INTRODUCTION:
Based on the permeability of drug,
the Biopharmaceutical Classification System (BCS) categorizes into two major
classes, viz. Class II and IV. Drugs which belong to class II of the biopharmaceutical
classification system (BCS) are characterized by high membrane permeability and
slow dissolution rate (due to low aqueous solubility) [1]. The solubility or
dissolution rate of a drug in this category is therefore a key factor in determining
the rate and extent of its absorption. Enhancement of the dissolution rate is
essential to obtain for therapeutic effect and rate-limiting step for
bioavailability. Several technological methods have been reported for
improvement of solubility and dissolution rate of poorly water-soluble drugs,
namely reducing particle size, solubilization in
surfactant systems, formation of water soluble complexes, strong electrolyte
salt formation that usually have higher dissolution rates[2], manipulation of
the solid state of the drug substance to improve drug dissolution[3]. Solid
dispersion can be defined as distribution of active ingredients in molecular,
amorphous, and/or crystalline forms surrounded by an inert carrier [4-5].
Formulation of poorly water-soluble drugs as solid dispersions leads to a
marked improvement in their dissolution rates and is often helped by an
increase in their relative bioavailability [6-7].
Nebivolol is a beta-adrenergic
receptor blocking agent, having very low water solubility, which results into
poor dissolution rates. The main objective of this work was to investigate the
possibility of improving the solubility and dissolution rate of NEB by
preparing solid dispersions with various carriers by two different methods viz.
fusion method and solvent evaporation method. Solid dispersions were evaluated
for solubility studies, in-vitro dissolution rate studies and
interaction between drug and carriers using FT-IR, DSC, SEM studies.
MATERIALS AND METHODS:
Materials:
Nebivolol HCl
was kindly gifted by Glenmark Generics Limited (Colvale) Goa. PEG 6000, PVP K 30 and methanol were obtained
from CDH Delhi.
Phase solubility study:
Phase solubility studies were carried out to evaluate the possible solubilizing effect of the carrier by adding an excess
amount of drug to flask containing 10 ml of aqueous solutions containing
increasing concentrations of PEG 6000 and PVP K 30 (0.11% w/v). The flasks
were placed in a mechanical shaker at 300 rpm and room temperature for 24 hour.
After 24 hour these solutions were filtered and analyzed by UV
spectrophotometer. The apparent stability constant (Ks) was calculated
according to the following equation [8]
The Gibbs free energy of transfer
(∆G0tr) of nebivolol from
pure water to the aqueous solution of carrier was calculated as follows:
Where S0/SS
is the ratio of molar solubility of nebivolol in
aqueous solutions of carrier to that of the same medium without carrier.
Determination of saturation solubility [2]:
Solubility study was
performed using agitation method and saturated solution of nebivolol
was prepared in respective solvent media and stirred for 24 hours. The solution
was then centrifuged for 15 min over 10,000 rpm and filtered through whatmann filter paper 0.45 mm. The concentration of nebivolol was determined using UV-visible spectrophotometer
(UV-1800, Shimadzu corporation) against respective solvent as blank.
Preparation of solid dispersions:
Fusion method [9]:
Solid dispersion was prepared by melting the weighed amount of either of
the different carriers separately in a glazed porcelain evaporating dish. The
carrier was heated to a temperature just sufficient to melt it completely. The
drug was then added to this melt, mixed thoroughly and continuously until a
uniform mix is obtained where upon the dish was instantly removed from the heat
source, and immediately cooled to 5°C using ice water mixture. The dish was
maintained at the specified temperature for period of 5 minutes or so until
solidified hard mass remained. Then allowed to air dry at
room temperature for 48 hr with intermittent mixing and agitation. The
dispersions after drying were pulverized using a glass mortar and pestle. The
pulverized mass was then sifted through a #40 sieve to obtain a uniform
particle size and stored in desiccators at room temperature until further use.
The dispersions were prepared in different ratios with respect to drug and
polymers as shown in Table 1.
Solvent evaporation method [10]:
Solid dispersions were prepared by dissolving weighed amount of either of
the different carriers separately in a glazed porcelain evaporating dish in a
quantity of methanol sufficient to dissolve it completely. The drug was then
added to this solution and mixed thoroughly and continuously until the major
portion of methanol used was volatilized and hard to semisolid mass remained. Then allowed to air dry at room temperature for 48 hr with
intermittent mixing and agitation. The dispersions after drying were
pulverized using a glass mortar and pestle. The pulverized mass was then sifted
through a #40 sieve to obtain a uniform particle size and stored in desiccators
at room temperature until further use. The dispersions were made in different
ratios with respect to drug and polymers as shown in Table 1.
Table
1: Composition of solid dispersion:
|
Formulation code |
Carrier |
Drug :carrier |
Method |
|
F1 |
PEG
6000 |
1:1 |
Fusion
method |
|
F2 |
|
1:3 |
|
|
F3 |
|
1:5 |
|
|
F4 |
PVP K
30 |
1:1 |
Solvent
evaporation |
|
F5 |
|
1:3 |
|
|
F5 |
|
1:5 |
|
Solid
state characterization:
Fourier Transform Infrared Spectroscopy (FTIR):
FT-IR spectrum
of the pure drug sample was recorded with Shimadzu 8400S. The interference study was carried out using FTIR analysis. IR spectrum of pure drug, pure polymer and its solid dispersions were performed for
polymer drug interaction studies between 4000 cm-1to 400 cm-1.
Differential scanning calorimetric
analysis (DSC):
The possibility of any interaction between the drugs and the carriers
during different approaches was assessed by carrying out thermal analysis of
drug as well as the optimized formulation, using DSC. DSC analysis was
performed using Shimadzu-Thermal Analyzer DSC 60 (Japan) on 1 to 5 mg samples.
Samples were heated in an open aluminum pan at a rate of 10°C/min conducted
over a temperature range of 50 to 300°C under a nitrogen atmosphere.
Scanning electron microscopy (SEM):
Morphology of prepared solid dispersion were examined by scanning electron
microscope (JSM-5610, Tokyo,
Japan)
operating at 10.0 kV accelerating voltage. For conventional imaging in the SEM,
specimens must be electrically conductive, at least at the surface, and
electrically grounded to prevent the accumulation of electrostatic charge at
the surface. Therefore the optimized solid dispersions were carbon coated
before being subject to electron scanning. The energy of electron beam was set
at 10 kV.
In-vitro drug release/dissolution studies:
The in vitro dissolution
study was performed in a USP Type II dissolution test apparatus using 900 ml of
phosphate buffer (pH 6.8) at 37+0.5 ◦C and stirred at
50 rpm for 30 min. Pure nebivolol
or its equivalent of SD was sprinkled into the dissolution flask. At
predetermined time intervals, samples of the dissolution medium were withdrawn,
filtered through a millipore membrane of 0.45 mm pore
diameter and analyzed spectrophotometrically against a blank formulation.
Drug content uniformity [2]:
Sample containing 10 mg of prepared solid dispersion was accurately weighed
and dissolved in freshly phosphate buffer pH 6.8 in a 100 ml volumetric flask.
Then the volume was made up to 100 ml with phosphate buffer pH 6.8. From this 1
ml was taken and suitable dilutions were made to get 1 μg/ml
with phosphate buffer pH 6.8. The absorbance of the resulting solution was
measured at 280 nm against blank (phosphate buffer pH 6.8).
Mathematical models for drug release
kinetics:
The in vitro drug release data were fitted to various release
kinetic models [11-14] viz. first-order, Higuchi, Hixson-Crowell
cube root, KorsemeyerPeppas and zero-order model
employing the following set of equations:
First-order model
(3)
Zero-order kinetic model
t (4)
Higuchi model
(5)
Hixson-Crowell cube root model
(6)
KorsemeyerPeppas model
(7)
Where Mo, Mt and M∞
correspond to the drug amount taken at time equal to zero, dissolved at a
particular time t and at infinite time, respectively. The terms Wo and Wt
refer to the weight of the drug taken initially and at time t, respectively.
Various other terms viz. k, ko,
k1, k1/3 and K refer to the release kinetic constants obtained
from the linear curves of KorsemeyerPeppas,
zero-order, first-order, Hixson-Crowell cube root law and Higuchi model,
respectively [15].
Statistical
analysis [16]:
All the results were expressed as
mean value ± standard deviation (SD). One way analysis of variance (ANOVA) by
using graph pad prism 6 XML was used to test for significance, at a 0.5%
significance level. Statistical difference dealing (P < 0.05) was considered
significant.
RESULTS AND DISCUSSION:
Phase solubility study:
Fig. 1 represents solubility of PEG
6000 and PVP K30 indicates a linear relationship (AL type of curve)
in the investigated polymer concentration range. The Gibbs free energy of
transfer (∆G0tr) and apparent stability constants
(Ks) derived from Fig. 1 are shown in Table 2. The
plots of drug solubility against the polymer concentration (Fig. 1).
Table 2 show that all values of ∆G0tr were negative
at all levels of carriers, demonstrating spontaneity of drug solubilization process. The values show a declining trend
with increase in the carrier concentration to construing that the process is
more favorable at higher carrier levels. Table 2 also indicates that PVP K30
interaction has a higher Ks value. The higher Ks value
indicates that the binding affinity between PVP K30 is more than that of PEG
6000. The results show that in both cases, the solubility of PVP K 30 increased
with increasing carrier concentration.
Table 2: Gibbs free energy values and apparent stability constants
(Ks) of PVP K30 and PEG 6000 interactions
|
Concentration of carrier (%w/v) |
G◦tr(j/mol)
for various water-soluble carriers at 37◦C |
|
|
PEG 6000 |
PVP K 30 |
|
|
0.1 |
-79.37 |
-250.74 |
|
0.25 |
-154.20 |
-458.47 |
|
0.50 |
-394.14 |
-971.68 |
|
0.75 |
-663.59 |
-1214.91 |
|
1 |
-835.17 |
-1532.25 |
|
Slope |
0.680 |
0.985 |
|
ks |
17.8571 |
564.327 |
|
R2 |
0.903 |
0.960 |
Fig. 1:Solubility of
nebivolol (g/100 ml) in aqueous solutions of PVP K30
and PEG 6000 in water at 37◦C. (Each point represents mean of
three determinations.)
Saturation solubility:
Saturation solubility of nebivolol was determined
in various aqueous media (distilled water, 0.1 N HCL, phosphate buffer pH 6.8
and methanol). In acidic pH, nebivolol has
appreciable solubility owing to its ionization and basic nature. It is obvious
that it dissolves less in the solutions of higher pH in which it remains in a
unionized form. From the solubility study data nebivolol
shows lower solubility in water.
Table 3: Solubility study data
|
Sr.
No. |
Solvent
media |
Concentration
(mg/ml) |
|
1 |
0.1N HCL |
0.215 |
|
2 |
pH 6.8 phosphate buffer |
0.115 |
|
3 |
Water |
0.041 |
|
4 |
Methanol |
0.569 |
Fourier Transform Infrared Spectroscopy (FTIR) analysis:
Fig. 2-6 shows the FTIR spectrum of NEB and its optimized solid
dispersions. Characteristic peaks of NEB at 1595.02 cm-1 (N-H stretching),
3203.54 cm-1 (O-H stretching), and 1101.20 cm-1 (cyclic ether C-O stretch),
3003.01 cm-1(aryl substituted C=C), 1303 cm-1 (C-N stretch) and 2923.88 (C-H
stretch) were observed.
Important vibrations in the spectrum of PEG 6000 are the OH stretching at
3442.70 cm-1 and CH stretching at 2887.24 cm-1. The spectra of PEG6000 (MA
1:1) solid dispersion (Fig. 4) can be simply regarded as the superposition of
NEB. The intensities of some peaks in PEG had doubled in its solid dispersion
indicating the summation of intensities of drug and carrier at these peaks and
probably a slight increase in the crystallinity of
PEG. This slight increase in crystallinity of PEG
might be a reason for decrease in solubility with increase in PEG concentration.
Slight difference was seen in the position of the absorption bands of PEG
whereas the minor peaks due to NEB were absent indicating trapping of NEB
inside the PEG matrix. However, the major characteristic peaks for NEB were
still present.
Important vibrations in the spectrum of PVP K30 (Fig. 5) are the C-N
stretching (tertiary amide) at 1541.02 cm−1, CH stretching at 2954.74
cm−1, C=O stretching at 1662.52 cm−1 and C-C stretching at 1290.29
cm−1.Increase in crystallinity of PVP K30 might
be a reason for increase in solubility with increase in PVP K30 concentration.
Slight difference was seen in the position of the absorption bands of PVP
whereas the minor peaks due to NEB were absent indicating NEB was dissolved
inside the PVP matrix. The major characteristic peaks for NEB were still
present. All optimized solid dispersions showed characteristic peaks of NEB
drug and carriers. These results indicated that there is no chemical
interaction between drug and carrier when formed as solid dispersion.
Fig. 2: FT-IR spectra of Nebivolol
Fig. 3: FT-IR spectra of PEG 6000
Fig. 4: FT-IR spectra of PEG 6000 SD
Fig. 5: FT-IR spectra of PVP K30
Fig. 6: FT-IR spectra of PVP K30 SD
Differential scanning calorimetric analysis
(DSC):
Thermal behavior of pure drug and corresponding drug-carrier system is
depicted in (Fig. 7) The DSC curve of NEB profiles a sharp endothermic peak at
228.78ΊC corresponding to its melting, indicating its crystalline nature.
However, the characteristic endothermic peak, corresponding to drug melting was
altered in the optimized solid dispersion.
A complete disappearance of the drug melting peak was observed in PVP K30
(SE 1:5) solid dispersion (Fig. 7C) which is attributable to the dissolution of
drug in the melted carrier before reaching its fusion temperature whereas one
endothermic peak at temperature slightly lower than that of the PVP K30 fusion
was observed which may be attributed to the fusion of an eutectic mixture
between NEB and PVP K30. It should also be noted that the incorporation of NEB
into PVP resulted in a change in the peak temperature of the endotherms displayed by the carrier, indicating that the
presence of higher polymer concentration and uniform distribution of drug in
the crust of polymer, resulted in complete miscibility of molten drug in
polymer.
Apart from this, no polymorphic changes were observed in any of the
optimized formulations.
Scanning electron microscopy (SEM):
For conventional imaging in the SEM, specimens must be electrically
conductive, at least at the surface, and electrically grounded to prevent the
accumulation of electrostatic charge at the surface. Therefore the optimized
solid dispersions were carbon coated before being subject to electron scanning.
The energy of electron beam was set at 10 kV.
Scanning electron micrograph of pure NEB shows needle shaped crystals
indicating the crystalline nature of the drug (Fig. 8A). The SEM images of
selected solid dispersions are shown in Fig. 8C. SEM
photomicrograph of SE PVP 1:5 shows that the drug particles are entrapped
within the carrier matrix, confirming FTIR and DSC data analyses. This surface
modification ensures the decrease in crystallinity of
the drug particle.
These
images indicate the change in surface morphology of drug particle due to
entrapment into the respective polymeric matrix.
Fig. 8: Scanning electron micrograph of (A)
Pure NEB (B) Pure PVP K30 (C) PVP K30 SD
In-vitro drug release/dissolution studies:
Fig. 9 summarizes the experimentally determined solubility and dissolution
of the pure NEB and its solid dispersions in phosphate buffer pH 6.8. All
drug-carrier combinations showed an increase in solubility and dissolution of
NEB as compared to pure NEB. Amongst, all dispersions PEG 6000 by fusion method
and PVP K30 by solvent evaporation method showed an exceptional increase in
solubility as well as dissolution of NEB as compared to plain drug. This might
be due to hydrophilic nature of the carriers. PEG6000 is a polymer of ethylene
oxide and water which entrap NEB into its matrix and enhances the solubility.
Correlating the solubility data with the concentration of carrier with respect
to drug, it was observed that with carriers PVP K30, solubility increased with
increasing concentration of the carrier whereas with PEG 6000 increase in
solubility of NEB was observed with decrease in concentration of carrier.
Dissolution profiles of all solid dispersion are shown in fig. 9 which
indicated that the SD ratio 1:5 of drug: PVP K30 gives fast dissolution of drug
as compared to other ratios. The
result of drug release it is concluded that the drug release in following order
F6>F3>F5>F4>F2>F1. Fig. 9 showed the dissolution profiles of
selected solid dispersions as compared to plain drug.
Fig. 9: Dissolution
profile of pure nebivolol and their solid dispersion
Drug content uniformity:
Drug content of the solid dispersions was found to be between 94.13-95.55 %
as shown in table 4. All the solid dispersions showed the presence of high drug
content and low standard deviations of the results. It indicates that the drug
is uniformly dispersed in the powder formulation. Therefore, the method used in
this study appears to be reproducible for preparation of solid dispersion.
Mathematical models for drug release
kinetics:
Solid dispersions of solvent evaporation
method tended to exhibit Fickian diffusional
characteristics, as the corresponding values of n were lower than the standard
value for declaring Fickian release behaviour, i.e.,
0.4500. The goodness of fit for various models investigated for binary systems
ranked in the order of Higuchi > Korsmeyer-peppas
> First-order > zero-order > Hixson-Crowell.
Table
4: % Drug content of nebivolol in solid dispersions
(mean± S.D.)
|
Batch code |
%Drug content |
|
F1 |
95.84+0.176 |
|
F2 |
96.25+0.028 |
|
F3 |
96.11+0.035 |
|
F4 |
95.25+0.042 |
|
F5 |
94.13+0.063 |
|
F6 |
95.55+0.622 |