Chapter 3 Experimental

CONTENTS:
The contents of this chapter are divided into three parts. Part I includes analytical method
development for the selected drugs by UV, HPLC and bioanalytical HPLC methods for
estimation of drugs in plasma. Part II deals with solubility enhancement approaches
investigated for telmisartan. Part III includes various strategies explored for solubility
enhancement of cefpodoxime proxetil. The figure and table nos. given in parenthesis are
presented in Chapter 4 (Results & Discussions) in respective sections.

PART I: ANALYTICAL METHOD DEVELOPMENT

Appropriate analytical methods are a prerequisite for quantification of the drugs at

various stages of the experimentation. The choice of the methodology depended on
convenience and suitability.

3.1. Analytical method development for TEL:

UV spectrophotometric and HPLC methods were developed for quantification of TEL in

various media as well as plasma for in vivo studies.

i] UV spectroscopy:

Preparation of calibration curve in distilled water:

TEL (10mg) was transferred to 10 ml volumetric flask containing 10 ml methanol. From

this stock solution 1 ml of aliquot was diluted to 10 ml using distilled water to give a
solution of concentration of 100µg/ml. This standard stock solution was then
appropriately diluted with distilled water to obtain series of TEL solutions in the range of
2-10 µg/ml. The absorbance of all the solutions was measured against blank as distilled
water at 296 nm using double beam spectrophotometer. A standard plot of absorbance v/s
concentration of drug was plotted. (Figure 4.1.1 & Table 4.1.1).

Preparation of calibration curve in 0.1N HCl:

TEL (10mg) was transferred to 10 ml volumetric flask containing 10 ml of 0.1N HCl.

From this stock solution 1 ml aliquot was diluted to 10 ml using 0.1N HCl to give a
solution of concentration of 100µg/ml. This standard stock solution was then

Solubility enhancement of BCS Class II/IV drugs 65

Chapter 3 Experimental

appropriately diluted with 0.1N HCl to obtain series of TEL solutions in the range of 2-
10µg/ml. The absorbance of all the solutions was measured against blank as 0.1N HCl at
296 nm using double beam spectrophotometer. A standard plot of absorbance v/s
concentration of drug was plotted. (Figure 4.1.2 & Table 4.1.2).

Preparation of calibration curve in pH 6.8 buffer:

TEL (10mg) was transferred to 10 ml volumetric flask containing 10 ml methanol. From

this stock solution 1 ml of aliquot was diluted to 10 ml using buffer pH 6.8 to give a
solution of concentration of 100µg/ml. This standard stock solution was then
appropriately diluted with buffer pH 6.8 to obtain series of TEL solutions in the range of
3-15 µg/ml. The absorbance of all the solutions was measured against blank as buffer
pH6.8 at 296 nm using double beam spectrophotometer. A standard plot of absorbance
v/s concentration of drug was plotted. (Figure 4.1.3 & Table 4.1.3).

Preparation of calibration curve in Fasting State Simulated Intestinal Fluid

(FaSSIF) & Fed State Simulated Intestinal fluid (FeSSIF):

10 mg of TEL was dissolved in 10 ml methanol and subsequently diluted 100 times using
methanol. This solution was further diluted with FaSSIF and FeSSIF to produce 0.5-4
µg/ml of TEL. Absorbances of these solutions were noted using UV/VIS
spectrophotometer (Jasco V-530, Japan) at the maximum wavelength of 298 nm using
FaSSIF and FeSSIF as blank.(Figures 4.1.4 & 4.1.5 and Tables 4.1.4 & 4.1.5).

ii] HPLC method:

HPLC is a well-documented analytical technique having greater accuracy and precision

as compared to UV spectrophotometry. For saturation solubility studies and drug content
determination, HPLC method was used for quantification.

The concentration of TEL was determined by RP-HPLC (Agilent Technologies 1120

series, Germany) using TC-C18 column (4.6× 250 mm, 5 µm). Acetonitrile and 0.05 M
potassium dihydrogen phosphate in a ratio 60: 40 (adjusted to pH 3.0 with o-phosphoric
acid) was used as the mobile phase at a flow rate of 1ml/min. The eluent (10 µl) was
analyzed at 271 nm by UV detector. Calibration curve was plotted by preparing standard

Solubility enhancement of BCS Class II/IV drugs 66

Chapter 3 Experimental

solutions of TEL in methanol in concentration range of 4-24µg/ml.(Table 4.1.6 & Figure

4.1.6).

Validation of method:

a) Linearity:

The linearity of the relationship between peak area and concentration was determined by
analyzing five standard solutions over the concentration range 4-24µg/µl. A standard
calibration curve was constructed from the data obtained and the linearity was checked
using the correlation coefficient and regression equation.(Tables 4.1.7 & 4.1.8)

b) Precision:
Method Precision
For checking method precision, standard solutions of 8, 16, 24 µg/µl was prepared and
subjected to analysis in three replicates. SD and % RSD were recorded.

Precision for repeatability

One set of three different concentrations (8, 16 and 24 µg/µl) of TEL was prepared. All
the solutions were analyzed thrice, in order to record any intra day variations in the
results. For inter day variations study, three different concentrations of standard solutions
were analyzed for three days.(Table 4.1.9).

c) Recovery studies:

Recovery studies were performed in triplicate for each three concentrations of TEL (8,
16, 24µg/µl), the response for each level being compared with that from the
corresponding standard solution.(Table 4.1.10).

d) Limit of detection (LOD):

Limit of detection is the minimum quantity of the drug which can be detected by the
method.
Limit of detection is calculated as
LOD= 3.3 (σ/S) Equation 3.1

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Chapter 3 Experimental

Where σ is the standard deviation of the response and S is the slope of the calibration
curve.

e) Limit of quantification (LOQ):

Limit of quantification is the minimum quantity of the drug that can be quantified by the
method.

Limit of quantification is calculated as

LOQ= 10 (σ/S) Equation 3.2

iii] HPLC (Bioanalytical method):

An isocratic reverse phase HPLC method (Jasco 2000 with PU-2080 plus pump) was
used to determine concentration of TEL in rat plasma. Plasma sample pretreatment
consisted of protein precipitation extraction with methanol. The mobile phase used was
sodium dihydrogen phosphate buffer (pH 3.0): acetonitrile (ACN) (42:58, v/v) on a
HiQSil C18HS (4.6mm x 250mm) at a flow rate of 1.2 ml/min. The method was validated
over the range of 2-5 μg/ml. Sample detection was carried out at 297 nm using UV-PDA
detector. (Table 4.1.11 & Figure 4.1.10).

Preparation of standard stock solutions of TEL

Standard stock solution of TEL (100 µg/ml) was prepared in methanol. For calibration,
1ml was taken from standard stock solution and diluted upto 2ml with blank plasma to
give standard spiked plasma stock solution of 50µg/ml. From the standard spiked plasma
stock solution different concentrations were prepared by taking 30, 60, 90, 120 and 150
µl and volume was made up to 300 µl with blank plasma. 1.2ml of precipitating agent
(Methanol) was added in all to obtain final concentration of 2, 3, 4 and 5 µg/ml,
respectively.

Sample extraction procedure

Protein precipitation was used for extraction of TEL from plasma using methanol as
precipitating agent. Plasma and methanol (1:4) were mixed together and centrifuged for
10min at 10,000rpm. The decanted clear supernatant liquid was injected to HPLC.

The methods were validated for precision, accuracy and recovery.

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Chapter 3 Experimental

3.2. Analytical method development for CP:

UV and HPLC methods were developed for quantification of CP in various media.

i] By UV spectroscopy:

Calibration curve in DW:

CP (10 mg) was dissolved in 10 ml methanol (1000 µg/ml). From this, 1 ml was diluted
to 10ml with DW to get 100 µg/ml solutions. Further dilutions were effected to get 20-
60 µg /ml dilutions and absorbances were measured at 263 nm. (Table 4.2.1 & Figure
4.2.1).

Calibration curve in 0.1N HCl:

CP (10 mg) was dissolved in 10 ml methanol and subsequently diluted 10 times using
methanol. This solution was further diluted with 0.1N HCl to produce 12-20 µg/ml of
CP. Absorbances of these solutions were noted using UV/VIS spectrophotometer (Jasco
V-530, Japan) at the maximum wavelength of 263 nm using 0.1 N HCl as blank. (Table
4.2.2 & Figure 4.2.2).

Calibration curve in pH 6.8 buffer:

CP (10 mg) was dissolved in 10 ml methanol and subsequently diluted 10 times using
methanol. This solution was further diluted with phosphate buffer pH 6.8 to produce 20-
60 µg/ml of CP. Absorbances of these solutions were noted using UV/VIS
spectrophotometer (Jasco V-530, Japan) at the maximum wavelength of 263 nm using
phosphate buffer pH6.8 as blank. (Table 4.2.3 & Figure 4.2.3).

Calibration curve in FaSSIF/FeSSIF:

CP (10 mg) was dissolved in 10 ml of ethanol (1000 µg/ml). From this solution, 2.5 ml
was diluted further upto 25 ml to get a solution of concentration 100µg/ml. This solution
was diluted with FaSSIF/FeSSIF to produce 10-50 µg/ml of CP and absorbance was
measured at 263 nm with FaSSIF/FeSSIF as blank. (Tables 4.2.4 & 4.2.5 and Figures
4.2.4 & 4.2.5).

Solubility enhancement of BCS Class II/IV drugs 69

Chapter 3 Experimental

Calibration curve in USP 30 dissolution medium:

Dissolution medium:
Glycine (54.5g) and 42.6 g of sodium chloride were dissolved in about 500 ml of water
in a 1000 ml volumetric flask. Hydrochloric acid (14.2 ml) was cautiously added, with
swirling and allowed to cool. The solution was diluted with water to volume, and mixed.
.From this stock solution 50 ml was transferred to a flask and diluted with water to 900
ml to obtain a solution having a pH of 3.0±0.1.

For calibration curve 10 mg of CP was dissolved in 100 ml of methanol to get 100 µg/ml
solution. This was further diluted with the above dissolution medium to get solutions of
concentrations 10,20,30,40 and 50 µg/ml. The absorbances were measured at 259 nm
using UV spectrophotometer.(Table 4.2.6 & Figure 4.2.6)

ii] HPLC method

The concentration of CP was determined by RP-HPLC (Agilent Technologies 1120
series, Germany) using TC-C18 column (4.6×250 mm, 5µm). Acetonitrile and 50 mM
potassium dihydrogen phosphate in a ratio 70: 30 (adjusted to pH 3.0 with 10 % o-
phosphoric acid) was used as the mobile phase at a flow rate of 1ml/min. The eluent was
analyzed at 228 nm by UV detector. Calibration curve was plotted by preparing standard
solutions of CP in methanol in concentration range of 70-350 µg/ml. The method was
validated for precision, accuracy and recovery. (Tables 4.2.7-4.2.11 & Figures 4.2.7-
4.2.8).

iii] HPLC (Bioanalytical method)

An isocratic reverse phase HPLC method (Jasco 2000 with PU-2080 plus pump) was
used to determine concentration of CP in human plasma. Plasma sample pretreatment
consisted of protein precipitation extraction with methanol. The mobile phase used was
acetonitrile (ACN): potassium dihydrogen phosphate (adjusted to pH 3.8 with 10%
orthophosphoric acid) (55:45 v/v) on a HiQSil C18HS (4.6mm x 250mm) at a flow rate
of 1 ml/min. Sample detection was carried out at 228 nm using UV detector (UV 2075
plus). (Table 4.2.12 & Figures 4.2.9-4.2.11)

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Chapter 3 Experimental

Preparation of standard stock solutions of CP:

Standard stock solution of CP (500 µg/ml) was prepared in acetonitrile. For calibration,
0.2ml was taken from standard stock solution and diluted upto 2ml with blank plasma to
give standard spiked plasma stock solution of 50µg/ml. This spiked plasma stock solution
was incubated at 37°C for 3h to allow hydrolysis of CP to release free Cefpodoxime.
From the standard spiked plasma stock solution different concentrations were prepared
by taking 120,150,240,300 µl and volume was made up to 300 µl with blank plasma.
1.2ml of precipitating agent (Acetonitrile) was added in all to obtain final concentration
of 2, 5, 8 and 10 µg/ml respectively.

Sample Extraction procedure:

Protein precipitation technique was used for extraction of Cefpodoxime from plasma
using Acetonitrile as precipitating agent. Plasma and acetonitrile l (1:4) were mixed
together and centrifuged for 10min at 10,000 rpm. The decanted clear supernatant liquid
was injected into HPLC.

The methods were validated for precision, accuracy and recovery over the linear
concentration range.

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Chapter 3 Experimental

PART II: SOLUBILITY ENHANCEMENT OF TELMISARTAN

3.3. Saturation solubility studies (in DW, pH 1.2, pH 6.8, FaSSIF, FeSSIF)

3.4. Formulation development of nanosponge:

3.4.1. Synthesis of NSs

3.4.2. Complexation of TEL with NS and β-CD

3.4.3. Evaluation of NS and complexes

3.4.4. Stability studies

3.5. Formulation development of nanosuspension/nanocrystals:

3.5.1. Determination of solubility of TEL

3.5.2. Preparation of nanosuspensions

3.5.3. Evaluation of nanosuspensions

3.5.4. Evaluation of freeze dried product

3.5.5. Preparation and evaluation of pellets

3.5.6. Stability studies

3.5.7. In vivo studies

3.5.8. Statistical studies

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Chapter 3 Experimental

3.3. Saturation solubility studies of TEL:

The extent of the solubility of a substance in a specific solvent is measured as the

saturation concentration where adding more solute does not increase the concentration of
the [Link] is poorly water soluble drug & to determine its solubility in water,
shake flask method was used. In 250 ml conical flask, 20 ml of distilled water (DW) was
taken to which an excess amount of a drug was added. Then the flask was kept in
thermostatically controlled orbital flask shaker (CIS-24 Remi, India) at 37°C for 48 h.
Various studies have been reported where the saturation solubility is determined after 24,
48 or 72 h of equilibration. Mixtures were filtered using Whatman filter paper no.41. The
filtrates were diluted suitably with distilled water and solutions were analyzed using
developed HPLC method. Similarly saturation solubility was determined in 0.1N HCl,
phosphate buffer pH 6.8, FaSSIF and FeSSIF (Table 4.3.1).

 Composition of Biorelevant Media: [Dressman J;1988,Marques M;2004]

The formulation and preparation instructions for the biorelevant media developed by Dr.
Dressman’s group were used and are detailed below:

Table 3.1. Formulae for FaSSIF and FeSSIF

[Link]. Ingredients Quantity for Quantity for

FaSSIF FeSSIF
01 Sodium taurocholate 3mM 15mM
02 Soy Lecithin 0.75 mM 3.75mM
03 NaOH pellets 0.174 g 4.04g
04 NaH2PO4.H2O 1.977 g --
05 Glacial acetic acid -- 8.65g
06 NaCl 3.093 g 11.874g
07 Purified water qs 500 ml 1L

Preparation of blank FaSSIF

NaOH pellets (1.74 g), 19.77 g of NaH2PO4.H2O or 17.19 g of anhydrous NaH2PO4, and
30.93g of NaCl were dissolved in 5 L of purified water. The pH was adjusted to exactly
6.5 using 1 N HCl.

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Chapter 3 Experimental

Preparation of FaSSIF

Sodium taurocholate (3.3g) was dissolved in 500 ml blank FaSSIF. 11.8 ml of a solution
containing 100 mg /ml lecithin in methylene chloride was added, forming an emulsion.
The methylene chloride was eliminated under vacuum at about 40°C. Vacuum was drawn
for 15 min at 250 mbar, followed by 15 min at 100 mbar. The resultant clear, micellar
solution, having no perceptible odor of methylene chloride was cooled to room
temperature, volume adjusted to 2 L with blank FaSSIF. Media had a pH of 6.50.

Preparation of blank FeSSIF

NaOH pellets (20.2g), glacial acetic acid (43.25g), and NaCl (59.37g) was dissolved in 5
L of purified water. The pH was adjusted to exactly 5.0 using 1 N NaOH.

Preparation of FeSSIF

Sodium taurocholate (16.5g) was dissolved in 500 ml of blank FeSSIF. Solution (59ml)
containing 100 mg/ml lecithin in methylene chloride was added, forming an emulsion.
The methylene chloride was eliminated under vacuum of 250 mbar for 15 min followed
by 100 mbar for 15 [Link] about 40°C. This resulted in a clear to slightly hazy, micellar
solution having no perceptible odor of methylene chloride. After cooling to room
temperature, the volume was adjusted to 2L with blank FeSSIF. The recommended
volume for simulating conditions in the upper small intestine after a meal is one liter.
Media had a pH of 5.00.

Preparation of Phosphate buffer (pH6.8)[IP’2007]

Potassium hydrogen phosphate solution (50 ml of 0.2M) was placed in 200 ml volumetric
flask; NaOH (22.4 ml of 0.2M) was added and followed by water to make up the volume.

Preparation of 0.1 N HCl (pH 1.2)[IP’2007]

Potassium chloride (50 ml of 0.2M) was placed in 200ml volumetric flask to which 85 ml
of 0.2 M HCl was added and volume made up with water.

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Chapter 3 Experimental

3.4. Formulation development of nanosponges

β-cyclodextrin (β-CD) based nanosponges are nanoporous colloidal carriers which are
prepared by cross-linking β-CD with carbonyl linkages using various cross linkers like
diphenyl carbonate and carbonyl diimidazole. The hypercrosslinked nanosponges enable
insoluble drugs to be dispersed at the molecular level and stabilizing them. They are
capable of carrying hydrophilic as well as lipophilic drugs and of improving the solubility
of poorly water-soluble molecules.

3.4.1. Synthesis of blank NSs

Series of three types of β-CD NSs (Table 3.2) was prepared using diphenyl carbonate
(DPC) for the cross-linking as previously reported [Trotta F;2009]. Briefly, anhydrous β-
CD (20g) and DPC (7.54g) all finely homogenized by trituration were placed in a 250 ml
conical flask. The amounts of β-CD and DPC specified above were for preparing NS1
having 1:2 β-CD:cross-linker ratio. The mixture was gradually heated to 100°C under
magnetic stirring, and left to react for 5 h. During the reaction crystals of phenol appeared
at the neck of the flask. The carbonyl functional group of DPC was responsible for cross-
linking the β-CD molecules and resulting in liberation of phenol. The reaction mixture
was left to cool, phenol crystals were carefully removed and the product was broken up
roughly. The solid was repeatedly washed (5 times) with 50 ml distilled water to remove
unreacted β-CD and then with 100 ml acetone, to remove the unreacted DPC and phenol
present as by-product of the reaction. The reaction was carried out at three different
molar ratios, e.g. 1:2, 1:4, 1:6 (β-CD:cross-linker).The quantities of β-CD:cross-linker
used for preparing NS2 was 20g of β-CD and 15.08g of DPC. The same for NS3 was 20g
of β-CD and 22.62g of DPC. The product obtained was a fine white powder which was
insoluble in water. NSs were stored at 25°C until further use.

Table 3.2: Different ratios of β-CD:cross-linker used for synthesizing NSs

Sr. No. Ratio of Code

β-CD:cross-linker
1. 1:2 NS1
2. 1:4 NS2
3. 1:6 NS3

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Chapter 3 Experimental

Percent Yield:
The prepared NS were weighed and the yield was calculated for each preparation using
following formula,

% Yield = (a/b) × 100 Equation 3.3

Where, ‘a’ is the practical weight of product obtained and ‘b’ is the theoretical weight.

3.4.2. Complexation of TEL with NS and β-CD:

A. Phase solubility studies:

Phase solubility equilibrium plots were obtained for binary systems at 25°C in distilled
water. The studies were performed according to the method reported by Higuchi and
Connors [1965]. Studies for binary system of TEL-NS were carried out by adding excess
amount of drug to 20 ml of aqueous solution containing increasing concentration of NS1,
NS2 and NS3(0.001-0.009 moles), separately. The contents were magnetically stirred for
48 h at ambient temperature. After equilibrium, the samples were filtered and absorbance
read at 296 nm using UV/Vis spectrophotometer (Make: Jasco V – 530, Japan).The phase
solubility plot is depicted in Figure 4.4.4.

The apparent stability constant was calculated from the initial straight portion of the
phase solubility diagram using the equation:

Equation 3.4

B. Solution state interaction studies:[Swaminathan S;2007]

Modification of UV-spectra in presence of CDs/NSs provides evidence of formation of

inclusion complex. Increasing concentrations of NS aqueous dispersions (10, 20, 30, 40,
50, 60, 70 and 80 ppm) were added to fixed concentrations of TEL (10 ppm). The
samples were then kept overnight for interaction. The dispersions were filtered and the
filtrates were scanned for λ max and absorbances were measured. The parameter studied
was spectral shift. This study was carried to find out the interaction between TEL and
NS. (Figure 4.4.5).

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Chapter 3 Experimental

C. Drug incorporation:
TEL (1g) was dissolved in 20 ml of dichloromethane (DCM) to form a solution. To this
solution β-CD (2g) was added and triturated until the solvent evaporated. The TEL and β-
CD were added in a ratio of 1:2 by weight. The obtained complex was dried overnight in
an oven (at 50°C at atmospheric pressure) to remove any traces of DCM. Hydrophilic
polymers have been used for enhancing the complexation efficiency of nanosponges
[Loftsson T;2005]. Modulation of pH in dosage forms is a promising way to modify the
release rate of pH dependent and ionizable drugs. The microenvironmental pH (pHM) is
defined as the pH of the saturated solution in the immediate vicinity of the drug particles.
TEL is readily ionizable and studies have been carried out to evaluate the effect of
alkalizers like MgO, NaOH, KOH and Na2CO3 in solid dispersions of PEG 6000 [Tran
P;2008]. The same rationale was applied for preparing ternary complexes of TEL with β-
CD and NSs. In the present study sodium bicarbonate was used as the alkalizer. Ternary
complexes with β-CD were prepared by dissolving TEL (1g) in DCM. To this solution,
2g of β-CD and 0.25g sodium bicarbonate were added under magnetic stirring to form a
homogenous dispersion. The dispersion was then triturated till the solvent evaporated.
The moist mass was dried overnight at 50°C at atmospheric pressure. The obtained
powder was sieved through 60 # and used for further work. Similar procedure was used
to prepare binary and ternary complexes with NSs and sodium bicarbonate. The sodium
bicarbonate, TEL and NS were added in a ratio of [Link] 16 by weight (Table 3.3).

Table 3.3: Coding for different inclusion complexes

Sr. No. Inclusion complexes Code

1. Inclusion complex of NS1 and TEL IC1
2. Inclusion complex of NS2 and TEL IC2
3. Inclusion complex of NS3 and TEL IC3
4. Inclusion complex of β-CD and TEL IC4
5. NaHCO3 containing Inclusion complex of IC5
NS1 and TEL
6. NaHCO3 containing Inclusion complex of IC6
NS2 and TEL
7. NaHCO3 containing Inclusion complex of IC7
NS3 and TEL
8. NaHCO3 containing Inclusion complex of IC8
β-CD and TEL

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Chapter 3 Experimental

3.4.3. Evaluation of NS and complexes:

The complexes were evaluated for appearance, particle size, drug content, % yield,
saturation solubility studies, pH dependent solubility studies, solubility in biorelevant
media and in-vitro dissolution studies.

A. Drug content and % yield:

Inclusion complexes equivalent to 40 mg of TEL were weighed accurately and dissolved

in 10ml of methanol. From this,1 ml was diluted to 100 ml in methanol. The drug content
was analyzed by previously developed HPLC method. (Table 4.4.2).

The inclusion complexes were weighed and the yield was calculated for each preparation
using following formula,

% Yield = (a/b) × 100 Equation 3.5

Where, ‘a’ is the practical weight of products obtained and ‘b’ is the theoretical weight.

B. Saturation solubility studies:

Saturation solubility of the complexes in DW, 0.1N HCl and pH 6.8 buffers and
biorelevant media was determined. Procedure similar to that for TEL was used for
determining the saturation solubility. Briefly, 20 ml of dissolution media was taken in a
conical flask and complexes equivalent to 100 mg of TEL were added. The flasks were
kept in an orbital shaker at 37°C for 48h. The resultant dispersions were than filtered and
the filtrates were subjected to quantification by the developed HPLC method.(Table
4.4.3-4.4.5 & Figure 4.4.10).

C. In vitro dissolution studies:

During drug development, in vitro dissolution testing is an important tool in the

evaluation of the best formulation and also in the understanding of possible risks related
to specific GI environment, dose dumping, food effects on bioavailability and interaction
with other drugs. Dissolution studies are the most frequently used tools in the
development and characterization of all types of formulations [Beyssac E;2005].

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Chapter 3 Experimental

Dissolution studies on the formulations were performed in triplicate in 900 ml of

different media i.e. 0.1N HCl, DW and pH 6.8 buffer using USP paddle type dissolution
apparatus at 37±0.5ºC which was stirred at 50 rpm. Aliquots (5ml) were withdrawn at
specified time intervals and filtered through Whatman filter paper no. 41. An equal
volume of fresh dissolution medium was replaced to maintain the volume of dissolution
medium. The filtered samples were analyzed spectrophotometrically at 296 nm. Physical
mixtures of TEL with the nanosponges prepared in a ratio of 1:1 were also subjected to in
vitro dissolution studies. The results are presented in Tables 4.4.6-4.4.8 and Figures
4.4.15-4.4.17).

D. Physicochemical evaluation:

1. Porosity:

Since nanosponges are colloidal carriers having numerous nanochannels created due to
the cross linking, the porosity of the nanosponges was determined to confirm the
presence of nanochanels.
Porosity of β-CD and NS2 were found out by using Helium Pcynometer (Make- S. P.
Consultant Mumbai, Model- HP-2000). Helium gas has the ability to penetrate inter and
intra particulate void spaces of material due to its small size. Using the helium
pcynometer true volume of material can be determined and % porosity can be calculated
by following Equation 3.6 [Sinko P;2006].The results are presented in Table 4.4.1.

Equation 3.6

2. Fourier Transform Infrared Spectroscopy (FTIR):

FTIR spectroscopy is a powerful tool for identifying types of chemical bonds (functional
groups). The wavelength of light absorbed is characteristic of the chemical bond. FTIR
spectra of pure compounds are so unique that they are like a molecular "fingerprint" and
hence any changes in the spectra will indicate changes in the chemical structure of the
substance. The IR spectra were recorded using FT-IR spectrophotometer (Jasco-450 plus,
Japan) with diffuse reflectance principle. Sample preparation involved mixing the sample
with KBr, triturating in glass mortar and finally placing in the sample holder. The spectra

Solubility enhancement of BCS Class II/IV drugs 79

Chapter 3 Experimental

were scanned over a frequency range 4000 – 400 [Link] characteristic functional
groups were identified from the spectra depicted in Figure 4.4.11.

3. Differential Scanning Calorimetry studies (DSC):

DSC is a thermoanalytical technique which is used to investigate various endothermic

and exothermic physical transformations that occur in the test sample. It has wide
applicability as a quality control tool used for measuring sample purity and interactions
with other substances. The DSC thermograms were recorded using Differential scanning
calorimeter (DSC 823e, Mettler Toledo, Japan). Approximately 2-5 mg of each sample
was heated in a pierced aluminum pan from 30° C to 300°C at a heating rate of 10°C/min
under a stream of nitrogen at flow rate of 50ml/min. Thermal data analyses of the DSC
thermograms were conducted using STARe software (version 5.21) and the thermograms
are depicted in Figure 4.4.12.

4. Powder X-ray Diffraction studies (PXRD):

PXRD is a rapid analytical technique primarily used for phase identification of crystalline
materials. X rays are generated in a cathode ray tube, filtered to produce monochromatic
radiation, collimated to concentrate and are focused on the samples. The incidents X rays
produce a characteristic diffraction pattern when the diffracted rays are directed towards
the detector. The PXRD spectra of samples were recorded using high power powder X-
ray diffractometer (Ru-200B, Pune, India) with Cu as target filter having a
voltage/current of 40 KV/40 mA at a scan speed of 4°/min. The samples were analyzed at
2θ angle range of 5° to 50°. Step time was 0.5 seconds and time of acquisition was 1 h
(Figure 4.4.13).

5. C13 Nuclear Magnetic Resonance studies (NMR):

Nuclear magnetic resonance spectroscopy, most commonly known as NMR

spectroscopy, is a research technique that exploits the magnetic properties of certain
atomic nuclei to determine physical and chemical properties of atoms or the molecules in
which they are contained. It relies on the phenomenon of nuclear magnetic resonance and
can provide detailed information about the structure, dynamics, reaction state, and

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Chapter 3 Experimental

chemical environment of molecules. C13 NMR spectra of NS2 and β-CD were taken by
using Verian-NMR-mercury 300 spectrometer. The stock solutions of NS and β-CD were
prepared in De-DMSO. The results are depicted in Figure 4.4.2.

6. Determination of particle size and zeta potential:

The NS2 samples were dispersed in filtered glass distilled water and sonicated in a bath
sonicator for 20 min. The obtained dispersion was then diluted suitably distilled water
and particle size was measured using DelsaTM Nano Beckmann coulter counter. The
instrument uses photon correlation spectroscopy (PCS), which determines particle size by
measuring the rate of fluctuations in laser light intensity scattered by particles as they
diffuse through a fluid, for size analysis measurements and electrophoretic light
scattering (ELS), which determines electrophoretic movement of charged particles under
an applied electric field from the Doppler shift of scattered light, for zeta potential
determination. Zeta potential was measured in same instrument, by placing plain NS
dispersed in double distilled water in the flow cell. Average electric field of about 16.25
V/cm and average current of about 0.05 mA was applied. The results are presented in
Section 4.4.2D.

7. Scanning Electron Microscope Studies (SEM):

The surface morphology of samples was determined using analytical scanning electron
microscope (JSM-6360A, JEOL, Tokyo, Japan). The samples were lightly sprinkled on a
double adhesive tape stuck to an aluminum stub. The stubs were then coated with gold to
a thickness of about 10 A° under an argon atmosphere using a gold sputter module in a
high-vacuum evaporator. Afterwards, the stub containing the coated samples was placed
in the scanning electron microscope chamber and bombarded with a focused beam of
high energy electrons. The signals derived from electron-sample interactions over a
selected area of surface display spatial variations in the external morphology of the
sample (Figure 4.4.14).

3.4.4. Stability studies [Cartensen J;1995]:

Based on the results of physicochemical evaluations, selected formulation (IC6) was

subjected to stability studies at 40°C /75% RH and ambient conditions for 6 months.

Solubility enhancement of BCS Class II/IV drugs 81

Chapter 3 Experimental

Samples were evaluated at three month intervals for physical appearance, drug content
and in vitro dissolution studies (Table 4.4.9-4.4.10).

The results and observations are discussed in Chapter 4: Section [Link] Results and
Discussions.

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Chapter 3 Experimental

3.5. Formulation development of nanosuspension/nanocrystals:

Bottom-up technology was used to prepare nanosuspensions of TEL. Preliminary studies

involved identification of suitable solvent (singly or in combination) systems and
stabilizers for the [Link] for selection of solvents was low boiling point
and high solubilization ability for TEL. Screening of stabilizers was done by preparing
solid dispersions of TEL with various surfactants and polymeric stabilizers and
evaluating their saturation solubilities. Different mechanical stirring equipments that were
investigated included ultrasonicator, overhead stirrer and tissue homogenizer.

3.5.1. Determination of solubility of TEL

The solubility of TEL in water and water miscible or partially water miscible organic
solvents such as acetone, acetonitrile (ACN), dichloromethane (DCM), petroleum ether,
chloroform and N- methyl-2-pyrrolidone (NMP) and mixtures thereof, was determined
by adding an excess of the drug in the solvents. TEL (100 mg) was added to 20 ml of the
solvents. The suspensions were stirred for 24 h using a magnetic stirrer, filtered and the
drug content in the filtrate was determined by HPLC. Each sample was analyzed in
triplicate. (Results in Table 4.5.1)

3.5.2. Preparation of nanosuspensions

Preliminary studies were carried out to optimize the conditions for preparing the
nanosuspensions. The procedure involved dissolving required quantity of TEL and
polymers separately in suitable solvents and slowly mixing the two solutions with stirring
at 5000 rpm using tissue homogenizer. After complete addition of the solutions, stirring
was continued at 35000 rpm for 30 min. The dispersion was centrifuged and the sediment
was subjected to particle size analysis using optical microscopy. Micron size particles
were obtained. Further studies were carried out to investigate the use of sonicator probe
for preparing the nanosuspensions and the time of mixing was increased to 1h and then
2h. A range of nanosuspensions were prepared using different solvent and stabilizer
combinations. These were evaluated for particle size distribution using Malvern zetasizer
and nanosize particles were obtained when time of stirring was increased to 2h. However
fine, black particles were evident in the nanosuspensions which were attributed to

Solubility enhancement of BCS Class II/IV drugs 83

Chapter 3 Experimental

metallic contamination from the sonicator probe and tissue homogenizer. Hence
further studies were conducted using a robust overhead stirrer.

TEL nanocrystals were prepared by antisolvent precipitation technique. Required amount

(2g) of TEL was dissolved in sufficient volume (~10-15ml) of solvents like NMP and
DCM. Different stabilizers such as Tocopherol polyethylene glycol succinate 1000
(TPGS), PEG 6000, Poloxamer 188, PVPK30 and Gelucire 43/01 were dissolved in 200
ml water/warm water (40°C) separately and resulting mixture stirred at 4000 rpm. After
formation of a homogenous solution, drug solution was added using a micro pipette with
continuous stirring at 4000 rpm. After complete addition of the drug solution, stirring was
continued for 2 h at 10,000 rpm. The suspension was kept for 1-2 h to allow the foam to
dissipate. The different ratios of polymer-surfactant used for preparing nanosuspensions
are listed in Table 3.4. Control sample of suspension containing drug alone was prepared
using similar conditions as above and subjected to particle size analysis.

Table 3.4. Formulae for nanosuspensions of TEL

Sr. No. Batch code Components Ratio

01 T0 TEL -
02 T1 TEL:P188 1:1
03 T2 TEL:P188: TPGS [Link]
04 T3 TEL:P188: TPGS [Link]
05 T4 TEL:PEG6000 1:1
06 T5 TEL:PEG6000: TPGS [Link]
07 T6 TEL:PEG6000: TPGS [Link]
08 T7 TEL:PVPK30 1:1
09 T8 TEL:PVPK30: TPGS [Link]
10 T9 TEL:PVPK30: TPGS [Link]

3.5.3. Evaluation of nanosuspensions

The suspensions were subjected to particle size analysis and measurement of zeta
potential to confirm the formation of nanosuspensions.

A. Particle size measurement:

The nanosuspensions were subjected to particle size analysis using Malvern Zetasizer
based on dynamic light scattering. The measurement was made in triplicate and Zav,

Solubility enhancement of BCS Class II/IV drugs 84

Chapter 3 Experimental

polydispersity index and zeta potential were measured. (Results in Table 4.5.2 and
Figures 4.5.1-4.5.10)

B. Ultracentrifugation:

To obtain the nanocrystals, the nanosuspension which showed minimum particle size was
subjected to ultracentrifugation using Beckmann Coulter (Model: OptimaxXL100K with
rotor: SW32Ti) operated at 20,000 rpm and 20°C for 45 min.

C. Freeze drying:

The residue obtained by ultracentrifugation was subjected to freeze drying at -45 to -51°C
and pressure of 0.08 mbar using LABCONCO Freezone, 2.5, Kansas, USA. The residue
was carefully transferred onto a butter paper and placed inside the freeze-drying chamber
which was pre-set to required temperature and pressure. The freeze drying process was
completed in a span of 4h. The supernatant obtained after ultracentrifugation and freeze
dried product were evaluated for drug content. Since a free-flowing crystalline product
was obtained and spectral studies showed no evidence of instability, no further studies
were done to include cryoprotectants.

3.5.4. Evaluation of freeze dried product

A. Percent drug content:

The freeze dried product was analyzed for % yield and drug content by HPLC method.
The percent yield was calculated from the theoretical and practical weight of the product.
For estimation of drug content, 10 mg of the product was dissolved in 10 ml of methanol
and 1 ml from this was further diluted to 100 ml with methanol. This solution (10 µl) was
injected into the HPLC column and analyzed as described in Section 3.1 (ii).

B. Saturation solubility:

Saturation solubility of freeze dried nanocrystals was determined. Excess product (500
mg)was placed in 20 ml deionized water in a capped flask and stirred on an orbital shaker
for [Link] suspension was double-filtered using 0.22 µm filter and 1ml of filtrate was
diluted to 10 ml and 10µl was injected into HPLC system and analyzed as previously

Solubility enhancement of BCS Class II/IV drugs 85

Chapter 3 Experimental

described. The studies were also conducted in 0.1 N HCl, pH6.8 buffer and biorelevant
media i.e. FaSSIF and FeSSIF.(See Tables 4.5.3-4.5.5 and Figure 4.5.14).

C. Wettability:

Tablets of plain TEL and TEL nanocrystals (100 mg each) were prepared using hydraulic
press at pressure of 5 tons and contact angle between water and tablet surface was
determined by static sessile drop method. It involved placing 10 µl of water on surface of
tablet using micropipette. Photographs of the drop were taken after 10 s. It was carefully
superimposed on tracing paper and contact angle was measured. Amaranth red was added
to water to ensure proper visibility of the drop.(Figure 4.5.22)

D. Specific surface area:

Specific surface area of plain TEL and nanocrystals was measured using BET surface
area analyzer (Model: SAA 2000, Make: SP Consultants, Mumbai) using Nitrogen as the
adsorbent gas at 26°C. Surface area was determined by measuring the quantity of
adsorbate gas desorbed from a solid surface, by sensing change in thermal conductivity of
a flowing mixture of nitrogen (adsorbate 30%) and helium (inert carrier 70%) gas. The
change in thermal conductivity brings about a proportional change in electrical
conductivity of the mixture which is measured by the instrument and converted into
volume of desorbed gas. Calibration counts (Nc) are the counts displayed by the
instrument on injection of a known volume of adsorbate gas (N2) into the chamber
without the sample. Sample counts (Sc) are the counts displayed by the instrument when
a known volume of adsorbate gas is injected into the chamber containing the sample. The
Brunauer Emmett Teller (BET) equation was used to calculate the specific surface area of
the sample as follows:

4.38 Sc. Vn 273

S. A. = X X X (1-p/p0) Equation 3.7
W Nc (273+t)

Where W = weight of dry /regenerated sample

Sc = sample counts

Solubility enhancement of BCS Class II/IV drugs 86

Chapter 3 Experimental

Vn = volume of N2 gas injected

Nc = calibration counts
p = partial pressure of nitrogen in cylinder
p0= total partial pressure of gas mixture
t = room temperature in ° centrigrade.
The specific surface area is expressed in meters square per gram of a sample. It is a
measure of area covered by nitrogen gas adsorbed in a mono-layer form.(See Figure
4.5.21)

E. Gas chromatography:

Solvent residue was determined using gas chromatography (GC).Shimadzu-GC-2014

system was used with helium as the carrier gas at a temperature range of 50-300°C and
pressure of 20kPa. RTX-Biodiesel TG was used as the column and flame ionization
detector was used. The freeze dried sample was dispersed in dimethyl sulfoxide (DMSO)
and filtered before injecting into the GC column. Since DCM was used to prepare the
nanosuspensions, plain DCM was also injected into the GC column. The retention time
was displayed by the instrument. (See Figure 4.5.24)

F. Atomic absorption spectroscopy:

The metal content due to the processing conditions was determined using atomic
absorption spectroscopy (Thermoscientific Chemito AA 201). One microliter of
concentrated nitric acid was mixed with 5 ml of sample and the mixture aspirated into the
instrument with the reference sample. The sample was tested for iron (Fe) and chromium
(Cr).The measurement was done at room temperature and absorbances were measured at
248.3 nm for Fe and 357.9 nm for Cr. The reference samples were Cr in the form of
potassium dichromate (2 ppm and 4 ppm) and Fe (1ppm and 2 ppm).

The FTIR, DSC, PXRD and SEM studies using instruments and procedures similar to
those described for nanosponges of TEL were performed. These studies were performed
to evaluate the nanocrystals for chemical, thermodynamic and physical transformations
and to study their surface morphology.

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Chapter 3 Experimental

3.5.5. Pelletization of nanosuspension:

A. Preparation of pellets

Nanosuspensions can be used directly or they can be converted into solid form using
techniques like spray drying, freeze drying and pelletization. The products obtained by
these techniques require further processing to make them patient-friendly. In the present
work spray coating of the nanosuspension on preformed pellets was done which can be
used directly without any additional processing. For optimizing the conditions for
coating, blank vehicle was prepared which contained PVPK30 and TPGS (1g each)
dissolved in 100 ml of water. This solution was sprayed onto Espheres under different
conditions of temperature, pellet bed weight and pan speed. The intention was also to
study the adhesivity of solution on the Espheres. The nanosuspension (Batch T8) of TEL
showed the least particle size as compared to all batches and hence was spray coated on
Espheres (Microcrystalline Cellulose nonpareil seeds 18-20#) using an R & D coater
(Model: Instacoat, Ideal Cures Pvt.,Ltd., Mumbai). Nanosuspension (80 ml) was sprayed
on 20 g of Espheres at 1ml/min with atomizing air pressure of 2 lb/[Link] pan speed
was 25 rpm and pellet bed temperature was 60-70° C. Talc was used as an adsorbent to
facilitate the drying process. Conventional suspension of TEL was also prepared by
dispersing TEL in water containing TPGS in similar quantities as the nanosuspension and
spray coated onto the pellets.

B. Dissolution rate studies:

The dissolution rate studies for the nanosuspension coated Espheres was performed in
triplicate using the dissolution rate apparatus (Electrolab TDT-08L USP standard). The
dissolution studies were carried out in 0.1N HCl as dissolution medium. The test was
performed using USP type I apparatus at 50 rpm and maintaining the temperature at 37 ±
0.5°C. Freeze dried product/pellets equivalent to 40 mg of TEL were placed in 900 ml of
dissolution media (DW, 0.1N HCl and pH 6.8 buffer). Aliquots of 5 ml were periodically
withdrawn and the volume was replaced with fresh dissolution medium. The aliquots
were analyzed spectrophotometrically at 296 nm. Cumulative percentage of labeled

Solubility enhancement of BCS Class II/IV drugs 88

Chapter 3 Experimental

amount of drug released at each time point was calculated. Model fitting was done using
PCP Disso software. (Tables 4.5.6-4.5.8 and Figures 4.5.26-4.5.27)

3.5.6. Stability studies:

The nanosuspensions/pellets were placed in screw-capped vials under ambient

conditions, at 40°C &75%RH and under refrigeration (4°C) for a period of 6 months with
evaluation at 3 month intervals. The samples were visually inspected for discoloration,
flocculation, sedimentation, percent drug content and particle size (after 6 months for
nanosuspension). The effect of monovalent and bivalent electrolytes as well as pH on the
physical stability of the nanosuspensions was evaluated by visual observation and
measurement of % transmittance at 600nm. Percent transmittance studies were performed
to detect the optical density of the nanosuspensions. The optical density would be
affected by the particle size of the dispersions. Any increase in particle size would be
reflected in lowered percent transmittance. For studying the effect of electrolytes, 1 ml of
nanosuspension was diluted to 10 ml with deionized water. To this, incremental
concentrations (0.5-1.5%) and volume (0.5-2.5 ml) of monovalent electrolyte solutions
(NaCl) were added. In case of bivalent (CaCl2) electrolyte solutions, 0.5 -2.5 ml of 0.1-
0.5 % solutions were added and observed visually after 4 h and 24 h for any changes as
well as percent transmission was measured. The stability of nanosuspension was also
evaluated at pHs 1.2, 4.6, 6.8 & 7.4. The results are given in Tables 4.5.11-4.5.12 &
Figures 4.5.28-4.5.30.

3.5.7. In vivo studies:

All animal studies were performed as per the CPCSEA guidelines of the Institutional
Animal Ethical Committee to investigate the effect of reduced particle size on oral
bioavailability of TEL. Pharmacokinetic and pharmacodynamic parameters were studied.
Organ distribution and sub acute oral toxicity studies were done to address safety
concerns arising out of nanosizing of TEL.

i] Pharmacokinetic studies
In vivo studies were carried out as per the guidelines of the Institutional Animal Ethical
Committee of Sinhagad Institute of Pharmacy, Pune (SIOP/IAEC/2011/24). Adult

Solubility enhancement of BCS Class II/IV drugs 89

Chapter 3 Experimental

Wistar rats of either sex were used for the study. The rats were weighed (200-250g),
labeled and randomly divided into 03 groups’ viz. control group which was administered
plain vehicle, 2nd group which was administered TEL suspension prepared using 1%
sodium carboxy methyl cellulose as suspending agent and 3rd group which was given
TEL nanosuspension. They were fasted overnight and water was provided ad-libitum.
The preparations were (equivalent to 4 mg/kg bodyweight) administered to rats using oral
feeding tube. The animals were anesthetized with ether before collection of blood which
was collected from retro orbital plexus in EDTA treated tubes. The time intervals for
blood withdrawal were of 10 min for the first 30 min followed by 30 min interval for the
next 2h and hourly intervals up to 8h followed by withdrawal at 12h and 24h. Plasma was
separated by centrifuging the blood at 3000 rpm for 10 min at -4°C. The plasma was
subjected to protein precipitation by addition of methanol in a ratio of 1:4. It was then
centrifuged at 10, 000 rpm for 10 min at -4°C. The supernatant was decanted and 20 l
was injected into HPLC column. Simultaneously calibration curve was plotted by
incubating known concentration of test drug into the rat plasma (spiking study). (Table
4.5.13 & Figure 4.5.31-4.5.32).The protocol for the animal studies are presented in Table
3.5. The data was analyzed by one way ANOVA followed by Dunnett’s test using
Graphpad Prism software. The level of significance was p<0.05.

Table [Link] for animal testing for pharmacokinetic parameters of TEL

nanosuspension

Group Group description Number of animals

number required

1 Normal control 6
(Untreated/vehicle)
2 TEL 6× 3=18
nanosuspension
3. Plain drug (TEL) 6x3 = 18
Total 42

ii] Organ distribution studies by Gamma scintigraphy:

The use of compounds labeled with radionuclides has increased considerably in the field
of medicine and biochemistry. Compounds with γ-emitting radionuclides have wider

Solubility enhancement of BCS Class II/IV drugs 90

Chapter 3 Experimental

applications particularly for in vivo imaging of different organs. In a radiolabelled

compound, atoms or group of atoms of a molecule are substituted by similar or different
radioactive atoms or groups for which a variety of physicochemical conditions can be
employed. One method of labeling is where a radionuclide is incorporated into a
molecule by a covalent or co-ordinate covalent bond. In many compounds chemical bond
is formed by chelation wherein one or more atoms donate a pair of electrons to the
99m
foreign acceptor atom which is usually a transition metal. Tc labeled compounds used
in nuclear medicine are formed by chelation.99mTc labeled compounds constitute > 80%
of all radiopharmaceuticals used in nuclear medicine. This is because it has favorable
physical and radiation characteristics; its physical half-life is 6h and emits small amounts
of electron emission which allows administration of millicurie amounts without
significant radiation dose. The monochromatic 140 keV photons are readily collimated to
give images of superior spatial resolution. It is readily available in sterile, pyrogen-free
state from 99Mo-99mTc generators.

Chemistry:

99m
The chemical form of Tc obtained from the Moly generator is sodium pertechnetate
[99mTc-NaTcO4]. Pertechnetate ion [99mTcO4] is a non reactive species and does not label
any compound by direct addition. It has to be reduced from the 7+ state to a lower
oxidation state. Various reducing agents that can be used include stannous chloride,
stannous citrate, stannous tartrate, conc. HCl and sodium borohydride of which stannous
chloride is most commonly used. The reaction between SnCl2 and pertechnetate is
undertaken in conc. HCl as follows:

3Sn+2 ↔ 3Sn4+ Equation 3.8

299mTcO4 + 16H+ + 6e- ↔ 299mTc4+ + 8H2O Equation 3.9

Combining previous equations:

299mTcO4 + 16H+ + 3Sn2+ ↔ 299mTc4+ + 3Sn4+ + 8H2O Equation 3.10

Solubility enhancement of BCS Class II/IV drugs 91

Chapter 3 Experimental

99m
The reduced Tc is chemically reactive and combine with variety of chelating agents
which usually donate a lone pair of electrons to form co-ordinate covalent bonds with
99m - -
[Link] containing COO , OH , NH2 and SH groups are capable of forming

such bonds [Saha G;2010].

 Radiolabelling of TEL:

TEL was labeled with Technetium-99m (99mTc) by stannous reduction method [Babbar
A; 2000].99mTc was chosen for radiolabelling because of its short half-life (6 h) and as it
allows very little electron emission. It can be administered in millicurie amounts,
99m
resulting in a very low radiation dose to the animal. Moreover, Tc is readily available
99m
in a sterile, pyrogen-free and carrier state. The Tc generated from the Moly generator
was subjected to Gamma camera to determine the actual concentration of sodium
pertechnetate. It was mixed with stannous chloride and mixed in a rotary shaker for 5 min
99m
to convert Tc into its reduced form. The solid drug was added to this mixture and
mixed on a rotary shaker and subjected to TLC plating every 5min to confirm the binding
99m
between drug and Tc and to optimize the time required for the binding. Acetone was
99m
used to develop the plates which were also spotted with pure Tc. The spots on the
plates were detected using gamma counter.

Optimization of radiolabelling:

The effect of varying pH of the reaction mixture on labeling efficiency was studied. In
another experiment, the pH of the reaction mixture was kept constant at 6.5 and the
quantity of stannous chloride was varied from 100 to 1000 μg. The labeling efficiency of
TEL was measured. (Tables 4.5.14-4.5.17)

 Organ distribution of TEL nanosuspension in rabbits:

Three New Zealand rabbits of either sex weighing 2.8 to 4.1 kg were used to evaluate
organ distribution of TEL nanosuspension. The rabbit was kept on a table without a
restraining box, its head supported by the experimenter’s hand. Whole Body Imaging
Scan for organ distribution of each formulation was taken after oral dosing of three
millicurie radiolabelled TEL nanosuspension (equivalent to 4mg/kg body weight) using

Solubility enhancement of BCS Class II/IV drugs 92

Chapter 3 Experimental

Siemens Symbia E Single Head system for one min (Low Energy High Resolution
Collimator).The percentage of radiolabelled formulation in the organs viz. kidney, liver
and heart was determined using SyngoDicom software. Optimized radiolabelling
99m 99m
conditions were used for drug Tc binding. Organ count of Tc was taken after oral
administration at 0 h and thereafter at 15 min interval for 3h.(Table 4.5.18 & Appendix 5)

iii] Pharmacodynamic studies:

 Principle:
Ischemia of the kidneys causes elevation of blood pressure by activation of the rennin-
angiotensin system. This classical Goldblatt [1995] technique can be used to induce both
acute and chronic hypertension. The one-kidney-one-clip method is used to induce
chronic hypertension. It involves clamping the left renal artery for 4h. After reopening
the vessel, accumulated renin is released into circulation. The protease renin catalyzes
the first and rate-limiting step in the formation of angiotensin II leading to acute
hypertension.

 Procedure:

Male Sprague-Dawley rats (300g) were anesthetized by intra-peritoneal injection of

1.3g/kg of urethane. The animals were divided into 06 animals per group and each group
was administered plain drug, test formulation and vehicle control. A PVC-coated
Dieffenbach clip was placed onto the left hilum of the kidney and fixed to the back
muscles. The renal artery was occluded for 4 h. The trachea was canulated to facilitate
spontaneous respiration. The animals were again anesthetized by intraperitoneal injection
of 1.3g/kg of urethane after [Link] systolic and diastolic blood pressure was measured by
connecting the canula (PE tube no.48) in the carotid artery to the pressure transducer of
Data Acquisition System (8 channel Powerlab AD Instruments, Australia).The
measurements were made using Labchart Pro software (MLU260/7, Labchart Pro V7
version CD # 53171). Removal of the renal arterial clip leads to rise in blood pressure
due to rise in plasma renin level. The cardiovascular parameters that were monitored
include heart rate, beats per minute, systolic and diastolic blood pressure, mean arterial
blood pressure and electrocardiogram and these were monitored before and after

Solubility enhancement of BCS Class II/IV drugs 93

Chapter 3 Experimental

administration of the test formulation. The jugular vein was cannulated for administration
of the test formulation. TEL nanosuspension (equivalent to 4mg/kg body weight) was
administered intraperitoneally (Tables 4.5.19-4.5.20 and Figure 4.5.32).

iv] Sub acute oral toxicity:

This test was conducted as per OECD [2001] guidelines. Sprague Dawley rats were
divided into 04 groups of 03 animals each and animals of both sexes were used for the
study. Two groups were assigned as negative and vehicle control. Plain drug suspension
and prepared nanosuspension was administered to the other groups at dose level of 10
mg/kg orally daily for a period of 28 days (Fixed dose procedure). The animals were
sacrificed on 28thday under halothane anesthesia and vital organs such as heart, liver and
kidney were isolated and subjected to histopathological studies. (Results in Table 4.5.21
and Figures 4.5.33-4.5.35)

3.5.8. Statistical studies:

Various statistical tools are available to evaluate the effect of different factors
(independent variables) on certain response parameters (dependent variables) during
formulation development. These tools facilitate quantification of the effects in terms of
various statistical equations and parameters and thereby aid develoment of optimum
formulations to meet predetermined specifications.

Experimental design

Box-Behnken design was used in this study to investigate the effect of variables on
specific [Link] design comprised of experimental trials/runs involving 3 factors at
3 levels totalling 17 trials/[Link] experimental design consisted of a set of points lying
at the midpoint of each edge and the replicated centre point of the multidimensional
[Link] independent variables selected for the study were concentration of TPGS, speed
of rotation and time of stirring and the dependent variable was particle size of the
[Link] analysis was carried out using Design Expert software version
[Link] levels of independent variables are listed in Table 3.6.

Solubility enhancement of BCS Class II/IV drugs 94

Chapter 3 Experimental

The polynomial equation generated by this experimental design [Design expert 7.1.6] is
as follows:
Y= K + aX1 + bX2 + cX3 + dX1X2 + eX1X3 + fX2X3 + gX1X1 + hX2X2 + iX3X3 Eqn. 3.11

Table [Link] values of independent variables for Box-Behnken design for TEL

Independent variables Levels

Low Medium High
Concentration of TPGS (%) (X1) 1 1.5 2
Speed of rotation (rpm) (X2) 5000 7500 10000
Time of stirring (min) (X3) 60 90 120
Transformed values -1 0 +1
Dependent variable (Y) Particle size (nm)

The results of statistical studies are presented in Section 4.5.7.

Solubility enhancement of BCS Class II/IV drugs 95

Chapter 3 Experimental

PART III: SOLUBILITY ENHANCEMENT OF CEFPODOXIME PROXETIL

3.6. Saturation solubility studies (in distilled water, pH 1.2, pH 6.8, FaSSIF, FeSSIF)

3.7. Formulation development of nanosponge

3.7.1. Synthesis of NSs

3.7.2. Complexation of CP with NS and β- CD

3.7.3. Evaluation of complexes

3.8. Formulation development of nanosuspensions/nanoparticles

3.8.1. Determination of solubility of CP

3.8.2. Preparation of nanosuspensions

3.8.3. Evaluation of nanosuspensions

3.8.4. Evaluation of freeze dried product:

3.8.5. Preparation and evaluation of pellets

3.9. Formulation development of self nanoemulsifying drug delivery systems

(SNEDDS)

3.9.1. Preparation of SNEDDS

3.9.2. Evaluation of SNEDDS

3.9.3. Preparation and Evaluation of solid SNEDDS

3.9.4. Stability studies

3.9.5. In vivo studies

3.9.6. Statistical studies

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Chapter 3 Experimental

3.6. Saturation solubility studies:

Saturation solubility studies of CP were carried out in DW, pH1.2 buffer, pH 6.8 buffer,
FaSSIF and FeSSIF as described previously in Section 3.1.1 for TEL. CP (100mg) was
placed in the 250 ml of dissolution media and subjected to orbital shaking for 48h at
37°C. The resulting dispersion was filtered using Whatman filter paper and 10 ml of
filtrate was diluted to 100ml and analyzed by developed HPLC method. The results are
depicted in Table 4.6.1.

3.7. Formulation development of nanosponge

3.7.1. Synthesis of NS:

Nanosponges were synthesized as previously reported for TEL in Section 3.4.1. NS was
prepared using β-CD & DPC in a ratio of 1:4 and subjected to phase solubility and
solution state interaction studies to confirm the interaction between CP and NS.

3.7.2. Complexation of CP with NS and β-CD:

A. Phase solubility studies:

Phase solubility equilibrium plots were obtained for binary systems at room temperature
in DW as described for TEL. Studies for binary system of drug-NS were carried out by
adding excess amount of drug (50mg) to 20 ml of aqueous solution containing increasing
concentration of NS (0.001-0.008 M). The contents were stirred for 48 h at 25±0.5°C.
After equilibrium, the samples were filtered and absorbance was read at 263 nm (UV/Vis
spectrophotometer). The apparent stability constant was calculated as given for TEL in
Section 3.4.2. (Figure 4.7.1)

B. Solution state interaction studies:

Increasing concentrations of NS solutions (5-50 ppm) were added to fixed concentrations

of CP (10 ppm).The samples were then kept overnight for interaction and were scanned
for λ max and absorbances were measured. The parameter studied was spectral shift. This
study was carried to find out the interaction between CP and NS. The shift in absorption

Solubility enhancement of BCS Class II/IV drugs 97

Chapter 3 Experimental

maxima of CP was studied to confirm the entrapment of CP in the NS structure.(Figure

4.7.2)

C. Drug incorporation:

Required quantity of CP was dissolved in DCM. Nanosponges were taken in evaporating

dish and CP solution was added to this with constant stirring till the solvent evaporated.
The ratio of CP:NS was 1:2. The damp solid product was dried overnight in a hot air
oven at 60°C. The product was then passed through a 60 # sieve.

3.7.3. Evaluation of drug loaded NS:

The complexes were evaluated for drug content, % yield, saturation solubility studies, pH
dependent solubility studies, solubility in biorelevant media and in-vitro dissolution
studies.
A. Drug content:

100 mg of the inclusion complex was dissolved in methanol and mixed under magnetic
stirring for 15 min. The dispersion was filtered using Whatman filter paper no.41 and 1ml
of filtrate was diluted to 10 ml and subjected to HPLC analysis.

B. Saturation solubility of nanosponge of CP:

Drug loaded nanosponges (62.5mg) was placed in 25 ml of medium and subjected to

saturation solubility studies as described for TEL. The dispersions were filtered using
Whatman filter paper no [Link] filtrate (1ml) was diluted to 10 ml with methanol and
subjected to HPLC analysis. The saturation solubility was determined in DW, pH 1.2
buffer, pH 6.8 buffer and biorelevant media. (See Table 4.7.1-4.7.2 and Figure 4.7.3)

C. In vitro dissolution studies:

Dissolution studies on the formulations were performed in triplicate in 900 ml of
different media i.e. 0.1N HCl, DW and pH 6.8 buffer using USP paddle type dissolution
apparatus at 37±0.5ºC which was stirred at 50 rpm. Aliquots (5ml) were withdrawn at
specified time intervals and filtered through Whatman filter paper no. 41. An equal
volume of fresh dissolution medium was replaced to maintain the volume of dissolution

Solubility enhancement of BCS Class II/IV drugs 98

Chapter 3 Experimental

medium. The filtered samples were analyzed spectrophotometrically at 263 nm. The
results are presented in Tables 4.7.3-4.7.4 & Figure 4.7.4.

D. Fourier Transform Infrared Spectroscopy (FTIR):

The IR spectra were recorded using FT-IR spectrophotometer (Jasco-450 plus, Japan)
with diffuse reflectance principle. Sample preparation involved mixing the sample with
KBr, triturating in glass mortar and finally placing it in the sample holder. The sample
was scanned over a frequency range 4000 – 400 [Link] characteristic functional groups
were identified from the spectra depicted in Figure 4.7.5.

E. Differential Scanning Calorimetry studies (DSC):

The DSC thermograms were recorded using Differential scanning calorimeter (DSC
823e, Mettler Toledo, Japan). Approximately 2-5 mg of each sample was heated in a
pierced aluminum pan from 30°C to 300°C at a heating rate of 10°C/min under a stream
of nitrogen at flow rate of 50ml/min. Thermal data analyses of the DSC thermograms
were conducted using STARe software (version 5.21) and the thermograms are depicted
in Figure 4.7.6.

F. Powder X-ray Diffraction studies (PXRD):

The crystallinity of the products was studied by PXRD. The PXRD spectra of samples
were recorded using high power powder x-ray diffractometer (Ru-200B, Pune, India)
with Cu as target filter having a voltage/current of 40 KV/40 mA at a scan speed of
4°/min. The samples were analyzed at 2θ angle range of 5° to 50°. Step time was 0.5
seconds and time of acquisition was 1 h.(Figure 4.7.7)

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Chapter 3 Experimental

3.8. Formulation development of nanosuspension/nanoparticles:

CP nanosuspensions were prepared using antisolvent precipitation method which is a

bottom-up technique involving controlled micro- or nano-precipitation. The critical
variables that affect the particle size in this process include nature of antisolvent,
stabilizers and properties of the drug.

3.8.1. Determination of solubility of CP

The solubility of CP in water and water miscible and partially water miscible organic
solvents such as acetone, ACN, DCM, petroleum ether, chloroform and NMP was
determined by adding an excess (100 mg) of the drug in 20 ml of solvents. The
suspensions were stirred for 24 h using a magnetic stirrer and filtered using Whatman
filter paper no.41.10 ml of filtrate was diluted to 100 ml and the drug content was
determined by HPLC. Each sample was analyzed in triplicate.

3.8.2. Preparation of nanosuspensions

Preliminary trials were conducted to identify the stabilizer(s) and solvent combinations
which were suitable for preparing the nanosuspensions. The batches were visually
evaluated for flocculation and sedimentation immediately after preparation and after 24h.
Flocculation and sedimentation could be considered as signs of formation of micro- or
coarse suspensions. Nanosuspensions, being sub colloidal in size will not settle except
when subjected to centrifugal forces. The procedure for preparing nanosuspensions
involved dissolving required amount of CP (100 mg) in sufficient volume of solvents(~20
ml). Different stabilizers such as TPGS, PEG 6000, Poloxamer 188, PVPK30 and
Gelucire 43/01 were dissolved in 100 ml of water/warm water (40°C) separately and
resulting mixture stirred at 4000 rpm. After formation of a homogenous solution, drug
solution was added using a micro pipette with continuous stirring. After complete
addition of the drug solution, stirring was continued for 2 h at 10,000 rpm at a
temperature of 15°C. Initial studies at room temperature resulted in discoloration of CP
nanosuspensions. It was inferred that the heat generated during high speed stirring
resulted in degradation of CP. Hence for further batches, stirring was done at 15°C and
no discoloration was evident. The suspension was kept for some time to allow the foam

Solubility enhancement of BCS Class II/IV drugs 100

Chapter 3 Experimental

to dissipate. The different ratios of polymer-surfactant used for preparing

nanosuspensions are listed in Table.3.7. Control sample of suspension containing drug
alone was prepared using similar conditions as above and subjected to particle size
analysis.

Table 3.7. Formulae for nanosuspensions of CP

Batch code Components Ratio

C0 CP -
C1 CP:PEG 6000 1:1
C2 CP:PEG 6000: TPGS [Link]
C3 CP:PEG 6000: TPGS [Link]
C4 CP:P188 1:1
C5 CP:P188:TPGS [Link]
C6 CP:P188:TPGS [Link]

[Link] of nanosuspensions

A. Particle size measurement:

The nanosuspensions were subjected to particle size analysis using Malvern Zetasizer
based on dynamic light scattering. The measurement was done in triplicate and Zav,
polydispersity index and zeta potential was measured.(Table 4.8.1 & Figures 4.8.1-4.8.8)

B. Ultracentrifugation:

The nanosuspension which showed maximum decrease in particle size was subjected to
ultracentrifugation using Beckmann Coulter (Model: OptimaxXL100K with rotor:
SW32Ti) operated at 20,000 rpm and 20°C for 45 min. The residue was subjected to
freeze drying at -45 to -51°C and pressure of 0.04-0.05 mbar using LABCONCO
Freezone, 2.5, Kansas, USA. The supernatant and freeze dried product were evaluated for
drug content.

3.8.4. Evaluation of freeze dried CP nanoparticles

The freeze dried product was evaluated in terms of percent drug content, saturation
solubility, FTIR, DSC, PXRD, wettability and specific surface area and particle size. The
results are given in Tables 4.8.2-4.8.3 and Figures 4.8.9-4.8.15.

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Chapter 3 Experimental

3.8.5. Preparation and evaluation of pellets

A. Pelletization of nanosuspension
The nanosuspension C5 containing CP, P188 and TPGS in a ratio of [Link] was spray
coated on Espheres (Microcrystalline Cellulose nonpareil seeds 18-20#) using an R & D
coater (Make: Ideal Cures Pvt.,Ltd., Mumbai ). Nanosuspension (80ml) was sprayed on
20 g of Espheres at 1ml/min with atomizing air pressure of 2 lb/[Link] pan speed was
25 rpm and pellet bed temperature was 50-60° C. Talc was used as an adsorbent to
facilitate the drying process.

B. Dissolution test

The dissolution test on the nanosuspension coated Espheres was performed in triplicate
using the dissolution test apparatus (Electrolab TDT-08L USP standard). The dissolution
test was carried out in 900 ml of 0.1N HCl as dissolution medium after placing pellets
equivalent to 120 mg of CP. The test was performed using USP type I apparatus at 50
rpm and maintaining the temperature at 37 ± 0.5°C. Aliquots of 5 ml were periodically
withdrawn and the volume was replaced with fresh dissolution medium. The aliquots
were analyzed spectrophotometrically at 263 nm. Cumulative percentage of drug released
at each time point was calculated. Model fitting was done using PCP Disso software. The
results are given in Tables 4.8.4-4.8.5 and Figures 4.8.16-4.8.17.

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Chapter 3 Experimental

3.9. Formulation development of self-nanoemulsifying drug delivery systems

3.9.1. Preparation of SNEDDS

A. Determination of solubility of CP in various oils, surfactants and co surfactants

The solubility of CP in different oils, surfactants and co surfactants was investigated. The
following oils and surfactants were used.
Oils: Capmul MCM C8, Capmul MCM, Sunflower oil, Labrafac lipophile WL 1349,
Labrafil M 1944 CS.
Surfactants/Co surfactant: Tween-20, TPGS, Tween-80, Polyethylene glycol 400
(PEG-400), Propylene glycol.

Procedure:
Known amount (500mg) of selected vehicles was added to each cap vial containing an
excess (200mg) of CP. After sealing, the mixture was heated to 40ºC in a water bath to
facilitate the solubilization. The systems were mixed using a magnetic stirrer. The
suspensions were then shaken in an orbital shaker at 25ºC for 24 h. The supernatant (1ml)
was diluted with methanol to 10 ml and analyzed at 263 nm using UV spectrophotometer.
CP was found to have maximum solubility in Capmul MCM, Capmul MCM C8, tween
80 (T80), propylene glycol (PG) , PEG 400 and TPGS, (Table 4.9.1 & Figure 4.9.1)
further studies were conducted using various combinations of these oils and surfactants to
identify the micro emulsion area. For this ternary phase diagrams were constructed as
given below.

B. Construction of ternary phase diagrams:

The pseudo-ternary phase diagrams of various combinations of oil, surfactant and co

surfactant were constructed using water titration method. The mixtures of oil and
surfactant/co surfactant at certain weight ratios were diluted with water in a drop wise
manner. For each phase diagram, the ratio of the Smix to oil was varied as 9:1, 8:2, 7:3,
6:4, 5:5, 4:6, 3:7, 2:8, 1:9(w/w). The Smix were taken at specific ratios of 1:1, 2:1, 3:1,
4:1 (w/w) for tween 80:propylene glycol and 1:0.25, 1:0.5 and 1:0.75 for tween 80:TPGS
as Smix. Required amount of TPGS as per the given ratios was first melted and tween 80

Solubility enhancement of BCS Class II/IV drugs 103

Chapter 3 Experimental

was incorporated into the molten TPGS with stirring. The oil was then added to this
Smix. Water was added drop wise to each mixture with vigorous stirring. After
equilibrium, the samples were visually checked and determined as being clear micro
emulsions or emulsions. This was indicated by the appearance of cloudiness in the
mixture. The phase diagrams were constructed using CHEMIX School Ver. 3.50 (USA,
Trial version). (Figures 4.9.3-4.9.4)

C. Drug loading:

Accurately weighed quantity of surfactant and co-surfactant was mixed in vial and
required quantity (120 mg) of drug was added. Accurately weighed quantity of oil was
added and heated to 40ºC till a transparent solution was obtained. Two sets of
formulations each comprising 07 batches were prepared containing increasing quantities
of oil with Smix of 4:1 of T80 and PG (F1-F7) (Table 3.8) and Smix of 1:0.5 of T80and
TPGS and Capmul MCM (C1-C7) (Table 3.9).

Table 3.8. Formulations containing T80: PG (4:1) and Capmul MCM

Code Smix Oil Drug

(gm) (gm) (mg)
F1 0.900 0.100 120
F2 0.880 0.120 120
F3 0.860 0.140 120
F4 0.840 0.160 120
F5 0.820 0.180 120
F6 0.800 0.200 120
F7 0.780 0.220 120

Table 3.9. Formulations containing T80: TPGS (1:0.5) and Capmul MCM

Code Smix Oil Drug

(gm) (gm) (mg)
C1 0.900 0.100 160
C2 0.880 0.120 160
C3 0.860 0.140 160
C4 0.840 0.160 160
C5 0.820 0.180 160
C6 0.800 0.200 160
C7 0.780 0.220 160

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Chapter 3 Experimental

3.9.2. Evaluation of SNEDDS

A. Percent transmission studies:

Percent transmission studies are carried out to evaluate the stability of the mixture on
dilution in the GI fluids. Appearance of turbidity is indicative of loss of solvent capacity
of the SNEDDS mixture for the drug. Hence a high value of percent transmission is
indicative of stability of the SNEDDS mixture on dilution. Stability of micro emulsion
formulation with respect to dilution was checked by measuring transmittance
using UV spectrophotometer (UV-1700,Shimadzu). Drug loaded SNEDDS (1g) was
dissolved in 250 ml distilled water and % transmission was determined at 530 nm.
Similar procedure was used to determine % transmission at various pHs since the pH of
the GI environment is dynamic. Results are presented in Tables 4.9.2-4.9.3.

B. Measurement of globule size and zeta potential:

A measure of the self-nanoemulsifying ability of the oil and Smix combination and
stability is the globule size and zeta potential of the microemulsion. The formulations C1-
C5 were subjected to globule size measurement using Malvern zeta sizer Nano ZS (model
Nano ZS, Malvern Instruments, Worcestershire, UK). The instrument is able to measure
sizes between 10 to 5000 nm. Helium-neon gas laser having intensity of 4 mW was the
light source. The instrument is based on the principle of dynamic light scattering (DLS).
Zetasizer measures the potential ranged from −120 to 120V. The samples were measured
at 37ºC after addition of 1 g of mixture in 250 ml double distilled water.(Table 4.9.4 &
Figures 4.9.5-4.9.11)

C. Cloud point determination:

Cloud point is the temperature at which the solubility of the surfactant is minimal. This
phenomena exhibited by most non ionic surfactants is characteristic for that surfactant.
However the presence of other components such as a co surfactant and oils may produce
a change in the cloud point of the surfactant. A low cloud point, especially below 37°C
will jeopardize the self emulsifying properties of the SNEDDS mixture. The SNEDDS
were compared for cloud point value. Each formulation was diluted with water in the

Solubility enhancement of BCS Class II/IV drugs 105

Chapter 3 Experimental

ratio of 1:100 and placed in a water bath with gradual increase in temperature. The
temperature at which cloudiness was visible was recorded. (Table 4.9.5)

D. Determination of self emulsification time:

Since SNEDDS formulations undergo emulsification due to GI movements, a key

performance criterion for SNEDDS is the ease of self emulsification i.e. emulsification
time which should be as less as possible. The emulsification time of SNEDDS was
determined using USP type 2 apparatus. 500 mg of each formulation was added drop
wise to 500ml of purified water at 37±5ºC. Gentle agitation was provided by a standard
stainless steel dissolution paddle rotating at 50 rpm. Emulsification time was assessed
visually. (Table 4.9.6 & Figure 4.9.12)

E. Determination of viscosity:

Viscosity of the CP SNEDDS is another important physical property which governs the
ease of emulsification as well as the handling properties of the SNEDDS mixture for
large scale processing. Brookfield digital viscometer was used to measure the viscosity of
the blank formulations (Model: CAP 2000+, Brookfield Engineering Labs, US). Blank
SNEDDS (10 ml) was placed in the small sample adaptor; temperature was set at room
temperature and speed was set at 50, 100 and 150 rpm. Zero number cylindrical spindle
was immersed in the liquid for measurement of viscosity. The percent torque generated
and viscosities of samples were recorded. Results are given in Table 4.9.7.

F. Saturation solubility studies:

Excess CP was added to 25 ml of DW to which 1g of blank SNEDDS mixture was

previously added and the flask was kept in thermostatically (37°C) controlled orbital
flask shaker (CIS-24 Remi, India) for 48 h. Mixtures were filtered using Whatman filter
paper no.41. The filtrates (1 ml) were diluted with 10 ml of methanol and solutions were
analyzed using developed HPLC method. Similarly saturation solubility was determined
in 0.1N HCl, phosphate buffer pH 6.8, FaSSIF and FeSSIF.(Table 4.9.8-4.9.10)

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Chapter 3 Experimental

G. In vitro dissolution studies:

Liquid SNEDDS mixtures were filled into hard gelatin capsules but the drug release was
found to be negligible. Investigation of the capsules revealed a sticky mass adhering to
the gelatin shell which could be attributed to interaction between gelatin and the oil/Smix
present in the SNEDDS. Hence SNEDDS were then filled in HPMC capsule (No.00).
The dissolution test was performed in triplicate using dissolution test apparatus
(Electrolab TDT-08L USP standard). The dissolution test was carried out in 0.1N HCl as
dissolution medium. The test was performed using USP type I apparatus at 50 rpm and 37
± 0.5°C. Aliquots of 5 ml were periodically withdrawn and the volume was replaced with
fresh dissolution medium. The aliquots were analyzed spectrophotometrically at 263 nm.
Cumulative percentage of drug released at each time point was calculated. Model fitting
was done using PCP Disso software. Similar studies were also performed in DW and pH
6.8 buffer. (Table 4.9.11-4.9.12)

H. Ex vivo permeation study:

Permeation studies were performed using Franz diffusion cell and goat intestine was used
as the barrier membrane. The intestine was cleaned using appropriate methods, cut to
required size and fixed between the two compartments of the diffusion cell with the help
of clips. Phosphate buffer pH 6.8 was used as the diffusion media. The sample (7 ml of
liquid SNEDDS) equivalent to 120 mg of CP in 250 ml was placed in the donor
compartment. The volume of both compartments was 10 ml and the temperature was
maintained at 37°C. The assembly was placed on magnetic stirrer. Aliquots (1 ml) were
withdrawn at time intervals of 1 h upto 8 h and continued till 24 h. The flux was
calculated using Ficks first law eqn (3.12):

J= dM/[Link] Equation 3.12

Where J = flux, dM/dT =amount diffusing per unit time, S=cross sectional area of the
barrier
Permeability (P) was calculated by plotting a graph of M vs t based on the following eqn
(3.13):
M=PSCdt Equation 3.13

Solubility enhancement of BCS Class II/IV drugs 107

Chapter 3 Experimental

where M=amount of permeant in receiving sink t= time

Cd =concentration in donor compartment S= cross sectional area of barrier
The results are presented in Table 4.9.13 & Figure 4.9.13.

3.9.3. Preparation of solid SNEDDS and evaluation of micromeritic properties:

Liquid SNEDDS (C5) were converted into solid state by adsorption on various adsorbents
such as aerosil, nanosponges and magnesium trisilicate. The liquid SNEDDS (1g) was
taken in a glass mortar and small quantities of the adsorbents were added with continuous
trituration till a free flowing powder was obtained. The powders were subjected to
evaluation of micromeritic properties (angle of repose/bulk density/tap density).(Table
4.9.14)
Another novel approach that was tried involved preparation of pellets of the SNEDDS
mixture. The liquid SNEDDS (C5) was dissolved in mixture of isopropyl alcohol and
dichloromethane (8:2) and spray coated on Espheres (Microcrystalline Cellulose
nonpareil seeds 18-20#) using an R & D coater. 80 ml of dispersion was sprayed on 20 g
of Espheres at 1ml/min with atomizing air pressure of 2 lb/[Link] pan speed was 25
rpm and pellet bed temperature was 60-70°C. Talc was used as an adsorbent to facilitate
the drying process. The pellets were subjected to evaluation of % drug loading, surface
morphology and in vitro dissolution studies in various media using the same conditions
as described for liquid SNEDDS. Dissolution studies were also carried out in the
dissolution media specified in USP 30.

Dissolution media:

Glycine (54.5g) and 42.6 g of sodium chloride were dissolved in about 500 ml of water
in a 1000 ml volumetric flask. Hydrochloric acid (14.2 ml) was cautiously added, with
swirling and allowed to cool. The solution was diluted with water to volume, and mixed.
.From this stock solution 50 ml was transferred to a flask and diluted with water to 900
ml to obtain a solution having a pH of 3.0±0.1. (Figures 4.9.14-4.9.16 & Tables 4.9.15-
4.9.19)

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Chapter 3 Experimental

3.9.4. Stability studies:

The liquid SNEDDS and SNEDDS coated pellets were kept in screw-capped vials under
ambient conditions, at 40°C &75%RH, ambient conditions and under refrigeration (4°C)
for a period of 6 months with evaluation at 3 month intervals. The samples were visually
inspected for discoloration, separation, percent drug content, self emulsification time and
% transmittance. (Table 4.9.20)

3.9.5. In vivo studies:

i] Pharmacokinetic studies:
In vivo studies were carried out as per the guidelines of the Institutional Animal Ethical
Committee (SIOP/IAEC/2011/24). Adult Wistar rats (240-300 g) of either sex were used
for the study. The rats were weighed, labeled and divided into 03 groups’ viz. control
group which was administered plain vehicle, 2nd group which was administered CP
suspension prepared using 1% sodium carboxy methyl cellulose as suspending agent and
3rd group which was given CP-SNEDDS. They were fasted overnight and water was
provided ad-libitum. The preparations were (10 mg/kg bodyweight) administered to rats
using oral feeding tube. The animals were anesthetized with ether before collection of
blood which was collected from retro orbital plexus in EDTA treated tubes. The time
intervals for blood withdrawal were of 10 min for the first 30 min followed by 30 min
interval for the next 2h and hourly intervals up to 8 h. Plasma was separated by
centrifuging the blood at the speed of 3000 rpm for 10 min at -4°C. The plasma was
subjected to protein precipitation by addition of cold acetonitrile in a ratio of 1:4. It was
then centrifuged at 10, 000 rpm for 10 min at -4°C. The supernatant was decanted and 20
l was injected into HPLC column. Simultaneously calibration curve was plotted by
incubating known concentration of test drug into the rat plasma (spiking study). (Table
4.9.21 & Figures 4.9.17-4.9.18)

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Chapter 3 Experimental

Table 3.10. Protocol for animal testing for pharmacokinetic parameters of CP

SNEDDS

Group Group description Number of animals

number required

1 Normal control 6
(Untreated/vehicle)
2 Plain drug 6x3= 18
3 CP SNEDDS 6x3= 18
Total 42

ii] Microbiological studies:

The aim of the present study was to improve the oral bioavailability of CP.
Microbiological studies were carried out to determine the effect of the formulation
components (Smix and oil) on the ability of CP to penetrate the bacterial cell membrane.
The broth tube dilution method [Cappucino J; 2008] was used to determine the minimum
inhibitory concentration (MIC) for CP. The MIC is the lowest concentration of an
antimicrobial agent that inhibits the growth of the test microorganism. The procedure
involved specific concentrations of the formulations prepared by means of a twofold
dilution technique in an enriched nutrient broth (double strength) [IP’2007] .The pH was
adjusted 7.0 with 10% sodium hydroxide and media was autoclaved at 121° C for 15 min.
The tubes containing the antibiotic dilutions were then inoculated with standardized
concentrations (106 cfu/ml) of the following test organisms:
1] Staphylococcus aureus ATCC 25922 2] Escherichia coli ATCC 29213
3] Bacillus subtilis ATCC 6633

Procedure for preparing nutrient broth:

The ingredients listed in Table 3.11 were dissolved in DW, mixed thoroughly and
autoclaved at 121°C for 15 min.
Table 3.11. Formula for enriched nutrient broth
[Link] Ingredients Quantity
1. Peptone 4g
2. Meat extract/beef extract 0.6g
3. Sodium chloride 1g
4. Distilled water (qs) 100ml

Solubility enhancement of BCS Class II/IV drugs 110

Chapter 3 Experimental

Preparation of stock solution:

Drug/formulation (100mg) was dispersed in 100 ml of distilled water. From this stock
solution, 10 µl was diluted to 100 ml with water resulting in 1 µg/ml solution.

Procedure:
Nutrient broth was prepared as per the procedure described above. One tube was marked
as UT i.e. uninoculated tube as no microbial inoculum was added to it and it served as
negative control. Inoculums (106 cfu/ml) were added to all other test tubes. The test
solution was added as per Table 3.12 in the tubes except in tube marked UT to give
solutions of strengths 50 ng/ml to 500 ng/ml. For MIC studies for B subtilis and E coli,
test solutions of strength 250-2500 ng/ml were prepared. Positive control tube was used
to check the suitability of medium for growth of culture. Volume was adjusted to 10 ml
using sterile water and mixed well. The samples were incubated at 37°C for 48 h. The
absorbance of all the samples was measured at 600 nm with UT as blank. The test
formulation was compared with marketed preparation i.e. CEPODEMTM (Ranbaxy)
containing cefpodoxime 100 mg/5ml, Batch No.9021389; [Link]/2010;
[Link]/2012.

Dilutions for marketed: Marketed formulation (10mg/0.5 ml) was diluted to 10 ml (1

mg/ml). From this, 1 ml was diluted to 10 ml giving 0.1 mg/ml and 1 ml was further
diluted to 10 ml to get a final concentration of 0.01 mg/ml (100 ng/ml). From this, 0.5,
1.0, 1.5 ml upto 5 ml were taken for microbiological studies.

Table 3.12. Dilution series for microbiological test of CP SNEDDS

Tube No. Medium (ml) Volume of test solution(ml) Sterile water
UTa 5 0.0 5.0
CTb 5 0.0 5.0
1 5 0.5 4.5
2 5 1.0 4.0
3 5 1.5 3.5
4 5 2.0 3.0
5 5 2.5 2.5
6 5 3.0 2.0
7 5 3.5 1.5
8 5 4.0 1.0
9 5 4.5 0.5
10 5 5.0 00
a
Uninoculated tube, bControl tube

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Chapter 3 Experimental

Cultures:
Saline suspension of 24 h trypticose soy broth cultures adjusted to 0.05 optical density at
a wavelength of 600 nm for E. coli and S. aureus was used for the studies.

Table 3.13 Formula for tryptic soy broth [IP, 2007]

[Link] Ingredients Quantity
1. Tryptone (Pancreatic digest of casein) 1.5 g
2 Soytone (Papaic digest of soybean meal) 0.5g
3. Sodium chloride 0.5g
4. Distilled water (qs) 100 ml

The above ingredients were dispersed in 100 ml of distilled water and boiled till the
solids dissolved completely. The solution was autoclaved at 121°C for 15 min and cooled
to room temperature.
Tryptic soy broth is the medium that supports the growth of many fastidious organisms
such as Streptococci, Neisseria, Brucella, Corynebacterium, Listeria, Pasteurella, Vibrio,
Erysipelothrix, etc.(Results in Tables 4.9.22-4.9.24)

iii] Organ distribution studies:

For studying the organ distribution pattern of CP in SNEDDS, radiolabelling studies were
undertaken. CP was labeled with Technetium-99m (99mTc) by stannous reduction method
99m
[Babbar A;2000]. Tc was chosen for radiolabelling because of its short half-life (6 h)
and as it allows very little electron emission. It can be administered in millicurie amounts,
resulting in a very low radiation dose to the animal. Moreover, 99mTc is readily available
in a sterile, pyrogen-free and carrier state.

 Optimization of radiolabelling:

The effect of varying pH of the reaction mixture on labeling efficiency was studies. In
another experiment, the pH of the reaction mixture was kept constant at 6.5 and the
quantity of stannous chloride was varied from 100 to 1000 μg. The labeling efficiency of
CP was measured. (Tables 4.9.25-4.9.28)

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Chapter 3 Experimental

 Organ distribution of CP SNEDDS in rabbits:

Three New Zealand rabbits of either sex weighing 2.8 to 4.1 kg were used to evaluate
organ distribution of CP-SNEDDS. The rabbits were kept on a table without a restraining
box, its head supported by the experimenter’s hand. Whole Body Imaging Scan for organ
distribution of each formulation was taken after oral dosing of three millicurie
radiolabelled CP-SNEDDS using Siemens Symbia E Single Head system for one min
(Low Energy High Resolution Collimator).The percentage of radiolabelled formulation in
the organs viz. kidney, liver and heart was determined using SyngoDicom software.
Optimized radiolabelling conditions were used for drug 99mTc binding. Organ count of
Tc was taken after oral administration at 0 h and thereafter at 15 min interval for 3h.
(Table 4.9.29 & Appendix 5)
iv] Sub acute toxicity studies:

The protocol for this study was similar to that of TEL. The doses administered to the
animals were 10 mg/kg of body weight.(Table 4.9.30 & Figures 4.9.19-4.9.21)

3.9.6. Statistical studies:

The CP-SNEDDS comprising tween 80 and TPGS were subjected to statistical evaluation
to assess the effect of the Smix combination on a key parameter in self emulsification.
Previous studies had indicated that the oil did not play as significant a role in self
emulsification as the Smix combination. Hence 2-component mixture design was selected
as the statistical tool to evaluate the effect of the Smix components. The trials were
conducted as per the data generated by Design Expert software (version) which consisted
of different levels between the selected low and high values of the two factors. The
response selected was time for self emulsification. The high and low levels selected for
the two factors are as follows:

Table 3.14. Levels of two variables for 2 component mixture design for CP

Factor Levels
TPGS concentration 2 18
Tween 80 concentration 54 70
Low High

The experimental design generated by the software consisted of 13 trials as follows:

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Chapter 3 Experimental

Table [Link] with various levels of the variables for CP SNEDDS

Trial No. TPGS Tween 80

Concentration(%) Concentration(%)
1 2 70
2 2 70
3 18 54
4 18 54
5 10 62
6 6 66
7 2 70
8 2 70
9 18 54
10 10 62
11 14 58
12 10 62
13 18 54

The results for the above studies are presented in Chapter 4; Section 4.9.7.

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PART IV

SCALE UP AND REPRODUCIBILITY:

For any pharmaceutical product to be successful, the product must be capable of being
processed and packaged on a large scale. Scale-up studies facilitate the transition of a
product from laboratory scale apparatus and equipments to production scale equipments.
This is mandatory as the operating conditions are vastly different and hence need to be
analyzed for ensuring reproducibility. Scale up is generally defined as the process of
increasing batch size and to identify key operating variables.

For TEL, the scale up studies were carried out for T8 batch of the nanosuspension and for
CP, the studies were carried out for C5 batch of the liquid SNEDDS.

1] Scale up of TEL nanosuspension

For preparing the nanosuspension of TEL, a stainless steel jacketed vessel (Capacity: 1L)
equipped with an overhead stirrer was used. Lab-scale batches (100 ml) were prepared in
a glass beaker with an overhead stirrer and the product was stirred for 2h at 10000 rpm
after addition of the drug solution. For the scale-up, 1L of the nanosuspension was
prepared containing 10g each of TEL, PVPK 30 and TPGS. TEL was previously
dissolved in DCM (100ml). Prior to this, TPGS was mixed with DW (250 ml) and
warmed to about 40°C to facilitate its solubilization. PVPK 30 was dissolved separately
in 250 ml DW and the 2 solutions were mixed in the jacketed vessel with stirring at 4000
rpm. DW was added to make up volume to 1 L followed by the slow addition of the drug
solution using a spray gun commonly used for spraying coating solutions. The stirring
was continued at 4000 rpm followed by gradient increase to 10000 rpm. The dispersion
was withdrawn after 2h and observed visually for sedimentation or flocculation. The final
nanosuspension was evaluated in terms of particle size.(Table 4.9.33)

2] Scale up of CP-SNEDDS

The preparation of liquid SNEDDS involved simple mixing of the oil-Smix and
incorporation of the drug into the mixture. Hence the spray coating of the liquid
SNEDDS onto Espheres was subjected to scale up studies. The C5 batch of liquid

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Chapter 3 Experimental

SNEDDS was used for the studies. This batch comprised of 160 mg of CP dissolved in
820 mg of Smix viz. tween 80 and TPGS in a ratio of 1:0.5 and 180 mg of Capmul
MCM. For the scale up studies 10 g of the SNEDDS mixture was prepared which
contained 1.6g of CP. The liquid SNEDDS was dissolved in 1000 ml of solvent mixture
prepared using IPA and DCM in a ratio of 8:2 and sprayed onto 100g of Espheres (18-
20#) in an R & D coater which was provided with coating pans of different capacities.
The pan speed was 40 rpm, atomizing air pressure was 2lb/inch2 and pellet bed
temperature was 60-70°C. The Espheres were evaluated for % drug content and in vitro
dissolution studies. (Table 4.9.34)

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