Saquinavir Sodgganga PDF

Chapter-III
3. PREPARATION AND EVALUATION OF SAQUINAVIR AND

RITONAVIR INCLUSION COMPLEXES
The most common pharmaceutical application of cyclodextrins is
to enhance drug solubility in aqueous solutions. Although prediction of
compound solubilization by cyclodextrins continues to be highly
empirical, various historical observations permit several general
statements. First, the lower the aqueous solubility of the pure drug, the
greater the relative solubility enhancement obtained through
cyclodextrin complexation.1 Second, type of cyclodextrin used for
complexation also influence solubility and dissolution rate
enhancement. Cyclodextrins of lower molar substitution are better
solubilizers than the same type of derivatives of higher molar
substitution. For example, RMβCD with molar substitution 0.6 provide
better solubilization than the same type of derivatives of higher molar
substitution.2 Third, charged cyclodextrins can be powerful
solubilizers. SBE7βCD cyclodextrin, where the anion been moved away
from the cavity by a butyl ether space group, is an excellent
solubilizer.3 Inclusion complexation of drug with cyclodextrins by
kneading, co precipitating, spray drying and freeze drying has also been
shown to dramatically increase drug solubility and dissolution rate.4
Hence, in the present investigation it is aimed to compare
different types of CD derivatives and methods of preparation in
enhancing the solubility, dissolution rate and bioavailability of two
protease inhibitors and BCS class II drugs, saquinavir and ritonavir.

3.1. Phase solubility studies
Phase solubility studies were performed as mentioned by
(Higuchi and Connors, 1965) 5 for saquinavir in pH 6.8 phosphate
buffer and for ritonavir in 0.1 N HCl. Solubility studies were carried out
by adding excess amounts of both drugs separately in quantities
exceeding its aqueous solubility to 50 mL of aqueous solutions of
buffers, containing increasing concentrations of cyclodextrins
(2-12 mM). The resulting suspensions were shaken at room
temperature for a period of 72 hrs, until equilibrium was established.
The samples were filtered through a 0.45 µm membrane filter (Millex-
HA filter units, Millipore) and suitably diluted with corresponding
buffer before analysis. All studies were performed in triplicate.
Apparent 1:1 stability constants were calculated from the
straight-line portion of the phase solubility diagrams, according to
Eq. 3.1.
Slope
K :
S 1 Slope
-Eq.3.1
where So is the solubility of the drug in examined buffer solution
in the absence of ligand.
3.2. Preparation of drug-CD complexes
In the present work, saquinavir-CD complexes were prepared
using physical mixing (PM), kneading (KN), co evaporation (COE), spray
drying (SD) and freeze drying (FD) and ritonavir-CD complexes were
prepared using physical mixing, co evaporation, spray drying and freeze
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drying. In the entire cases drug-cyclodextrin ratio was kept at 1:1 molar
ratio.
3.2.1. Physical mixture method6,7
Selected drugs and cyclodextrins at equimolar ratio (1:1) were
weighed and mixed in a mortar by geometric dilution method for
sufficient time (~5 min) to obtain a homogenous powder blend, passed
through sieve no. 80 and stored in a sealed glass vials and kept in
desiccator over fused calcium chloride until further use.
3.2.2. Kneading method7-9
Cyclodextrins were wetted in a mortar with minimum volume of
water-ethanol mixture (1:1) until a paste was obtained. The required
amount of drug was slowly added and the slurry was kneaded for about
45 minutes. During this process, a suitable quantity of solvent was
added to maintain optimum consistency. Further the products were
dried at 40°C to constant weight, passed through sieve no. 80 and
stored in a desiccator until further evaluation.
3.2.3. Solvent evaporation method9
Required quantities of drug and cyclodextrin were dissolved in
sufficient quantity of water-ethanol solvent mixture (1:1) and
evaporated on a water bath at 50°C with stirring. Each solid product
was sieved through #80 and stored in desiccator.
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3.2.4. Spray drying
In spray drying, characteristic properties of powders like particle
size and shape obtained are influenced by the nozzle, the viscosity of
the feeding solution, and the outlet temperature (Tout), the later being
dependent on the two spray drying process variables: inlet temperature
(Tin) and solution flow.10-12 To select best optimizing conditions, SQV
with βCD combination was used and the same process conditions were
applied with other CDs used in complex formation. For optimization,
influence of solution flow rate and Tin were studied and the solutions
were atomized at three different flow rates (2, 5 and 10 mL/min) using
fixed values for compressed air (500 L/h) and aspirator (40 m3/h). Two
Tin values were used: 55°C and 70°C. Tout was kept constant at a
temperature of 50°C.
Spray drying was performed using Labultima LU22 spray dryer
for the preparation of inclusion complexes of both drugs and
cyclodextrins. Equimolar ratio of drug and cyclodextrins were dissolved
in sufficient quantity of ethanol and water mixture (1:1). The solutions
were atomized at a flow rate 2 mL/min using fixed values for
compressed air (500 L/h) and aspirator 40-50 m3/hr with Tin value of
70°C, corresponding to a Tout of 48°C. After spray drying, each resulting
powder was collected by cyclone separation and transferred to glass
vials. After spray drying, all complexes were kept in an oven at 40°C for
24 hrs to remove traces of moisture present in the complexes.
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3.2.4.1. Determination of the moisture content
The residual moisture content of the spray dried complexes was
determined via loss on drying using a HR83 halogen moisture analyzer
(Mettler-Toledo, India). A sample of 0.5 g was dried at 105°C for 5 min.
3.2.4.2. Particle size measurement
Determination of average particle size of spray dried complexes
was carried out by optical microscopy. The eyepiece micrometer was
calibrated with the help stage micrometer. The sample of the spray
dried powder was prepared as dilute dispersion in glycerin. The
dispersion was spread on a clean glass slide and covered with a cover
slip and average size of 300 particles was determined.13
3.2.5. Freeze drying/lyophilization method14,15
Required quantity of cyclodextrin was dissolved in sufficient
quantity of water and required quantity of drug was added and
dispersed. The dispersion was kept under magnetic stirring at 200 rpm
for 72 hrs in closed vials for complex formation which resulted in the
formation of clear solution. The resulting solution was fast frozen at -
20°C using liquid nitrogen and dried at -50°C and 0.0070 mbar
pressure in a freeze dryer (Model MODUL YOD 230, Thermo Electron
Corporation, India) for 48 hrs. The obtained freeze dried product in the
glass vial was stored in desiccator.
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3.3. Drug content estimation
The drug-CD complex equivalent to 50 mg of the drug was
accurately weighed and transferred to 50 mL volumetric flask. 5 mL
methanol was added and continuously shaken for 10 min and final
volume was made up to the mark with pH 6.8 phosphate buffer for
saquinavir and 0.1 N HCl for ritonavir. Suitable dilutions were made
with respective drugs for both drugs. The solution was filtered through
0.45 µm Millipore nylon filter disc and the absorbance was measured at
240 nm using respective buffer for both the drugs as blank. Each study
was done in triplicate.
3.4. Dissolution characteristics16-18
Dissolution experiments were carried out in triplicate (n = 3) with
an USP XXIII paddle apparatus in 900 mL of pH 6.8 phosphate buffer
for SQV and 0.1N HCl for RTV at 37°C using a rotation speed of 50
rpm. In each study drug-CD complex equivalent to 100 mg of drug was
used. 5 mL sample was withdrawn at intervals of 5, 15, 30, 45, 60 min
using a syringe fitted with prefilter (0.45 µm). An equal amount of fresh
dissolution medium maintained at the same temperature was replaced
immediately after withdrawal of the test sample. Test samples were
suitably diluted wherever necessary and the absorbance was measured
as per the analytical procedures described. The mean percent of drug
dissolved and the standard deviations were calculated.
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3.5. Comparison of dissolution profiles
Mathematical comparison of dissolution data to quantify
observed differences in the rate and extent of drug release as influenced
by formulation and process variables, several approaches can be
followed such as analysis of variance (ANOVA) based, model
independent approaches.19 ANOVA methods are commonly used to
detect significant differences between groups and, thereby can be used
to detect statistically significant difference between dissolution profiles.
Model independent approaches are based on the ratio of area under the
dissolution curve (dissolution efficiency) or on mean dissolution time.
3.5.1. Model independent approaches20-25
Model independent approaches include T50 (time to dissolve 50%
of the drug from the dosage form), DE (dissolution efficiency), DP30
(percentage of drug dissolved after 30 min), and MDT (mean dissolution
time). Each of these procedures compares the dissolution profile of two
products at each time point.
3.5.1.1. Dissolution Efficiency (DE)
Dissolution Efficiency suggested by Khan20 was employed and is
defined as the area under the dissolution curve (AUC) up to the time t
expressed as a percentage of the area of the rectangle described by
100% dissolution in the same time. The DE is calculated by the
following Eq.3.2.
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y. dt
Dissolution Efficiency DE "
y! t
-Eq.3.2
Where y is the drug percent dissolved at time t.
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. For example, the index DE60 would relate to
the dissolution of the drug from a particular formulation after 60
minutes and could only be compared with %DE60 of other formulations.
Summation of the drug dissolution data into DE enables ready
comparison to be made between a large numbers of formulations.
%DE60 and DP30, %DE10, T50 and MDT values were calculated from the
dissolution data and compared.
3.5.1.2. Mean dissolution time (MDT)
By definition, the mean dissolution time (MDT) is the arithmetic
mean value of any dissolution profile. It is a measure of dissolution
process and inversely related to release rate. The MDT is calculated by
the following by the following equation Eq.3.3.
∑'+,
'+! t &'( ) ∆M
MDT
∑'+,
'+! ∆M
-Eq.3.3
Where n is the number of dissolution sample times, tmid is the
time at midpoint between two sample intervals, t2 and t1 and ∆M is the
additional amount of drug dissolved between t2 and t1.
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3.5.1.4. Statistical evaluation of experimental data
Different model independent parameters like T50, dissolution
efficiency %DE60, DP30 and MDT obtained for the inclusion complexes
were subjected for one way-ANOVA followed by the Tukey multiple
comparison test. Statistical analysis was performed using Graph Pad
Prism 5.03 (GraphPad Software, Inc.CA, USA) software (trial version)
considering p<0.05.
3.6. Drug-excipient interaction studies
Drug excipient interaction studies were conducted for both
drugs, saquinavir and ritonavir, pure CDs, physical mixtures and freeze
dried complexes to gain insight into interactions between drug and
respective CDs in the complexed form.
3.6.1. Fourier transform infrared spectroscopy (FTIR)
Fourier transform infrared spectra (FTIR) were used to identify
the formation of complex. Samples were analyzed by potassium
bromide pellet method in an IR spectrophotometer (Shimadzu, FTIR
1300) in the region between 400‐4000 cm‐1. Complex formation was
evaluated by comparing the IR spectra of the solid complex with drug.
For comparison purposes, FTIR spectra were obtained for pure
saquinavir and ritonavir, each βCD being studied as well as physical
mixtures of both drugs with each cyclodextrin.
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3.6.2. Differential scanning calorimetry (DSC)
The thermal behavior of saquinavir and cyclodextrin inclusion
complexes was studied using Differential scanning calorimetry in order
to confirm the formation of solid complex. When guest molecules are
incorporated in the cyclodextrin cavity or in the crystal lattice, their
melting, boiling and sublimation points are usually shifted to a
different temperature or disappear within the temperature range. The
samples were heated from 0 to 350°C at a heating rate of 10°C/min
under a nitrogen flow, flowing at a rate of 40 mL/min through the DSC
cell.
3.6.3. Powder X-Ray diffraction study (XRD)
X-Ray diffraction of inclusion complexes and the pure drugs were
performed to identify the interaction of the drug with cyclodextrins
using a PW 1720 X-ray generator and a PW 1710 diffractometer control
(Philips Electronic Instrument). The scanning range (2θ) was from 5° to
90°, and the scan step and scan speed were 0.04° and 0.02°/s,
respectively.
3.6.4. Nuclear magnetic resonance (NMR) spectroscopy
1H-NMR studies were conducted to determine the electronic
interactions between drug and cyclodextrins were obtained from a
Bruker AM-400 spectrophotometer. Samples were prepared by
dissolution in D2O for SQV (99.9% D) and methanol was used as
solvent for RTV. 1H-NMR spectra of inclusion complexes and pure
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components were recorded. Chemical shifts were reported in ppm (δ)
downfield from tetra methyl silane (TMS) (internal reference).
3.6.5. Scanning electron microscopy (SEM)
The morphological properties of the pure saquinavir and
ritonavir, physical mixture, spray dried and freeze dried powders of
SBE7βCD were characterized by scanning electronic microscopy (JEOL,
Model JSM 840 A). For ritonavir, the morphological properties of the
freeze dried powders of ritonavir and RMβCD complex and pure
ritonavir were characterized by scanning electronic microscopy
(Cambridge instrument: Stereoscan 360).
3.7. Results and discussion
The bioavailability of an orally administered drug depends on
general factors like solubility, dissolution rate and the rate of gastro
intestinal absorption. Following the oral administration of a drug alone,
the drug may dissolve slowly and incompletely in the gastrointestinal
tract. Since orally administered drugs must dissolve in the aqueous
medium of gastrointestinal tract prior to absorption, the improvement
of the solubility and the rate of dissolution of poorly soluble drugs can
be seen as first step towards an improvement in oral bioavailability. If
the drug itself is very soluble in water, absorption is the rate
determining step and improvement in the solubility of the drug does
not enhance absorption or may eventually decrease it. On the other
hand, if the drug is poorly soluble in water, solubility is the rate
determining step and the solubility could be increased by drug in the
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complex form to increase the bioavailability. As the two selected drugs
are having poor solubility it was proposed to enhance their solubility by
complexation with cyclodextrins for improving their oral bioavailability.
3.7.1. Phase solubility studies
To test the effect of the cyclodextrins in the improvement of
solubility of the selected drugs, phase solubility studies were conducted
in pH 6.8 phosphate buffer for saquinavir and 0.1N HCl for ritonavir as
suggested by Higuchi and Connors. Fig.3.1 shows phase solubility
diagrams for saquinavir in presence of four different cyclodextrins in
pH 6.8 phosphate buffer. The solubility of saquinavir increased linearly
as a function of cyclodextrin concentration and the values are
mentioned in Table 3.1. The phase solubility profiles showed linear
increase in complexation of saquinavir with increase in cyclodextrin
concentration with all four cyclodextrins, displaying type AL phase
diagrams according to the classification by Higuchi. The correlation
coefficient of the linear regression line for each of the phase solubility
curves was greater than 0.99, indicating a good fit for all complexes.
The slope of the lines was found to be 0.2855 for βCD, 0.5954 for
HPβCD, 0.7711 for RMβCD and 0.7762 for SBE7βCD (Fig. 3.1). As the
slope of the line is less than unity, it was assumed that the increase in
the solubility observed was due to the formation of a 1:1 molar
complex.
The stability constants (K1:1) of saquinavir with each cyclodextrin
were calculated using Eq. 3.1 and given in Table 3.2. The calculated
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values were 906.6 M-1 for βCD, 3706.2 M-1 for HPβCD, 6899.46 M-1 for
RMβCD and 8281.28 M-1 for SBE7βCD. The K1:1 value for saquinavir-
SBE7βCD complex was found to be 8281.28 M-1 which is 9 fold higher
than βCD, 2.23 fold higher than HPβCD and 1.2 fold higher than
RMβCD indicating SBE7βCD has superior saquinavir complexing
efficiency. This may potentially be due to saquinavir interacting with
the anionically charged moiety of the SBE7βCD.
Ritonavir is practically insoluble in water. The phase solubility
diagrams obtained were linear as shown in Fig. 3.2 and values are
represented in Table 3.3. Such profile according to Higuchi and
Connors are of AL type. Because these profiles were characterized by a
slope of less than one, it was assumed that the solubility increase was
due to the formation of a complex in 1:1 molar ratio. Stability constant
values obtained for ritonavir cyclodextrin complexes were 119.01 M-1
for HPβCD and 192.96 M-1 for RMβCD (Table 3.4). 1.6 fold higher
stability constant was obtained with RMβCD compared to HPβCD
proved that RMβCD is having higher complexation efficiency.
Complexation is generally due to the hydrophobic interaction
between poorly water soluble guest molecules such as saquinavir,
ritonavir and the apolar cavity of cyclodextrin molecule. The
hydrophobicity and geometry of the guest molecule as well as the cavity
size and the derivative groups of the cyclodextrin are important for
complex formation. In the present study, the enhancement of
saquinavir and ritonavir solubility was highly dependent on the type of
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cyclodextrin used. Based on the phase solubility diagrams, SBE7βCD is
more effective in solubilising saquinavir in pH 6.8 phosphate buffer
compared to other CDs. The order of solubilising capacity of CDs was
as follows: SBE7βCD>RMβCD>HPβCD>βCD. RMβCD is more effective
in solubilising ritonavir in 0.1N HCl than with HPβCD. The different
complexation constants found for different cyclodextrin molecules
indicated that the derivative groups on cyclodextrins appear to play an
important role in the incorporation of saquinavir and ritonavir into
cyclodextrin cavity, since much higher complexation constants were
obtained with RMβCD and SBE7βCD for saquinavir and with RMβCD
for ritonavir.
Table 3.1: Phase solubility studies of SQV in four CDs

Solubility of SQV (mM/L)
Concentration (mean±s.d.) (n=3)
of CD (mM/L)
βCD HPβCD RMβCD SBE7βCD
0 0.34±0.02 0.34±0.02 0.34±0.02 0.34±0.02
2 0.99±0.01 1.6±0.012 1.89±0.011 2.10±0.01
4 1.56±0.01 3.05±0.02 3.04±0.03 3.67±0.02
6 2.22±0.01 4.23±0.01 5.01±0.01 4.72±0.01
8 2.81±0.02 5.17±0.01 6.61±0.02 6.67±0.02
10 3.28±0.01 6.42±0.03 7.93±0.04 8.10±0.01
12 3.72±0.02 7.53±0.02 9.52±0.02 9.83±0.03
Table 3.2: Stability constants of SQV with four CDs
Type of CD K1:1 (M-1) Phase diagram
βCD 906.80 AL Type
HPβCD 3706.20 AL Type
RMβCD 6899.46 AL Type
SBE7βCD 8281.28 AL Type
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4.5 9
(A) (B)
Concentration of SQV in mM
4 8 y = 0.5954x + 0.4795
y = 0.2855x + 0.4216
r = 0.9973
3.5 r = 0.9957 7
3 6
2.5 5
2 4
1.5 3
1 2
0.5 1
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Concentration of HPβCD in
Concentration of βCD in mM mM
12 12
(C) (D)
y = 0.7711x + 0.2822 y = 0.7762x + 0.4081

10 r = 0.9977 10 r = 0.9973
8 8
6 6
4 4
2 2
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Concentration of RMβCD in Concentration of SBE7βCD in
mM mM
Fig. 3.1: Phase solubility diagrams of SQV A) βCD B) HPβCD

C) RMβCD D) SBE7βCD
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7 7 (B)
(A)
Concentration of RTV in mM
Concentration of RTV in mM
6 6
5 5
4 4
3 3
2 2 y = 0.4552x + 1.0389
y = 0.4052x + 0.9746
r = 0.9922 r = 0.9901
1 1
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Concentration of HPβCD in Concentration of RMβCD in
mM mM
Fig. 3.2: Phase solubility diagrams of RTV in A) HPβCD B) RMβCD
Table 3.3: Phase solubility studies of ritonavir in two CDs

Solubility of ritonavir (mM/L)
Concentration (mean±s.d) (n=3)
of CD (mM/L)
HPβCD RMβCD
0 0.7±0.01 0.7±0.01
2 1.95±0.01 3.03±0.02
4 2.75±0.02 3.68±0.02
6 3.45±0.04 4.36±0.01
8 4.24±0.01 4.72±0.01
10 5.05±0.04 5.11±0.01
12 5.70±0.02 5.56±0.02
Table 3.4: Stability constants of RTV with two CDs

Cyclodextrin K1:1 (M-1) Phase diagram
HPβCD 119.01 AL Type
RMβCD 192.96 AL Type
3.7.2. Preparation of drug-CD complexes
The phase solubility studies carried out with different
cyclodextrins on both drugs indicated the formation of complex in 1:1
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molar ratio. Drug-CD complexes were prepared with all the four CDs
for saquinavir whereas HPβCD and RMβCD were used for ritonavir
using four different methods of preparation. In each method, 4-5 g of
drug-CD complexes was prepared at equimolar ratio.
3.7.3. Study of spray drying process conditions
Spray drying is a process that can be used for the preparation of
CD complexes and also a widely used method for the preparation of
microparticle powders to be used as dry powder inhalers.26,27 The
particles obtained via spray drying are usually spherical, with a small
diameter (1–5 µm) and narrow particle-size distribution.28-30 Careful
selection of operating parameters can play a significant role in
obtaining high quality product during spray drying. The spray drying
conditions were optimized for SQV-βCD complexes and extended for
other drug-CD complexes. In the present study, aspiration rate was
kept at 40-50 m3/hr and Tout was kept constant at 50°C. The
complexes were prepared by spray drying at two Tin, 55°C and 70°C,
and three feeding solution flow rates: 2, 5 and 10 mL/min. This study
aimed at evaluating the influence of these variables on the particle size
and shape of the spray drying powder and the yield. The maximum Tin
was set to 70°C, because at higher temperatures, the evaporation of the
hydro alcoholic solvent takes place in the atomization cone with the
consequent powder deposition at this level. The lowest value of Tin,
55°C, corresponds to the minimum value that allows the use of a
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solution feeding flow of 11 mL/min without condensation in the
collector.
The result of the determination of the yield for each one of the
experimental conditions is shown in Table 3.5. The yield obtained
under varying conditions of Tin and flow rate was in the range of 37-
62%. Yield was significantly influenced by flow rate rather than Tin in
which lower flow rate resulted in higher yield. No significant difference
in yield was observed between the two Tin values. Earlier reports also
confirmed this result in which higher flow rates resulted in low yields in
the range of 40-50%.31-33 These low values could be justified by the
biggest number of atomised particles that in proportion adheres more
to the walls of the drying chamber, its removal for the cyclone collector
being more difficult, leading to lower yield values in the process.
Hence a flow rate of 2 mL/min with a Tin of 55ºC were found to
be optimized conditions which gave highest yield of 62% drug-CD
complexes and these two conditions were used for preparation of drug-
CD complexes by spray drying technique.
Table 3.5: % Yield of SQV-βCD complexes under different

experimental conditions
Solution flow
Air inlet
rate % Yield
temperature (°C)
(mL/min)
2 62.0
55 5 50.5
10 37.5
2 61.8
70 5 50.0
10 37.0
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3.7.3.1. Determination of moisture content
The final residual moisture of spray dried product is important in
preventing the adhesion of particles and hence the moisture content of
the spray dried drug-CD complexes was determined. Moisture content
for all the complexes were in the range of 0.9-2.5% (Table 3.6)
Saquinavir complex prepared with SBE7βCD showed slightly higher
moisture content compared to other CDs. Earlier reports also indicated
higher moisture content for complexes prepared with SBE7βCD due to
its hygroscopic nature.4
Table 3.6: Moisture content of spray dried complexes

Moisture content
Drug Type of CD
(mean±s.d.) (n=3)
Saquinavir βCD 1.01±0.50
HPβCD 1.23±0.45
RMβCD 1.15±0.50
SBE7βCD 2.51±0.42
Ritonavir HPβCD 0.82±0.20
RMβCD 0.91±0.15
3.7.3.2. Particle size measurement
Through spray drying, uniform particle size was achieved,
irrespective of the drug and cyclodextrin used for spray drying. All the
particles were in the mean size range of 1.5-3 µm. The complexes were
fine and uniform in size with spherical shape.
3.7.4. Drug content estimation
All the complexes prepared were found to be fine powders. The
percent drug content values of all the complexes of saquinavir are given
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in Table 3.7 and for ritonavir are represented in Table 3.8. Low s.d.
values in the percent drug content ensured drug content uniformity in
each batch and the method of preparation showed no effect on the
content uniformity. The drug content obtained in physical mixing,
kneading was found to be 98-99% for both the drugs. Co evaporation,
spray drying and freeze drying methods, drug content of 85-90% was
observed. Around 10-15% drug loss was observed in these methods for
all drug-cyclodextrin complexes which may be due to loss of the drug
during evaporation of solvent in the preparation and other processes.
Table 3.7: Drug content of prepared SQV inclusion complexes
Percentage drug content (mean±s.d.) (n=3)

Method of SQV- SQV-
SQV-βCD SQV-HPβCD
preparation RMβCD SBE7βCD
PM 99.54±0.42 99.75±0.34 99.32±0.64 99.60±0.32
KN 98.55±0.56 98.55±0.37 99.55±0.58 99.25±0.95
COE 86.44±0.47 88.61±0.29 84.55±0.89 88.78±0.78
SD 89.78±1.04 84.49±0.67 88.64±0.90 87.45±0.62
FD 88.56±0.15 85.11±0.45 87.42±0.69 89.46±0.88
Table 3.8: Drug content of prepared RTV inclusion complexes

Percentage drug content (mean±s.d.) (n=3)
Method of
RTV-HPβCD RTV-RMβCD
preparation
PM 99.60±1.01 99.68±0.85
COE 89.32±0.95 87.48±0.94
SD 85.12±0.92 86.24±1.56
FD 86.68±1.02 88.77±1.25
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3.7.5. In vitro dissolution studies
Dissolution testing is the measurement of the rate and extent of a
drug substance in an in vitro test system. For a given drug substance,
the quantity dissolved is a function of the solubility and physical
characteristics of the drug, composition, volume, temperature, and
dynamics of the test system and the input/state variables of the
apparatus selected. Dissolution testing can be a valuable tool as the
rate and/or extent of absorption of a drug can depend on its
dissolution rate. The dissolution testing can be used as a guide to a
formulator in the early stages of drug product design.
Dissolution characterizes of a dosage form have a direct bearing
on its efficacy especially when the medicament is poorly soluble or
insoluble in aqueous fluids. Generally for poorly soluble drugs
difficulties are usually encountered in selecting a suitable dissolution
medium with good discriminating power. Dissolution of drugs from
solid dosage form is a key parameter in assessing the release rate,
release mechanism and kinetics at product development stage.
Saquinavir capsules, tablets are official in IP and USP Citrate buffer
(pH 3.0) is mentioned as dissolution medium in all official books and
FDA listed drugs.34-37 But this medium is not suitable as discriminating
medium as saquinavir is highly soluble in this media as per studies
conducted by Pathak et al. 38 Simultaneously, ritonavir official medium
is 0.1N HCl with 25 mM polyoxyethylene 10 lauryl ether as per FDA
listed dissolution media and in USP.36,37,39 In such high surfactant
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conditions, ritonavir is highly soluble. So, pH of the dissolution media
were selected based on discriminating ability, and pH of upper GIT for
saquinavir i.e. pH 6.8 phosphate buffer and pH of stomach for
ritonavir, i.e. 0.1N HCl as per earlier reports.38,39
All dissolution studies were performed for a period of 60 minutes
as the objective of the present investigation was to achieve the complete
drug release within this period. Drug-CD complexes failing to achieve
this objective were not studied further. Drug-CD complex equivalent to
100 mg of the drug was used for dissolution study in each case and
each study was replicated for three times and average values with s.d.
are reported in the tables. Error bars are not included in figures due to
smudging of graphs.
3.7.5.1. Dissolution studies of SQV-CD complexes
The dissolution data of SQV, SQV-βCD, SQV-HPβCD, SQV-
RMβCD and SQV-SBE7βCD complexes prepared by PM, KN, COE, SD
and FD techniques at equimolar ratio are given in Table 3.9 to
Table 3.12.
Fig.3.3 shows the dissolution profiles of pure saquinavir and the
inclusion complexes prepared by PM, KN, COE, SD and FD techniques.
Fig.3.4 shows dissolution profiles of SQV and SQV inclusion complexes
prepared with four cyclodextrins, βCD, HPβCD, RMβCD and SBE7βCD
by spray drying and freeze drying techniques respectively.
From the dissolution studies, it was observed that only 20.82%
dissolution of pure saquinavir was observed in 60 min. The dissolution

135
of SQV with βCD, HPβCD, RMβCD and SBE7βCD physical mixtures
showed a release of 27.21-38.44% of SQV in 60 min. Complexes
prepared by kneading method showed drug release of 36.57-47.29%
and that are prepared with co evaporation method showed 37.6-48.05%
with all the four CDs. Among different methods of preparation, spray
dried (37.75-80.63%) and freeze dried (38.74-99.27%) complexes
showed higher drug release in 60 min of dissolution study. Among all
complexes, SQV-SBE7βCD complexes prepared by freeze drying
method showed 100% drug release in 60 min.
All inclusion complexes showed improvement of drug release
compared to pure saquinavir alone except for physical mixtures where
the enhancement is negligible. Complexes prepared by spray drying
and freeze drying methods showed marked enhancement in dissolution
compared to other methods for all CDs. The order of drug release from
complexes prepared by different methods was FD>SD>COE>KN>PM for
βCD and HPβCD. This may be due to the heat used in co evaporation
which helps in complex formation. In case of RMβCD and SBE7βCD,
kneading method showed higher drug release than co evaporation.
Similar reports were mentioned in literature.8 When the effect of type of
CD was observed, complexes prepared with SBE7βCD showed good
enhancement in dissolution. The improvement in dissolution follows
the order: SBE7βCD>RMβCD>HPβCD>βCD. Enhanced drug release
with SBE7βCD can be attributed to its charged groups which are
appropriately spaced from the cavity and also increase in the
hydrophobicity around the cavity due to the presence of alkyl chains.

136
Table 3.9: Cumulative percent of SQV released from βCD complexes
Mean percentage drug release (n=3) (mean±s.d.)
Time Pure Drug PM KN COE SD FD
5 11.86±0.83 19.14±0.57 28.47±1.72 27.01±0.35 23.32±1.11 24.51±0.81
10 14.37±1.55 21.53±0.52 33.35±1.30 31.55±1.23 32.52±1.52 26.49±0.65
15 16.84±1.04 22.41±0.55 33.48±1.75 34.4±0.38 35.11±1.45 27.54±1.15
30 19.12±0.72 23.83±1.11 35.67±0.75 36.67±0.86 36.52±0.82 30.49±0.18
45 20.13±0.83 25.97±1.15 36.25±0.32 37.35±0.36 37.47±0.35 32.68±1.53
60 20.82±0.41 27.21±0.44 36.57±0.14 37.82±0.55 37.75±0.16 38.74±1.19
Table 3.10: Cumulative percent of SQV released from HPβCD complexes

5 11.86±0.83 22.82±0.74 35.87±0.51 36.78±0.41 24.41±1.52 35.86±0.95
10 14.37±1.55 26.37±0.65 36.78±0.65 38.62±0.49 32.46±1.85 37.17±0.82
15 16.84±1.04 28.50±0.73 38.04±0.68 39.48±0.37 37.95±0.55 38.44±0.75
30 19.12±0.72 31.09±0.65 39.29±1.06 40.74±0.29 39.93±0.75 39.99±0.67
45 20.13±0.83 33.26±0.54 39.86±0.41 41.39±0.31 43.81±0.62 41.95±0.81
60 20.82±0.41 34.78±0.49 40.43±0.18 42.07±0.19 46.18±1.21 46.81±1.23
137
Table 3.11: Cumulative percent of SQV released from RMβCD complexes
5 11.86±0.83 19.29±0.35 32.20±0.46 21.96±0.51 28.10±1.20 24.90±0.61
10 14.37±1.55 22.2±0.54 35.05±0.52 22.93±1.2 39.68±1.40 39.55±0.74
15 16.84±1.04 23.75±0.65 37.16±0.85 24.01±1.01 42.95±1.05 45.02±0.88
30 19.12±0.72 26.48±0.36 38.48±0.69 24.81±1.4 52.08±0.82 56.45±1.03
45 20.13±0.83 28.78±0.47 39.57±0.38 25.18±1.48 61.56±0.59 67.09±1.04
60 20.82±0.41 31.95±0.52 40.26±0.31 25.65±1.49 80.38±1.12 85.17±0.55
Table 3.12: Cumulative percent of SQV released from SBE7βCD complexes
5 11.86±0.83 25.58±0.64 36.47±1.31 24.76±1.51 38.55±1.25 36.65±1.05
10 14.37±1.55 27.53±1.20 40.65±0.70 33.82±1.80 42.75±1.65 49.02±1.51
15 16.84±1.04 32.93±0.50 41.94±0.60 38.29±0.62 55.54±0.45 55.22±0.75
30 19.12±0.72 35.64±0.31 43.14±0.64 41.80±0.82 59.97±0.35 61.32±0.65
45 20.13±0.83 37.29±0.52 44.49±0.39 45.27±0.51 72.73±0.75 86.05±1.12
60 20.82±0.41 38.44±0.47 48.59±0.62 47.05±1.52 84.63±0.25 99.70±0.45
138
100 KN Pure drug 100 COE Pure drug
PM COE PM KN
(A) SD FD
(B)
SD FD
80 80
Mean % drug release
Mean % drug release

60 60
40 40
20 20
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time in min Time in min
Pure drug PM Pure drug PM

100 100 KN COE
KN COE
SD FD SD FD
Mean % drug release
80 (C) 80 (D)
Mean % drug release
60 60
40 40
20 20
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time in min
Time in min
Fig. 3.3: Dissolution profiles of SQV-CD complexes prepared by

different methods A) βCD B) HPβCD C) RMβCD D) SBE7βCD
139
(A) (B)
Pure drug Pure drug
100 100
SQV-βCD SQV-βCD
SQV-HPβCD SQV-HPβCD
Mean % drug release
Mean % drug release

SQV-RMβCD SQV-RMβCD
80 80
SQV-SBE7βCD SQV-SBE7βCD
60 60
40 40
20 20
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time in min
Time in min
Fig. 3.4: Comparative dissolution profiles of SQV-CD complexes
prepared by A) spray drying B) freeze drying
3.7.5.2. Dissolution studies of RTV-CD complexes
The dissolution data of pure ritonavir and complexes prepared
with HPβCD and RMβCD by physical mixing, co evaporation, spray
drying and freeze drying are given in Table 3.13 and 3.14.
The dissolution profiles of pure ritonavir and the complexes
prepared by PM, COE, SD and FD techniques are shown in Fig.3.5.
Dissolution profiles of RTV complexes prepared with both cyclodextrins
by freeze drying is represented in Fig. 3.6. 25.86% ritonavir dissolution
was observed in 60 min. Physical mixtures with both CDs showed
57.89% and 62.45% drug release. Co evaporated and spray dried
products exhibited 64.45-64.49% and 64.45-72% respectively.
Complete drug release was obtained with freeze dried complex of RTV-
RMβCD within 10 min and RTV- HPβCD complex released 98.87% RTV
within 20 min. A marked enhancement in dissolution was observed in
complexes prepared by freeze drying technique like saquinavir-CD

140
systems. The order of drug release based on method of preparation was
as follows, FD>SD>COE>PM. RMβCD complexes of ritonavir gave
higher dissolution compared to HPβCD. This may be due to RMβCD
reduced the interfacial tension between the solid particles of RTV and
the dissolution medium, leading to a greater rate of dissolution.
The higher drug release observed with inclusion complexes
prepared by freeze drying method may be due to better interaction of
drugs and cyclodextrins. When two selected drugs were compared,
saquinavir with SBE7βCD showed 100% drug release in 60 min,
whereas, ritonavir with RMβCD showed 100% drug release within 10
min. Compared to SQV, RTV is more hydrophobic. RTV might have
interacted well with the hydrophobic capacity of RMβCD and hence
exposed to the dissolution medium more effectively compared to SQV
because of which higher dissolution of drug.
For saquinavir, four cyclodextrins were tested to find out best
CD derivative which gives maximum release in stipulated dissolution
time of 60 min. Based on SQV studies, βCD was not tested in case of
RTV, as it gave minimal improvement in drug release. As 100% drug
release of RTV was achieved with RMβCD itself within 10 min,
SBE7βCD was not tested in the study. However, the study was
compared with HPβCD.
141
Table 3.13: Cumulative percent of RTV released from HPβCD complexes
Time
Pure drug PM COE SD FD
(min)
5 6.50±0.22 25.57±0.21 32.86±0.12 28.45±0.24 85.24±0.22
10 12.81±0.12 32.52±0.32 55.84±0.16 41.67±0.48 95.54±0.16
20 13.01±0.29 36.51±0.43 56.89±0.35 42.33±0.37 98.87±0.26
30 16.84±0.43 42.13±0.47 58.73±0.41 45.55±0.26
45 21.02±0.41 45.44±0.29 59.62±0.32 61.57±0.21
60 25.61±0.32 47.89±0.37 62.54±0.29 64.45±0.40
Table 3.14: Cumulative percent of RTV released from RMβCD complexes
Time Pure drug PM COE SD FD
(min)
5 6.50±0.22 30.66±0.22 40.56±0.34 35.56±0.43 90.55±0.12
10 12.80±0.12 59.68±0.27 53.11±0.21 45.10±0.16 99.45±0.25
20 13.01±0.29 60.61±0.49 56.82±0.43 45.31±0.18
30 16.84±0.43 60.62±0.12 59.25±0.16 61.54±0.12
45 21.02±0.41 61.34±0.22 61.13±0.12 71.05±0.10
60 25.60±0.32 62.45±0.26 64.49±0.20 72.00±0.13
142
(A) (B)
Pure drug 100 Pure drug
100 PM
PM
COE COE
SD SD
Mean % drug release

80 80 FD
Mean % drug relese
FD
60 60
40 40
20 20
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (min)
Time (min)
Fig. 3.5: Dissolution profiles of RTV prepared by different methods

A) HPβCD B) RMβCD
100
Pure drug
RTV-HPβCD
Mean % drug releasee
80
RTV-RMβCD
60
40
20
0
0 10 20 30 40 50 60
Time (min)
Fig. 3.6: Comparative dissolution profiles of RTV inclusion

complexes prepared by freeze drying
143
3.7.6. Comparison of dissolution profiles
Dissolution profiles of saquinavir and ritonavir complexes
prepared by various methods with each cyclodextrin were compared
and dissolution profiles of complexes prepared by freeze drying with
cyclodextrins used for the study were compared by model dependent
and model independent methods.
3.7.6.1. Model independent methods-Dissolution parameters
Dissolution parameters %DE60, DP30, %DE10, T50 and MDT were
calculated for both the drugs and are represented in Table 3.15 and
3.16. Bar diagrams of dissolution parameters for both the drugs are
shown in Fig. 3.7 to Fig.3.12.
In case of SQV, %DE10, T50 were calculated only for spray dried
and freeze dried products as the drug release was very slow for other
methods, physical mixing, kneading and co evaporation and some of
the complexes did not show even 50% of drug release. All drug-CD
complexes showed higher %DE60, DP30 values compared to pure drug.
Among different cyclodextrins used in the study, complexes prepared
with SBE7βCD showed higher %DE60, DP30. When method of
preparation was observed, freeze dried and spray dried products
exhibited higher %DE60, DP30 values than the physical mixture,
kneaded and co evaporated products and pure saquinavir. Kneaded
products showed higher %DE60, DP30 compared to co evaporated
products with SQV-RMβCD and SBE7βCD. On the contrary, co
evaporated products showed higher %DE60, DP30 than kneaded
144
products with βCD and HPβCD complexes. Higher %DE10, lower T50
values were observed with spray dried and freeze dried complexes
prepared with SBE7βCD. The extent of the enhancement of the
dissolution was found to be dependent on the preparation method, and
the type of CD, since SQV-SBE7βCD complex prepared by freeze drying
exhibited 4.14 fold higher DE60 value and two fold increase in DP30
values compared to pure SQV.
MDT values were calculated for freeze dried complexes as they
gave maximum drug release. All freeze dried complexes showed
decrease in MDT compared to saquinavir alone. The lowest MDT was
observed with complexes prepared by freeze drying with SBE7βCD. The
lower the MDT value, the faster the drug release. Very high MDT was
found for saquinavir, 238.01 which was decreased to 20.67 with
SBE7βCD indicated that complexation of SQV with SBE7βCD and
using freeze drying method enhanced the dissolution rate.
Similar way, the dissolution efficiencies were 9 fold higher with
RMβCD and 8.16 fold higher with HPβCD in %DE10 compared to RTV.
8.2 fold with HPβCD and 1.96 fold with RMβCD higher values in %DE10
of freeze dried products corresponding physical mixture were observed.
High dissolution efficiency and low T50 values were obtained with freeze
dried complexes of RTV-RMβCD. Both RTV-CD complexes prepared by
co evaporation showed higher DE60, DE10, DP30 and low T50 values
compared to spray dried products. Spray dried products showed very
slow release but co evaporated products showed initial quick release
145
followed by slowing down of release of drug. This may be due to long
heating time in co evaporation could help in complex formation. Higher
DE60 and DE10 and lower T50 were observed with freeze dried complexes
of RTV-RMβCD and HPβCD complexes.
A drastic decrease in MDT values was observed with freeze dried
complexes of ritonavir with both HPβCD and RMβCD. Ritonavir had
shown higher MDT value of 200.6. The reduction in MDT to 3.0 was
found with RTV-RMβCD complex indicated that effective complexation
of RTV with RMβCD has been taken place. Like saquinavir, freeze
drying method proved in enhancing dissolution rate.
The enhancement with freeze dried complexes of both drugs may
be due to increased interaction and conversion of drug into highly
amorphous state in freeze drying method. Increase in dissolution
parameters of SQV complex with SBE7βCD and RTV complex with
RMβCD has been attributed to the formation of an inclusion complex in
the solid state and formation of readily soluble complexes in the
dissolution medium with these cyclodextrins.
146
Table 3.15: Dissolution parameters of SQV inclusion complexes
Type of Method of DE60 DP30 DE10 T50(min) MDT
complex preparation
Pure Drug --- 18.16±0.91 19.12±0.32 238.01±7.5
SQV-βCD PM 26.28±0.42 23.83±0.22
KN 33.87±0.85 35.67±0.56
COE 34.68±0.65 35.67±0.58
SD 34.54±1.20 36.52±1.52
FD 31.00±1.42 30.49±1.24 109.22±5.6
SQV-HPβCD PM 35.41±1.43 31.59±0.53
KN 37.62±1.78 39.29±0.87
COE 39.09±0.98 40.74±0.47
SD 39.36±0.87 39.93±0.57
FD 39.98±0.64 39.99±0.34 79.84±8.1
SQV-RMβCD PM 34.21±1.25 26.48±0.96
KN 36.93±1.51 38.48±0.86
COE 23.73±0.66 24.81±0.88
SD 57.56±0.80 52.08±0.81 26.8±0.55 27.5±0.20
FD 62.22±0.67 56.45±1.35 26.0±0.67 21.5±0.45 37.48±5.9
SQV- PM 33.94±0.92 35.64±1.10
SBE7βCD KN 42.13±0.95 43.14±1.28
COE 40.81±1.43 41.82±1.23
SD 63.12±1.22 59.97±1.56 30.9±0.58 13.0±0.30
FD 75.25±0.98 61.32±1.75 33.6±0.45 10.5±0.25 20.67±6.2
147
Table 3.16: Dissolution parameters of RTV inclusion complexes
Type of Method of DE60 DP30 DE10 T50(min) MDT
complex preparation
Pure --- 17.97±0.95 16.80±0.55 8.02±0.35 --- 200.46±4.5
Drug
RTV- PM 46.76±0.58 52.13±0.56 8.40±0.30 27.5±0.15
HPβCD
COE 55.83±0.78 58.73±0.69 36.13±0.55 7.50±0.25
SD 50.81±0.75 42.96±0.45 27.97±0.56 35.0±0.25
FD 93.80±0.65 84.69±0.52 69.08±0.45 2.50±0.15 3.67±5.2
RTV- PM 41.05±0.86 59.29±0.55 37.50±0.91 8.50±0.12
RMβCD
COE 56.86±0.75 59.25±0.45 36.69±0.95 8.01±0.32
SD 58.81±0.42 61.54±0.60 31.44±0.85 25.00±0.15
FD 94.76±0.35 92.86±0.42 72.40±0.75 2.00±0.15 3.00±4.9
148
SQV-βCD SQV-HPβCD
SQV-RMβCD SQV-SBE7βCD
80
70
60
50
DE60
40
30
20
SQV-…
10
SQV-RMβCD
0
SQV-HPβCD
PM
KN SQV-βCD
COE
SD
FD
SQV-βCD SQV-HPβCD
SQV-RMβCD SQV-SBE7βCD
70
60
50
40
DP30
30
20
10 SQV-SBE7βCD
SQV-RMβCD
0 SQV-HPβCD
PM SQV-βCD
KN
COE
SD
FD
Fig.3.7: Bar diagrams of A) DE60 B) DP30 for SQV-CD complexes

prepared by different methods using a) βCD b) HPβCD
c) RMβCD d) SBE7βCD
149
(A) SQV-RMβCD
30
SQV-SBE7βCD
20
T50(min)
10
SQV-SBE7βCD
0 SQV-RMβCD
SD
FD
40 SQV-RMβCD
(B)
SQV-SBE7βCD
30
DE10
20
10
SQV-…
0 SQV-RMβCD
SD
FD
Fig. 3.8: Bar diagrams of A) T50 B) DE10 for SQV-CD complexes

prepared by spray drying and freeze drying methods using
a) RMβCD b) SBE7βCD
250
200
150
100
50
SQV SQV-βCD SQV-HPβCD SQV-RMβCD SQV-SBE7βCD
Fig. 3.9: Bar diagram of MDT values for SQV-CD complexes

prepared by freeze drying method
150
(A) RTV-HPβCD RTV-RMβCD
100
80
60
DE60
40
20
0 RTV-RMβCD
RTV-HPβCD
PM
COE SD
FD
(B)
100
80
60
DE10
40
20
0 RTV-RMβCD
RTV-HPβCD
PM
COE
SD
FD
Fig. 3.10: Bar diagram of A) DE60 B) DE10 for RTV-CD complexes

prepared by different methods using A) HPβCD B) RMβCD
151
35
30
25
T50(min)
20
15
10
5
0 RTV-RMβCD
PM RTV-HPβCD
COE
SD
FD
Fig. 3.11: Bar diagram of T50 values for RTV-CD complexes

prepared by different methods using a) HPβCD b) RMβCD
200
150
100
50
RTV RTV-HPβCD RTV-RMβCD
Fig.3.12: Bar diagram of MDT values for RTV-CD complexes

prepared by freeze drying
3.6.7.2. Statistical analysis
Two dissolution parameters %DE60, DP30 of the complexes
prepared by different methods were compared by the one-way ANOVA.
The results are shown in Table 3.17 to 3.20.
Null hypothesis was rejected in the one way ANOVA test as p
value is less than 0.05. Therefore it indicated statistically a significant
152
difference in their dissolution profiles prepared by different methods
and using different cyclodextrins.
To evaluate the difference among the complexes prepared by
different methods, the Tukey multiple comparison test was performed
on the results of ANOVA and the results are given in Table 3.21 to
3.24. Tukey multiple comparison test for saquinavir and different
complexes showed significant difference in dissolution parameters
between SQV and different methods of preparation. However, no
significant difference was observed between some of the methods
especially with βCD and HPβCD and the details are given in
Table 3.25.
One way ANOVA was performed for freeze dried complexes of
SQV with four CDs and the results are shown in Table 3.26. To test
the effect of different cyclodextrins on dissolution rate, for complexes
prepared by freeze drying, dissolution parameters %DE60, DP30 and
MDT values were subjected to Tukey multiple comparison test
(Table 3.27). It showed significant difference between different
cyclodextrin complexes of saquinavir prepared by freeze drying, which
clearly indicated type of cyclodextrin used for complexation influenced
dissolution rate.
153
Table 3.17: ANOVA for dissolution parameters of SQV-βCD
complexes
Null hypothesis: Ho= there is no significant difference in dissolution
parameters between products prepared by various methods
Parameter Source df SS MS F ratio P
of
variation
DE60 Between 5 623.6 124.7 257.5- <0.0001
products
Within 12 5.813 0.4844
products
Total 17 629.4
DP30 Between 5 792.8 158.6 210.0* <0.0001
products
Within 12 9.061 0.7551
products
Total 17 801.8
Table 3.18: ANOVA for dissolution parameters of

SQV-HPβCD complexes
of
variation
DE60 Between 5 1039 207.8 182.6* <0.0001
products
Within 12 13.65 1.138
products
Total 17 1053
DP30 Between 5 1094 218.9 858.8* <0.0001
products
Within 12 3.059 0.2549
products
Total 17 1098
df: Degrees of freedom, SS: Sum of square, MS: Mean square,
p: Significance level.
*Significant difference (Null hypothesis rejected)
154
SQV-RMβCD complexes
of
variation
DE60 Between 5 4437 887.3 1257* <0.0001
products
Within 12 8.474 0.7062
products
Total 17 4445
DP30 Between 5 3554 710.8 934.9* <0.0001
products
Within 12 9.639 0.8033
products
Total 17 3564

SQV- SBE7βCD complexes
of
variation
DE60 Between 5 6239 1248 1698* <0.0001
products
Within 12 8.818 0.7348
products
Total 17 6248
DP30 Between 5 3703 740.5 686.6* <0.0001
products
Within 12 12.94 1.079
products
Total 17 3716
df: Degrees of freedom, SS: Sum of square, MS: Mean square,
p: Significance level.
155
Table 3.21: Tukey multiple comparison test for DE60 and DP30
values of SQV-βCD complexes
Parameter Comparison Mean q Significant 95% 95%
of products diff (P<0.05) LCL UCL
DE60 SQV vs. PM -7.95 19.80 s* -9.86 -6.04
SQV vs. KN -15.65 38.96 s* -17.56 -13.74
SQV vs. COE -16.03 39.89 s* -17.94 -14.12
SQV vs. SD -16.28 40.51 s* -18.19 -14.37
SQV vs. FD -13.08 32.54 s* -14.99 -11.17
PM vs. KN -7.69 19.15 s* -9.60 -5.78
PM vs. COE -8.07 20.09 s* -9.98 -6.16
PM vs. SD -8.32 20.71 s* -10.23 -6.41
PM vs. FD -5.12 12.74 s* -7.02 -3.21
KN vs. COE -0.37 0.93 n.s* -2.28 1.53
KN vs. SD -0.62 1.55 n.s* -2.53 1.28
KN vs. FD 2.57 6.41 s* 0.66 4.48
COE vs. SD -0.24 0.61 n.s* -2.15 1.66
COE vs. FD 2.95 7.35 s* 1.04 4.86
SD vs. FD 3.20 7.96 s* 1.29 5.10
DP30 SQV vs. PM -4.72 9.421 s* -7.11 -2.34
SQV vs. KN -16.54 32.96 s* -18.92 -14.15
SQV vs. COE -16.65 33.19 s* -19.04 -14.27
SQV vs. SD -17.42 34.73 s* -19.81 -15.04
SQV vs. FD -11.33 22.58 s* -13.71 -8.94
PM vs. KN -11.81 23.54 s* -14.19 -9.42
PM vs. COE -11.93 23.77 s* -14.31 -9.54
PM vs. SD -12.70 25.31 s* -15.08 -10.31
PM vs. FD -6.60 13.16 s* -8.98 -4.21
KN vs. COE -0.11 0.23 n.s* -2.50 2.26
KN vs. SD -0.88 1.76 n.s* -3.27 1.49
KN vs. FD 5.21 10.38 s* 2.82 7.59
COE vs. SD -0.77 1.53 n.s* -3.15 1.61
COE vs. FD 5.32 10.62 s* 2.94 7.71
SD vs. FD 6.09 12.15 s* 3.71 8.48
s*-significant difference n.s*-not significant
156
values of SQV-HPβCD complexes
DE60 SQV vs. PM -17.10 27.77 s* -20.03 -14.17
SQV vs. KN -19.20 31.18 s* -22.13 -16.28
SQV vs. COE -20.83 33.82 s* -23.75 -17.90
SQV vs. SD -21.02 34.13 s* -23.95 -18.09
SQV vs. FD -21.73 35.29 s* -24.66 -18.80
PM vs. KN -2.10 3.41 n.s* -5.02 -0.82
PM vs. COE -3.72 6.05 s* -6.65 -0.80
PM vs. SD -3.92 6.36 s* -6.84 -0.99
PM vs. FD -4.63 7.51 s* -7.55 -1.70
KN vs. COE -1.62 2.63 n.s* -4.54 1.30
KN vs. SD -1.81 2.95 n.s* -4.74 1.10
KN vs. FD -2.52 4.10 n.s* -5.45 0.39
COE vs. SD -0.193 0.31 n.s* -3.11 2.73
COE vs. FD -0.903 1.46 n.s* -3.82 2.02
SD vs. FD -0.71 1.15 n.s* -3.63 2.21
DP30 SQV vs. PM -12.50 42.90 s* -13.89 -11.12
SQV vs. KN -20.21 69.34 s* -21.59 -18.83
SQV vs. COE -21.65 74.28 s* -23.03 -20.27
SQV vs. SD -20.83 71.47 s* -22.22 -19.45
SQV vs. FD -20.86 71.55 s* -22.24 -19.47
PM vs. KN -7.707 26.44 s* -9.09 -6.32
PM vs. COE -9.147 31.38 s* -10.53 -7.76
PM vs. SD -8.33 28.58 s* -9.71 -6.94
PM vs. FD -8.35 28.66 s* -9.73 -6.96
KN vs. COE -1.44 4.94 s* -2.82 -0.05
KN vs. SD -0.62 2.13 n.s* -2.00 0.76
KN vs. FD -0.64 2.21 n.s* -2.03 0.73
COE vs. SD 0.81 2.80 n.s* -0.56 2.20
COE vs. FD 0.79 2.72 n.s* -0.59 2.17
SD vs. FD -0.02 0.08 n.s* -1.40 1.36
157
values of SQV-RMβCD complexes
DE60 SQV vs. PM -16.03 33.04 s* -18.34 -13.72
SQV vs. KN -18.34 37.80 s* -20.65 -16.03
SQV vs. COE -5.447 11.23 s* -7.75 -3.14
SQV vs. SD -39.09 80.57 s* -41.40 -36.78
SQV vs. FD -42.01 86.58 s* -44.31 -39.70
PM vs. KN -2.310 4.761 s* -4.61 -0.00
PM vs. COE 10.58 21.81 s* 8.27 12.89
PM vs. SD -23.06 47.53 s* -25.37 -20.75
PM vs. FD -25.98 53.54 s* -28.28 -23.67
KN vs. COE 12.89 26.57 s* 10.59 15.20
KN vs. SD -20.75 42.77 s* -23.06 -18.44
KN vs. FD -23.67 48.78 s* -25.97 -21.36
COE vs. SD -33.64 69.34 s* -35.95 -31.34
COE vs. FD -36.56 75.35 s* -38.87 -34.25
SD vs. FD -2.91 6.012 s* -5.22 -0.61
DP30 SQV vs. PM -7.323 14.15 s* -9.78 -4.86
SQV vs. KN -19.29 37.27 s* -21.75 -16.83
SQV vs. COE -5.703 11.02 s* -8.16 -3.24
SQV vs. SD -32.98 63.73 s* -35.44 -30.52
SQV vs. FD -37.35 72.17 s* -39.81 -34.89
PM vs. KN -11.96 23.12 s* -14.42 -9.50
PM vs. COE 1.620 3.131 n.s* -0.83 4.07
PM vs. SD -25.65 49.58 s* -28.11 -23.19
PM vs. FD -30.02 58.02 s* -32.48 -27.56
KN vs. COE 13.58 26.25 s* 11.12 16.04
KN vs. SD -13.69 26.46 s* -16.15 -11.23
KN vs. FD -18.06 34.90 s* -20.52 -15.60
COE vs. SD -27.27 52.71 s* -29.73 -24.81
COE vs. FD -31.64 61.15 s* -34.10 -29.18
SD vs. FD -4.370 8.44 s* -6.82 -1.91
158
values of SQV-SBE7βCD complexes
DE60 SQV vs. PM -15.68 28.5 s* -18.29 -13.07
SQV vs. KN -24.16 5.03 s* -26.77 -21.55
SQV vs. COE -22.60 43.98 s* -25.21 -19.99
SQV vs. SD -45.02 41.14 s* -41.63 -42.41
SQV vs. FD -57.09 97.24 s* -59.88 -54.30
PM vs. KN -8.47 88.39 s* -11.08 -5.86
PM vs. COE -6.91 15.43 s* -9.52 -4.30
PM vs. SD -29.33 12.59 s* -31.94 -26.72
PM vs. FD -32.87 53.40 s* -35.48 -30.26
KN vs. COE 1.55 59.84 n.s* -1.05 -4.16
KN vs. SD -20.86 2.83 s* -23.47 -18.25
KN vs. FD -24.40 37.97 s* -27.01 -21.79
COE vs. SD -22.42 44.42 s* -25.03 -19.81
COE vs. FD -34.41 58.61 s* -37.20 -31.62
SD vs. FD -12.00 20.43 s* -14.79 -9.20
DP30 SQV vs. PM -16.52 27.56 s* -19.37 -13.67
SQV vs. KN -24.40 40.69 s* -27.25 -21.55
SQV vs. COE -22.71 37.88 s* -25.56 -19.86
SQV vs. SD -40.52 67.58 s* -43.37 -37.67
SQV vs. FD -42.12 70.25 s* -44.97 -39.27
PM vs. KN -7.87 13.14 s* -10.73 -5.02
PM vs. COE -6.19 10.32 s* -9.03 -3.34
PM vs. SD -24.00 40.03 s* -26.85 -21.15
PM vs. FD -25.60 42.69 s* -28.45 -22.75
KN vs. COE 1.68 2.81 n.s* -1.16 4.53
KN vs. SD -16.12 26.89 s* -18.97 -13-27
KN vs. FD -17.72 29.55 s* -20.57 -14.87
COE vs. SD -17.81 29.70 s* -20.66 -14.96
COE vs. FD -19.41 32.37 s* -22.26 -16.56
SD vs. FD -1.59 2.66 n.s* -4.44 1.25
159
Table 3.25: Result of Tukey multiple comparison test
Type of CD used DE60 DP30
βCD KN vs. COE* KN vs. COE*
KN vs. SD* KN vs. SD*
COE vs. SD* COE vs. SD*
HPβCD PM vs. KN* ---
KN vs. SD* KN vs. SD*
COE vs. SD* COE vs. SD*
COE vs. FD* COE vs. FD*
SD vs. FD* SD vs. FD*
RMβCD PM vs. COE* PM vs. COE*
SBE7βCD KN vs. COE* KN vs. COE*
---- SD vs. FD*
*no significant difference
160
Table 3.26: ANOVA for dissolution parameters of SQV-CD
of
variation
DE60 Between 4 6295 1574 2456* <0.0001
products
Within 10 6.409 0.6409
products
Total 14 6301
DP30 Between 4 3714 928.4 2644* <0.0001
products
Within 10 3.512 0.3512
products
Total 14 3717
MDT Between 4 87397 21849 263646* <0.0001
products
Within 10 0.8287 0.0828
products
Total 14 87398
df: Degrees of freedom, SS: Sum of square, MS: Mean square, p:
Significance level.
161
Table 3.27: Tukey multiple comparison test for DE60, DP30 and MDT values of
SQV-CD complexes prepared by freeze drying
Parameter Comparison of products Mean diff q Significant 95% LCI 95% UCI
(P<0.05)
DE60 SQV vs. βCD complex -13.18 39.31 s* -14.74 -11.62
SQV vs. HPβCD complex -21.51 64.14 s* -23.07 -19.95
SQV vs. RMβCD complex -42.29 126.1 s* -43.85 -40.73
SQV vs. SBE7βCD complex -57.39 124.6 s* -59.54 -55.24
βCD vs. HPβCD complex -8.32 24.83 s* -9.88 -6.76
βCD vs. RMβCD complex -29.11 86.81 s* -30.67 -27.55
βCD vs. SBE7βCD complex -35.63 106.3 s* -37.19 -34.07
HPβCD vs. RMβCD complex -20.78 61.98 s* -22.34 -19.22
HPβCD vs. SBE7βCD complex -35.58 76.97 s* -37.73 -33.42
RMβCD vs. SBE7βCD complex -15.30 33.10 s* -17.45 -13.15
DP30 SQV vs. βCD complex -11.53 33.7 s* -21.91 -18.72
SQV vs. HPβCD complex -20.31 59.37 s* -38.88 -35.69
SQV vs. RMβCD complex -37.28 109 s* -43.8 -40.61
SQV vs. SBE7βCD complex -42.2 123.4 s* -10.38 -7.191
βCD vs. HPβCD complex -8.78 25.67 s* -27.35 -24.16
βCD vs. RMβCD complex -25.75 75.27 s* -32.27 -29.08
βCD vs. SBE7βCD complex -30.67 89.65 s* -18.56 -15.38
HPβCD vs. SBE7 βCD complex -21.89 63.98 s* -6.51 -3.32
RMβCD vs. SBE7βCD complex -4.92 14.38 s* -28.86 -25.74
contd….
162
Table 3.27 contd….
MDT SQV vs. βCD complex 129.1 777 s* 128.4 129.9

SQV vs. HPβCD complex 158.9 955.8 s* 158.1 159.6
SQV vs. RMβCD complex 201.0 1209 s* 200.2 201.7
SQV vs. SBE7βCD complex 213.1 1282 s* 212.3 213.9
βCD vs. HPβCD complex 29.71 178.8 s* 28.94 30.48
βCD vs. RMβCD complex 71.81 432.1 s* 71.04 72.59
βCD vs. SBE7βCD complex 83.96 505.2 s* 83.19 84.73
HPβCD vs. RMβCD complex 42.1 253.3 s* 41.33 42.88
HPβCD vs. SBE7 βCD complex 54.25 326.4 s* 53.48 55.02
RMβCD vs. SBE7βCD complex 12.15 73.05 s* 11.37 12.92
s*-significant difference
163
ANOVA results for dissolution parameters DE60, DE10, DP30 and
T50 of RTV-CD complexes prepared by different methods are given in
Table 3.28 and 3.29. Tukey multiple comparison test was performed
on the results of ANOVA and the results are given in Table 3.30 to
3.31. One way ANOVA and Tukey multiple comparison test was
performed for freeze dried complexes with two CDS and the results are
shown in Table 3.32 and 3.33.
Null hypothesis was rejected in the one way ANOVA test as p
value is less than 0.05. Therefore it indicated statistically a significant
difference in the dissolution profiles of RTV complexes.
The results showed that there is significant difference among
RTV-CD complexes prepared with four methods compared to ritonavir
alone. But, no significant difference was observed between methods
physical mixing and co evaporation. T50 values showed no significant
difference as all complexes showed quick release compared to pure
RTV.
Tukey multiple comparison test showed significant difference
between two cyclodextrins, HPβCD and RMβCD for %DE10, DP30 and T50
values whereas DE60 values showed no significant difference. This may
be due to complexes prepared by all methods showed faster release
compared to RTV alone.
164
RTV- HPβCD complexes
Parameter Source of df SS MS F ratio P
variation
DE60 Between 4 9466 2366 4743* <0.0001
products
Within 10 4.989 0.4989
products
Total 14 9471
DP30 Between 4 9600 2400 4379* <0.0001
products
Within 10 5.481 0.5481
products
Total 14 9605
DE10 Between 4 7572 1893 4181* <0.0001
products
Within 10 4.527 0.4527
products
Total 14 7576
T50 Between 3 2189 729.7 21620*
products
Within 8 0.270 0.0337
products
Total 11 2189
df: Degrees of freedom, SS: Sum of square, MS: Mean square
p: Significance level, *Significant difference (Null hypothesis rejected)
165
RTV- RMβCD complexes
variation
DE60 Between 4 9483 2371 4960* <0.0001
products
Within 10 4.780 0.4780
products
Total 14 9488
DP30 Between 4 11483 2871 6303* <0.0001
products
Within 10 4.554 0.4554
products
Total 14 11488
DE10 Between 4 6372 1593 2552* <0.0001
products
Within 10 6.242 0.6242
products
Total 14 6378
T50 Between 3 879.3 293.1 13027*
products
Within 8 0.180 0.0225
products
Total 11 879.5
166
Table 3.30: Tukey multiple comparison test for DE60, DP30, DE10,
and T50 values of RTV- HPβCD complexes
of products Diff (p<0.05) LCL UCL
DE60 RTV vs. PM -18.79 46.08 s* -20.69 -16.89
RTV vs. COE -37.86 92.85 s* -39.76 -35.97
RTV vs. SD -32.88 80.63 s* -34.78 -30.98
RTV vs. FD -75.9 186.1 s* -77.8 -74.01
PM vs. COE -19.07 46.77 s* -20.97 -17.18
PM vs. SD -14.09 34.55 s* -15.99 -12.19
PM vs. FD -57.11 140.1 s* -59.01 -55.22
COE vs. SD 4.98 12.22 s* 3.08 6.88
COE vs. FD -38.04 93.28 s* -39.94 -36.14
SD vs. FD -43.02 105.5 s* -44.92 -41.13
DP30 RTV vs. PM -45.33 106.1 s* -47.32 43.34
RTV vs. COE 51.53 120.6 s* -53.52 49.54
RTV vs. SD 36.16 84.6 s* -38.15 34.17
RTV vs. FD 78.06 182.6 s* -80.05 76.07
PM vs. COE 6.2 14.51 n.s* -8.18 4.21
PM vs. SD 9.17 21.45 s* 7.18 11.16
PM vs. FD -32.73 76.57 s* -34.72 30.74
COE vs. SD 15.37 35.96 s* 13.38 17.36
COE vs. FD -26.53 62.06 s* -28.52 24.54
SD vs. FD -41.9 98.02 s* -43.89 39.91
DE10 RTV vs. PM -0.38 0.97 s* -2.18 1.42
RTV vs. COE -28.77 74.05 s* -30.57 -26.96
RTV vs. SD -19.61 50.49 s* -21.42 -17.81
RTV vs. FD -61.00 157.0 s* -62.81 -59.19
PM vs. COE -28.39 73.08 n.s* -30.19 -26.58
PM vs. SD -19.23 49.51 s* -21.04 -17.43
PM vs. FD -60.62 156.1 s* -62.43 -58.81
COE vs. SD 9.15 23.56 s* 7.34 10.96
COE vs. FD -32.23 82.98 s* -34.04 -30.43
SD vs. FD -41.39 106.5 s* -43.19 -39.58
T50 PM vs. COE 20.00 188.6 n.s* 19.52 20.48
PM vs. SD -7.50 70.71 n.s* -7.98 -7.02
PM vs. FD 25.00 235.7 n.s* 24.52 24.52
COE vs. SD -27.5 259.3 n.s* -27.98 -27.98
COE vs. FD 5.0 47.14 n.s* 4.52 4.52
SD vs. FD 32.50 306.4 n.s* 32.02 32.02
167
Table 3.31: Tukey multiple comparison test for DE60, DP30, DE10
and T50 values of RTV-RMβCD complexes
of products Diff (p<0.05) LCL UCL
DE60 RTV vs. PM -23.12 57.93 s* -24.98 -21.27
RTV vs. COE -38.93 97.53 s* -40.79 -37.07
RTV vs. SD -40.86 102.4 s* -42.72 -39.01
RTV vs. FD -76.83 192.5 s* -78.69 -74.97
PM vs. COE -15.81 39.6 s* -17.66 -13.95
PM vs. SD -17.74 44.44 s* -19.6 -15.88
PM vs. FD -53.71 134.5 s* -55.56 -51.85
COE vs. SD -1.933 4.843 s* -3.791 -0.075
COE vs. FD -37.90 94.94 s* -39.76 -36.04
SD vs. FD -35.97 90.10 s* -37.82 -34.11
DP30 RTV vs. PM -52.49 134.7 s* -54.3 -50.68
RTV vs. COE -52.45 134.6 s* -54.26 -50.64
RTV vs. SD -54.41 139.6 s* -56.22 -52.59
RTV vs. FD -86.06 220.9 s* -87.87 -84.25
PM vs. COE -0.04 0.102 n.s* -1.77 -1.85
PM vs. SD -1.91 4.919 s* -3.73 -0.103
PM vs. FD -33.57 86.16 s* -35.38 -31.76
COE vs. SD -1.957 5.022 s* -3.77 -0.143
COE vs. FD -33.61 86.26 s* -35.42 -31.8
SD vs. FD -31.65 81.24 s* -33.47 -29.84
DE10 RTV vs. PM -29.51 64.7 s* -31.64 -27.39
RTV vs. COE -28.67 62.85 s* -30.79 -26.55
RTV vs. SD -23.42 51.34 s* -25.54 -21.30
RTV vs. FD -64.38 141.1 s* -66.5 -62.26
PM vs. COE -0.843 1.849 n.s* -1.27 2.96
PM vs. SD 6.093 13.36 s* -3.97 8.21
PM vs. FD -34.87 76.44 s* -36.99 -32.74
COE vs. SD 5.25 11.51 s* 3.12 7.373
COE vs. FD -35.71 78.29 s* -37.83 -33.59
SD vs. FD -40.96 89.8 s* -43.08 -38.84
T50 PM vs. COE 0.50 0.013 n.s* -172.9 173.9
PM vs. SD -93.17 2.43 n.s* -266.6 80.28
PM vs. FD 6.50 0.16 n.s* -166.9 179.9
COE vs. SD -93.67 2.44 n.s* -267.1 79.78
COE vs. FD 6.00 0.15 n.s* -167.4 179.4
SD vs. FD 99.67 2.60 n.s* -73.78 273.1
168
Table 3.32: ANOVA for dissolution parameters of RTV complexes
with HPβCD and RMβCD by freeze drying
variation
DE60 Between 2 11633 5817 14388* <0.0001
products
Within 6 2.422 0.404
products
Total 8 11633
DP30 Between 2 10440 5220 17593* <0.0001
products
Within 6 1.788 0.296
products
Total 8 10442
DE10 Between 2 7884 3942 38863* <0.0001
products
Within 6 0.608 0.101
products
Total 8 7885
T50 Between 2 77850 38925 601321* <0.0001
products
Within 6 0.388 0.064
products
Total 8 77851
169
Table 3.33: Tukey multiple comparison test for DE60, DP30, DE10 and MDT values of RTV
Mean Significant
Parameter Comparison of products q 95% LCL 95% UCL
Diff (p<0.05)
RTV vs. HPβCD complex -75.75 206.4 s* -77.34 -74.16
DE60 RTV vs. RMβCD complex -76.77 209.1 s* -78.37 -75.18
HPβCD vs. RMβCD complex -1.023 2.788 n.s* -2.616 0.569
DP30 RTV vs. RMβCD complex -75.71 240.7 s* -77.07 -74.34
DE10 RTV vs. RMβCD complex -64.25 349.4 s* -65.05 -63.45
RTV vs. HPβCD complex 196.9 1340 s* 196.3 197.5
MDT RTV vs. RMβCD complex 197.7 1346 s* 197.1 198.3
HPβCD vs. RMβCD complex 0.7933 5.401 s* 0.1559 1.431
s*-significant difference, n.s*-not significant
170
Based on the result of Tukey multiple comparison test, SQV
complex with SBE7βCD, by freeze drying showed highest mean
difference, 57.39 in %DE60, 42.2 in DP30 and 213.1 in MDT values
compared to SQV. Similarly, freeze dried complex of RTV with RMβCD
showed highest mean difference, 75.9 in %DE60, 78.6 in DP30, 61 in
%DE10 and 197.7 in MDT values. Based on all these values, SQV-
SBE7βCD and RTV- RMβCD complexes prepared by freeze drying were
found to be optimized complexes for achieving the complete drug
release in the contemplated 60 min. SQV- SBE7βCD release the drug in
60 min whereas RTV- RMβCD released the drug in 10 min.
3.7.6.3. Model dependent methods
Model dependent methods like zero and first order for
establishing drug release kinetics and Higuchi diffusion model, Hixon-
Crowell erosion model for establishing mechanism were applied for the
dissolution data of drug-CD complexes. However, none of the models
gave correlation coefficient values>0.90 for good linear fit for any of the
models. The dissolution data indicated initial slow release of drug
followed by rapid release during the near completion of drug release for
many drug-CD complexes. Some of the drug-CD complexes failed to
release the complete drug in 60 min. In case of ritonavir, drug release
was completed in 10 min for the optimized drug-CD complexes. Hence
further analysis of the drug release kinetics and drug release
mechanism was not done.
171
3.7.7. FTIR studies
Fourier infrared spectrophotometry (FTIR) has been employed as
a useful tool to identify drug excipient interactions.40,41 In the present
study, the use of FTIR spectroscopy to provide important information
regarding confirmation of inclusion complexation formation of CDs with
both selected drugs. FTIR patterns of SQV-βCD, HPβCD, RMβCD and
SBE7βCD freeze dried complexes, physical mixture, SQV and
corresponding cyclodextrins are represented in Fig. 3.13 to 3.16.
FTIR studies of SQV exhibited peaks at 3529 cm-1 for OH
stretching, 3100 cm-1 for NH2 stretching, 1622 cm-1 for C=O stretching
and 3381 cm-1 for NH stretching which confirms the structure of SQV.
The spectra of βCD showed prominent peaks at 3420.08 cm-1 for
O-H stretch, 2925 cm-1 for C-H aliphatic stretch. IR spectra of physical
mixture and freeze dried complex of SQV-βCD showed broadening of
peak from 3200-3400 cm-1.
The spectra of HPβCD showed prominent peaks at 3433.95 cm-1
for O-H stretch, 2923.9 cm-1 for C-H aliphatic stretch. IR spectra of
physical mixture and freeze dried complex of SQV-HPβCD showed
broadening of peak from 3100-3500 cm-1.
The spectra of RMβCD showed prominent peaks at 3400.02 cm-1
for O-H stretch, 2923.07 cm-1 for C-H aliphatic stretch. IR spectra of
physical mixture of SQV-RMβCD showed broadening of peak from
3150-3500 cm-1 and increase in the intensity of C-H aliphatic stretch at
2935.76 cm-1 indicates interaction between SQV and RMβCD.

172
The spectra of SBE7βCD showed prominent peaks at 3425 cm-1
for O-H stretch, 2936.7 cm-1 for C-H stretch and 1647.2 cm-1 for C=O
stretch. The peaks of physical mixture of SBE7βCD were smoothened
compared to that of SQV indicating complexation between SQV and
SBE7βCD. FTIR spectrum of SQV-SBE7βCD freeze dried complex
shows characteristic broad peak at 3291.89 cm-1. This shift and
broadening of peak indicated complexation between SQV and
SBE7βCD.
The FTIR spectrums of complexes showed substantial decrease in
intensity and broadening compared to that of SQV. This result
suggested the formation of a new supramolecular compound.
Additionally, no new peaks were observed in the spectra of SQV-CD
complex systems, indicated no new chemical compounds were created
in the complex formation. Thus the FTIR spectra indicated that
saquinavir is partially included into the CD to form inclusion complex.
FTIR patterns of ritonavir complexes with HPβCD and RMβCD
are represented in Fig. 3.17 and 3.18 respectively.
When FTIR was performed, the drug spectrum indicated
characteristic peaks at 3344.88 cm−1 (N–H stretching amide group),
2951 cm−1 (hydrogen-bonded acid within the molecule), 1714 cm−1
(ester linkage), 1627.97 and 1525.74 cm−1 (–C=C– stretching aromatic
carbons).
The FTIR spectra of ritonavir cyclodextrin complexes showed
considerable difference compared to pure ritonavir and cyclodextrin
173
spectra and showed decrease in frequency of a specific peak which is
generally seen in complexation. Physical mixtures showed broadening
of peak and shifting from 3344.68 cm-1 to 3338.89 cm-1 (NH stretching),
2952 cm-1 to 2922 cm-1 hydrogen bonded acid peak, 1734 cm-1 (ester
linkage) and 1639 cm-1 C=C group with HPβCD. RMβCD physical
mixture showed peaks with less intensity and broadening at 3452.39
cm-1, 2939 cm-1, 1716 and at 1631 cm-1 regions. Freeze dried
complexes showed similar pattern with widening. Freeze dried
complexes with RMβCD showed one small additional peak at 2407.24
cm-1. Intermolecular hydrogen bonding was observed in ritonavir
cyclodextrin complexes prepared by freeze drying, due to broad
absorption at 3389.04 cm-1. The broadening and widening of peak at
1730 cm-1 region may be due to the interaction between cyclodextrins
at the aldehydic link. The results confirmed the formation of stable
hydrogen bonds and interaction at the aldehydic group was responsible
in complex formation, giving increase in solubility of the drug.
174
Fig. 3.13: FTIR spectra of a) SQV b) βCD c) SQV-βCD physical
mixture d) SQV-βCD complex prepared by freeze drying
175
Fig. 3.14: FTIR spectra of a) SQV b) HPβCD c) SQV-HPβCD physical
mixture d) SQV-HPβCD complex prepared by freeze drying
176
Fig. 3.15: FTIR spectra of a) SQV b) RMβCD c) SQV-RMβCD
physical mixture d) SQV-RMβCD complex prepared by freeze drying
177
Fig. 3.16: FTIR spectra of a) SQV b) SBE7βCD c) SQV-SBE7βCD
physical mixture d) SQV-SBE7βCD complex prepared
by freeze drying
178
Fig 3.17: FTIR spectra of a) RTV b) HPβCD c) RTV-HPβCD physical
mixture d) RTV-HPβCD complex prepared by freeze drying
179
Fig. 3.18: FTIR spectra of a) RTV b) RMβCD c) RTV-RMβCD
physical mixture d) RTV- RMβCD complex prepared by freeze
drying
180
3.7.8. DSC studies
Differential scanning calorimetry (DSC) has been one of the most
widely used calorimetric techniques for the studies of the interactions
between drugs and cyclodextrins in the solid state.30 In this study, DSC
was applied to evaluate the interaction between saquinavir and
ritonavir with cyclodextrins used in the study in the solid state.
DSC thermograms of saquinavir, cyclodextrins and the
complexes are presented in Fig.3.19 to 3.22. The DSC thermogram of
SQV showed an endothermic peak at 239°C which was corresponding
to its melting point.
DSC thermogram of βCD showed a broad endothermic peak at
101°C due to loss of water molecule. The SQV complex of βCD prepared
by physical mixing showed a less intense endothermic peak of
saquinavir at 237°C and was nearly identical to that of pure saquinavir.
SQV complex with βCD prepared by freeze drying showed a greater
reduction of intensity in endothermic peak of saquinavir at 239°C
(Fig. 3.19).
DSC thermogram of HPβCD showed a broad endothermic peak
from 60-80°C. In case of SQV and HPβCD physical mixture showed an
endothermic peak at 239°C and slight decrease in intensity. But freeze
dried complex of saquinavir showed a sharp reduction in the intensity
of peak indicates strong interaction between SQV and HPβCD
(Fig. 3.20).
181
The DSC spectra of RMβCD showed a broad endothermic peak in
the range of 80-100°C which can be attributed to desolvation. The
physical mixture of saquinavir and RMβCD showed an endothermic
peak at 233°C and freeze dried complex showed broadening of
endothermic peak in the range 226-260°C respectively due to formation
of inclusion complex (Fig. 3.21).
The DSC thermogram for SBE7βCD showed at 270°C where as
the physical mixture of SQV and SBE7βCD showed very less intense
peak which indicated good interaction happened in physical mixing
itself with SBE7βCD. Freeze dried complex showed reduced intensity of
endothermic peak and shifted to 237.05°C due to the carrier induced
drug amorphorization (Fig. 3.22).
The thermograms of ritonavir, cyclodextrins and the complexes
are presented in Fig. 3.23 and 3.24. The DSC thermogram of ritonavir
showed an endothermic peak at 124.55°C, which is corresponding to
its melting point.
Thermogram of HPβCD showed a broad endothermic peak from
60-80°C. In case of RTV and HPβCD complex prepared by physical
mixture showed and endothermic peak showed at 118.99°C and freeze
dried complex showed broad endothermic peak at 107.19°C that
strongly indicates strong interaction between ritonavir and HPβCD and
hence confirms complexation.
Thermogram of RMβCD showed a broad endotherm in the range
of 80-100°C which can be attributed to desolvation. Physical mixture of
182
RTV and RMβCD showed broadening of endothermic peak in the range
of 100.41°C and freeze dried complex showed a broad endothermic
peak at 100.63°C due to formation of inclusion complex.
The results of the DSC studies showed that the complexed form
of saquinavir and ritonavir was the major product in the solid mixture.
Fig. 3.19: DSC spectra of a) SQV b) βCD c) SQV-βCD physical

mixture d) SQV-βCD complex prepared by freeze drying
183
Fig. 3.20: DSC spectra of a) SQV b) HPβCD c) SQV-HPβCD physical
mixture d) SQV-HPβCD complex prepared by freeze drying
184
Fig. 3.21: DSC spectra of a) SQV b) RMβCD c) SQV-RMβCD physical
mixture d) SQV-RMβCD complex prepared by freeze drying
185
Fig. 3.22: DSC spectra of a) SQV b) SBE7βCD c) SQV-SBE7βCD
physical mixture d) SQV-SBE7βCD complex prepared by freeze
drying
186
Fig. 3.23: DSC spectra of a) RTV b) HPβCD c) RTV- HPβCD physical
mixture d) RTV- HPβCD complex prepared by freeze drying
187
Fig. 3.24: DSC spectra of a) RTV b) RMβCD c) RTV- RMβCD
physical mixture d) RTV- RMβCD complex prepared by freeze
drying
188
3.7.9. Nuclear magnetic Resonance spectroscopy (NMR)
Only 1H-NMR spectroscopy can afford the most direct evidence
for a true inclusion complex formation by evidencing interactions
between guest molecules and the host cyclodextrins.
1H-NMR spectra of saquinavir, cyclodextrins and freeze dried
complexes were performed and are shown in Fig.3.25 to 3.28. All the
proton signals in the 1H-NMR spectrum of the pure saquinavir, proton
signals were observed at 1.26 ppm which represents aliphatic amine,
multiplet aromatic protons 6.7-6.9 ppm and other prominent chemical
shifts at 2.76 and 4.7 ppm were observed. Proton signals for benzene
rings in saquinavir were represented δH at 6.7 ppm, 6.9 ppm, 7.1 ppm,
7.75 ppm, 7.9 ppm, 8.05 ppm, 8.1 ppm, 8.14 ppm and 8.5 ppm.42 δH at
4.7 ppm represented the signal of water.
The insertion of saquinavir molecule into CD cavity was
demonstrated by changes in 1H-NMR chemical shift values. NMR
spectra clearly indicated interactions between saquinavir and H-3 and
H-5 protons belonging to the host CD and pointed towards the interior
of the cavity. Significant changes were observed in the signal due to H-
3 and H-5, whereas H-1, H-2, H-4 and H-6 (located outside the cavity)
were relatively unshielded by saquinavir. The shifts observed for the CD
protons were indicative of the occurrence of an inclusion of saquinavir
into the cyclodextrin cavity. In case of SQV, a significant upfield shift
for the resonance of the protons of the two aromatic cycles of SQV
(isoquinoleine ring and phenyl ring) was observed in presence of CD.
189
This upfield shift noted for the resonance of aromatic protons indicated
that the two aromatic cycles of SQV were mainly involved in the
formation of the complex suggesting that both isoquinoleine and phenyl
ring moieties would be expected to be included with in CD cavity due to
their hydrophobicity and satisfactory geometry. The study clearly
showed significant reduction in signal intensity with SQV-SBE7βCD
complex and shifting of peaks indicating SQV completely included with
SBE7βCD than with other CDs.
1H-NMR spectra of ritonavir, cyclodextrins, physical mixtures and
its freeze dried complexes were carried out and are presented in
Fig.3.29 to 3.30. All the proton signals in the 1H-NMR spectrum of the
pure ritonavir are essentially located in a narrow range 1.0 to 5.2 ppm
including the aliphatic amine peak at 3.2 ppm and other prominent
peaks at 7.0 and 7.3 ppm. 1H-NMR signals reported for HPβCD was
found at 1.12, 3.8, 4.7 and 5.19 ppm. 1H-NMR spectrum of RMβCD
showed proton signals at 3.48, 3.78, and 4.65 ppm.
In NMR spectra of ritonavir, NH and OH groups must have
undergone hydrogen bonding and other molecular forces of attraction
which can be shown by NMR data by disappearance of many OH, NH
signals between 3.6-5.2 ppm and disappearance of few signals near
7.0-7.3 ppm that indicates strong interaction between ritonavir and
cyclodextrins. Around 3.0 ppm, N-H in ritonavir has intense peak
where as in freeze drying of ritonavir with HPβCD and RMβCD complex,
190
peak was disappeared indicating complexation between ritonavir and
cyclodextrins.
(a)
Fig. 3.25: 1H NMR spectra of a) SQV b) βCD c) SQV-βCD complex

191
Fig. 3.26: 1H NMR spectra of a) SQV b)HPβCD c) SQV-HPβCD
complex prepared by freeze drying
192
Fig. 3.27: 1H NMR spectra of a) SQV b) RMβCD c) SQV-RMβCD
193
Fig. 3.28: 1H NMR spectra of a) SQV b) SBE7βCD c) SQV-SBE7βCD
194
Fig. 3.29: 1H NMR spectra of a) RTV b) HPβCD c) RTV- HPβCD
physical mixture d) RTV- HPβCD complex prepared
by freeze drying
195
Fig 3.30: 1H NMR spectra of a) RTV b) RMβCD c) RTV- RMβCD
physical mixture d) RTV- RMβCD complex prepared
by freeze drying
196
3.7.10. X-RAY DIFFRACTION STUDIES
The X-ray powder diffraction studies were carried out for SQV,
CDs and freeze dried complexes and are represented in Fig. 3.31 to
3.34.
In the X-Ray diffractogram of saquinavir, sharp peaks at a
diffraction angle (2θ) of 12°, 16°, 17°, 18°, 19° and 20° were present
which suggested that the drug is present as a crystalline material.
In the freeze dried complexes prepared with βCD, HPβCD,
RMβCD and SBE7βCD, the presence of free crystalline drug was
revealed by few sharp peaks of low intensity at 21°, which emerged on
the diffuse background due to the amorphous cyclodextrins, indicating
loss of crystallinity in of the drug and also due to reduction of size of
particles during the process of freeze drying which indicated the
formation of inclusion complex.
It was observed that the intensity of crystalline peaks of freeze
dried complex of saquinavir-SBE7βCD was reduced to a greater extent
compared to other CD derivatives. This may be due to the better
interaction between the drug and SBE7βCD which converted the drug
into amorphous form.
XRD of ritonavir, cyclodextrins, physical mixture and freeze dried
complexes were performed and are shown in Fig. 3.35 and 3.36. In the
X-Ray diffractogram of ritonavir, peaks at a diffraction angle (2θ) of 17°,
18° and 22° were present but the peak intensity was less in pure
197
ritonavir which suggested that the drug is not completely crystalline
but it is a mixture of both crystalline and amorphous forms.
In the physical mixture complexes, prepared with HPβCD and
RMβCD, the presence of drug was revealed by peaks of low intensity at
21°. Peaks were not observed in freeze dried complexes of ritonavir with
HPβCD and RMβCD indicating that the drug is completely converted
into amorphous form. A similar behavior was previously reported for
ketoprofen and ibuprofen.43
198
Fig. 3.31: XRD patterns of a) SQV b) βCD c) SQV-βCD complex
199
Fig. 3.32: XRD patterns of a) SQV b) HPβCD c) SQV-HPβCD
200
Fig. 3.33: XRD patterns of a) SQV b) RMβCD c) SQV-RMβCD
201
Fig. 3.34: XRD patterns of a) SQV b) SBE7βCD c) SQV-SBE7βCD
202
Fig. 3.35: XRD patterns of a) RTV b) HPβCD c) RTV- HPβCD
physical mixture d) RTV-HPβCD complex prepared by freeze drying
203
Fig.3.36: XRD patterns of a) RTV b) RMβCD c) RTV- RMβCD
physical mixture d) RTV- RMβCD complex prepared
by freeze drying
204
3.7.11. Scanning Electron Microscopy (SEM)
The SEM of saquinavir, SQV-SBE7βCD complexes prepared by
spray drying and freeze drying are shown in Fig. 3.37. Saquinavir has
appeared as irregular shaped crystals and were constituted by
relatively bulky particles, with smaller ones adhered on its surface.
Spray dried samples appeared with uniform spherical shaped particles
and freeze dried samples appeared as elongated particles. The drastic
change of the particle’s shape and aspect in the spray dried and freeze
dried samples was indicative of the presence of a single phase, thus
correlating with PXRD observations.
The SEM images of RTV, RTV complexes prepared with HPβCD
and RMβCD by freeze drying method are shown in Fig. 3.38. Ritonavir
has appeared as needle shaped crystals. Freeze dried samples appeared
as elongated particles. The change of the particle’s shape and
disappearance of needle structure of pure RTV in freeze dried
complexes confirmed single phase existence and loss of needle
structure indicates loss of crystallinity, thus supporting with XRD
studies.
205
(A)
(B)
(C)
Fig. 3.37: SEM images of A) SQV B) SQV-SBE7βCD complex

prepared by spray drying C) SQV-SBE7βCD complex prepared by
freeze drying
206
(A)
(B) (C)
Fig. 3.38: SEM images of A) RTV B) RTV- HPβCD complex C) RTV-

RMβCD complex by freeze drying method
The drug-excipient interaction studies were performed for freeze
dried complexes with all CDs used in the study. FTIR studies showed
good interaction between drugs and CDs and no structural change was
observed in drug molecules. DSC data showed broadening of peak with
decrease in intensity, confirming better interaction between drug and
CDs. NMR data proved complexation between drug and CDs, giving
evidence of inclusion of drug into CD cavity. X-ray diffrractograms
showed significant reduction in crystalline nature of drug, indicating

207
conversion of drug into amorphous form after complexation. SEM
studies showed formation of single solid state complexes with elongated
particle shape in freeze drying. Based on this study, freeze drying
technique was proved as effective technique in formation of complex in
improving solubility and high dissolution.
3.8. Preparation of capsules
Optimized SQV-SBE7βCD and RTV-RMβCD complexes prepared
by freeze drying were filled into empty hard gelatin capsules (size 1)
with weighed amount of drug equivalent to 100 mg (total of SQV-
SBE7βCD complex-223.66 mg, RTV-RMβCD complex-225.30 mg) and
evaluated for uniformity of weight and in vitro dissolution studies.
3.8.1. Uniformity of weight44
According to IP, 20 capsules were selected at random, weighed
individually and the average weight was calculated for the
determination of weight variation of capsules. Then mean and percent
deviations were determined. If the average weight of capsule content is
less than 300 mg then ±10% deviation can be acceptable.
3.8.2. In vitro dissolution studies
The filled hard gelatin capsules were tested for the dissolution
studies. The same procedure was followed as mentioned in Sec. 3.4 for
both drugs.
208
3.9. Results and discussion
To find out the suitability of converting the powdered complex
into dosage form, they were filled into empty hard gelatin capsules.
They were not compressed into tablets as the process may affect drug
release characteristics. Both optimized complexes were fine and free
flowing powders. Drug equivalent to 100 mg were filled and evaluated
for uniformity of weight and dissolution studies. No excipients were
added to the powdered complexes due to their free flowing nature. Drug
content was not estimated for filled capsules as estimated powdered
complex was only used for the study.
3.9.1. Evaluation of capsules for uniformity in weight
The test of uniformity of weight for filled capsules of both SQV
and RTV were conducted and the results are shown Table 3.34. The
weight variation of all the filled capsules was within the limits. These
results indicated that the method of filling of drug-CD complexes into
the hard gelatin capsules was uniform.
Table 3.34: Uniformity of weight for prepared capsules

of SQV and RTV complexes
Weight (mg)
Formulation
(mean±s.d.) (n=20)
SQV 100.00±0.75
RTV 100.00±0.80
SQV-SBE7βCD 223.66±0.95
RTV-RMβCD 225.30±0.70
209
3.9.2. Dissolution studies
The dissolution was carried out for optimized complexes. The
capsule shell was dissolved within 15 min and releasing full of its
contents. No change was observed in the drug release profiles of
complexes filled into capsules. The dissolution profiles are given in
Table 3.35 and 3.36. The comparative dissolution profiles of powdered
complex form and from capsule are shown in Fig.3.44 and 3.45.
Table3.35: Dissolution data of SQV capsules

Mean percentage drug release (n=3)
(mean±s.d.)
SQV-
Time SQV SBE7βCD
5 11.75±0.50 36.60±0.95
10 14.25±0.65 48.95±0.75
15 16.75±0.91 54.82±0.82
30 19.10±0.85 61.01±0.64
45 20.5±1.05 86.05±0.65
60 20.60±0.61 99.75±0.65
100
Mean % drug released
80
60
40
20 Powder
Capsule
0
0 10 20 30 40 50 60
Time in min
Fig.3.39: Dissolution profiles of complex of

SQV-SBE7βCD powder and capsule
210
Table3.36: Dissolution data of RTV capsules
Mean percentage drug release (n=3)
(mean±s.d.)
RTV-
Time RTV RMβCD
5 6.45±0.50 90.61±0.52
10 12.61±0.15 99.50±0.65
15 13.80±0.55
30 16.75±0.65
45 21.25±0.44
60 25.75±0.80
120
100
Mean % drug released
80
60
40
Powder
20 Capsule
0
0 10 20 30 40 50 60
Time in min
Fig.3.40: Dissolution profiles of complex of

RTV-RMβCD powder and from capsule
From the results, it was concluded that the prepared complexes
can be successfully formulated into a capsule dosage form since no
change was observed in dissolution profiles compared to powder form.
3.9. Conclusion
In the present investigation, the enhancement of saquinavir and
ritonavir solubility is highly dependent on the type of βCD molecule
and the method employed in the preparation of complex. The different
211
inclusion constants found for the different cyclodextrin molecules
studied indicated that the derivative groups in the cyclodextrin play an
important role in the complexation of saquinavir into the CD cavity.
Dissolution of saquinavir and ritonavir complexes was found to be
rapid compared to pure drugs. Statistical analysis showed significant
difference among various methods and different CD derivatives used in
the study. Drug-excipient interaction studies proved complexation
between the drugs and CDs. Good enhancement was observed with
freeze dried complexes of SBE7βCD for saquinavir and freeze dried
complexes of RTV-RMβCD. So, these formulations were optimized and
filled into capsules and evaluated. No change in drug release profiles
was observed. So, these capsules were selected for further stability and
in vivo studies.
212
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216

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Chapter-III

3. PREPARATION AND EVALUATION OF SAQUINAVIR AND

The most common pharmaceutical application of cyclodextrins is

to enhance drug solubility in aqueous solutions. Although prediction of

compound solubilization by cyclodextrins continues to be highly

empirical, various historical observations permit several general

greater the relative solubility enhancement obtained through

cyclodextrin complexation.1 Second, type of cyclodextrin used for

complexation also influence solubility and dissolution rate

enhancement. Cyclodextrins of lower molar substitution are better

solubilizers than the same type of derivatives of higher molar

substitution. For example, RMβCD with molar substitution 0.6 provide

better solubilization than the same type of derivatives of higher molar

substitution.2 Third, charged cyclodextrins can be powerful

solubilizers. SBE7βCD cyclodextrin, where the anion been moved away

from the cavity by a butyl ether space group, is an excellent

solubilizer.3 Inclusion complexation of drug with cyclodextrins by

shown to dramatically increase drug solubility and dissolution rate.4

Hence, in the present investigation it is aimed to compare

different types of CD derivatives and methods of preparation in

enhancing the solubility, dissolution rate and bioavailability of two

protease inhibitors and BCS class II drugs, saquinavir and ritonavir.

Phase solubility studies were performed as mentioned by

(Higuchi and Connors, 1965) 5 for saquinavir in pH 6.8 phosphate

by adding excess amounts of both drugs separately in quantities

exceeding its aqueous solubility to 50 mL of aqueous solutions of

buffers, containing increasing concentrations of cyclodextrins

(2-12 mM). The resulting suspensions were shaken at room

temperature for a period of 72 hrs, until equilibrium was established.

The samples were filtered through a 0.45 µm membrane filter (Millex-

HA filter units, Millipore) and suitably diluted with corresponding

buffer before analysis. All studies were performed in triplicate.

Apparent 1:1 stability constants were calculated from the

straight-line portion of the phase solubility diagrams, according to

where So is the solubility of the drug in examined buffer solution

in the absence of ligand.

3.2. Preparation of drug-CD complexes

In the present work, saquinavir-CD complexes were prepared

using physical mixing (PM), kneading (KN), co evaporation (COE), spray

prepared using physical mixing, co evaporation, spray drying and freeze

3.2.1. Physical mixture method6,7

Selected drugs and cyclodextrins at equimolar ratio (1:1) were

weighed and mixed in a mortar by geometric dilution method for

sufficient time (~5 min) to obtain a homogenous powder blend, passed

desiccator over fused calcium chloride until further use.

3.2.2. Kneading method7-9

Cyclodextrins were wetted in a mortar with minimum volume of

water-ethanol mixture (1:1) until a paste was obtained. The required

45 minutes. During this process, a suitable quantity of solvent was

added to maintain optimum consistency. Further the products were

dried at 40°C to constant weight, passed through sieve no. 80 and

stored in a desiccator until further evaluation.

3.2.3. Solvent evaporation method9

Required quantities of drug and cyclodextrin were dissolved in

sufficient quantity of water-ethanol solvent mixture (1:1) and

evaporated on a water bath at 50°C with stirring. Each solid product

was sieved through #80 and stored in desiccator.

In spray drying, characteristic properties of powders like particle

dependent on the two spray drying process variables: inlet temperature

(Tin) and solution flow.10-12 To select best optimizing conditions, SQV

applied with other CDs used in complex formation. For optimization,

Spray drying was performed using Labultima LU22 spray dryer