihydropyridine ring is involved in the first step of drug photo degradation. In docking of felodipine with cyclodextrin derivative(HPβ CD), a hypothetical structures of complex also supported inclusion of dihydropyridine ring into CD cavity.

 

KEYWORDS: Felodipine, Water soluble polymers, HP β-Cyclodextrin, freeze drying, molecular modeling photostability.

 


 

INTRODUCTION:

The increasing interest in optimizing the efficiency of drug activity   through the use of rationally designed drug carrier molecules. Cyclodextrins (CD’s) are strong candidates for modifying physical, chemical and biological properties of the drug molecules through formation of inclusion complexes. 1, 2

 

Beta cyclodextrins are cyclic (α 1-4) linked oligosaccarides of α D-glucopyranose, containing a relatively hydrophilic outer surface. Owing to lack of free rotation about the bond connecting the glucopyranose units are not perfectly cylindrical molecules but are toroidal or cone shaped.

 

Based on this architecture the primary hydroxyl groups are located on narrow side of the cone shape, while the secondary hydroxyl groups are located on wider edge3. Many advantages of drug complexed with cyclodextrins have been reported in scientific literature which includes increased solubility, enhanced bioavailability, improved stability, taste masking of bad taste or odor, reduced volatility, transformation  of liquid or gas into solid form, reduced side effect and the possibility of drug release system.4,5

 

For variety of reasons including cost, production capability and toxicology, the amount that can be incorporated limited.6 Therefore it is important to develop methods which can be applied in order to enhance the efficiency of drug: CD complexation, as low complex efficiency would required a large amount of CD than that acceptable for solid or liquid dosage form. Therefore it is of practical importance to add small amount of suitable water soluble polymer to drug-CD system in improving both complexing and solublizing efficiencies of CD’s.7 Polymers are known to interact with CDs, enhancing drug availability in aqueous solutions8. The favorable effect of polyvinyl pyrrolidone  K-259 and polyethylene glycols10 on solubility and dissolution rate of naproxen has been demonstrated. A strong increase in naproxen-hydroxyl propyl β CD binding constant is observed when 0.1٪ w/v of PVP is added.11

 

Felodipine is dihydropyridine calcium antagonist widely used as potent antihypertensive drug. However the oral bioavailability of felodipine is very low because of 1st pass-effect.12 Felodipine is poorly water soluble; the dissolution would be rate limiting process for absorption of drug. Many technological methods regarding enhancement of dissolution characteristics of drug with low water solubility has been reported such as micronisation, formation of solvates, complexes, micro spear and solid dispersions.13 In present work, the effect of water soluble polymers viz HPMC, PVP and PEG6000 on complexation of felodipine with HP β cyclodextrins was investigated. The effect of watersoluble polymer on solublising efficiency of cyclodextrins and dissolution of felodipine from CD complexes were also investigated.         

 

MATERIALS AND METHODS:

Materials:

Felodipine was obtained as gift sample from Cipla Private Limited Mumbai, HP β- CD was obtained from Rouquet Service Techniques ET Laboratories France, HPMC E- 5, PVP K-25 and PEG 6000 were obtained from Loba Chemie, India. All other chemicals used were of pharmaceutical or analytical grade.

 

Method:

Phase solubility studies:

Solubility studies were performed according to the method reported by Higuchi and Conner’s.14  Excess drug was added to 10 ml double distilled water containing various concentration of HP β CD (0 – 15Mm) in series of 50 ml of conical flask and mixture were shaken for 72 Hr at room temperature on rotary shaker. After equilibrium 2ml aliquots were withdrawn at 1 Hr interval and filtered immediately using 0.45 μm nylon disc filters. The filtered sample were diluted suitably and assayed for felodipine at wavelength 364nm against blank prepared in the same concentration of HP β- CD in water so as to cancel absorbance that may be exhibited by cyclodextrin molecules. Shaking was continued until three consecutive estimations were same. Phase solubility studies were conducted in each case with addition of, HPMC, PVP and PEG 6000, at concentration of 0.25 %w/v to solution containing CD. The solubility experiments were conducted in triplicate.

 

The apparent stability constant (Kc) according to the hypothesis of 1:1stochiometric ratio of complex was calculated from phase solubility diagram using following equation:

K1:1=Slope/S0 (1-slope)……………..1

Preparation of Inclusion Complex:

Solid inclusion complex of felodipine-HP β CD were prepared in 1:1 ratio by freeze drying method with or without addition of PVP, HPMC, PEG 6000. 50 ml methanolic solution of felodipine of concentration1×10-3mole/lit was mixed with 50ml of 1×10-3mole/lit water solution of HP β CD.PVP,15  HPMC and PEG6000 were added at  concentration of 10%w/w of solid complex and mixed for 30 min with magnetic stirrer at constant rate of revolution. The samples were freeze dried at - 40 C  for   48 hrs.

 

Powder X -Ray Diffractometry:

The powder X- ray diffractometer pattern of drug and complex were recorded by using an automated Philips PW 3710, Cu radiation over the interval 5-990/2Ө. The operation data were as follows: voltage 40 kV, current 30 mA.

 

 Differential Scanning Calorimetry:

Thermal analysis was performed on DSC-60 230 V Shimadzu TA60WS. Samples (10mg) of solid complexes containing same amount of felodipine were heated at scanning rate of 200C/min from 400C up to 2500C. Temperature calibration was performed using Indium as standard.

 

Infrared Spectroscopy:

Complex formation was evaluated by comparing IR spectra of solid complexes, by   analyzing on Shimadzu FT-IR equipment. Blends corresponding to 1.5mg of samples and 150 mg of KBr were compressed and IR spectra were recorded in region of 4000-400cm-1.

 

Dissolution Rate Study:

100 mg samples of felodipine and equivalent amount of inclusion complexes were placed in container in order to determine their dissolution rates according to USP paddle method using 500ml double distilled water (370C) as a solvent. The whole sample was agitated at 100 rpm and after specific time intervals of 10 min, for one hour, 5ml of solution was withdrawn and volume was made up at each time by adding double distilled water (370C) to 500ml. Withdrawn 5 ml sample filtered through membrane filter of 0.45 µm and was assayed spectrophotometrically (364nm). All samples were analyzed in triplicate.

 

Photo-stability studies:

The photostability of felodipine and complexes were assessed in distilled water. Complex equivalent to 0.03mg/ml (9×10-5 moles/lit) felodipine was added in excess to prepare solution. The suspensions were filtered through the 0.45 µm membrane filter (Whatman Ltd., USA) to obtain a clear solution. These solutions were positioned 30 cm away from ultraviolet radiation light source (continuous 254-nm UV lamp Camag UV cabinet). The solutions were sampled at the specified time intervals (0.33, 0.66, 1.0, 2.0, 3.0 months). The aliquot of solution removed at predetermined check points. The remaining felodipine in solution was assayed using UV spectrophotometer at   364 nm.

In order to study kinetically the photodegradation reaction, a first order reaction is assumed to obtain the predicted shelf-life (t0.1), by using Eqs. (1) and (2):

nC= lnC0-Kt                                                      (1)

t0.1 = (100 - 90)/ K                                             (2)

Where C0 is the initial percentage of felodipine i.e (C0  =100%), C is the ratio (%) of the amounts of felodipine at time t to the initial amount and K is the rate constant. 16

 

Molecular modeling/Docking:

Study protocol:

The 3D structure of hydroxyl propyl βCD was built using the builder module of Avagadro-1.0.0. The obtained models were subjected to optimization with MMFF94 force field, a protocol of steepest descent and 500 steps of conjugate gradients to avoid Steric hindrance and clashes that can appear in the building process.

 

The most probable structure of the felodipine/ HPβ-CD inclusion complex was determined using the ArgusLab (Version 4.0) program (Planaria Software). Autodock 3.0.518 with Lamarkian Genetic Algorithm (LGA) was used to generate the starting complexes. The parameters used for the global search was an initial population of 50 individuals, with a maximal generations of 1000 as an end criterion. An elitism value of 5 was used, and a probability of mutation and crossing-over of 0.2 and 0.8 was used, respectively. From the best solutions obtained according to these parameters, some of them defined by the user as the best probabilities in our case 0.06 were further refined by a local search method.

 

The following procedure was employed on the CD docking simulations: 298 runs were done for CD. At the end of each run, the solutions were separated into clusters according to their lowest RMSD and the best score value based on a free empiric energy function. Cluster solutions whose average score was not over 1 kcal mol-1 with respect to the best energy obtained in the respective run were selected.

 

RESULTS AND DISCUSSION:

Phase Solubility Study:

The phase solubility diagram for complex formulation between felodipine HP β CD in absence and presence of water soluble polymers is presented in figure 1. The plot showed that the aqueous solubility of the drug increases linearly as a function of HP β CD. The linear host guest co-relation with slope of less than 1 suggested that formation of 1:1 complex was with respect to concentrations of HP β-CD. The presence of water soluble polymers elicited that the formation of 1:1 ternary complex of felodipine with HPβ CD. The apparent stability constants (Kc) obtained from the slope of linear phase solubility diagram were as in table 1. The values of stability constant were found to be higher in presence of water soluble polymers, with HPβ CD indicating higher complexation efficiency.

 

Fig.1 felodipine/HP β-cd phase solubility curves

 

Powder X-Ray Diffraction:

The diffraction pattern of complex is supposed to be clearly distinct rather than the superimposion of each component in the true inclusion complex. Crystallinity was determined by comparing some respective peak heights in diffraction pattern of binary and ternary with those of reference. The powder X-ray diffraction pattern of complex and felodipine were presented in figure 2. The relationship used for the calculation of crystallinity is relative degree of crystallinity (RDC) = I sam/ I ref. Where  Isam= the peak height of sample under investigation and I ref= The peak height at same angle for reference with highest intensity17. Pure drug peak at 23.2300(2θ)was used for calculating the RDS of Freeze dried binary and ternary systems. The RDC values of corresponding binary and ternary systems are given in the table-2. RDC values obtained by using felodipine as reference indicate that crystallinity of binary and ternary system was lower than felodipine.


 

 

Table1. Data Obtained From Phase Solubility Analysis: Apparent stability constant (Ks) and fold increase in solubility.

No.

Formulation

Stability Constant

M-1

Fold increase in stability constant

Solubility μg/ml

Fold increase in solubility

1

Felodipine

-

-

2

-

2

HP β CD+ Felodipine

467.44

1.40

8.83

4.41

3

HP βCD + PVP+ Felodipine

1003

3

18.04

9.02

4

HP βCD + PEG6000 + Felodipine

710

2.12

13.05

6.52

5

HP βCD + HPMC + Felodipine

768.45

2.30

13.82

6.91

 


 

Fig. 2 x-ray diffraction pattern of pure substance and their freeze dried complexes (A) felodipine (F) felodipine HP β-cd (G) felodipine HP β-cd PVP (H) felodipine  HP β-cd PEG 6000 (I) felodipine HP β-cd HPMC.

 

Table 2.Relative degree of crystallinity and peak intensities of felodipine and HP β-CD complexes in presence of water soluble polymers.

Sr. No.

Formulation

Peak Intensity

Relative degree of crystallinity   (RDC)

1

Felodipine

296

-

2

HP β-CD + Felodipine

48

0.162

3

HP β-CD + PVP + Felodipine

117

0.397

4

HP β-CD + PEG6000 + Felodipine

164

0.554

5

HP β-CD + HPMC + Felodipine

164

0.554

 

Differential Scanning Calorimetry:

The thermal behavior of binary and ternary freeze dried complex was different to untreated drug sample, indicating that freeze drying process substantially affect their solid state properties. The DSC thermogram for felodipine, binary and ternary complexes are shown in figure 3. Felodipine exhibits a characteristic endoderm fusion peak at 148.76 0C hence no polymorph of felodipine could be found. Data obtained from DSC was as per Table 3. The broad endothermic peak at the beginning of DSC curve has been related to liberation of water molecule from HP β CD grooves. Both characteristic peaks due to water liberation and drug melting were clearly visible in freeze dried mixture of felodipine, even if melting endotherm were slightly shifted. This may be due to interaction between the two species occurring during their mixing. From ∆H values obtained it was easy to obtain percentage of free drug from following equation.18

%Free Drug =

[∆H complex × 100]/ [∆H pure drug × (DC)/100] …..2

 From equation 2 we could able to estimate that percentage of complexed drug (100 - % Free Drug).

 

Ternary complex of felodipine, HP β CD and HPMC showed disappearance of characteristic endothermic fusion peak at 148.76 0C. A disappearance of endothermic peak may be attributed to amorphous state and /or inclusion complexes.

 

Fig. 3 DSC thermograms of felodipine and its freeze dried complexes (1) felodipine (2) felodipine HP β-cd (3) felodipine HP β-cd PVP (4) felodipine  HP β-cd PEG 6000 (5) felodipine HP β-cd HPMC

 

FT-IR Spectroscopy:

The complexation process of felodipine, HP β-CD, in presence or absence of water soluble polymer had been confirmed by IR spectroscopy. The IR spectra of complexes were significantly different from pure drug as in Figure 4. In IR spectra of inclusion compounds of felodipine studied, the bands were in the range of 3050-3370 cm-1due to the stretching vibration of NH bonds in dihyropyridine ring which were much broader. The difference also was appeared in band corresponding to vibration of carbonyl groups in ester bonds. The spectra of corresponding inclusion complexes were broadened and shifted toward shorter waves. Differences were also noted in range 1500-1600 cm-1 assigned to vibrations of multiple bonds in unsaturated DHP ring.

 


Table 3Thermal data from DSC studies

Sr. No.

Formulation

Peak ( 0C)

Onset

( 0C)

∆ H

J/g

% Drug content

% free drug

Entrapment efficiency in %

1

Felodipine

148.76

143.56

-95.50

-24.85

-

-

2

HP β- CD+ Felodipine

145.32

142.77

-6.07

21.76

29.2

70.8

3

HP β-CD + PVP+ Felodipine

146.79

143.30

-16.57

19.79

88.1

11.9

4

HP β-CD + PEG 6000+ Felodipine

148.85

143.41

-15.02

19.79

79

21.0

5

HP β-CD + HPMC +felodipine

Peak disappeared

-

-

19.79

-

100

 

 


fig. 4   FT-IR spectra of felodipine and its freeze dried complexes (A) felodipine (F) felodipine HP β-cd (G) felodipine HP β-cd PVP (H) felodipine  HP β-cd PEG 6000 (I) felodipine HP β-cd HPMC.

 

Fig. 5 Dissolution profile obtained from felodipine and its freeze dried complexes with HP-β-cd.

 

DISSOLUTION:

One aim of this study was to form complexation of felodipine HP β-CD with or without water soluble polymer to increase the drug solubility, therefore the dissolution rate of all obtained complexes were evaluated. Dissolution profiles of pure drug felodipine, their 1:1mole ratio freeze dried binary and ternary inclusion complexes with HP β-CD in presence and absence of water soluble polymer were presented in Figure 5. From these curves, binary and ternary complex systems of felodipine displayed an increase in dissolution rate with respect to pure drug. The mark increase in dissolution of felodipine may be explained by mean of greater solubility of drug in aqueous solution of cyclodextrin because of hydrophilic environment surrounding the drug resulting in better wettability of drug. However the increment in dissolution from felodipine-HP β-CD binary and ternary systems was greater as compare to felodipine alone.

 

Photo-stability studies:

Aqueous solutions containing felodipine and methanol - aqueous solutions containing the felodipine-CD inclusion complex, all at the same felodipine concentration were exposed to ultraviolet radiation light source. The effect of inclusion complexation of  felodipine with HP β CDs on the photostability of the drug  was explored by studying the decomposition kinetics of the drug. Felodipine was highly protected against photo degradation by inclusion complexation. The determined values of degradation rate constants of felodipine and complexes are presented ask, t0.1  upon irradiation by light sources are also shown in Table 4. It is clear that inclusion complexation of felodipine with HP β CD showed dramatic decreases in degradation rate constants and increases in the values of  t0.1 (Table 4). It was interesting to notice that inclusion complexation of felodipine with  HP β CD offered much higher protection. Protection of complexed felodipine against photo degradation could be due to inclusion of dihydropyridine ring into CD cavity where, dihydropyridine ring is involved in the first step of drug photo degradation19.

 

The results of this study indicated that it is possible to successfully prepare felodipine HP βCD inclusion complex in solid state by freez drying. The formed inclusion complexes can efficiently retard the photodegradation of felodipine upon exposure to UV light (Fig.-6).

 

Table 4.Photo degradation data of felodipine and its complexes.

Formulation

Kinetic parameters of photochemical decomposition

Rate constant  K (day -1)

Shelf life      ( t 0.1 ) days

Felodipine

- 1.42× 10-3

40.2

HP β-CD + Felodipine

-1.25× 10-3

60.3

HP β-CD + Felodipine+PVP

- 1.11× 10-3

81.6

HP β-CD + Felodipine+HPMC

 - 1.12× 10-3

71.1

HP β-CD + Felodipine+PEG6000

 - 1.07× 10-3

73.5

 

Fig 6.  First order kinetic plot of photodegradation

 

Molecular modeling /Docking:

In docking of felodipine with cyclodextrin derivatives, a hypothetical structures of complex formed is presented in Fig 7.As illustrated,1,4 dihydropyridine ring is enclosed by  HP β cyclodextrin and aromatic phenyl ring of felodipine oriented at the rim of wider side of cyclodextrin cavity. As a result of previous studies and literature data suggested changes in spectra of irradiated felodipine due to formation of 2,3-dichlorophenyl pyridine as degradation compounds20. This suggests that the complexation of felodipine and  HP cyclodextrin should increase the photostability of felodipine since 1,4 dihydropyridine that is prone enclosed by cyclodextrin cavity. The photo-stability data indicated that photo degradation was delayed in presence of studied binary and ternary systems of cyclodextrin. For felodipine and CD complexes, the total computed energy shown in table 5.

 

Table 5.  Total computed energy of felodipine and HPβ CD

Complex

 Computed Total Energy(kcals/mol)

Felodipine-HP βCD

      - 3.6359

 

Fig 7. Hypothetical structure of felodipine HP- β cyclodextrin.

For the felodipine HP β CD complex, computed total energy (kcals/mol) was found comparatively less. This result indicated that the interaction between felodipine-cyclodextrin derivatives might be stronger which was consistent with the results of phase solubility study.

 

A previous studies in literature also presented similar findings that molecular modeling could predict accurately the differences in binding affinity between acetretin and various chemically modified βCD. This  suggest that in development of new chemically modified cyclodextrins or formulations of cyclodextrin complexes, the cyclodextrin molecules and complexes can be assessed computationally for the feasibility even before actual synthesis and experiments to be carried out. This will expedite the process and save the cost in the development21.

 

ACKNOWLEDGEMENT:

We are thankful, the Principal Govt.  College of Pharmacy, Karad (India) for  providing facilities to our research work. The authors are also thankful to Roquette Services Techniques ET Laboratories France for providing gift sample of HP βCD.

 

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Received on 06.07.2011          Modified on 26.07.2011

Accepted on 07.08.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1809-1815