Chapter 5
Methodology
CHAPTER-V
METHODOLOGY
5.1.
LIST OF INSTRUMENTS USED:
Table 5.1. List of Instruments used
S. No.
Instruments / Equipments
Model/Make
1
HPLC with PDA detector
Shimadzu LC-2010AHT/ SPD-M20 A, Japan
2
Analytical balance
3
pH meter
Model AL 204, Mettler and Toledo,
Switzerland
Mettler and Toledo, Switzerland
4
Magnetic stirrer
Daihan Labtech Co. Ltd., Korea
5
Ultra sonic bath
6
Wrist action shaker
7
Centrifuge
Barnstead International Aquawave Ultrasonic
cleaners, Fisher scientific, USA
IKA-Werke shaker, Model 501-Digital,
Germany
AccuSpin 400, Fisher scientific, USA
8
Millipore, USA
9
Millipore filtration unit with
vacuum pump
FT-IR spectrophotometer
10
Karl fisher titrator
Metrohm, Germany
11
UV-spectrophotometer
12
Refrigerator
Agilent 8453, Agilent Technologies, Inc.
Wayne, PA
Sanyo corporation, Japan
13
Hot air oven
Sanyo corporation, Japan
Shimadzu, Japan
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14
Stability chamber
Thermo Electron corporation, USA
15
DSC-60, Shimadzu, Japan
16
Differential scanning
calorimeter
Disintegration tester
17
Dissolution apparatus
Electrolab, India
18
Sieves
Endecotts Ltd., England
19
Moisture analyzer
Sartorius, Germany
20
Texture analyzer
21
Mixer
TA.XT2i, Texture Technologies Corp,
Scarsdale, NY
KevLab, India
22
Granulator
KevLab, India
23
Double-cone blender
HBD-100Z Single arm mixer, Canaan, China
24
ZP S Rotary tablet press
STC, Shangai, China
25
Tablet Hardness tester
26
Friabilator
Schleuniger hardness tester 6 D, Schleuniger
Pharmatron AG, Solothurn, Switzerland
Electrolab, India
Electrolab, India
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5.2.
Methodology
LIST OF CHEMICALS AND REAGENTS USED
Table 5.2. List of chemicals and Reagents
Materials
S. No.
Source
1
Pantoprazole sodium sesquihydrate
Dr. Reddy’s laboratories, India
2
Dr. Reddy’s laboratories, India
5
Pantoprazole sodium related
impurities A, B, C, D & E
Sodium dihydrogen ortho phosphate
(NaH2PO4)
Disodium hydrogen ortho phosphate
(Na2HPO4)
Orthophosphoric acid (85% w/w)
6
Acetonitrile
Fisher scientific, Germany
7
Panreac Quimica S.A.Barcelona, Spain
8
Potassium dihydrogen ortho
phosphate (KH2PO4)
Sodium acetate
9
Boric acid
Panreac Quimica S.A.Barcelona, Spain
10
Sodium starch glycolate
JRS Pharma, Patterson, NY
11
L-HPC LH21, Shin-Etsu, Japan
12
Low substituted hydroxypropyl
cellulose
Pregelatinized starch
13
Croscarmellose sodium
Ac-Di-Sol, FMC Biopolymers, Belgium
14
Crospovidone NF
Kollidon CL, BASF, UK
15
Hydroxypropyl cellulose
Klucel EXF, Ashland Aqualon, USA
16
Povidone K 30
Dongying Hua’an Chemical Co. Ltd.,
China
3
4
Panreac Quimica S.A.Barcelona, Spain
Panreac Quimica S.A.Barcelona, Spain
Mallinckrodt, Paris, KY
Panreac Quimica S.A.Barcelona, Spain
Starch 1500, Colorcon, West Point, PA
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17
Sodium carbonate
Panreac Quimica S.A.Barcelona, Spain
18
Panreac Quimica S.A.Barcelona, Spain
19
Magnesium oxide (MgO), heavy
powder
Magnesium hydroxide (MgOH2)
20
Trisodium phosphate
Panreac Quimica S.A.Barcelona, Spain
21
Sodium bicarbonate (NaHCO3)
Panreac Quimica S.A.Barcelona, Spain
22
Calcium carbonate (CaCO3)
Panreac Quimica S.A.Barcelona, Spain
23
Tromethamine (TRIS Buffer)
Panreac Quimica S.A.Barcelona, Spain
24
Mannitol
Mannitol-DC, Merck, UK
25
Sucrose
Merck, UK
26
Lactose monohydrate
27
Panreac Quimica S.A.Barcelona, Spain
Quest International, Sheffield Products,
Norwich, NY
Dibasic calcium phosphate anhydrous JRS Pharma, Patterson, NY
FMC corp., Philadelphia, PA
29
Microcrystalline cellulose (MCC PH
102)
Ludipress
30
Magnesium stearate
BASF, UK
31
Colloidal silicondioxide
BASF, UK
32
0.1 M HCl titrimietric solution
33
0.2 M NaOH titrimietric solution
34
Hydrochloric acid
Riedel-de Haën, Seelze-Hanover,
Germany
Riedel-de Haën, Seelze-Hanover,
Germany
Mallinckrodt, Paris, KY
35
Potassium bromide discs
28
BASF, UK
KBr, FT-IR grade, Sigma-Aldrich, Inc.,
Saint Louis, MO
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5.3.
Methodology
PURITY PROFILE OF ACTIVE DRUG PANTOPRAZOLE
SODIUM SESQUIHYDRATE
State
:
Solid
Identification :
IR spectrum matches with reference standard
Description
:
A White to almost white powder.
Solubility
:
Freely soluble in water and ethanol (96 per cent)
Water
:
6.7% w/w
Figure 5.1. IR Spectrum of Pantoprazole sodium sesquihydrate
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Table 5.3. IR Interpretation for Pantoprazole sodium sesquihydrate
S. No.
Wave Number cm-1
Functional group
1.
3483.56 cm-1
N-H
2.
3358.18 cm-1
O-H
3.
3176.87 cm-1
CH2
4.
2942.20 cm-1
CH3
5.
1589.20 cm-1
C-O
6.
1362.60 cm-1
C-F
7.
1042.50 cm-1
S=O
The IR spectrum of the drug Pantoprazole sodium sesquihydrate was
compared with the reference spectrum and was found to be similar. The functional
groups assigned in the wave numbers exhibited same wave length and had similar
intensities to that of the reference spectrum.
5.4.
HPLC METHOD DETAILS AND VALIDATION
Analytical methods development and validation play important roles in the
discovery, development, and manufacture of pharmaceuticals.
5.4.1. Stability indicating assay and related impurities method by HPLC
Preparation of reagents
Buffer Preparation: About 2.75 g of Sodium dihydrogen orthophosphate and 0.4258
g of Disodium hydrogen orthophosphate was weighed and transferred into a 1000 mL
volumetric flask. It was dissolved and diluted to 1000 mL with water and mix.
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Mobile phase: 700 volumes of Buffer and 300 volumes of Acetonitrile (70:30, v/v)
was measured separately into a dry glass bottle and mixed. The apparent pH was
adjusted to 6.0 with orthophosphoric acid (85% w/w). The contents were subjected to
sonication in a bath type sonicator for degassing the solution. The solution was
filtered by passing through 0.45 µm membrane filter under vacuum.
Diluent: A filtered and degassed mixture of Acetonitrile and Water (50:50, v/v) was
used.
Table 5.4. Chromatographic conditions:
1
Column
Symmetry C8, 150 x 3.9mm, 5 µm (Waters,
USA)
2
Flow rate
1.0 mL/min
3
Wavelength
290 nm
4
Injection volume
20 µl
5
Column Temperature
Ambient
6
Run time
12 min for assay & 25 min for related
impurities
Standard preparation:
About 45.70 mg of Pantoprazole sodium sesquihydrate reference/working
standard equivalent to 40 mg of Pantoprazole base was weighed and transferred
accurately into a 50 mL volumetric flask. About 30 mL of diluent was added and
sonicated to dissolve. The solution was cooled to room temperature and diluted to
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volume with diluent and mixed. Further 5 mL of this stock solution was transferred
by pipette into a 50 mL volumetric flask and made up to volume with diluent to
obtain a solution of 0.08 mg of Pantoprazole base per mL. The solution was filtered
through 0.45µm membrane filter and 20 µl was injected five times in replicate into
the HPLC system.
Sample preparation:
About 45.70 mg of Pantoprazole sodium sesquihydrate raw material equivalent
to 40 mg of Pantoprazole base was weighed and transferred accurately into a 50 mL
volumetric flask. About 30 mL of diluent was added and sonicated to dissolve. The
solution was cooled to room temperature and diluted to volume with diluent and
mixed. This solution contained 0.8 mg of Pantoprazole base per mL and was used as
test solution for related impurities. Further 5 mL of this stock solution was transferred
by pipette into a 50 mL volumetric flask and made up to volume with diluent to
obtain a solution of 0.08 mg of Pantoprazole base per mL. This solution was used as
test solution for assay.
Both the solutions were filtered through 0.45µm membrane filter and 20 µl was
injected in duplicate into the HPLC system.
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Figure 5.2. HPLC chromatogram of Pantoprazole sodium sesquihydrate
(top) and related impurities (bottom)
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5.4.2. Validation of Stability indicating method by HPLC
In order to confirm method suitability during routine quality control use, the
proposed method was checked critically for the following validation characteristics as
per ICH guidelines.
5.4.2.1. Specificity & Selectivity: Specificity and selectivity are established by
spiking with appropriate levels of impurities into Pantoprazole and demonstrating that
the determination is unaffected by the presence of Impurities.
Standard Pantoprazole impurities Stock solution:
Accurately 4.0 mg of each of, Impurity A, Impurity B, Impurity C, Impurity D
and Impurity E working standards were weighed into five different 20 mL volumetric
flasks. Dissolved and made up to volume with diluent and mixed.
Pantoprazole standard stock solution:
Accurately 91.4 mg of Pantoprazole sodium reference standard equivalent to
80 mg of Pantoprazole base was weighed into a 100 mL volumetric flask. 50 mL of
diluent was added and sonicated to dissolve, cooled to room temperature and made up
to volume with diluent and mixed.
Identification Solution:
1. Pantoprazole Impurity A:
2.5 mL of 'Pantoprazole Impurity A' stock was transferred by pipette into a
100 mL volumetric flask. Dissolved and made up to volume with diluent and mixed.
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2. Pantoprazole Impurity B:
2.5 mL of 'Pantoprazole Impurity B' stock was transferred by pipette into a
100 mL volumetric flask. Dissolved and made up to volume with diluent and mixed.
3. Pantoprazole Impurity C:
2.5 mL of 'Pantoprazole Impurity C' stock was transferred by pipette into a
100 mL volumetric flask. Dissolved and made up to volume with diluent and mixed.
4. Pantoprazole Impurity D:
2.5 mL of 'Pantoprazole Impurity D' stock was transferred by pipette into a
100 mL volumetric flask. Dissolved and made up to volume with diluent and mixed.
5. Pantoprazole Impurity E:
2.5 mL of 'Pantoprazole Impurity E' stock was transferred by pipette into a
100 mL volumetric flask. Dissolved and made up to volume with diluent and mixed.
6. Mixture of Pantoprazole standard and Impurities:
10 mL of Pantoprazole Standard stock solution and 2.5 mL of each of
Standard Pantoprazole impurities Stock solution was transferred by pipette into a 100
mL volumetric flask. Dissolved and made up to volume with diluent and mixed.
7. Placebo preparation:
Accurately weighed quantity of Placebo of Pantoprazole formulation was
transferred into a 50 mL volumetric flask. 30 mL of diluent was added, sonicated for
10 min and shaken for 15 min on a wrist action shaker. Then the contents were made
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up to volume with diluent and mixed. A portion was centrifuged at 4000 rpm for 8
minutes. The clear supernatant was filtered and injected into the HPLC system.
Procedure: 20 µl of mobile phase, diluent and solution 1, 2, 3, 4, 5, 6 and 7 was
injected. Method should be capable to separate the Pantoprazole impurities and
Pantoprazole from peaks due to mobile phase, diluent, placebo & from each other.
5.4.2.2. Linearity: Linearity for Pantoprazole was determined in the concentration
range from 0.064 to 0.096 mg/mL and linearity for Pantoprazole impurities A, B, C,
D and E were determined in the concentration range from 0.0010 to 0.0048 mg/mL.
The peak area responses were plotted against the corresponding concentrations and
the r2 values were calculated.
5.4.2.3. Precision
System precision: Six replicate injections of standard solution at the concentration of
0.08 mg/mL of Pantoprazole and 0.004 mg/mL of Pantoprazole impurities were
injected into HPLC system. The percentage relative standard deviations (% RSD)
were calculated.
Method precision: Six replicate samples of Pantoprazole formulation were analyzed
as per the method. The mean percentage of drug content as per label claim,
percentage of impurities and % RSD were calculated.
Intermediate precision or inter-day precision: The intermediate or inter-day
precision of the method was determined by six replicate analysis of Pantoprazole and
impurities from sample, as per the proposed method by different instruments
(Shimadzu LC2010AHT and Shimadzu SPD-10 A VP), by same analyst on different
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days. The average drug content, percentage of impurities and the % RSD were
calculated.
5.4.2.4. Accuracy (recovery studies): Recovery studies were performed by standard
addition method at three levels i.e. 80.0 %, 100.0 % and 120.0 % for assay and 50.0
%, 100.0 % and 150.0 % for related impurities. Known amounts of standard
Pantoprazole and impurities were added to placebo and they were subjected to
proposed HPLC method.
5.4.2.5. Stability of analytical solution: The standard and sample solutions were
prepared and kept at room temperature to evaluate the solution stability. The solutions
were injected into the system and analyzed as per the proposed method, initially and
at 6 h time intervals up to 24 h.
5.4.2.6. Robustness (system suitability): The robustness study was done by making
small changes in the optimized method parameters. System suitability parameters
were evaluated after making small deliberate variations.
5.5.
PREFORMULATION STUDIES: ANALYTICAL INVESTIGATION
OF PANTOPRAZOLE AND SYSTEMATIC
EXCIPIENT
SELECTION
5.5.1. Analytical Studies of Pantoprazole, an Acid-Labile Model Drug
Pharmaceutical characterization of drug and excipients plays a critical role in
understanding the properties of drug molecules and further development of a suitable
dosage form.
The aim of this section is to apply different analytical methods for
characterization of Pantoprazole.
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Two different methods were employed for both quantitative and qualitative
analysis in this section, a spectrometric method, using ultraviolet (UV) absorbance
spectroscopy, and a chromatographic method, using High Performance Liquid
Chromatography (HPLC). UV-spectroscopy is a useful and convenient technique in
analytical investigations of different pharmaceutical active ingredients based on their
absorptivity at a defined detection wavelength. This technique, however, poses some
limitations. For instance, the analyte should possess absorptivity within the UV
wavelength range, and depending on this value, the solutions of the drug molecules
might be diluted prior to UV detection in order to comply with Beer’s law. UVspectroscopy can be used for detecting two or more analytes simultaneously, using
specific technologies such as application of multiple-wavelength detection or
derivative spectroscopy. This, however, depends on the difference in absorption
characteristics of the analytes when tested at different wavelengths. In addition, in
some cases, UV spectroscopy may not be sufficient in the analysis of the active
ingredients due to certain peak overlapping that may interfere with accurate
determination of the analytes within the sample (Takumura and Machida, 2001).
HPLC analysis, on the other hand, has been extensively used since its
introduction in the mid century. HPLC has become a versatile and powerful analytical
technique in the arena of pharmaceutical sciences. HPLC analysis offers advantages
over UV spectroscopy in simultaneous determination of different analytes within a
mixed sample solution. However, prior to HPLC analysis, often appropriate sample
preparation steps are required for extraction of the active ingredient from the dosage
form in order to omit the background interference for detection of the analyte.
In this section, a UV-spectroscopy method and an HPLC method were
developed for determination of Pantoprazole and its possible degradation products in
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different solutions. Moreover, the applicability of UV-spectroscopy as a simple and
rapid method in analytical studies of Pantoprazole solutions in comparison with
HPLC analysis was investigated.
5.5.1.1. pH Stability analysis of Pantoprazole in Different Solutions using UVSpectroscopy
Materials and Methods
Pantoprazole sodium was purchased from Dr. Reddy’s lab, India. Upon
receipt, the bulk powder of Pantoprazole was preserved in a tight container and stored
in the refrigerator, in accordance with the USP-32 for further analysis. For this study,
several solutions were prepared, as the working media, at different pH values of 1.2,
3.2, 6.8, 8.5, 10.0, using respective USP buffer solutions of hydrochloric acid (pH=1
.2), acetate buffer (pH=3.2), phosphate buffer (pH=6.8), and alkaline borate buffer
(pH=8.5 and 10.0). A pH meter was used to adjust the pH of the solutions.
Bulk solutions of Pantoprazole, at the concentration of 0.08 mg/mL, were
prepared in each working medium in order to investigate the possible change in
physical appearance and variations in UV-absorbance of the solutions of Pantoprazole
overtime. Samples were stored at room temperature under regular light and at 2-5°C
in the refrigerator.
The samples were analyzed using a UV-spectrophotometer with 1-cm quartz
cells. The maximum UV-absorbance values and the corresponding wavelengths were
recorded per sample and compared with the samples stored over the period of 2
weeks.
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5.5.1.2. pH Stability analysis of Pantoprazole in Different Solutions using HPLC
Materials and Methods
Instrument
The HPLC system was model Shimadzu LC-2010AHT composing quaternary
pump, autosampler, mobile phase degasser, heated column thermostat, and variable
UV detector (Chromatographic and Spectrophotometric Division, Kyoto, Japan). The
mobile phase was eluted in an isocratic mode at a flow rate of 1.0 mL/min. The UV
detector was operated at a wavelength of 290 nm. Chromatographic separations were
performed at ambient temperature on a Symmetry C8 column (150 cm × 3.9 mm,
5μm) (waters, USA), and the injection volume was 20 μl. The data processing
software was LC solution 1.22 SP1 (Shimadzu, Kyoto, Japan).
The mobile phase contained 70 portions of buffer (2.75 g of Sodium
dihydrogen orthophosphate and 0.4258 g of Disodium hydrogen orthophosphate in
1000 mL water) and 30 portions of acetonitrile. The apparent pH of the solution was
adjusted to 6.0, using phosphoric acid. The mobile phase was then passed through a
0.45-µm membrane filter. The prepared mobile phase was degassed using a sonicator
bath. A pH meter was used to adjust the pH of the solutions.
Sample preparation
Pantoprazole sample solutions, at the concentration of 0.08 mg/mL, were
prepared by dissolving Pantoprazole powder, accurately weighed, in different USP
buffer solutions in a volumetric flask. The buffer solutions used in this study were
hydrochloric acid buffer (pH=l.2), acetate buffer (pH=3.2), phosphate buffer
(pH=6.8), and alkaline borate buffer (pH= 8.5 & 10.0). The Pantoprazole solution
prepared in diluent was regarded as standard.
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The HPLC analysis was performed on both fresh and aged solutions of
Pantoprazole. The samples of the latter were stored at ambient conditions for 2
weeks. Prior to HPLC analysis, all solutions were passed through a 0.45-µm
membrane filter. The injection volume was 20 μl and the retention time of
Pantoprazole was recorded at about 7.8 minutes.
5.5.2. Excipient Selection for Subsequent Formulation Development of
Pantoprazole
As cited earlier, Pantoprazole molecules are extremely acid-labile and are also
susceptible to heat, moisture, and, to some extent, to light and organic solvents. Thus,
in order to design a stable solid dosage form of Pantoprazole, different factors should
be taken into consideration, including selection of suitable excipients, manufacturing
techniques, and process variables.
In general, due to their different functionalities, excipients are considered an
integral part of the formulation and should be selected cautiously. The performance of
the final dosage form depends upon the selected excipients, the potential
incompatibility and interrelationship between various excipients within the
formulation and the impact that the individual excipients or their combination may
have on the active drug (Rowe et al., 2003). The International Pharmaceutical
Excipients Council (IPEC) has defined excipients as “substances, other than the
active drug substance or finished dosage form, which have been appropriately
evaluated for safety and are included in a drug delivery system to either aid the
processing of the drug delivery system during its manufacture, protect, support,
enhance stability, bioavailability, or patient acceptability, assist in product
identification, or enhance any other attributes of the overall safety and effectiveness
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of the drug delivery system during storage or use” (Robertson, 1999). Therefore, in
order to design and develop a stable and effective drug product, it is essential to know
the properties of the active ingredient alone and in combination with all other
ingredients based on the requirements of the dosage form and the applied processes
(Gohel and Jogani, 2005).
If the drug molecules and the selected excipients are not compatible, the
stability and bioavailability are altered which may further affect the safety and
efficacy of the drug. Therefore, the study of drug-excipient interaction is an important
prerequisite to the development of stable dosage forms. In spite of the significance of
drug-excipient compatibility testing, there is no universally accepted protocol to
achieve this purpose (Verma and Garg, 2005). Several methods have been suggested
and used in the literature including, but not limited to, thermal analysis, infrared
spectroscopy, X-Ray Diffractometry (XRD), Nuclear Magnetic Resonance (NMR),
Scanning
Electron
Microscopy
(SEM),
and
High
Performance
Liquid
Chromatography (HPLC), as well as observation of any change in the appearance of
mixtures of drug-excipient (Arias et al., 2000; Mendes and De Sousa, 2000; Sarisuta
and Kumpugdee, 2000; Bruni et al., 2002; Araujo et al., 2003.).
In the case of Pantoprazole, the selected excipients should be compatible with
Pantoprazole and improve its stability within the formulation. In order to achieve this,
several excipients were examined as per their physical and chemical properties and
for possible interactions with Pantoprazole molecule. Different techniques were
employed for this purpose, including:
1. Visual observation
2. Infrared spectroscopy
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3. Thermal analysis (Differential Scanning Calorimetry)
4. HPLC analysis
5.5.2.1. Stress testing by Visual Observation
Materials and Methods
Among the excipients to be considered in formulation of Pantoprazole solid
dosage forms is Mannitol DC, due to its known multi-functional characteristics and
its widespread use in tablet and capsule formulations (Rowe et al, 2003). Among
different commercial grades of Mannitol, Mannitol DC was initially selected due to
its low moisture content which is specifically indicated for moisture-sensitive drugs.
As cited earlier, the powder particles of Pantoprazole are very fine. The
powder is of a fluffy and cohesive nature and the particles easily form aggregates.
Thus, handling of Pantoprazole powder poses difficulties and may specially cause
problems when scale-up procedures are concerned. Different manufacturing methods,
such as wet granulation or direct compression, can generally be employed to aid
powder handling and improve flowability. Owing to the inherent susceptibilities of
Pantoprazole molecule to water and various organic solvents and for the ease of
manufacturing process, the application of dry blending and direct compression
method were primarily investigated in the design of Pantoprazole solid dosage form.
Hence, to investigate the solid-state stability of Pantoprazole, a binary
physical mixture of Pantoprazole and Mannitol DC was prepared via dry blending in
a mortar and pestle, with the weight ratio of 1:2 (Pantoprazole: Mannitol DC) which
was considered as the potential ratio to be used in Pantoprazole formulation. The
powder blend was then poured in uncapped glass vials and placed in four different
storage conditions, as follows, for further observation and analysis:
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1. Ambient condition
2. Oven at 50°C (dry heat)
3. Stress condition of 40°C / 75% Relative Humidity (RH) (moist heat)
4. Refrigerator at 2°C
In addition, individual samples of plain Pantoprazole powder and pure powder
of Mannitol DC were placed at each storage condition as ‘control vials’.
In order to select the suitable excipients for further inclusion in Pantoprazole
formulations, the same procedure was repeated for the following materials. In all
instances, Pantoprazole was blended with the excipient in the weight ratio of 1:1
(Pantoprazole: selected excipient), unless otherwise noted. In addition, ‘control vial’
of each excipient was also examined under the same testing conditions.
Disintegrating agents:
— Sodium starch glycolate
— Low substituted hydroxypropyl cellulose
— Pregelatinized starch
— Croscarmellose sodium
— Crospovidone NF
Binders:
— Klucel EXF
— Povidone K 30
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Alkalizing agents to be used as a pH stabilizer or buffering agents (blended with
drug in the weight ratio of 1:2):
— Sodium carbonate
— Magnesium oxide (MgO), heavy powder
— Magnesium hydroxide (MgOH2)
— Trisodium phosphate
—Sodium bicarbonate (NaHCO3)
—Calcium carbonate (CaCO3)
—Tromethamine (TRIS Buffer)
Diluents (blended with drug in the weight ratio of 1:2):
— Mannitol (DC-Mannitol)
— Lactose monohydrate
— Dibasic calcium phosphate anhydrous
— Microcrystalline cellulose (MCC PH 102)
— Ludipress
Lubricants and glidants (blended with drug in the weight ratio of 5:1):
— Magnesium stearate
— Colloidal silicondioxide
Prior to preparation of physical mixtures, the respective excipients were
passed through a 40-mesh sieve.
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Since degradation of Pantoprazole molecule takes place with distinct
discoloration, all samples were examined at different time points for possible signs of
degradation over the period of 4 weeks. For this purpose, all vials were placed
uncapped in order to expedite the probable degradation of Pantoprazole within the
powder blends. At each predetermined time point, the physical mixtures were
removed from their respective storage chambers and visually examined against a
white background in normal daylight. Any signs of discoloration or other physical
change were noted.
Depending on the nature of the excipients, their respective physicochemical
properties, and their probable reaction with Pantoprazole molecule, various degrees of
discoloration was observed at different time points. For instance, at the storage
condition of 40°C/ 75% RH, the blend of Pantoprazole and MCC PH 102 (1:2) which
was originally off-white, exhibited a distinct brown discoloration within 24 hours
which gradually turned dark brown within a week. This rapid discoloration appears to
be due to the degradation of Pantoprazole in contact with MCC PH 102.
5.5.2.2. Stress testing by Infrared Spectroscopy
IR-spectroscopy is commonly used in order to detect the possible drugexcipient interactions.
The infrared (1R) region of the electromagnetic spectrum is from the red end
of the visible range to the beginning of the microwave region. The region of the
infrared spectrum which is of greatest interest in organic chemistry is the wavelength
range of 2.5 - 15 µm (corresponding to 4000 to 600 cm-1). In practice, the use of wave
number (cm-1) rather than wavelength, is more prevalent, which is proportional to
frequency. Infrared spectroscopy involves examinations of twisting, stretching,
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bending, rotating, and vibrational motions of atoms in a molecule (Talukder, 2004).
When photons of the infrared spectrum are passed through a sample, certain
frequencies are absorbed, while the rest are transmitted. The absorbed photons lead to
molecular vibrational frequencies and an absorption spectrum for the material. For
instance, carboxylic acid absorbs at 2500-3200 cm-1 C-N amines at 1180-1360 cm-1,
and aromatic rings at 3000-3100 cm-1. Therefore depending on the molecules, various
peaks may be observed in the IR spectra of the samples.
Fourier Transform (FT) is a linear transformation which is widely used in
many fields of science as a mathematical or physical tool to alter a problem into one
that can be more easily solved. FT, in essence, decomposes or separates a waveform
or function into sinusoids of a different frequency which sum to the original
waveform and identifies or distinguishes different frequency sinusoids and their
respective amplitudes. In combination with infrared spectroscopy, FT is utilized to
enhance absorptions of real peaks of interest (Borenstein, 2001).
The aim of this section is to investigate the possible interactions and
incompatibilities between Pantoprazole molecule and selected excipients, using IRspectroscopy.
Materials and Methods
Based on the results obtained from the previous section 5.5.2.1 in stress
testing by visual observation, the set of excipients that did not show obvious
discoloration such as Crospovidone NF, L-hydroxy propyl cellulose, Mannitol DC,
Dicalcium phosphate, Lactose, Colloidal silicon dioxide and Magnesium Stearate
were selected for further analysis via IR-spectroscopy. Similar to the previous
methodology, physical mixtures of Pantoprazole and the individual excipients were
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prepared in the weight ratio of 1:2 via dry blending. Prior to preparation of physical
mixtures, the respective excipients were passed through a 40-mesh sieve. Each
powder blend was placed in uncapped glass vials and further stored at the harsh
condition of 40°C/75% RH for a period of 2 weeks. The plain powder of refrigerated
Pantoprazole was also stored under identical conditions and further evaluated.
After 2 weeks, the physical mixtures were subjected to IR analysis. For further
comparison, the IR spectra of each powder blend were compared against their
respective freshly prepared blends in the same ratio (t=0 days). The IR spectra
pertaining to the plain excipients were also recorded. The obtained spectra were
evaluated for any drug excipient interaction.
Sample Preparation for IR Analysis
The powder samples of the study were prepared using potassium bromide
discs. KBr was dried and further kept in a desiccator to remove moisture. In order to
prepare the samples for IR analysis, KBr was initially ground using a mortar and
pestle to achieve fine particles. The powder samples of the study were then
individually dispersed in the ground KBr at 2% w/w and blended further. The mixture
was compressed to a translucent disk and subjected to IR analysis.
Instrument
The IR spectra of the powder samples were recorded using a Shimadzu 8700
Series FT-IR spectrophotometer. IR analysis was performed over the range of 4000500 cm-1. For each sample, 20 scans were taken and averaged with the resolution of
4.0 cm-1. Prior to the analysis the system was calibrated using the standard
polystyrene film.
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5.5.2.3. Differential Scanning Calorimetry (DSC)
Thermal analysis has been used for rapid evaluation of physicochemical
interactions between different ingredients of the formulation and selection of
compatible excipients. Differential Scanning Calorimetry (DSC) is a thermo
analytical tool in which the difference in the amount of heat required to increase the
temperature of a sample and reference is measured as a function of increasing
temperature. Thus, the energy associated with various thermal events such as melting,
glass transition temperature and crystallization can be evaluated (Araujo et al., 2003).
In general, the incompatibilities between the drug and excipients can be
identified from the respective DSC thermograms through appearance, shift, or
disappearance of endotherms or exotherms and/or variation in the pertinent enthalpy
values. However, interpretation of the thermograms may be difficult at times and the
conclusions based on the DSC result alone are sometimes misleading (Verma and
Garg, 2005).
Materials and Methods
For the purpose of DSC analysis of the powder blends, the same set of
excipients, as determined by visual observation, was selected. The physical mixtures
of Pantoprazole and the individual excipients were prepared following the same
procedures, as explained in section 5.5.2.2. A sample of plain Pantoprazole powder
stored at 40°C/75% RH for a period of 2 weeks was also taken for further evaluation.
The physical mixtures were subjected to DSC analysis. The obtained thermograms
were evaluated for any signs of drug-excipient interaction.
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Instrument
Thermal analysis of the powder samples was carried out using a differential
scanning calorimeter. The powder samples were analyzed in the DSC aluminum pans.
The typical sample size was 2-8 mg. The thermal behaviour of the samples was
investigated at a heating rate of 10°C /min. The samples were scanned over the
temperature range of 40-300°C, under nitrogen gas purge.
5.5.2.4. Stress testing by HPLC Analysis
The stability of Pantoprazole in combination with various excipients was
investigated using HPLC analysis. Since this could be a powerful tool in detecting
potential drug excipient compatibilities, the same set of excipients, as mentioned
earlier, were selected for further analysis. The focus of this study was on selection of
the most suitable excipients.
Materials and Methods
The physical mixtures of Pantoprazole and the individual excipients were
prepared, as given in Table 5.5, in amber colored glass vial (n = 2) and mixed on a
vortex mixer for 2 min. Each vial was sealed using a teflon lined screw cap and stored
at 50°C and 40°C/75% RH for 4 weeks. These samples were periodically examined
for any unusual color change. After 4 weeks of storage at the above conditions,
samples were quantitatively analyzed using HPLC.
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Table 5.5. Drug – Excipient ratio for incompatibility studies by HPLC
S.No.
Drug/Excipient
Ratio
1.
2.
3.
4.
Pantoprazole/Crospovidone NF
Pantoprazole/Sodium starch glycolate
Pantoprazole/ Starch 1500
Pantoprazole/ Croscarmellose sodium
1:1
1:1
1:1
1:1
5.
6.
7.
8.
9.
10.
11.
12.
13.
Pantoprazole/ L-HPC
Pantoprazole/Magnesium oxide
Pantoprazole/Sodium bicarbonate
Pantoprazole/ Magnesium hydroxide
Pantoprazole/ Tris buffer
Pantoprazole/ Trisodium phosphate
Pantoprazole/ Sodium carbonate
Pantoprazole/ MCC PH102
Pantoprazole/ Ludipress
1:1
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2
14.
15.
16
17.
18.
19.
20.
Pantoprazole/ Lactose
Pantoprazole/ Mannitol
Pantoprazole/ Dicalcium phosphate
Pantoprazole/ Klucel EXF
Pantoprazole/ Povidone K30
Pantoprazole/ Magnesium stearate
Pantoprazole/ Colloidal silicon dioxide
1:2
1:2
1:2
1:1
1:1
5:1
5:1
For sample preparation, 2 mL of diluent was added into each vial. The
mixture was vortexed and transferred to a suitable volumetric flask. Vials were rinsed
twice with diluent and the volume made up. The samples were centrifuged and the
supernatant was filtered through 0.45-μm nylon membrane filters. After appropriate
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dilutions, samples were analyzed using HPLC and drug content was determined by
the comparison of standard and sample area.
For the analysis of drug-excipient mixtures, Shimadzu HPLC system equipped
with LC-2O AT pump, DGU-20 AS on-line degasser, SIL-20A auto injector, CTO2O A column oven, and SPDM20A, photo diode array detector was used for peak
purity testing. Shimadzu LC-solution software (Version 1.22 SP1) was used for data
acquisition
and
mathematical
calculations.
Chromatographic
separation
of
Pantoprazole was performed on a C8 Symmetry column (3.9 mm x 150 mm; 5 μm
particle size; Waters, USA). Mobile phase used was acetonitrile, phosphate buffer
[2.75 g of sodium dihydrogen orthophosphate (NaH2PO4) and 0.4258 g of disodium
hydrogen orthophosphate (Na2HPO4)], in the ratio of 30:70, v/v, pH 6.0 with
orthophosphoric acid, at a flow rate of 1 mL/min. Temperature of the column oven
was maintained at ambient. Diluent used was acetonitrile and water in the ratio of
50:50, v/v for preparation of all the samples. Standard solutions and drug-excipient
samples (20 μl) were injected and analyzed at 290 nm using a UV detector. For peak
purity testing, PDA detector in the range of 200-800 nm was used.
5.5.3. Selection of Buffers for Subsequent Formulation Development
As cited earlier, prazoles are acid labile drugs and are prone to degradation in
the acidic medium of the stomach unless protected by an enteric coating or in the
presence of alkalising agents. Buffering capacity is the ability of the buffer to resist
changes in pH. It increases as the molar concentration of the buffer salt/acid solution
increases. Buffering capacity of different alkalising agents varies and depends on the
molecular nature and pKa of that particular compound.
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5.5.3.1. Determination of neutralization capacity of different buffers
A number of buffers, including Tri sodium phosphate, Calcium carbonate,
Sodium bicarbonate, Tromethamine (TRIS buffer), Heavy Magnesium oxide and
Magnesium hydroxide were tested so as to select a buffer of good acid neutralizing
capacity. 25 mL of 0.1 M HCl titrimietric solution was transferred to a 250 mL
beaker with the help of a pipette. The quantity of buffer equivalent to about 0.10 g
was added into the beaker and mixed on magnetic stirrer for two minutes (200 rpm).
After the addition of buffer, the mixing procedure was continued (200 rpm),
accurately timed, for 10 minutes. Excess hydrochloric acid was titrated with 0.2 M
NaOH titrimietric solution to attain pH 7.0 stable for 15 seconds. The obtained result
is expressed in mEq of acid neutralised/per g of buffer.
5.5.3.2. Selection of buffers based on acid neutralizing capacity
Based on their acid neutralizing capacity, buffers, both individual and
combination, were evaluated for their behaviour at the excess secretion of acid. The
technique involved consists of adding an excess quantity of the buffer to a sample of
artificial gastric juice. The basal stomach fluid contains 9.6 mL of 0.1 N HCl and
releases 0.5 mL of 0.1N HCl per minute (Lentner C, 1999). The model was simulated
by adding a known quantity of buffer into a 400 mL beaker containing 9.6 mL of
0.1N HCl + 210 mL of water (basal stomach fluid) and titrated with excess acid (0.1N
HCl) at the rate of 0.5 mL per minute for a period of 1 hour. A pH meter was attached
to the assembly to continuously monitor the change in pH with time. The buffer(s)
which gave an immediate rise in pH and maintained a pH above 6.0 at the excess
secretion of acid was selected.
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5.5.4. Disintegration Studies
The selected combination of buffers, such as Magnesium oxide heavy and
Trisodium phosphate (MgO and TSP), Magnesium hydroxide and Trisodium
phosphate (MgOH2 and TSP), Sodium bicarbonate and Magnesium oxide heavy
(NaHCO3 and MgO), Tromethamine and Magnesium hydroxide (Tris and MgOH2)
were compressed into tablets and the disintegration of these buffers as such was
tested in 0.1 N HCl. All the buffers were sifted through suitable mesh and mixed
together in a suitable mixer. The resultant powder mix was mixed with magnesium
stearate. The final blend is compressed into tablets using rotary press fitted with 11.0
mm punches. The tablets were subjected for disintegration studies.
Various disintegrants such as the croscarmellose sodium, sodium starch
glycolate, crospovidone were added to the buffer blend to evaluate the disintegration
of buffering agents. Alternatively, soluble sugars such as the mannitol and sucrose
were also tried to enhance the release of buffering agents in acid medium.
5.5.5. Dissolution studies
5.5.5.1. Dissolution studies of API with and without buffer
The buffer combination which gave an optimum pH rise within few minutes
and maintained a pH above 6.0 was selected. The release profile of the API
Pantoprazole sodium sesquihydrate with selected buffers Magnesium oxide heavy
and Trisodium phosphate (MgO and TSP) was tested against API without buffers.
The API without buffers when added to the dissolution vessel containing simulated
gastric fluid, the active ingredient degraded immediately. When the API and required
amount of buffer was mixed and added simultaneously to the dissolution vessel
containing SGF, the color of the dissolution medium turned slightly yellow indicating
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a partial degradation of the active ingredient. In-vitro drug release was determined
using the USP Type II apparatus (250 mL SGF pH 1.7; 37°C; 50 rpm; n=6). At
predetermined time intervals, 5 mL samples were withdrawn (not replaced), filtered
and assayed for drug content and related impurities. The amount of Pantoprazole
released was measured by a stability indicating method as mentioned in section 5.4.1.,
with a computer connected Shimadzu-HPLC system. Pantoprazole solutions of
known concentration were used to calculate the amount of drug released.
Pantoprazole was stable in the dissolution medium at 37oC for at least 12 h as
indicated with the stability indicating HPLC method.
5.5.5.2. Concept of creating a Macroenvironment pH
The partial degradation of API and buffer mixture in the SGF medium, as
cited in the above experiment, evolved into a concept of creating a macroenvironment
pH. To achieve this, the required amount of buffers was added initially to the SGF
medium and neutralized for 2 minutes followed by the addition of accurately weighed
amount of API into the safe high pH environment which did not result in any color
change of the solution. The amount of drug dissolved and analysis by HPLC was
performed as mentioned in section 5.5.5.1.
5.5.5.3. Dissolution studies of buffers in Simulated Gastric Fluid (pH profile of
buffers)
Dissolution of the buffers in SGF was evaluated to understand the release
behaviour of buffers to attain the required pH with respect to time. For this purpose,
the selected combination of buffers, such as Magnesium oxide heavy and Trisodium
phosphate (MgO and TSP), Magnesium hydroxide and Trisodium phosphate (MgOH2
and TSP), Sodium bicarbonate and Magnesium oxide heavy (NaHCO3 and MgO),
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Tromethamine and Magnesium hydroxide (Tris and MgOH2), and mannitol were
sifted through suitable mesh and mixed together in a suitable mixer. The resultant
powder mix was lubricated with magnesium stearate. The final blend is compacted
and sized through 20 mesh sieve. The resultant buffer granules were filled in capsules
or compressed into tablets and the dissolution of these buffers was tested in simulated
gastric fluid. The capsules/tablets were subjected for dissolution using a USP Type-I
dissolution apparatus. The dissolution media consisted of 250 mL of SGF (40 mL of
0.1 N HCl+ 210 mL of purified water; pH 1.70). The baskets were operated at 100
rpm and the bath temperature was maintained at 37± 0.5°C using a temperature
controller. A pH meter was attached to one of the dissolution vessel to continuously
monitor the change in pH with time and evaluated for 30 minutes.
5.5.6. Evaluation and Comparison of Physicomechanical characteristics of
gelatin and hypromellose capsules
5.5.6.1. Introduction
The term ‘capsule’ derives from the Latin word ‘capsula’, meaning a small
box. The use of capsules as a means of drug delivery was originated in the first half of
the nineteenth century. A French pharmacy student, F. A. B. Mothes devised a onepiece soft gelatin capsule and in 1834 filed for a patent in Paris. Following on from
this, the hard two-piece capsules were invented by J. C. Lehuby, a Parisian
pharmacist who was granted a patent in 1846 (Podczeck and Jones, 2004). These
capsules were manufactured using a decoction of tapioca or starch. Three additions to
Lehuby’s original patent were granted within a few years, extending the range of the
raw materials in capsule preparation to carrageen, gums and gelatins.
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Pharmaceutical capsules consist of a container filled with the active ingredient
and, if needed, the appropriate excipients. Capsules are usually made of gelatin and
are manufactured in different forms which can be divided into two categories of ‘soft
gelatin capsules’ and ‘hard gelatin capsules’. While soft capsules are made as a onepiece container, the hard capsules consist of two separate parts, the cap and the body.
The hard capsules have been widely used for over a century due to their various
advantages, including ease of ingestion, masking unpleasant taste and odor, aesthetic
properties, and versatility to accommodate incompressible or compression sensitive
drugs (Podczeck and Jones, 2004).
Gelatin has a widespread use in pharmacy and has been used as a material of
choice for hard capsules, mainly due to its relative ease of manufacturing. A solution
of gelatin is able to form a gel just above ambient temperature conditions and further
results in a rapid formation of a homogeneous film which is an essential factor in the
manufacturing process of capsules. However, gelatin presents certain problems and
disadvantages. Gelatin is obtained through denaturation of collagen (Podczeck and
Jones, 2004). As an amphoteric substance, gelatin reacts with both acids and bases.
Moreover, as a protein, gelatin exhibits chemical properties which are characteristic
of such materials, for instance, gelatin may easily be hydrolyzed by most proteolytic
systems to yield amino acid components. In addition, gelatin reacts with aldehydes
and aldehydic sugars, anionic and cationic polymers, metal ions, electrolytes,
plasticizers, and preservatives (Rowe et al, 2003). Moreover, the properties of gelatin
change when subjected to Gamma-radiation (Fassihi and Parker, 1988). Upon
exposure to severe storing conditions (40°C/ 75% RH) for about 6 months, gelatin
capsules undergo a cross-linking reaction which further reduces the solubility of the
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capsule shells and the dissolution rate of the active drug within them (Digenis et al.,
1994; Podczeck and Jones, 2004).
Gelatin capsule shells generally have a moisture content of about 13-15%
w/w, however, depending on the atmospheric conditions to which the capsules have
been exposed, the moisture content may vary. Such high water content makes gelatin
capsules unsuitable for moisture-sensitive drugs (Podczeck and Jones, 2004). On the
other hand, the presence of a strongly hygroscopic ingredient within the gelatin shells
may lead to the loss of moisture content of the shells which may cause the capsule
shells to lose their mechanical strength and become more brittle. Furthermore, gelatin
is derived from animal sources, mainly bovine or porcine, the former of which poses
the risk of transmitting mad cow disease or BSE (Bovine Spongiform
Encephalopathy) (U.S. Department of Health and Human Services, 1997). Gelatin
products from animal sources are sometimes avoided due to the religious or
vegetarian dietary restrictions. Therefore, the use of animal gelatin especially in the
recent past has been associated with a number of technical, regulatory, commercial
and consumer concerns (Bowtle, 2002).
Thus, several new materials have been examined as possible substitutes for
gelatin in manufacturing hard capsules, among which hypromellose, formerly
regarded as hydroxypropyl methylcellulose (HPMC), has gained popularity and is
commercially available worldwide from various capsule shell manufacturers,
including Shionogi Qualicaps Co., Ltd. (Quali-V® Capsules), Capsugel Division of
Pfizer Inc. (Vcaps® Capsules), and Natural Capsules Ltd. (Cellulose Capsules),
among others.
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Hypromellose is a tasteless, odorless and inert hydrophilic polymer with no
ionic charge. It belongs to a group of compounds commonly known as cellulose
ethers which is derived from non-animal sources. It contains varying ratios of
hydroxypropyl and methyl substitution, a factor which affects its solubility and
thermal gelation temperature. Various hypromellose grades are suitable for forming
hard capsule shells and are included in the pharmacopoeias in the U.S., Europe and
Japan (Bowtle, 2002).
As a versatile material, hypromellose is widely used in different
pharmaceutical applications, based on the physicochemical characteristics of its
respective grades. In oral products, hypromellose has been employed as a tablet
binder in either wet or dry granulation processes. It also possesses film coating
properties and has been utilized in various coating applications either from organic or
aqueous compositions. Hypromellose can also be used in the fabrication of
hydrophilic matrix systems in order to prolong the drug release from tablets or
capsules (Pillay and Fassihi, 2001; Rowe et al., 2003; Li et al, 2005). Furthermore, in
topical formulations, hypromellose is used as a suspending and thickening agent,
stabilizing agent and an emulsifier. Hypromellose has been also used as an adhesive
in plastic bandages and as a wetting/viscous agent in ophthalmic and tear replacement
solutions. It also offers a variety of applications in cosmetics and food products
(Rowe et al., 2003).
As a raw material for capsule shells, hypromellose is chemically stable and
compatible with most active drugs and a variety of solid, semi-solid and liquid
excipients. The only known incompatibility for hypromellose is the interaction with
some oxidizing agents (Rowe et al., 2003). The inherent nature of hypromellose
diminishes the potential of cross-linking which further provides a more consistent
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release of the active ingredient in vitro and in vivo. Under identical storing
conditions, the moisture content of hypromellose shells, 2-5%, is much lower than
gelatin capsules, 13-15%, which makes them more suitable for water-sensitive drugs
(Nagata, 2002; Quali-V® HPMC capsules, Technical Manual, 2004). Moreover,
hypromellose shells maintain their mechanical integrity and remain elastic even under
very low moisture conditions (Ogura et al., 1998).
Table 5.6. Comparison of physical characteristics of gelatin and hypromellose
capsule shells (Ogura et al., 1998)
Properties
Gelatin
Hypromellose
Moisture content (% w/w)
13-15%
2-5%
Water vapour permeability
Low
Low
Substrate for Protease
Yes
No
Maillard reaction with drug fill
Yes
No
Deformation by heat
>60°C
>80°C
Aqueous dissolution at room temperature
Insoluble
Soluble
Static charge
High
Low
Light degradation
Possible
No
In fabrication of solid oral dosage forms, it may be necessary for capsules,
similar to tablets, to be coated either to improve the aesthetic properties of the capsule
shells and/or to impart desirable functionality to the encapsulated dosage form. Cole
et al. (2002) have demonstrated that in contrast to the smooth and lustrous surface of
gelatin shells, hypromellose capsules possesses a rough and matte surface, which
further provides for desirable adhesion between the capsule shell and the film-coat
layer. Such property eliminates the need for application of a sub-coat layer which is
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often required with gelatin shells especially when functional coating is to be applied.
Additionally, since hypromellose is derived from non-animal sources, it eliminates
the issues pertaining to regulatory, religious and vegetarian dietary restrictions.
In terms of biopharmaceutical properties of gelatin and hypromellose
capsules, studies have been conducted to evaluate whether these capsules can be
considered interchangeable. The overall conclusion was that hypromellose could be
regarded as a noteworthy alternative to gelatin (Ogura et al., 1998; Honkanen, 2004).
In regard with manufacturing the capsule shells, gelatin, itself, is a gel
promoting material, whereas gelling aids should be added to hypromellose to ensure
consistent capsule formation. For instance, hypromellose capsules (Quali-V®- consist
of hypromellose as the base, and a small quantity of carrageenan and potassium
chloride as gelling agent and gelling promoter, respectively (Quali-V® HPMC
capsules, Technical Manual, 2004). Carrageenan is a hydrocolloid that under the
influence of potassium chloride forms a three-dimensional network which further
results in the gel formation. The other difference in manufacturing the capsule shells
is that the gelling process takes longer compared to gelatin and, therefore, production
speeds are slightly slower for hypromellose shells.
Nevertheless, hypromellose capsules are manufactured by the same dipping
and forming method that is employed in the manufacturing of hard gelatin capsules
and are suited to all current capsule filling machines. These shells are produced in a
variety of sizes and colors comparable to the gelatin capsules.
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The objective of this study is two-fold:
(1) Evaluation and comparison of physical and disintegration properties of
hypromellose and gelatin capsules
(2) Investigation of inter-variability between the capsules for each type, since capsule
shell morphology plays a critical role in the manufacture of reproducible products
with desired attributes.
Materials and Methods
Materials
The empty capsules of hypromellose (Quali-V®, Shionogi Qualicaps Co.,
Ltd., Whitsett, NC) and gelatin (Coni-Snap®, Capsugel Division of Pfizer Inc.,
Morris Plains, NJ) were obtained from the respective manufacturers. Both capsules
were of size ‘0’ and possessed a white color.
In order to investigate the characteristics of the capsules, both empty and
filled capsule shells were examined. For this purpose, the capsule shells were
manually filled with powdered cellulose lubricated with 0.5% magnesium stearate.
Both empty and filled capsules were evaluated for physical characteristics and
disintegration properties.
5.5.6.2. Physical Characteristics
Hypromellose and gelatin capsules were tested as received with regard to
weight variation and capsule dimensions. For each capsule type, 10 samples of empty
and filled capsules were randomly selected and examined for weight variation, using
an analytical balance. The dimensions of these capsules (length, width and body wall
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thickness) were also measured and recorded using a texture analyzer which has
accuracy and distance resolution of 2.5 µm.
5.5.6.3. Disintegration Properties
The filled capsules of hypromellose and gelatin were evaluated and compared
in regard with the time required for capsule disintegration. As described in the USP
(2005), a disintegration apparatus was utilized, with the basket-rack assembly,
oscillating at a frequency rate of 30 cycles per minute. The capsules were tested in
immersion fluids of different compositions at 37°C. The selected media were
deionized water, hydrochloric acid solution (pH=1 .7), USP alkaline borate buffer
(pH= 10.0), and two phosphate buffer systems (PBS) at pH=6.8, potassium phosphate
monobasic buffer (USP, 2005) and sodium phosphate monobasic buffer, which will
be herein referred to as K-PBS and Na-PBS, respectively. As per the USP, the
disintegration time is recorded as the time that “all of the capsules have disintegrated
except for fragments from the capsule shell” (USP, 2005). The disintegration
properties of at least 6 capsules were examined in this study.
5.5.6.4. Effect of Temperature on Physical Properties of Capsules
The empty shells of hypromellose and gelatin capsules were placed in an oven
at 45°C for various periods of time. The capsules were removed from the oven at
predetermined time intervals, 1, 24, and 72 hours and the effect of temperature on
their physical properties was investigated. An average of 20 capsules was tested for a
corresponding weight loss upon exposure to the elevated temperature of 45°C at each
time point.
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5.6. Design and Development of Stable Immediate Release Formulations for
Acid-Labile Compound: Dosage Form Design and Performance Evaluation for a
model drug, Pantoprazole
5.6.1. Design Strategy
The aim of this chapter is to design and develop immediate release
pharmaceutical oral solid dosage forms of Pantoprazole, employing suitable
excipients and different manufacturing technologies relative to what is currently
known. The dosage forms should be designed in a way to raise the gastric pH above
6.0 within minutes so as to withstand the gastric environment and completely release
the Pantoprazole content in the stomach so as to achieve the desired therapeutic effect
within a short period of time. Furthermore, the final dosage forms of Pantoprazole
should remain stable during the manufacturing process and under storage conditions.
For this purpose, after a careful review of the current patents/publications
outlined in Chapter 2, it became apparent that in general, the designed dosage forms
of Pantoprazole had several layers of film-coating and/or alkaline stabilizers for
protecting the active drug and improving its stability. However, various published
work indicates that film-coating is a complex and multi-step process which depends
on many different variables. In addition, the reported immediate release omeprazole
formulations consisted of huge quantity of alkalising agents (Philips et al., 2003)
which, in turn, add to the complexity of the designed dosage forms.
Therefore, the aim of this study was to investigate the possibility of applying
new approaches with fewer quantity of alkalising agents without compromising the
stability and performance of the final Pantoprazole dosage form. To achieve this,
three different formulation strategies were considered and further investigated:
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(1) Pantoprazole core tablet filled in a capsule containing buffers which can
provide for immediate delivery of acid-labile compounds in various dose
ranges.
(2) Pantoprazole core tablet embedded inside the buffer core (Inlay tablets) which
can also provide for immediate delivery of acid-labile compounds.
(3) Pantoprazole pellets and buffer granules filled into a capsule which can
provide for immediate delivery of acid-labile compounds.
The choice of materials and the preparation steps involved in manufacturing of
these dosage forms are described in detail in the following sections.
5.6.2. Portraying the Real Issue associated with Microenvironment pH concept
The micro environmental or virtual pH can be said as the pH of the immediate
solution when the solid is dissolved in water. This virtual membrane pH determines
the extent of drug ionization and hence drug dissolution and absorption. Thus the
concept of microenvironment pH questions the basics of pH partition hypothesis. It
has been demonstrated that the pH of the diffusion layer at the surface of the dosage
form resembles that of a saturated solution of drug and excipients in a dissolution
media and represents the microenvironment pH of the system (Bramhankar and
Jaiswal, 1998). During dissolution, medium that may eventually penetrate into the
core, or during storage moisture may penetrate into the core resulting in a saturated
solution of drug and excipients. If the microenvironment pH is low, it will lead to
ultimate degradation of the drug. Hence; it is seen that the compositions of acid labile
drugs of prior art either use an enteric coating or high concentration of buffers or are
liable to degradation in the microenvironment pH.
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The aim of this experiment is to investigate the problem associated with
Microenvironment pH concept.
5.6.2.1 Materials and Methods
The selected combination of buffers, Magnesium oxide (MgO) and Trisodium
phosphate (TSP), in an amount sufficient to neutralize the stomach acid, active
ingredient Pantoprazole and other excipients were granulated and filled into size ‘0’
HPMC capsules. The prepared formulation was evaluated for its dissolution in SGF.
Table 5.7 shows the list of ingredients used in preparing the formulation as per the
prior art (Microenvironment pH concept).
In vitro drug release from the formulation was determined using the USP
Type I apparatus (250 mL SGF pH 1.7; 37°C; 100 rpm; n=6) (Vankel Dissolution
System, USA). At predetermined time intervals, 5 mL samples were withdrawn (not
replaced), filtered and assayed for drug content and related impurities. The amount of
Pantoprazole released was measured with a computer connected Shimadzu-HPLC
system (LC2010 AHT integrated with UV detector and LC solution software,
Shimadzu Corp., Japan).
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Table 5.7. List of ingredients used in preparing the formulation as per
Microenvironment pH concept
Ingredients
Amount per capsule (mg)
Pantoprazole sodium sesquihydrate
45.1
Magnesium oxide
100
Trisodium phosphate
250
Crospovidone NF
8
Sodium carbonate
15
Mannitol
28.4
Colloidal silicon dioxide
0.5
Magnesium stearate
3
Total weight
450.0
5.6.3. Formulation and Evaluation of Pantoprazole Tablet Cores
Tablets of Pantoprazole were formulated using the following ingredients.
Pantoprazole sodium sesquihydrate was purchased from Dr. Reddy’s Laboratories,
India. All the other tablet excipients such as Crospovidone NF, Sodium carbonate,
Mannitol, Colloidal silicon dioxide, Magnesium stearate were purchased from
different manufacturers. Table 5.8 displays the formula used in preparation of
Pantoprazole tablets.
Core tablets containing 40 mg of Pantoprazole as active, magnesium stearate
as lubricant were prepared by direct compression. The respective powders (drug and
additives, for compositions see Table 5.8) were passed through a 20 mesh sieve and
blended in a double cone blender.
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The tablets were prepared by using an eight station tabletting machine
equipped with 6.0 mm punches. The tablet weight was kept constant at 100 mg and
the hardness of the core tablets was kept constant at 6 Kp if not otherwise mentioned.
Table 5.8. Composition of Pantoprazole core tablet
Ingredients
mg per tablet
Pantoprazole sodium sesquihydrate eq. to 40 mg
45.1
of PantoprazoleNF
Crospovidone
8
Sodium carbonate
15
Mannitol
28.4
Colloidal silicon dioxide
0.5
Magnesium stearate
3
Total weight
100 mg
The core tablets were evaluated for physical tests such as thickness, hardness,
friability, moisture content, average weight, and chemical tests such as disintegration,
dissolution in pH 6.8 buffer, assay, uniformity of dosage units and related impurities.
Thickness:
Randomly 10 tablets were taken from the representative sample. The
individual tablet thickness was checked and recorded using Vernier calliper.
Hardness:
Randomly 10 tablets were taken from the representative sample. The hardness
of tablets were evaluated using the instrument Tablet Hardness Tester.
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Friability:
A sample of 6.5 g of tablets was taken. The tablets were dedusted carefully
prior to testing. The tablet sample was accurately weighed (W1), and placed in the
drum. The drum was rotated 100 times and the tablets were removed. Any loose dust
from the tablets was removed as before and weighed (W2). The weight loss was
calculated due to friability (%F) by the formula: % F = (W1-W2)/W1 x100
Moisture content:
The moisture content is determined on about 1 g of sample at 105°C on a
moisture analyzer.
Average weight:
Randomly 20 tablets were taken from the representative sample and weighed
on an analytical balance. The weight of individual tablets was recorded and the
average weight was determined.
Disintegration:
The tablets were subjected to disintegration studies in a disintegration tester
using purified water as the medium.
Dissolution:
The tablets were subjected for dissolution using a USP Type-II dissolution
apparatus. The dissolution media consisted of 1000 mL of pH 6.8 phosphate buffer.
The paddles were operated at 100 rpm and the bath temperature was maintained at
37± 0.5°C using a temperature controller. The samples were withdrawn at specified
intervals and analysed using an online UV visible spectrophotometer.
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Assay, content uniformity and related impurities:
The core tablets were evaluated for drug content, uniformity of dosage and
related impurities by a stability indicating HPLC method. For the analysis, Shimadzu
HPLC system equipped with LC-2O AT pump, DGU-20 AS on-line degasser, SIL20A auto injector, CTO-2O A column oven, and SPDM20A, photo diode array
detector was used for peak purity testing. Shimadzu LC-solution software (Version
1.22 SP1) was used for data acquisition and mathematical calculations.
Chromatographic separation of Pantoprazole was performed on a C8 Symmetry
column (3.9 mm x 150 mm; 5 μm particle size; Waters, USA). Mobile phase used
was acetonitrile, phosphate buffer [2.75 g of sodium dihydrogen orthophosphate
(NaH2PO4) and 0.4258 g of disodium hydrogen orthophosphate (Na2HPO4)], in the
ratio of 30:70, v/v, pH 6.0 with orthophosphoric acid, at a flow rate of 1 mL/min.
Temperature of the column oven was maintained at ambient. Diluent used was
acetonitrile and water in the ratio of 50:50, v/v for preparation of all the samples.
Preparation of solutions:
Standard Solution (for Assay & content uniformity):
An accurately weighed quantity of Pantoprazole sodium sesquihydrate
working standard equivalent to 40 mg of Pantoprazole was weighed into a 50 mL
volumetric flask. 30.0 mL of diluent was added, sonicated to dissolve and diluted to
volume with diluent and mixed well (standard stock solution). 5 mL of this solution
was transferred by pipette into a 50 mL volumetric flask and made up the volume
with diluent and mixed. This solution contains 0.08 mg/mL of Pantoprazole base. The
solution was filtered through 0.45µm membrane filter and injected into the HPLC
system.
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Standard Solution (for related impurities):
2 mL of standard solution (for assay) was transferred by pipette into a 100 mL
volumetric flask and made up the volume with diluent and mixed. This solution
contains 0.0016 mg/mL of Pantoprazole base. The solution was filtered through
0.45µm membrane filter and injected into the HPLC system.
Test Solution (for Assay & related impurities):
5 tablets were weighed and transferred into a 250 mL volumetric flask. 150
mL of diluent was added and sonicated for 30 minutes swirling the flask occasionally.
The flask was shaken on a wrist action shaker for 10 minutes, cooled to room
temperature and diluted to volume with diluent and mixed well. A portion of this
solution was centrifuged at 4000 rpm for 8 minutes. The supernatant solution was
filtered through 0.45 µm membrane filter and used as test solution for related
impurities. This solution contains 0.8 mg/mL of Pantoprazole. 5 mL of the clear
supernatant solution was transferred by pipette into a separate 50 mL volumetric flask
and diluted to volume with diluent and mixed. This solution contains 0.08 mg/mL of
Pantoprazole and used as test solution for assay. The solution was filtered through
0.45µm membrane filter and injected into the HPLC system.
Test Solution (for content uniformity):
10 tablets were assayed individually. 1 tablet was transferred into a 50 mL
volumetric flask. 30 mL of diluent was added and sonicated for 10 minutes swirling
the flask occasionally. The flask was shaken on a wrist action shaker for 10 minutes,
cooled to room temperature and diluted to volume with diluent and mixed well. A
portion of this solution was centrifuged at 4000 rpm for 8 minutes. 5 mL of the clear
supernatant solution was transferred by pipette into a separate 50 mL volumetric flask
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and diluted to volume with diluent and mixed. This solution contains 0.08 mg/mL of
Pantoprazole and used as test solution for content uniformity. The solution was
filtered through 0.45µm membrane filter and injected into the HPLC system.
Standard solutions and formulation samples (20 μl) were injected and
analyzed at 290 nm using a UV detector. For peak purity testing, PDA detector in the
range of 200-800 nm was used.
5.6.4. Formulation Design using Macroenvironment pH concept
The concept of creating a macroenvironment pH is based on the direct pH
neutralization of the stomach acid by the rapid release of the buffers in the stomach
creating a safe pH environment, followed by the release of Pantoprazole and thus
protecting the active ingredient from degradation by the stomach acid.
In an in-vitro model, the capsule disintegrates in the simulated gastric fluid
and releases the buffer which rapidly increases the pH of the medium to greater than
6.0 within 2-4 minutes and sustains this pH environment for approximately 1 hour.
This is followed by the release of the active PPI and attains 100% release within 30
minutes. This pharmacological synergy of the buffers protects the active ingredient
from gastric acid degradation, allows it to be rapidly absorbed, and eliminates the
need for an enteric coating.
5.6.4.1. Core tablet filled in capsule containing buffers
The selected buffer components and mannitol were passed through suitable
mesh and mixed together. The resultant powder mix was mixed with magnesium
stearate. The lubricated powder mix was subjected for slugging process and milled to
produce suitable sized granules. Weighed amount of buffer components and a core
tablet was filled into a size ‘0’ hydroxy propyl methyl cellulose (HPMC) capsule.
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The compositions of the formulations F1 to F4 using the above technique
containing different selected buffer combinations are given in Table 5.9.
Table-5.9. Composition of formulations F1 to F4 (core tablet filled in capsule
containing buffers)
Ingredients for core tablet
(mg/tablet)
Formulation code
F1
F2
F3
F4
45.1
45.1
45.1
45.1
Crospovidone
8
8
8
8
Sodium carbonate
15
15
15
15
Mannitol
28.4
28.4
28.4
28.4
Colloidal silicon dioxide
0.5
0.5
0.5
0.5
3
3
3
3
100
100
100
100
F1
F2
F3
F4
Trisodium Phosphate
250
250
--
--
Magnesium oxide heavy
100
--
100
--
Magnesium hydroxide
--
250
--
250
Sodium bicarbonate
--
--
350
--
Tromethamine
--
--
--
500
100
100
100
100
3
3
3
3
453
603
553
853
Pantoprazole
sodium
sesquihydrate
Magnesium stearate
Quantity per tablet (mg)
Ingredients
for
Buffer
composition (mg/capsule)
Mannitol
Magnesium stearate
Quantity per capsule (mg)
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5.6.4.2. Core tablet embedded inside the buffer core (Inlay tablets)
In this technique a core tablet of the active is sandwiched between a layer of
buffers and compressed into tablets. 200 mg of the buffer composition was filled into
die cavity of the rotary press, core tablet is placed at the center and the remaining 600
mg of buffer composition is filled over that and compressed into tablet. The
compositions of the formulations F5 to F8 using the above technique containing
different selected buffer combinations are given in Table 5.10.
Table-5.10. Composition of formulations F5 to F8 [Core tablet embedded inside
the buffer core (Inlay tablets)]
Ingredients for core tablet
(mg/tablet)
Formulation code
F5
F6
F7
F8
45.1
45.1
45.1
45.1
sesquihydrate
Crospovidone
8
8
8
8
Sodium carbonate
15
15
15
15
Mannitol
28.4
28.4
28.4
28.4
Colloidal silicon dioxide
0.5
0.5
0.5
0.5
3
3
3
3
Quantity per tablet (mg)
100
100
100
100
Ingredients
F5
F6
F7
F8
Trisodium Phosphate
250
250
--
--
Magnesium oxide heavy
100
--
100
--
--
250
--
250
Pantoprazole
sodium
Magnesium stearate
for Buffer
composition (mg/tablet)
Magnesium hydroxide
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Sodium bicarbonate
--
--
350
--
Tromethamine
--
--
--
500
Mannitol
200
200
200
200
Hydroxy propyl cellulose
200
200
200
200
Colloidal silicon diioxide
4
4
4
4
Magnesium stearate
6
6
6
6
760
910
860
1160
Quantity per tablet (mg)
5.6.4.3. Pellets and buffer granules filled into capsule
In this technique Pantoprazole and polyethylene glycol 8000 were passed
through suitable mesh and mixed together. The powder mix was transferred to a
suitable container, melt granulated and passed through 10 mesh sieve to get suitable
sized pellets. Weighed amount of buffer components and pellets were filled into
hydroxy propylmethyl cellulose capsules. The compositions of the formulations F9 to
F12 using the above technique containing different selected buffer combinations are
given in Table 5.11.
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Table-5.11. Composition of formulations F9 to F12 (Pellets filled in capsule
containing buffers)
Ingredients
for pellets
(mg/capsule)
Formulation code
F9
F10
F11
F12
45.1
45.1
45.1
45.1
Polyethylene glycol 8000
29.9
29.9
29.9
29.9
Quantity per capsule (mg)
75
75
75
75
Ingredients
F9
F10
F11
F12
Trisodium Phosphate
250
250
--
--
Magnesium oxide heavy
100
--
100
--
Magnesium hydroxide
--
250
--
250
Sodium bicarbonate
--
--
350
--
Tromethamine
--
--
--
500
100
100
100
100
3
3
3
3
453
603
553
853
Pantoprazole
sodium
sesquihydrate
for Buffer
composition (mg/capsule)
Mannitol
Magnesium stearate
Quantity per capsule (mg)
5.6.5. Evaluation of Immediate Release Formulations
5.6.5.1. Physical parameters
Average weight and moisture content of the various formulations F1 to F12
were determined as mentioned earlier in section 5.6.3.
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5.6.5.2. Disintegration Studies of Immediate Release Formulations in 0.1N HCl
The formulations were subjected to disintegration studies in 0.1N HCl for 30
minutes maintaining the temperature at 37±0.5°C.
5.6.5.3. Drug content and Related Impurities
The formulations F1 to F12 were evaluated for drug content, uniformity of
dosage and related impurities by a stability indicating HPLC method. For the
analysis, Shimadzu HPLC system equipped with LC-2O AT pump, DGU-20 AS online degasser, SIL-20A auto injector, CTO-2O A column oven, and SPDM20A, photo
diode array detector was used for peak purity testing. Shimadzu LC-solution software
(Version 1.22 SP1) was used for data acquisition and mathematical calculations.
Chromatographic separation of Pantoprazole was performed on a C8 Symmetry
column (3.9 mm x 150 mm; 5 μm particle size; Waters, USA). Mobile phase used
was acetonitrile, phosphate buffer [2.75 g of sodium dihydrogen orthophosphate
(NaH2PO4) and 0.4258 g of disodium hydrogen orthophosphate (Na2HPO4)], in the
ratio of 30:70, v/v, pH 6.0 with orthophosphoric acid, at a flow rate of 1 mL/min.
Temperature of the column oven was maintained at ambient. Diluent used was
acetonitrile and water in the ratio of 50:50, v/v for preparation of all the samples.
Standard solutions and formulation samples (20 μl) were injected and analyzed at 290
nm using a UV detector. For peak purity testing, PDA detector in the range of 200800 nm was used.
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5.6.5.4. In-vitro release of Pantoprazole Immediate Release Formulations in SGF
Materials and Methods
In vitro drug release from the formulations F1 to F12 was determined using
the USP Type I apparatus (250 mL SGF pH 1.7; 37°C; 100 rpm; n=6). At
predetermined time intervals, 5 mL samples were withdrawn (not replaced), filtered
and assayed for drug content and related impurities. The amount of Pantoprazole
released was measured with a computer connected Shimadzu-HPLC system using the
HPLC method as mentioned for assay.
Pantoprazole solutions of known
concentration were used to calculate the amount of drug released.
5.6.5.4.1 Effect of varying dissolution parameters on selected formulation
The drug release from the formulation F1 was also evaluated by varying
different parameters such as change in rpm, volume of medium and increasing the
content of acid in SGF.
In order to observe the effect of variation in gastric motility, in-vitro, the
speed of rotation was reduced to 75 rpm to evaluate its effect on release
characteristics of the formulation.
The volume of medium was increased to 900 mL and the apparent changes in
drug release were noted.
As per the literature, the basal stomach fluid contains 9.6 mL of 0.1 N HCl
and releases 0.5 mL of 0.1N HCl per minute. The quantity of acid produced per hour
in a normal human being would be approximately 30 mL. Keeping this in mind, for
the current study, SGF was prepared by adding excess quantity of acid, 40 mL of
0.1N HCl, to 210 mL of water (Total volume=250 mL) whose apparent pH was 1.70
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which was similar to the gastric fluid. In order to simulate the condition of extremely
acidic patients, the acid content in the SGF was increased to 50 mL in a total volume
of 250 mL of medium. The drug release and related impurities was observed for any
changes.
5.6.5.5. pH profile of Pantoprazole Immediate Release Formulations in SGF
A mobile pH meter was attached to the dissolution assembly in one of the
vessel to continuously monitor the change in pH of the medium with time. The probe
of a precalibrated pH meter was inserted in one of the vessel and the rise in pH with
time was recorded at every 2 minute intervals during dissolution of the different
formulations in SGF. Medium in other vessels were also checked for pH periodically
in order to evaluate the uniformity in pH rise in all the vessels. An average of six
vessels pH readings for formulations F1 to F12 was plotted against time.
5.6.5.6. In-vitro release profile of the Innovator’s Delayed release formulation
In order to successfully design and develop suitable Pantoprazole immediate
release dosage forms in this research work, the commercial products of delayed
release Pantoprazole which are currently available on the market were evaluated in
terms of their release characteristics as detailed below.
Initial studies involved the evaluation of release characteristics of delayed
release Pantozol® tablets. Each Pantozol® tablet contains 40 mg of Pantoprazole
which is enterically coated with pH sensitive polymers.
Pantozol® dosage form is expected to remain intact in the gastric fluid while
immediately release the active ingredient upon reaching the proximal part of the small
intestine.
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The aim of this study was to evaluate the release pattern of Pantozol® dosage
form, which will subsequently be utilized in design and development of Pantoprazole
immediate release dosage forms of the present research work.
Materials and Methods
Pantozol® tablets (Altana Pharma, Germany) were purchased from the local
market. Pantoprazole powder was purchased from Dr. Reddy’s Lab, India.
The dosage form was evaluated for in-vitro drug release by means of a
dissolution tester using USP apparatus II, paddle system, operated at the agitation rate
of 100 rpm (revolution per minute). Dissolution studies were conducted in accordance
with the USP 34 guidelines for delayed release (enteric-coated) articles. For this
purpose, the dosage form was tested at both acid stage and buffer stage. For the first 2
hours, the dosage units were placed in 1000 mL of 0.1 N hydrochloric acid solution
(pH of about 1.2) maintained at 37±0.5°C. After 2 hours, the dosage forms were
removed from the acid medium and transferred to the vessel containing 1000 mL of
phosphate buffer of pH=6.8. The phosphate buffer was prepared by mixing 0.1 N
hydrochloric acid with 0.2 M tribasic sodium phosphate in the ratio of 3:1.
At each stage of the test, samples were analysed online periodically from each
dissolution vessel, and their respective absorbance values were measured at the
wavelength of 288 nm, using a UV-spectrophotometer with 1-cm quartz cells.
The concentration of the liberated drug was quantitatively determined, using
the constructed standard plot. For this purpose, the stock solution of Pantoprazole (0.5
mg/mL) was prepared in the USP alkaline borate buffer, pH= 10.0, which provides
satisfactory stability of the drug. Standard solutions over the range of desired
concentrations were prepared by appropriate dilutions of the stock solution in
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phosphate buffer (pH=6.8). In construction of the standard plots, the wavelength of
288 nm was selected since the absorbance spectra of Pantoprazole obtained at pH
values of 10.0 and 6.8 cross at this wavelength. The absorbance data were acquired
against the blank solutions pH=6.8. The linearity range was 10-70 µg/mL for all
solutions, and the correlation coefficient was greater than 0.999. The standard plots,
hence, can be used to calculate the extent of Pantoprazole release into the dissolution
media. Based upon the USP acceptance criteria, the amount of the drug released in
the acid stage must not exceed 10% of the total drug content of the dosage form. All
dissolution tests were performed in triplicates.
The intra-day absorbance values obtained for the samples under the described
testing conditions exhibited an RSD value of less than 1%.
5.7.
COMPARATIVE BIOAVAILABILITY STUDIES IN HEALTHY
HUMAN VOLUNTEERS
The study was conducted according to ICH- GCP and the Declaration of
Helsinki as amended by the 59th WMA General Assembly, Seoul, October 2008. The
study was approved by Institutional Review Board (IRB)/Independent Ethics
Committee (IEC) at Pharmaquest JO (Jordan) and Jordan FDA.
5.7.1. Study Objectives and Purpose
The objectives of the study are as follows:
To compare the rate and extent of absorption of two oral formulations of
Pantoprazole 40 mg. The test product (PANTOPRAZOLE Immediate Release
Capsules) will be compared with the reference product (PANTOZOL® 40 mg
Gastro-Resistant Tablets) of Nycomed GmbH, D-78467 Konstanz, Germany after a
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single oral dose (40 mg) administered to 12 healthy adult participants under fasting
condition.
The Pharmacokinetic Parameters that best characterize the rate and extent of
absorption are Cmax, AUC0→inf and AUC0→last for Pantoprazole. These primary
pharmacokinetic parameters in addition to tmax, t1/2, ke, MRTinf and residual area (%)
will be calculated for Pantoprazole from its plasma concentrations determined by a
validated analytical method.
5.7.2. Study design
The study was an open label, single-dose, randomized, two-treatment, twoperiod, two-sequence, fasting, crossover pilot comparative bioavailability study
including 12 participants with confinement periods 12 hours before dosing, and 23
hours after dosing in each period.
Washout Period
An adequate washout period (e.g., more than 5 half lives of the moieties to be
measured) should separate study periods to avoid drug carryover effects (U.S
Department of Health and Human Services; 2003). In our study the dosing periods
was separated by a washout period of one week that will be left between doses.
Randomization
Randomization was performed using SAS statistical package version 6.12
(Chunqin Deng and Julia Graz, Statistics and Data Analysis).
Before the first drug administration each participant received a badge
identifying the study code together with his random number. Equal number of
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participants was randomly assigned to either sequence (TR) or (RT) at two dosing
periods. In this study 6 participants received product T and the other 6 received
product R in each period.
Participants, within the TR sequence, received the test formulation in the first
dosing period and the reference formulation in the second dosing period. Participants,
within the
RT sequence, however, received the reference formulation in the first dosing
period and the test formulation in the second dosing period.
Figure 5.3. Schematic diagram detailing the study design
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5.7.3. Study Population
General Considerations
The study population consists of 12 healthy adult participants. The
participants were selected from Caucasian population, between the ages of 18 and 50
inclusive, body-mass index 19 to 30 Kg/m2 inclusive, non-smokers or moderate
smokers (smokers of less than 10 cigarettes per day).
Screening Procedures
Each participant completed screening procedures within not more than 2
weeks prior to period I. Demography, medical history, physical examination, ECG
examination and laboratory examination (including drugs of abuse test, and alcohol
consumption test) was carried out prior to participation.
Inclusion Criteria
Participants were expected to meet all the following criteria:
1) Ethnic Group (Caucasian).
2) Age 18-50 years inclusive.
3) Body-mass index 19 to 30 Kg/m2 inclusive.
4) Participant is fully aware of the study details and gave written informed consent.
5) Physical examination being assessed and accepted by the attending physician.
6) Systolic blood pressure within the normal range (90-140 mmHg).
7) Diastolic blood pressure within the normal range (60-90 mmHg).
8) Heart rate within the normal range (60-100 beat/min.).
9) Oral body temperature within the normal range (35.9 – 37.6 ºC).
10) All laboratory screening results within the normal reference range.
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Exclusion Criteria
Participants with any of the following were excluded from the study:
1) Women of child bearing potential.
2) Ethnic group (Non Caucasian).
3) History of hypersensitivity to the drug or similar compound.
4) Known history or presence of food allergies or intolerability (e.g diary product or
gluten containing food), or any known condition that could interfere with the
absorption, distribution, metabolism or excretion of drugs.
5) Vegetarian
6) Exhausting physical exercise in the last 48 hours (e.g. weight lifting).
7) History of serious illness that can impact fate of drugs or clinically significant
illness 4 weeks before study Period I
8) Participant HBsAg, HCV, and HIV positive.
9) History of drug or alcohol abuse, smoking more than 10 cigarettes per day
10) Regular use of medication.
11) Use of any known enzyme inducers or inhibitors (e.g. Barbiturates,
Carbamazepine, Phenytoin, Rifampin) within 30 days prior to study entry.
12) Use of any prescription or non prescription (OTC) medication within 2 weeks
prior to study.
13) Donation of 1) at least 400 mL of blood within 60 days, or 2) more than 150 mL
of blood within 30 days, or 3) more than 100 mL blood plasma or platelets within
14 days before study Period I.
14) Participation in another bioequivalence study within 60 days prior to the start of
this study Period I.
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15) Obvious signs of renal, gastrointestinal, cardiovascular, hepatic, respiratory,
neurological, musculoskeletal, endocrine disorders as evidenced by physical
examination, and/or clinical laboratory tests.
16) Participant with ALT, ALP, or AST elevated above the normal reference range.
5.7.4. Dosage Formulations
One tablet of Pantoprazole 40-mg delayed-release (Pantozol, Nycomed
GmbH, D-78467 Konstanz, Germany) and one Capsule of Pantoprazole 40 mg
Immediate Release were taken orally, with 240 mL of water at dosing.
5.7.5. Sample Collection and analysis
The time of drug administration was established as 0 minutes. 16 mL of blood
samples was collected predose and 8 mL were obtained at 0.25, 0.50, 0.75, 1.00, 1.25,
1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.33, 3.67, 4.00, 4.50, 5.00, 5.50, 6.00, 7.00,
8.00, 10.0, 12.0, 14.0, and 16.0 hours post dose during each treatment period. The
blood samples were collected via indwelling catheter into the labeled heparin blood
tubes (10.0 mL) and centrifuged (4000 rpm/4.0 minutes). The plasma samples were
transferred, using disposable polypropylene droppers into the labeled polypropylene
tubes containing a base to increase the plasma pH to ≥8, then capped and stored at -80
°C until analysis. Pantoprazole in plasma was measured using a validated analytical
method.
5.7.6. Comparative Bioavailability Assessment
The assessment of comparative bioavailability was based on the
Pharmacokinetic parameters derived from the concentrations of Pantoprazole
determined in individual plasma samples, harvested from each participant. The CPMP
Note for Guidance on the Investigation of Bioavailability and Bioequivalence (CPMP
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and
the
Methodology
recommendations
of
“Bio-International,
Bioavailability,
Bioequivalence and Pharmacokinetics” (Midha and Blume, 1992) was observed
during data evaluation.
The following Pharmacokinetic parameters (variables) of Pantoprazole were
determined and calculated: Cmax, tmax, Terminal half-life (t1/2), terminal rate constant
(ke), AUC0→last, AUC0→inf, Residual area (%) and MRT0→inf.
• Cmax and tmax:
The maximum plasma concentration (Cmax) and the time of the peak
concentration (tmax) will be taken directly from the raw plasma concentration-time
data. The units of Cmax and tmax are μg/mL or ng/mL and hour (h), respectively.
• Terminal half-life (t1/2), terminal rate constant ke:
The terminal half-life (t1/2) values will be estimated from the slope (terminal
rate constant ke) of linear regression of the semi-logarithmic plot of the terminal
phase of the plasma concentration curve (t1/2= ln2/ke). An assumption is that the
terminal elimination phase is reached within sampling period. The unit is hour. The
time interval used for the determination of the terminal elimination rate constant ke,
will be reported.
• AUC0→last:
The area under the plasma concentration-time curve (AUC0→last) will be
calculated by the linear trapezoidal rule from measured data points from time of
administration until the time of last quantified concentration, where Clast is the last
point.
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• AUC0→inf:
The area under the plasma concentration-time curve (AUC0→inf) will be
estimated by trapezoidal rule (AUC0→last) and extrapolation to infinity (AUClast→inf).
The extrapolation is performed by dividing the last measurable plasma concentration
Clast by terminal rate constant ke (AUClast→inf = Clast / ke). The AUC0→inf is the sum of
the estimated and extrapolated parts (AUC0→inf = AUC0→last + AUClast→inf).
• Residual area (%):
The residual areas will be determined in % by the following equation:
{(AUC0→inf - AUC0→last) /AUC0→inf}* 100. The unit is %.
• MRT0→inf:
Mean residence time from 0 to infinity. The parameters were derived
individually for each participant from the Pantoprazole concentrations in plasma. All
concentrations below the LLOQ was reported as zero when they occurred before the
first quantifiable concentration and was reported as (-) no value when they occurred
after the last quantifiable concentration. The arithmetic means, medians, minimum
and maximum values, geometric mean, standard deviations, and coefficients of
variations for all parameters were reported.
The Pharmacokinetic (PK) Parameters was calculated using the WIN
NONLIN (Version 1.5) a commercially available Software package, using the
compiled WIN NONLIN Model 200 for extra vascular input. All plasma
concentration-time profiles were drawn with WIN NONLIN.
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5.7.7. Statistical Analysis
Pharmacokinetic parameters derived from measures of concentration, e.g.
AUC, Cmax was analyzed using ANOVA procedures and methods. The data was
transformed prior to analysis using a log transformation. tmax was adopted
nonparametric and was applied to untransformed data. For all Pharmacokinetic
parameters of interest in addition to the appropriate 90% confidence intervals for the
comparison of the two formulations, a summary statistics including median,
minimum and maximum was given (OECD Principles of Good Laboratory Practice.
1992) SAS statistical package version 6.12 was used for evaluating all the reported
evaluations.
5.8.
STABILITY STUDIES
The aim of stability testing is to provide evidence on how the quality of a drug
substance or drug product varies with time upon exposure to different environmental
factors such as temperature, humidity, etc. (ICH, 2003).
Based on the desired release characteristics and minimum amount of buffer
required to maintain a stable composition, the selected formulation (F1, F5 & F9)
from each dosage form design was subjected to stability studies as per the ICH
guidelines.
In order to assess the effects of such factors on Pantoprazole dosage forms, the
designed tablets and capsules of Pantoprazole were packaged in alu-alu blisters since
the product is highly sensitive to moisture. The blisters were kept in a carton as a
secondary packaging material. The packs were then placed at two different storage
conditions, ambient environment 30°C/ 65% RH and stress condition of 40°C/ 75%
RH. The stability of the dosage forms in regard with their appearance, the remaining
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Pantoprazole content within each formulation, related impurities and their dissolution
performance was investigated and compared at predetermined time intervals, namely,
0, 90 and 180 days. The stability testing was performed in duplicates for each dosage
form.
5.8.1. Physical stability
At each sampling point, the designed tablets and capsules of Pantoprazole
were removed from their respective stability chambers and visually examined as a
whole against a white background in normal daylight. Moreover, the appearance of
the capsule contents and of the inner cores of the tablets was evaluated in a similar
manner. This was achieved by opening the capsules or through cross- sectioning of
the inlay tablets. Any sign of discoloration or other physical change was noted.
5.8.2. Assessing drug content and related impurities
The Pantoprazole assay and related impurities was carried out using HPLC
analysis as described in section 5.6.5.3.
5.8.3. Dissolution Performance of various Immediate Release formulations upon
Storage
In order to investigate the effects of stability testing on dissolution
performance of the designed formulations, at the predetermined time points, the
samples of each dosage form (n=6) were removed from the stability chambers and
subjected to dissolution testing, as described in section 5.6.5.4. The main purpose of
this study was twofold:
(1) To evaluate the efficiency of the buffers in protecting the Pantoprazole
content of the dosage forms and the release characteristics of buffers upon
storage.
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(2) To investigate the adequacy of the dosage forms as a whole in fully
releasing the Pantoprazole content to the dissolution media upon
exposure to SGF.
To fulfill this purpose, the dosage forms were directly exposed to the
respective dissolution media. The obtained results at each sampling point, over the
180-day period of this study, were further compared to the initial dissolution
behaviour of the respective dosage forms, assessed immediately after production.
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