Manimala M PDF

DEVELOPMENT AND VALIDATION OF IN-VITRO
AND IN-VIVO CORRELATIONS FOR

PREGABALIN AND PRAMIPEXOLE
FORMULATIONS
A THESIS
Submitted by
M. MANIMALA
in partial fulfillment for the award of the degree
of
DOCTOR OF PHILOSOPHY
Department of Biotechnology
Interdisciplinary of Chemistry / Biotechnology
FACULTY OF HUMANITIES AND SCIENCES
Dr.M.G.R.
EDUCATIONAL AND RESEARCH INSTITUTE
UNIVERSITY
(Declared as Deemed to be University u/s. 3 of UGC Act 1956)
CHENNAI 600095
FEBRUARY 2016
DECLARATION BY THE CANDIDATE
I declare that the thesis entitled “DEVELOPMENT AND VALIDATION

OF IN-VITRO AND IN-VIVO CORRELATIONS FOR PREGABALIN AND
PRAMIPEXOLE FORMULATIONS” submitted by me for the Doctor of
Philosophy is a bonafide record of work carried out by me during the period from
August 2007 to August 2014 under the guidance of Dr. Karpagam.S and has not
formed the basis for the award of any degree, diploma, associate-ship, fellowship,
titles in this or any other University or other similar institution of higher learning and
without any plagiarism.
I have also published my papers in International Journals (Scopus rated) as

per list of publications in the Annexure.
Signature of Research Scholar
ii
BONAFIDE CERTIFICATE
Certified that the thesis entitled “DEVELOPMENT AND VALIDATION OF

IN•VITRO AND IN-VIVO CORRELATIONS FOR PREGABALIN AND
PRAMIPEXOLE FORMULATIONS” is the bonafide work of
Mrs. M. MANIMALA who had carried out the research under my supervision and
without any plagiarism to the best of my knowledge. Certified further, that to the best
of my knowledge, the work reported herein does not form part of any other thesis or
dissertation on the basis of which a degree or diploma was conferred on an earlier
occasion on this or any other scholar.
Signature of Supervisor
Dr. S. Karpagam, Ph.D.,
Associate Professor
Queen Mary’s College
Chennai 600 004
iii
ABSTRACT
In recent years, the concept and application of the in-vitro and in-vivo
correlation (IVIVC) for pharmaceutical dosage forms have been a main focus of
attention of pharmaceutical industry, academia, and regulatory sectors. Development
and optimization of formulation is an integral part of manufacturing and marketing
of any therapeutic agent which is indeed a time consuming and costly process.
Optimization process may require alteration in formulation composition,
manufacturing process, equipment and batch sizes. Certainly, implementation of
these requirements not only halts the marketing of the new formulation but also
increases the cost of the optimization processes. It would be, desirable, therefore, to
develop in-vitro tests that reflect bioavailability data. The main objective of an
IVIVC is to serve as a surrogate for in-vivo bioavailability and to support bio-
waivers. Thus the need for a tool to reliably correlate in-vitro and in-vivo drug
release data has exceedingly increased.
The drug candidates Pramipexole and Pregabalin are water soluble drugs that
are predominantly ionized in gastrointestinal pH ranges and are well absorbed after
oral administration and are categorized as high solubility/high permeability drugs
under the proposed Class I Biopharmaceutical Classification System (BCS). Hence it
becomes necessary to determine the in-vitro and in-vivo correlations for Pramipexole
and Pregabalin. A proliferation of modified-release products of Pramipexole and
Pregabalin, it becomes necessary to examine the concept of IVIVC of these drugs in
greater depth.
A single dose, randomized, complete, two treatment cross over study was
conducted in healthy human subjects for each drug formulation. The subjects were
selected and were screened based on the inclusion criteria of the study. On the basis
of this preliminary screening, 24 volunteers were selected and their liver and renal
iv
functions and hematological parameters such as hemoglobin content, RBC and WBC
counts, blood sugar, cholesterol, bilirubin and ECG were examined by standard
clinical and biochemical investigations.
In each dosing session, volunteers received either immediate or modified

release formulations. The order of treatment administration was randomized in three
sequences (AB, BA) in blocks of two. Blood samples (4 ml) were collected using
disposable syringes in pre-heparinised centrifugal tubes at 0 (before drug
administration), 0.50, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, 18.0 and 24.0 h post
dosing. The samples were centrifuged at 3500 rpm for 10 minutes to separate plasma.
A similar procedure adopting cross over design in drug treatment was repeated after
7 days of wash out period.
The LC-MS/MS instrument was calibrated with polypropylene glycol standard

in positive and negative ion mode. The sensitive and selective LC-MS/MS method
was developed and validated. The Method development involves evaluation and
optimization of the various stages of sample preparation, chromatographic
separation, detection and quantification. The source parameters namely gas
temperature, gas flow, nebulizer, sheath gas temperatures, sheath gas flow, capillary
voltage, charging voltage were optimized. Different reverse phase columns, different
mobile phases with different ratios and mobile phase flow rates were optimized.
Extraction of the drugs from the plasma sample was tried with Liquid-Liquid
Extraction (LLE) and Solid Phase Extraction procedure with various cartridges.
The standard stock solutions, standard solutions, Calibration curve samples

(CC), Quality control (QC) Samples, blank plasma samples were prepared. The
standard solutions, CC samples, QC samples and plasma sample solutions were
injected with the optimised chromatographic conditions and the chromatograms were
recorded. The quantification of the chromatogram was performed using peak area
ratios (response factor) of the drug to internal standard. The calibration curves are
constructed routinely for spiked plasma containing drug and internal standard during
the process of pre-study validation and in-study validation.
5
The Pharmacokinetic parameters were determined for individual drug
treatments from the observed plasma concentration-time data. The area under the
plasma concentration-time curves (AUC) were calculated by trapezoidal rule from
time zero to the last observed concentration. The statistical analysis was carried out
for Cmax, AUC (0-t) and AUC (0-∞).
The release characteristics of selected drugs in their formulations is carried out

using USP XXIII dissolution apparatus (type I, basket; type II, paddle), at different
rpm, with the media of pH 1.2, 4.5, 6.8, distilled water and 7.4 buffers maintained at
37±0.5˚C. Dissolution tests were performed on twelve tablets. Percentage drug
release at various time intervals was calculated and compared. Difference factor (f1)
and a similarity factor (f2) were calculated.
The Wagner-Nelson method was used to calculate the percentage of the

selected drugs dose absorbed. The percent absorbed is determined by dividing the
amount absorbed at any time by the plateau value, ke,AUC (0-ω) and multiplying this
ratio by 100. The percentage of drug dissolved was determined using the
aforementioned dissolution testing method and the fraction of drug absorbed was
determined using the method of Wagner-Nelson. Linear regression analysis was used
to examine the relationship between percentage of drug dissolved and the percentage
of drug absorbed. The percentage of drug un-absorbed was calculated from the
percentage absorbed. The slope of the best-fit line for the semi-log treatment of this
data was taken as the first order rate constant for absorption. The dissolution rate
constants were determined from percentage released Vs the square root of time.
Linear regression analysis was applied to the in-vitro and in-vivo correlation plots
2
and coefficient of determination (r ), slope and intercept values were calculated.
The predictability of the IVIVC was examined by using the mean in-vitro
dissolution data and mean in-vivo pharmacokinetics of the selected modified release
formulations. These two data points, along with the zero-zero intercept were used to
calculate the expected absorption rate constants and predicted plasma concentration.
To further assess the predictability and the validity of the correlations, IVIVC
model-predicted Cmax and AUC values were determined for each formulation. The
6
percent prediction errors for Cmax and AUC were calculated. Level A correlation was
observed at the in-vitro dissolution conditions developed. These dissolution methods
predicted also the best absorption rate for the selected modified release formulations.
The target to find out a predictive in-vitro dissolution method was reached gradually.
The validity of the correlation was also assessed by determining how well the IVIVC
model could predict the rate and extent of absorption as characterized by Cmax and
AUC. The percent prediction error of ≤ 10 % for Cmax and AUC was obtained, which
establishes the predictability of the developed IVIVC model. The developed
dissolution methods using Apparatus I, pH 6.8 at 75 rpm for Pregabalin and
Apparatus I, pH 1.2 at 75 rpm for Pramipexole found to yield acceptable IVIVC. The
developed dissolution methods can surrogate for human bioequivalence studies and
also to discriminate batches which are non-bioequivalent.
vii
ACKNOWLEDGEMENT
First and foremost, praises and thanks to the God Almighty, for his love and
showers of blessings throughout my life and to complete my research work
successfully.
I extend my profound thanks to Thiru. A.C. Shanmugam, Founder,
Mr. A.C.S. Arun Kumar, President, Dr. Meer Mustafa Hussain,
Vice Chancellor, Dr. C.B. Palanivelu, Registrar, Dr. A. Thirunavukkarasu,
Dean Research and Dr. T.S. Saravanan, Research Coordinator and
Dr. Rajeshwari Hari, Head of Department, Biotechnology of Dr. MGR
Educational and Research Institute University for providing the wonderful
opportunity and for supporting me all through my research work.
My deepest gratitude is to my Guide, Dr. S. Karpagam, Associate Professor,
Queen Mary’s College, Chennai for providing invaluable guidance throughout this
research.
I would like to express my special thanks to my Co-guide,
Prof. Dr. M. Deecaraman, Senior Professor, Department of Biotechnology for his
guidance and encouragement.
I heartfelt thanks to my research committee member
Dr. M. Vijayalakshmi, Professor, Department of Biotechnology for her comments
and suggestions.
I am extending my heartfelt thanks to my beloved husband
Dr. M. Vasudevan, my children Mr. V. Yeshwanth and Ms. V. Geethanjali and
also all my family members and friends for their love, prayers, understanding and
support to complete this research work and in general.
Though only my name appears on the cover of this dissertation, a great many
people have contributed to its production. I owe my gratitude to all those people who
have made this dissertation possible and because of whom my graduate experience
has been one that I will cherish forever.
8
88
TABLE OF CONTENTS
Chapter No. Title Page No.
ABSTRACT iv
LIST OF TABLES xiv
LIST OF FIGURES xviii
LIST OF ABBREVATIONS AND SYMBOLS xxi
1 INTRODUCTION 1
1.1 In-vitro and in-vivo Correlations 1
1.2 Bio-pharmaceutics Classification System (BCS) 2
1.3 Bio-availability Studies for Development of IVIVC 3
1.4 In-vitro dissolution 5
1.5 IVIVC Models 7
1.5.1 Level A Correlation 7
1.5.2 Level B Correlation 8
1.5.3 Level C Correlation 8
1.5.4 Multiple-level C correlation 8
1.5.5 Level D correlation 9
1.5.6 IVIVC Model Development 9
1.5.7 IVIVC Model Validation 9
1.6 Drug Profile 11
1.6.1 Pregabalin 11
1.6.2 Pramipexole 12
1.7 Scope and Object of the Present Study 13
1.7.1 Bioequivalence study design and data handling 14
9
1.7.2 Development of LC-MS/MS methods for the 14

estimation of selected drugs in plasma samples.
1.7.3 Development of in-vitro dissolution studies 15
1.7.4 In-vitro and in-vivo data analysis 15
1.7.5 Development of IVIVC correlations 16
2 LITERATURE SURVEY 17
3 MATERIALS AND METHODS 40
3.1 Reagents, Chemicals and Instruments 40
3.1.1 Reagents and Chemicals used 40
3.1.2 Instruments used 40
3.2 Bioequivalence study 41
3.2.1 Study Design 41
3.2.2 Product for Evaluation 41
3.2.3 Subjects 41
3.2.4 Ethics Review Procedure 42
3.2.5 Drug Administration 42
3.2.6 Subject Monitoring 43
3.2.7 Meals and Food Restrictions 43
3.2.8 Extraction of drugs from plasma 43
3.2.9 Estimation of Pharmacokinetic Parameters 44
3.3 Analytical Method Development 44
3.3.1 Selection of Molecular Ions 44
3.3.2 Selection of Source Parameters 44
3.3.3 Column Selection 46
3.3.4 Selection of Mobile Phase 46
3.3.5 Effect of Injection Volume 46
3.3.6 Effect of Flow Rate 46
3.3.7 Selection of Internal standard 47
3.3.8 Selection of Extraction Procedure 47
3.4 Method Validation 49
1
0
3.4.1 System Suitability 49

3.4.2 Linearity 50
3.4.3 Accuracy and Precision 50
3.4.4 Recovery 50
3.4.5 Selectivity 51
3.4.6 Sensitivity 51
3.4.7 Matrix Effect 51
3.4.8Carry over Test 51
3.4.9 Stability 52
3.5 Estimation of Pregabalin in Plasma Samples 53
3.5.1 Preparation of standard Stock Solution 53
3.5.2 Preparation of Calibration Curve Samples Stock 53
Dilutions
3.5.3 Internal standard stock solution 53
3.5.4 Preparation of QC Samples 55
3.6 Estimation of Pramipexole in Plasma Samples 57
3.6.1 Preparation of Pramipexole Calibration Curve 57
Samples Stock Dilutions
3.6.2 Stock Dilution for Pramipexole Quality Control 58
Sample
3.6.3 Preparation of Quetiapine Stock Solution 58
3.6.4 Preparation of Quetiapine Stock Dilutions 58
3.6.5 Pramipexole Calibration Standards in Human 59
plasma
3.6.6 Preparation of Quality Control Samples of 60
Pramipexole
3.7 Determination of Pharmacokinetic Parameters 60
3.8 Development of in-vitro dissolution studies 61
3.9 In-vitro and in-vivo correlation 61
3.9.1 Validation of IVIVC 63
1
1
4 RESULTS AND DISCUSSION 65

4.1 Bioequivalence study design and data handling 65
4.2 Development of LC-MS/MS Methods for the Estimation of 66
Pregabalin and Pramipexole in Plasma Samples
4.3 Method Validation for Pregabalin 69
4.3.1 Linearity 69
4.3.3 Matrix Selectivity 71
4.3.5 Matrix effect 71
4.3.6 Carry over Test 72
4.3.7 Stability 72
4.4 Validation of Pramipexole 82
4.4.1 Linearity 82
4.4.3 Matrix Selectivity 83
4.4.5 Matrix effect 84
4.4.6 Carry over Test 84
4.4.7 Stability 84
4.5 Estimation of Pregabalin and Pramipexole in plasma 96
samples
4.6 In-vitro and in-vivo correlations for Pregabalin 107
4.6.1 In-vivo data analysis 107

4.6.2 In-vitro data analysis 109
4.6.3 In-vitro and in-vivo correlations 109
4.6.4 Internal validation 110
4.6.5 External validation 111
4.7 In-vitro and in-vivo correlations for Pramipexole 135
4.7.1 In-vivo data analysis 135
xii
4.7.2 In-vitro data analysis 137

4.7.3 In-vitro and in-vivo correlations 137
4.7.4 Internal validation 139
4.7.5 External validation 139
5 SUMMARY AND CONCLUSION 164
REFERENCES 168
LIST OF PUBLICATIONS 174
13
LIST OF TABLES
Table No. Title Page No.
3.1 Source Parameters for Pregabalin 45

3.2 Source Parameters for Pramipexole 45
3.3 Liquid – Liquid Extraction of Pregabalin 47
3.4 Preparation of Pregabalin Standards for Calibration Curve 54
(Dilution I)
3.5 Preparation of Pregabalin Standards for Calibration Curve 55
(Dilution II)
3.6 Preparation of Pregabalin QC Samples (Dilution-I) 56
3.7 Preparation of Pregabalin QC Samples (Dilution-II) 56
3.8 Stock Dilution for Pramipexole Calibration Standard 57
3.9 Stock Dilution for Pramipexole Quality control Samples 58
3.10 Pramipexole Calibration standards in Human plasma 59
3.11 Pramipexole Quality Control samples in Human Plasma 60
4.1 Intercept, Slope and Correlation Coefficient Values for 73
Pregabalin Calibration curve
4.2 Intra-batch Accuracy and Precision of Pregabalin 74
4.3 Inter-batch Accuracy and Precision of Pregabalin 75
4.4 Recovery of Pregabalin 76
4.5 Recovery of Tramadol (lnternal Standard) 77
4.6 Matrix Selectivity of Pregabalin 78
4.7 Lower Limit of Quantitation (LLOQ) 78
4.8 Matrix Effect of Pregabalin 79
4.9 Carry over test of Pregabalin 79
4.10 Freeze and Thaw Stability of Pregabalin 80
4.11 Bench Top stability of Pregabalin 81
14
4.12 Intercept, Slope and Correlation coefficient values for 86

Pramipexole Calibration curve
4.13 Intra-batch Accuracy and Precision of Pramipexole 87
4.14 Inter-batch Accuracy and Precision of Pramipexole 88
4.15 Recovery of Pramipexole 89
4.16 Recovery of Quetiapine (Internal Standard) 90
4.17 Matrix Selectivity of Pramipexole 91
4.18 Lower Limit of Quantitation (LLOQ) of Pramipexole 92
4.19 Matrix Effect of Pramipexole 93
4.20 Carry over test of Pramipexole 94
4.21 Freeze and Thaw stability of Pramipexole 94
4.22 Bench Top stability of Pramipexole 95
4.23 Individual Plasma Concentrations (mcg/ml) and 113
Pharmacokinetic Parameters for Pregabalin Immediate Release
Formulation
4.24 Individual Plasma Concentrations (Mcg/Ml) and Pharmacokinetic 115
Parameters for Pregabalin Modified Release Formulation
4.25 Mean Plasma concentrations (mcg/ml) for Pregabalin 117
4.26 Pharmacokinetic Profile of Pregabalin 119
4.27 Statistical data for Pregabalin Immediate Release Vs Modified 120
Release Formulations
4.30 Paired Sample Test for Pregabalin 123
4.31 Cumulative percentage dissolved at 50 rpm for Pregabalin 124
formulations
4.32 Cumulative percentage dissolved at 75 rpm for Pregabalin 126
formulations
15
4.33 Similarity factors for Pregabalin modified release dosage forms 128
in Various dissolution condition
4.34 IVIVC model regression of % absorbed vs. % dissolved for 129
Pregabalin Formulation using pH 6.8 at 50 and 75 rpm
4.35 Observed and IVIVC model predicted Cmax and AUC values 132
for Pregabalin
4.36 Prediction errors (%) associated with Cmax and AUC for 132
Pregabalin
4.37 IVIVC model linear regression plots of % absorbed vs % 134
dissolved for Pregabalin tablets using pH 6.8, at 75 rpm
4.38 Individual plasma concentrations (mcg/ml) and pharmacokinetic 141
parameters for Pramipexole Immediate Release Formulation
4.39 Individual plasma concentrations (mcg/ml) and pharmacokinetic 143
parameters for Pramipexole Modified Release Formulation
4.40 Plasma concentrations (mcg/ml) of Pramipexole 145
4.41 Pharmacokinetic profile of Pramipexole 147
4.42 Statistical data for Pramipexole Immediate Release Vs Modified 148
Release Formulation
Release Formulation
Release Formulation
4.45 Paired Sample Test for Pramipexole 151
4.46 Cumulative percentage dissolved at 50 rpm for Pramipexole test 152
formulations
4.47 Cumulative percentage dissolved at 75 rpm for Pramipexole test 154
formulations
4.48 Similarity Factors for Pramipexole Modified Release 156
Formulations in Various Dissolution Conditions
16

Pramipexole Formulations using pH 1.2 and 4.5 at 50 rpm
4.51 Observed and IVIVC model predicted Cmax and AUC values 161
for Pramipexole
4.52 Prediction errors (%) associated with Cmax and AUC for 163
Pramipexole
xvi
i
LIST OF FIGURES
Figure No. Title Page No.
4.1 Calibration Curve of Pregabalin 73

4.2 Calibration Curve of Pramipexole 86
4.3 Mass Spectrum of Pregabalin (Parent Ion) 97
4.4 Mass Spectrum of Pregabalin (Product Ion) 97
4.5 Mass Spectrum of Tramadol (Parent Ion) 98
4.6 Mass Spectrum of Tramadol (Product Ion) 98
4.7 Representative chromatogram of processed blank plasma 99
4.8 Typical chromatogram obtained from LLOQ 99
4.9 Typical chromatogram obtained from LQC 100
4.10 Typical chromatogram obtained from MOQ 100
4.11 Typical chromatogram obtained from HQC 101
4.12 Typical chromatogram obtained from ULOQ 101
4.13 Mass Spectrum ofPramipexole(Parent Ion) 102
4.14 Mass Spectrum of Pramipexole(Product Ion) 102
4.15 Mass Spectrum of Quetiapine (Parent Ion) 103
4.16 Mass Spectrum of Quetiapine (Product Ion) 103
4.17 Representative chromatogram of processed blank plasma 104
4.18 Typical chromatogram obtained from LLOQ 104
4.19 Typical chromatogram obtained from LQC 105
4.20 Typical chromatogram obtained from MOQ 105
4.21 Typical chromatogram obtained from HQC 106
4.22 Typical chromatogram obtained from ULOQ 106
4.23 Mean Concentration Time Curve for Pregabalin 118
4.24 Cumulative Pregabalin Release Vs Time Profile for 125
Immediate and Modified Release Formulations at 50 RPM
181
818

4.26 IVIVC model Linear Regression Plot of % Absorbed Vs % 130
Dissolved for Immediate and Modified Release Pregabalin
Formulations using pH 6.8 Buffer at 50 RPM
Dissolved for Immediate and Modified Release Pregabalin
4.28 Cumulative Pregabalin Release Vs Square Root of Time 131
Profile for Immediate and Modified Release Pregabalin
4.29 Cumulative Pregabalin Release Vs Square Root of Time 131
Profile for Immediate and Modified Release Pregabalin
4.30 Observed and Predicted Pregabalin Plasma Concentration for 133
the Immediate Release Pregabalin Formulations using IVIVC
Modeland Modified Release Formulations at 50RPM
the Modified Release Pregabalin Formulations using IVIVC
Model
4.32 Mean Concentration Time Curve for Pramipexole 146
Dissolved for Immediate and Modified Release Pramipexole
19

the Immediate Release Pregabalin Formulations using IVIVC
Model
the Modified Release Pregabalin Formulations using IVIVC
Model
20
LIST OF ABBREVATIONS AND SYMBOLS
ANOVA : Analysis of variance

AQ : Aqueous
AR : Analytical reagent
BA : Bioavailability
BCS : Bio-pharmaceutics Classification System
BE : Bioequivalence CC
: Calibration curve CS :
Comparison sample
CV : Coefficient of variation
c(t) : plasma concentration
oC : Degree centigrade
F : Fraction absorbed
FAB : Fast atom bombardment
FD : Field desorption
FT : Freeze thaw
g : Gram
GC : Gas chromatography
GC-MS : Gas chromatography-mass spectrometry
GR : General reagent
HPLC : High performance liquid chromatography
HQC : High Quality control
ICH : International conference on harmonization
ID : Identity
IS : Internal standard
IV : Intravenous
IVIVC : In-vitro and in-vivo Correlations
ka : absorption rate constant
21
ke : elimination rate constant
K2EDTA : Dipotassium ethylene Diamine tetra acetic acid
LC-MS : Liquid Chromatography-Mass Spectrometry
LLOQ : Lower limit of quantification
LOD : Loss on drying
LOQ : Limit of quantification
LOQQC : Limit of quantification of quality control
LQC : Lower quality control
m/z : mass-to-charge ratio
MALDI : Matrix assisted laser desorption ionisatoin
µg/mL : Microgram per mille litre
µm : Micrometer
mg : milligram
mL : milliliter
msec : Millisecond
mM : milli Molar
mm : millimeter
MQC : Middle quality control
MRM : Multiple Reaction Monitoring
MS : Mass Spectrometry
NDA : New drug application
ng/mL : nanogram per milliliter
NMR : Nuclear magnetic resonance
P&A : Precision and accuracy
% : Percentage
Pg/mL : pictogram/milliliter
ppm : Parts per million
QC : Quality Control
QMS : Quadrupole mass spectrometer
R.S.D. : Relative standard deviation
RIA : Radio immune assay
RP : Reverse phase
xxi
i
Rpm : Revolutions per minute
rabs : Absorption rate time course
SAS : Statistical analysis system
SD : Standard Deviation
SS : Stock solution
STD : Standard
tmax : Time to reach maximum peak plasma
t1/2 : Half life
UPLC : Ultra performance liquid chromatography
USFDA : United States Food and Drug Adminstration
USP : United States of Pharmacopoeia
UV : Ultra violet
Vd : volume of distribution
V/V : Volume by volume W
% : Weight percentage
WS : Working standard
µL/min : Micro liter per minute
xxii
i
CHAPTER 1
INTRODUCTION
1.1 In-vitro and in-vivo Correlations
In recent years, the concept and application of the in-vitro and in-vivo
Correlation (IVIVC) for pharmaceutical dosage forms have been a main focus of
attention of pharmaceutical industry, academia, and regulatory sectors. Development
and optimization of formulation is an integral part of manufacturing and marketing
of any therapeutic agent which is indeed a time consuming and costly process.
Optimization process may require alteration in formulation composition,
manufacturing process, equipment and batch sizes (Dickinson et al. 2008). If these
types of changes are applied to a formulation, studies in human healthy volunteers
may be required to prove that the new formulation is bioequivalent with the old one.
Certainly, implementation of these requirements not only halts the marketing of the
new formulation but also increases the cost of the optimization processes. It would
be, desirable, therefore, to develop in-vitro tests that reflect bioavailability data. A
regulatory guidance for both immediate and modified release dosage forms has been,
therefore, developed by the regulatory authorities to minimize the need for
bioavailability studies as part of the formulation design and optimization.
IVIVC can be used in the development of new pharmaceuticals to reduce the

number of human studies during the formulation development (Chakshu
Bhatia et al. 2012). The main objective of an IVIVC is to serve as a surrogate for
in•vivo bioavailability and to support bio-waivers. IVIVCs could also be employed
to establish dissolution specifications and to support and/or validate the use of
dissolution methods. This is because the IVIVC includes in-vivo relevance to in-vitro
dissolution specifications. It can also assist in quality control for certain scale-up and
post-approval changes, for instance, to improve formulations or to change production
processes (Jost 2010). There must be some in-vitro means of assuring that each batch
of the same product will perform identically in-vivo.
The term correlation is frequently employed within the pharmaceutical and

related sciences to describe the relationship that exists between variables.
Mathematically, the term correlation means interdependence between quantitative or
qualitative data or relationship between measurable variables and ranks. From
biopharmaceutical standpoint, correlation could be referred to as the relationship
between appropriate in-vitro release characteristics and in-vivo bioavailability
parameters.
IVIVC is a predictive mathematical model describing the relationship between

an in-vitro property of a dosage form and a relevant in-vivo response (Elena Soto et
al. 2010). Generally, the in-vitro property is the rate or extent of drug dissolution or
release while the in-vivo response is the plasma drug concentration or amount of drug
absorbed.
A successful IVIVC model can be developed, if in-vitro dissolution is the rate-

limiting step in the sequence of events leading to the appearance of the drug in the
systemic circulation following oral or other routes of administration. Thus, the
dissolution test can be utilized as a surrogate for bioequivalence studies (involving
human subjects) if the developed IVIVC is predictive of in-vivo performance of the
product (Koenen-Bergmann et al. 2008). For orally administered drugs, IVIVC is
expected for highly permeable drugs or drugs under dissolution rate-limiting
conditions as supported by the Biopharmaceutical Classification System (BCS).
1.2 Bio-pharmaceutics Classification System (BCS)
Bio-pharmaceutics Classification System (BCS) is a fundamental guideline

for determining the conditions under which in-vivo and in-vitro correlations are
expected. In the BCS, a drug is classified as one of the following four classes based
solely on its solubility and intestinal permeability;
2
Class I(High solubility/High permeability) drugs exhibit a high Absorption
number and a high Dissolution number. The rate limiting step to drug absorption is
the drug dissolution or gastric emptying rate if dissolution is very rapid.
Class II (Low solubility/High permeability) drugs have a high Absorption

number but a low Dissolution number. In-vivo drug dissolution is then a rate limiting
step for absorption (except at very high Dose number). The absorption for Class II
drugs are usually slower than Class I and occur over a longer period of time.
Class III (High solubility/Low permeability) drugs exhibit a high variability in

rate and extent of drug absorbed. Since the dissolution is rapid, the variation is due to
alteration of GI physiological properties and membrane permeation rather than
dosage form factors.
Class IV (Low solubility/Low permeability) drugs have low solubility and low
permeability and exhibit a lot of problems for effective oral administration.
IVIVC are usually expected for Class I and Class II drugs.
1.3 Bio-availability Studies for Development of IVIVC
A bioavailability study should be performed to characterize the plasma

concentration versus time profile for each of the formulation. Bioavailability studies
for IVIVC development should be performed with sufficient number of subjects to
characterize adequately the performance of the drug product under study (Quinones
et al. 2010). In prior acceptable data sets, the number of subjects has ranged from 6
to 36. Although crossover studies are preferred, parallel studies or cross-study
analyses may be acceptable. The latter may involve normalization with a common
reference treatment. The reference product in developing an IVIVC may be an
intravenous solution, an aqueous oral solution, or an immediate release product.
IVIVCs are usually developed in the fasted state. When a drug is not tolerated in the
fasted state, studies may be conducted in the fed state.
Drug absorption from GI tract following ingestion of an oral dosage form could
be influenced by a number of in-vivo variables. For the determination of reproducible
3
in-vivo parameters and consequently useful in-vitro and in-vivo relationship, it is
imperative that such variables be identified. As a result, the study should be designed
appropriately that as many variables as possible be eliminated or controlled to
prevent or minimize their disturbance of IVIVC. Control or standardization of a
number of variables including subject selection criteria such as age, gender, physical
condition, etc., and the abstinence by the subject from coffee and other xanthene’s
containing beverages or food, alcohol, irregular diets and smoking before and during
the study should be taken in to consideration. Food, posture and exercise may
influence hepatic blood flow which in turn may substantially affect the absorption of
drugs possessing high hepatic extraction ratio.
Liquid chromatographic coupled with tandem mass spectrometer (LC-MS/MS)

have become the methods of choice for measuring drugs in biological fluids, yielding
concentration versus time data for drug compounds from in-vivo samples such as
plasma (Alessandro Musenga et al. 2008).
LC-MS/MS instrument consists of three major components
 LC (to resolve a complex mixture of components)

 An interface (to transport the analyte in to the ion source) of a mass
spectrometer
 Mass spectrometer (to ionize and mass analyze the individually
resolved components)
The quadrapole mass spectrometer is the most common mass analyzer. Its
compact size, fast scan rate, high transmission efficiency and modest vacuum
requirements are ideal for small inexpensive instruments. Most quadrupole
instruments are limited to unit m/z resolution and have a mass range of m/z 1000.
Many bench top instruments have mass range of m/z 500 but research instruments
are available with mass range upto m/z 4000.
4
Bioavailability studies can be accessed via plasma or urine data using the
following parameters:
 Area under the plasma time curve (AUC), or the cumulative amount of
drug excreted in urine,
 Maximum concentration (Cmax), or rate of drug excretion in urine and
 Time of maximum concentration (Tmax).
Several approaches can be employed for determining the in-vivo absorption.

Wagner-Nelson, Loo-Riegelman and numerical de-convolution are such methods.
Wagner Nelson andLoo-Riegelman are both model dependent methods in which the
former is used for a one compartment model and the latter is for a multi-
compartment system.
The Wagner Nelson method is less complicated than the LooRiegelman, as

there is no requirement for intravenous data. However, misinterpretation on the
terminal phase of the plasma profile is likely in the occurrence of a flip-flop
phenomenon in which the rate of absorption is slower than the rate of elimination.
De-convolution is a numerical method used to estimate the time course of drug

input using a mathematical model based on the convolution integral. For example,
abs
the absorption rate time course (r ) that resulted in plasma concentration (c(t)) may
be estimated by solving the convolution integral equation for absorption rate time
course. De-convolution is a model independent method which can be employed for
either one-or multiple-compartment models.
1.4 In-vitro dissolution
Drug absorption from a solid dosage form following oral administration

depends on the release of the drug substance from the drug product, the dissolution
or solubilization of the drug under physiological conditions, and the permeability
across the gastrointestinal tract (Jose David et al. 2013). Because of the critical
nature of the first two of these steps, in-vitro dissolution may be relevant to the
prediction of in- vivo performance.
5
The purpose of in-vitro dissolution studies in drug development process is to
assess the lot-to-lot quality of a drug product, guide development of new
formulations; and ensure continuing product quality and performance after certain
changes, such as changes in the formulation, the manufacturing process, the site of
manufacture, and the scale-up of the manufacturing process (Pui Shan Chana et al.
2014).
Modified release dosage forms typically require dissolution testing over

multiple time points, and IVIVC plays an important role in setting these
specifications. Specification time points are usually chosen in the early, middle and
late stages of the dissolution profiles. In the absence of an IVIVC, the range of the
dissolution specification rarely exceeds ± 10% of the dissolution of the pivotal
clinical batch. In the presence of IVIVC, however, wider specifications may be
applicable based on the predicted concentration-time profiles of test batches being
bioequivalent to the reference batch.
However, for the IVIVC perspective, dissolution is proposed to be a surrogate

of drug bioavailability. Thus, a more rigorous dissolution standard may be necessary
for the in-vivo waiver. Generally, a dissolution methodology, which is able to
discriminate between the study formulations and which best, reflects the in-vivo
behavior would be selected.
The utilization of in-vitro dissolution data for predicting in-vivo performance

requires a meaningful method of transformation of the data (Lake et al.1999). A
direct comparison between in-vitro and in-vivo data is not possible since the
measurement of in-vivo release/absorption profiles is not straightforward. There are
also arguments about appropriateness of using classical single (experimental) point
pharmacokinetic parameters like Cmax and Tmax to assess
bioavailability/bioequivalence of modified release preparations. Various
mathematical models and equations have been described in literature for conversion
of directly measurable pharmacokinetic data to release/absorption characteristic of
the drug from the dosage form for comparison with in-vitro dissolution data.
6
1.5 IVIVC Models
IVIVC models are developed to explore the relationships between in-vitro

dissolution/release and in-vivo absorption profiles. This model relationship facilitates
the rational development and evaluation of immediate/extended release dosage forms
as a tool for formulation screening, in setting dissolution specifications and as a
surrogate for bioequivalence testing.
IVIVC modeling involves three stages, which are,
 model development,
 model validation, and
 model application to different scenarios.
Developing a predictable IVIVC depends upon the complexity of the delivery

system, its formulation composition, method of manufacture, physicochemical
properties of the drug and the dissolution method.
Five correlation levels have been defined in the IVIVC FDA guidance. The
concept of correlation level is based upon the ability of the correlation to reflect the
complete plasma drug level-time profile which will result from administration of the
given dosage form.
1.5.1 Level A Correlation
This level of correlation is the highest category of correlation and represents a

point-to-point relationship between in-vitro dissolution rate and in-vivo input rate of
the drug from the dosage form (Kortejarvi et al. 2002). Generally, percent of drug
absorbed may be calculated by means of model dependent techniques such as
Wagner-Nelson procedure or Loo-Riegelman method or by model-independent
numerical de-convolution. The purpose of Level A correlation is to define a direct
relationship between in-vivo data such that measurement of in-vitro dissolution rate
alone is sufficient to determine the biopharmaceutical rate of the dosage form. In the
case of a level A correlation, an in-vitro dissolution curve can serve as a surrogate for
in-vivo performance (Malte Selch Larsen et al. 2015). Therefore, a change in
7
manufacturing site, method of manufacture, raw material supplies, minor formulation
modification, and even product strength using the same formulation can be justified
without the need for additional human studies.
1.5.2 Level B Correlation
A level B IVIVC utilizes the principles of statistical moment analysis. In this

level of correlation, the mean in-vitro dissolution time (MDT vitro) of the product is
compared to either mean in-vivo residence time (MRT) or the mean in-vivo
dissolution time (MDT vivo). Although a level B correlation uses all of the in-vitro
and in-vivo data, it is not considered to be a point-to-point correlation, since there are
a number of different in-vivo curves that will produce similar mean residence time
values. A level B correlation does not uniquely reflect the actual in-vivo plasma level
curves. Therefore, one cannot rely upon a level B correlation alone to justify
formulation modification, manufacturing site change, excipient source change, etc.,
1.5.3 Level C Correlation
In this level of correlation, one dissolution time point (t50%, t90%, etc.) is
compared to one mean pharmacokinetic parameter such as AUC, t max or Cmax.
Therefore, it represents a single point correlation and does not reflect the entire shape
of the plasma drug concentration curve, which is indeed a crucial factor that is a
good indicative of the performance of modified-release products. This is the weakest
level of correlation as partial relationship between absorption and dissolution is
established. Due to its obvious limitations, the usefulness of a Level C correlation is
limited in predicting in-vivo drug performance.
1.5.4 Multiple-level C correlation
A multiple level C correlation relates one or several pharmacokinetic

parameters of interest (Cmax, AUC, or any other suitable parameters) to the amount of
drug dissolved at several time points of the dissolution profile
(Gregor Bender et al. 2009). A multiple point level C correlation may be used to
justify a bio-waiver, provided that the correlation has been established over the entire
dissolution profile with one or more pharmacokinetic parameters of interest. A
8
relationship should be demonstrated at each time point at the same parameter such
that the effect on the in-vivo performance of any change in dissolution can be
assessed. If such a multiple level C correlation is achievable, then the development of
a level A correlation is also likely. A multiple Level C correlation should be based on
at least three dissolution time points covering the early, middle, and late stages of the
dissolution profile.
1.5.5 Level D correlation
Level D correlation is a rank order and qualitative analysis and is not

considered useful for regulatory purposes. It is not a formal correlation but serves as
an aid in the development of a formulation or processing procedure.
1.5.6 IVIVC Model Development
Development of an IVIVC consists of model development and model

validation (Rossi et al. 2007). A number of methods are available to probe the in-vivo
and in-vitro relationships. Among the earliest methods are the two stage de-
convolution methods that involve estimation of the in-vivo absorption profile from
the concentration-time data using the Wagner-Nelson or Loo-Riegelman methods
(Stage 1). Subsequent to the estimation of the in-vivo absorption profile, the
relationship with in-vitro dissolution is evaluated (Stage 2). More recently, one stage
convolution-based approaches for IVIVC have been investigated. The one stage
convolution methods compute the in-vivo absorption and simultaneously model the
in-vivo and in-vitro data. While the two stage method allows for systematic model
development, the one stage method obviates the need for the administration of an
intravenous, oral solution or immediate release bolus dose (Soto et al. 2010).
1.5.7 IVIVC Model Validation
The objective of any mathematical predictive tool is to successfully predict the

outcome (in-vivo profile) with a given model and test condition (in-vitro profile).
Integral to the model development exercise is model validation, which can be
accomplished using data from the formulations used to build the model (internal
validation) or using data obtained from a different (new) formulation (external
9
validation). While internal validation serves the purpose of providing the basis for
the acceptability of the model, external validation is superior and affords greater
“confidence” in the model.
Internal Validation
Using the IVIVC model, for each formulation, the relevant exposure
parameters (Cmax and AUC) are predicted and compared to the actual (observed)
values. The prediction errors are calculated (Jantratid et al. 2009).
The criteria set in the FDA guidance on IVIVC are as follows: For Cmax and
AUC, the mean absolute percent prediction error (% PE) should not exceed 10%, and
the prediction error for individual formulations should not exceed 15%.
External Validation
For establishing external predictability, the exposure parameters for a new

formulation are predicted using its in-vitro dissolution profile and the IVIVC model
and the predicted parameters are compared to the observed parameters (Gomez-
Mantilla et al. 2014). The prediction errors are computed as for the internal
validation. For Cmax and AUC, the prediction error for the external validation
formulation should not exceed 10%. A prediction error of 10% to 20% indicates
inconclusive predictability and illustrates the need for further study using additional
data sets. For drugs with narrow therapeutic index, external validation is required
despite acceptable internal validation, whereas internal validation is usually
sufficient with non-narrow therapeutic index drugs.
10
1.6 Drug Profile
For the present study, based on the solubility and permeability, Pregabalin and
Pramipexole were selected for developing IVIVC correlation.
1.6.1 Pregabalin
Chemical Name : (3S)-3-aminomethyl-5-methyl hexanoic acid.
Molecular Formula : C8H17NO2
Solubility : It is freely soluble in water and basic and acidic

aqueous solutions.
Molecular structure :
Mechanism of Action
An anticonvulsant activity of Pregabalin is mediated through an

alpha 2-delta site (an auxiliary subunit of voltage-gated calcium channels) in central
nervous system tissues (Charles et al. 2007). In-vitro Pregabalin reduces the calcium
dependent release of several neurotransmitters, possibly by modulation of calcium
channel function (Robert Lee et al. 2008).
Pharmacokinetics
Pregabalin is rapidly absorbed when administered on an empty stomach, peak

plasma concentrations occurring within one hour. Pregabalin oral bioavailability is
estimated to be greater than or equal to 90% and is independent of dose
(Daniel et al. 2007). The volume of distribution of Pregabalin for an orally
administered dose is approximately 0.56 L/kg and is not bound to plasma proteins.
11
Pregabalin undergoes negligible metabolism in humans (Meihua Rose Feng et al.
2001). Approximately 98% of the Pregabalin recovered in the urine was unchanged.
The N-methyl Pregabalin is the major metabolite (Thrasivoulos et al. 2008).

Pregabalin is eliminated from the systemic circulation primarily by renal excretion as
unchanged drug. Renal clearance of Pregabalin is 73 mL/minute.
1.6.2 Pramipexole
Chemical Name : (S)-2-amino-4,5,6,7-tetrahydro-6 (propylamino)

benzothiazole dihydrochloride monohydrate.
Molecular Formula : C10H17N3S·2HCl·H2O
Solubility : It is freely soluble in water
Molecular Structure:
Mechanism of Action
Pramipexole is used for the treatment of Parkinson’s disease

(Salin et al.2009;Chwieduk and Curran 2010 and Trond Kvernmo et al. 2006). It is a
non-ergot dopamine agonist with high relative in-vitro specificity and full intrinsic
activity at the D2 subfamily of dopamine receptors, binding with higher affinity to
D3 than to D2 or D4 receptor subtypes (Schapira et al. 2009;Grosset et al. 2005 and
Wolfram Eisenreich et al. 2010). It stimulates dopamine receptors in the striatum
(Dziedzicka-Wasylewska et al. 2001 and Rascol et al. 2010).
12
Pharmacokinetics
Pramipexole is rapidly absorbed, reaching peak concentrations in

approximately two hours (Poewe et al. 2009 and Dansirikul et al. 2009). The
absolute bioavailability of Pramipexole is greater than 90%, indicating that it is well
absorbed and undergoes little pre-systemic metabolism (Abib et al. 2012 and Hauser
et al. 2009). Pramipexole distributes into red blood cells (Jenner et al. 2009). Urinary
excretion is the major route of Pramipexole elimination, with 90% of a Pramipexole
dose recovered in urine, almost all as unchanged drug (Peter Jenner et al. 2009)
1.7 Scope and Object of the Present Study
IVIVC can be used in the development of new pharmaceuticals to reduce the

number of human studies during the formulation development, as the main objective
of an IVIVC is to serve as a surrogate for in-vivo bioavailability and to support
biowaivers. Thus the need for a tool to reliably correlate in-vitro and in-vivo drug
release data has exceedingly increased. Such a tool shortens the drug development
period, economizes the resources and leads to improved product quality. With the
proliferation of modified-release products, it becomes necessary to examine the
concept of IVIVC in greater depth. Investigations of IVIVC are increasingly
becoming an integral part of extended release drug development. There must be
some in-vitro means of assuring that each batch of the same product will perform
identically in-vivo.
The aim of IVIVC is thus to enable the dissolution test to be used as a

surrogate for bioequivalence studies. Dissolution testing of modified release
formulations poses many challenges. These challenges include developing and
validating the test method, ensuring that the method is appropriately discriminatory
and addressing the potential for an IVIVC.
Bioavailability and bioequivalence studies involve mathematical analyses of

plasma level vs time curves which permits the estimations of half-life, absorption and
excretion rates, extent of absorption (area under the curve), and other constants that
are useful in describing the fate of a given drug in an organism. It should be noted,
however, that neither bioavailability nor bioequivalence data could be generated
13
without analytic methodology to accurately measure drugs in biological fluids
(Kasawar and Farooqui 2010 ; Pathare et al. 2006 ;Reza Ahmadkhaniha et al. 2014
and Yi Lau Yau et al. 1996).
For the estimation of the drugs present in the biological fluid, LC-MS/MS
method is considered to be more suitable since these are the powerful and rugged
method and also extremely specific, linear, precise, accurate, sensitive and rapid.
The present study, therefore, aims to develop and validate IVIVC of

Pramipexole and Pregabalin. An extensive literature survey carried out by the
writer revealed that there are however, no reports of IVIVC.
In detail, the aims of the study described here were as set out below:
1.7.1 Bioequivalence study design and data handling
It is proposed to conduct a randomized, two treatment, two period, two

sequence, single dose, crossover bioequivalence study for the immediate release
formulation and modified release formulation in twenty four healthy, adult, male,
human subjects under fasting conditions.
1.7.2 Development of LC-MS/MS methods for the estimation of selected drugs

in plasma samples.
The main objective of this work is to develop rapid, selective and sensitive
LC-MS/MS methods that have short and simple extraction procedures, consume
small amounts of solvent and biological fluid for extraction and a short turn-around
time.
For the present study propose to optimize the following chromatographic conditions,
 Selection of Mass range

 Selection of initial separation conditions
 Nature of the stationary phase
 Nature of the mobile phase (pH, peak modifier, solvent strength, ratio and
flow rate)
14
 Sensitivity and
 Selection of internal standard
The developed method is also proposed to be validated using the various

validation parameters such as,
 Accuracy
 Precision
 Linearity and Range
 Limit of detection (LOD)/Limit of Quantitation (LOQ)
 Selectivity/specificity,
 Robustness/ruggedness
1.7.3 Development of in-vitro dissolution studies
It is proposed to carryout in-vitro dissolution methods for selected formulations

using five different mediums with two different agitations and calculates the
cumulative percentage release of the drug in the formulations.
1.7.4 In-vitro and in-vivo data analysis
After estimating the selected drugs in biological fluids the following

pharmacokinetic parameters are proposed to be calculated;
 Cmax Maximum Plasma Concentration

 tmax Time of Maximum Plasma Concentration
 AUC (0-t) Area under plasma concentrations time curve 0 to 24 hrs
 AUC (0- ∞) Area under plasma concentrations time curve 0 to  hrs

 t 1/2 Elimination half life
 keli Elimination constant
The In –transformed pharmacokinetic parameters are proposed to be analysed

by an Analysis of Variance (ANOVA) and two sided ‘T’ tests for 95% confidence
intervals for the difference between treatments.
15
From the cumulative percentage release of the drug in the formulations, it is
proposed to calculate dissolution rate constants and similarity factor for selected
formulations.
1.7.5 Development of IVIVC correlations
After carrying out an in-vivo and in-vitro data analysis, it is proposed to

develop level A IVIVC for the selected drugs and validate the correlation internally
and externally.
16
CHAPTER 2
LITERATURE REVIEW
Abib et al. (2012) reported comparative bioavailability studies of two

Pramipexole formulations in healthy volunteers after a single dose administration
under fasting conditions. The study was conducted with randomized two period
crossover design and 8 days wash out period in 48 volunteers of both sexes. Plasma
samples were obtained over a 48 hour interval. Pramipexole was analyzed by LC-
MS/MS in the presence of Tansulosina as internal standard. The mean ratio of
parameters Cmax and AUC0-t and 90% confidence intervals of correspondents were
calculated to determine the bioequivalence. The means AUC0-t for test and reference
formulation were 8201.90 pg.h/ml and 7891.56 pg.h/ml, for AUC0-inf were 8574.71
pg.h/ml and 8288.01 pg.h/ml and, for Cmax were 642.09 pg/ml and 633.94 pg/ml,
respectively. Geometric mean ratio was 103.61 % for AUC0-t, 103.13% for AUC0-inf
and 100.81% for Cmax. The 90 % confidence intervals were 98.02 - 109.51%, 97.95-
108.59%, 93.06-109.21%, respectively. Since the 90 % confidence intervals for Cmax,
AUC0-t and AUC0-inf were within 80–125% interval proposed by Food and Drug
Administration, it was concluded that test formulation was bioequivalent to reference
formulation according to both the rate and extent of absorption.
Alessandro Musenga et al. (2008) reported an analysis of the anti-parkinson

drug Pramipexole in human urine by capillary electrophoresis with laser-induced
fluorescence detection. Separation was carried out in uncoated fused silica capillaries
(75 μm internal diameter, 75.0 and 60.0 cm total and effective length, respectively),
with a background electrolyte composed of borate buffer (50 mM, pH 10.3), tetra
butyl ammonium bromide (30 mM), and acetone (15%, v/v). Applying a 20 kV
voltage, the electrophoretic run is completed within 12 min. A sample pre-treatment
17
procedure based on liquid/liquid extraction with ethyl acetate, followed by
derivatisation of Pramipexole with fluoresce in iso thiocyanate at pH 9, allows the
complete removal of biological interferences, with extraction yields always higher
than 94.5%. Method validation gave good linearity (r2 = 0.9992) in the 25.0–1000
ng/ml range; limit of detection and limit of quantitation were 10.0 and 25.0 ng/ml,
respectively; precision was ≤ 6.8 R.S.D%, accuracy expressed as recovery
percentage was > 90.0. The method was applied to the analysis of urine samples
from patients undergoing therapy with Pramipexole.
Chakshu Bhatia et al. (2012) reported the formulation and evaluation of

transdermal patch of Pregabalin. Matrix type transdermal drug delivery system
(TDDS) of Pregabalin was prepared by the solvent evaporation technique. Several
batches were prepared by using combination of HPMC and PVP; PVA and PVP;
Eudragit RL-100 and Eudragit RS-100; HPMC and EC in different ratios. Propylene
glycol was used as plasticizer and DMSO was incorporated as a permeation enhancer.
Formulated transdermal patches were charachterised for their physicochemical
parameters like thickness, weight variation, flatness, tensile strength, folding
endurance, moisture content, moisture uptake and drug content uniformity. Patches
were evaluated for their in-vitro drug release profile and ex-vivo skin permeation
studies. Patches were also subjected to stability studies and skin irritation studies to
determine their compatibility with skin. Formulation P1 containing HPMC and PVP
in the ratio of 3:1 and propylene glycol, 5% w/v and DMSO, 6% w/v was found to be
the most optimum formulation. P1 was also found to exhibit maximum
in-vitro drug release of about 81.70%. Result of evaluation studies revealed that
Pregabalin can be administered as a controlled drug delivery system to reduce
frequency of drug administration. But this hypothesis requires further confirmation
via in-vivo pharmacodynamic and pharmacokinetic studies in animal and human
models.
Charles et al. (2007) reported the pharmacology and mechanism of action of

Pregabalin. Pregabalin is approved in US and Europe for adjunctive therapy of
partial seizures in adults, and also has been approved for the treatment of pain from
diabetic neuropathy or post-herpetic neuralgia in adults. Recently, it has been
approved for treatment of anxiety disorders in Europe. Pregabalin is structurally
18
related to the antiepileptic drug gabapentin and the site of action of both drugs is
similar, the alpha2–delta (α2–δ) protein, an auxiliary subunit of voltage-gated
calcium channels. Pregabalin subtly reduces the synaptic release of several
neurotransmitters, apparently by binding to α2–δ subunits, and possibly accounting
for its actions in-vivo to reduce neuronal excitability and seizures. Several studies
indicate that the pharmacology of Pregabalin requires binding to α2–δ subunits,
including structure-activity analyses of compounds binding to α2–δ subunits and
pharmacology in mice deficient in binding at the α2–δ Type 1 protein. The
preclinical findings to date are consistent with a mechanism that may entail reduction
of abnormal neuronal excitability through reduced neurotransmitter release. This
review addresses the preclinical pharmacology of Pregabalin, and also the biology of
the high affinity binding site, and presumed site of action.
Chwieduk and Curran (2010) investigated the use of once-daily Pramipexole

extended release formulation for the treatment of early and advanced idiopathic
Parkinson’s disease. Once-daily Pramipexole ER and three times-daily Pramipexole
immediate release (IR) have similar exposure over 24 hours. The ER formulation is
associated with fewer fluctuations in plasma Pramipexole concentrations over this
period. Pramipexole ER improved the symptoms of Parkinson's disease in three well
designed trials in adults with early or advanced disease, as measured by changes
from baseline in the sum of the Unified Parkinson's Disease Rating Scale (UPDRS)
parts II and III subtotal scores. In a nine week study, the majority of patients with
early Parkinson's disease who were receiving stable Pramipexole IR treatment were
successfully switched to Pramipexole ER. Relative to placebo at week 18,
Pramipexole ER 0.375-4.5 mg (of the salt) once daily significantly decreased the
sum of the UPDRS parts II and III subtotal scores from baseline in two trials in
patients with early or advanced Parkinson's disease, and also reduced the percentage
of off-time during waking hours in patients with advanced disease. The efficacy of
Pramipexole ER was maintained after 33 weeks of treatment in the trials in patients
with early or advanced Parkinson's disease. Pramipexole ER was generally well
tolerated in patients with Parkinson's disease, with the rate of adverse events being
generally similar to that with Pramipexole IR.
19
Daniel et al. (2007) investigated the pharmacology, pharmacokinetics, efficacy,
and tolerability of Pregabalin. In 4 clinical trials in a total of 1068 patients with
diabetic peripheral neuropathy, the patients receiving Pregabalin 300 to 600 mg/d
had significantly greater improvement in mean pain scores than placebo recipients (P
≤ 0.01). Patients with post herpetic neuralgia receiving Pregabalin 450 to 600 mg/d
had significantly greater improvement in relief of pain and pain-related sleep
interference than placebo recipients (P ≤ 0.002). Patients with refractory partial-onset
seizures who received Pregabalin 150 to 600 mg/d (divided into 2 or 3 doses)
concomitantly with antiepileptic drugs had significantly fewer seizures than placebo
recipients (P ≤ 0.001). In the 3 studies that evaluated the efficacy of Pregabalin in
patients with GAD or SAD, the patients receiving Pregabalin 200 to 600 mg/d
(divided into 2 or 3 daily doses) had a significantly greater reduction in mean pain
scores on the Hamilton Anxiety Scale than placebo recipients (P ≤ 0.01). Across all
the reviewed clinical trials, the most commonly reported adverse effects (AEs) were
those affecting the central nervous system, including somnolence (≤ 50%), dizziness
(≤ 49%), and headache (≤ 29%). AEs resulted in withdrawal from the study in ≤ 32%
of patients. Pregabalin appears to be an effective therapy in patients with diabetic
peripheral neuropathy, post therapeutic neuralgia, and adults with refractory partial-
onset seizures. The available data suggest that Pregabalin may be beneficial as an
adjunctive therapy in adult patients with GAD or SAD.
Dansirikul et al. (2009) reported the relative bioavailability comparing

extended release Pramipexole and immediate release Pramipexole in identical daily
doses, titration steps, and titration intervals. In a phase III study on patients with
early Parkinson’s disease, plasma concentrations were determined before taking the
morning dose, and 1, 2, and 4 hours later after a stable final dose of Pramipexole had
been established. Aided by population-centered pharmacokinetic procedures, this
enabled the simulation of mean consecutive time profiles of Pramipexole plasma
concentrations in Parkinson patients. It illustrates the desired 24 hours steady
exposure in patients after the application of the extended-release Pramipexole tablet.
Dickinson et al. (2008) have reported a Quality by Design (QbD) in

pharmaceutical product development in a regulatory context and the process of
implementing such concepts in the drug approval process. This has the potential to
20
allow for a more flexible regulatory approach based on understanding and
optimization of how design of a product and its manufacturing process may affect
product quality. Thus, adding restrictions to manufacturing beyond what can be
motivated by clinical quality brings no benefits but only additional costs. This leads
to a challenge for biopharmaceutical scientists to link clinical product performance to
critical manufacturing attributes. In vitro dissolution testing is clearly a key tool for
this purpose and the present bioequivalence guidelines and biopharmaceutical
classification system (BCS) provides a platform for regulatory applications of in-
vitro dissolution as a marker for consistency in clinical outcomes. However, the
application of these concepts might need to be further developed in the context of
QbD to take advantage of the higher level of understanding that is implied and
displayed in regulatory documentation utilizing QbD concepts. Aspects that should
be considered include identification of rate limiting steps in the absorption process
that can be linked to pharmacokinetic variables and used for prediction of
bioavailability variables, in-vivo relevance of in-vitro dissolution test conditions and
performance/interpretation of specific bioavailability studies on critical
formulation/process variables. This article will give some examples and suggestions
how clinical relevance of dissolution testing can be achieved in the context of QbD
derived from a specific case study for a BCS II compound.
Dziedzicka et al. (2001) reported the indication of Pramipexole as selective

dopamine D2 receptor agonist for the symptomatic treatment of Parkinson’s disease,
either alone (without levodopa) or in combination with levodopa, that is, during the
entire progress of disease up to the advanced stage. Pramipexole is a full non
ergoline dopamine agonist with selective affinity for dopamine receptors of the D2
subfamily. This substance shows a 7 to 10 fold higher affinity to D3 than to D2
receptors. Pramipexole acts on presynaptic as well as on postsynaptic receptors. In
intact dopaminergic systems, though, the effects of Pramipexole are primarily stirred
via presynaptic auto receptors of the D3 and D2 type, thereby reducing the synthesis
and synaptic release of dopamine. Effects on postsynaptic receptors are only
observed at higher doses, and they are marked by prolonged latency periods. With
reduced dopamine release due to loss or damage of the presynaptic termini, however,
the postsynaptic D2 and D3 receptors are additionally and immediately stimulated.
21
Elena Soto et al. (2010) reported an in-vitro and in-vivo correlation model for
Pramipexole slow-release oral formulations. The IVIVC was developed based on
data from an immediate-release (IR) and three slow-release (SR) formulations of
Pramipexole, a fourth SR formulation was used for validation purposes. In vitro
dissolution profiles were obtained from all SR formulations. Fifteen volunteers
received all Pramipexole formulations in a randomized cross-over trial. Data were
analyzed using the population modeling approach. Dissolution profiles of the SR
formulations were described by the Weibull model. The pharmacokinetics of the IR
formulation was described by a two-compartment disposition model with first-order
absorption. Difference between the in-vivo and in-vitro estimates of the release rate
constants (k(d)) from the Weibull function suggests the release process occurs faster
in-vivo. Pharmacokinetic profiles for SR formulations were described based on the
in-vitro release model with k (d) increased in 0.058/h and the population
pharmacokinetic model developed from the IR formulation. A level A IVIVC was
established and evaluated for the Pramipexole SR formulations, which can be used in
the future as a surrogate to avoid certain bioequivalence studies.
Gomez-Mantilla et al. (2014) reported a statistical comparison of dissolution

profiles to predict the bioequivalence of extended release formulations. Appropriate
setting of dissolution specification of extended release (ER) formulations should
include precise definition of a multidimensional space of complex definition and
interpretation, including limits in dissolution parameters, lag time (t-lag), variability,
and goodness of fit. This study aimed to set dissolution specifications of ER by
developing drug-specific dissolution profile comparison tests (DPC tests) that are
able to detect differences in release profiles between ER formulations that represent a
lack of bioequivalence (BE). Dissolution profiles of test formulations were simulated
using the Weibull and Hill models. Differential equations based in-vivo and in-vitro
correlation (IVIVC) models were used to simulate plasma concentrations. BE trial
simulations were employed to find the formulations likely to be declared
bioequivalent and non-bioequivalent (BE space). Customization of DPC tests was
made by adjusting the delta of a recently described tolerated difference test (TDT) or
the limits of rejection of f2. Drug ka (especially if ka is small), formulation lag time
(t-lag), the number of subjects included in the BE studies, and the number of sampled
22
time points in the DPC test were the factors that affected the most these setups of
dissolution specifications. Another recently described DPC test, permutation test
(PT), showed excellent statistical power. All the formulations declared as similar
with PT were also bioequivalent. Similar case-specific studies may support the bio-
waiving of ER drug formulations based on customized DPC tests
Gregor Bender et al. (2009) reported the population pharmacokinetic model of

the Pregabalin-sildenafil interaction in rats, application of simulation to preclinical
PK- PD study design. Preliminary evidence has suggested a synergistic interaction
between Pregabalin and sildenafil for the treatment of neuropathic pain. The focus of
this study was to determine the influence of sildenafil on the pharmacokinetics (PK)
of Pregabalin with the objective of informing the design of a quantitative
pharmacodynamic (PD) study. The pharmacokinetics were determined in rats
following 2-hr intravenous infusions of Pregabalin at doses of 4 mg/kg/hr and
10 mg/kg/hr with and without a sildenafil bolus (2.2 mg) and steady state infusion
(12 mg/kg/hr for 6 h). This PK model was utilized in a preclinical trial simulation
with the aim of selecting the optimal sampling strategy to characterize the PK-PD
profile in a future study. Eight logistically feasible PK sampling strategies were
simulated in NONMEM and examined through trial simulation techniques. A two-
compartment population PK model best described Pregabalin pharmacokinetics.
Significant model covariates included either a binary effect of sildenafil
administration (30.2% decreases in clearance) or a concentration-dependent effect
due to sildenafil’s active metabolite. Analysis of simulations indicated that three
post-PD samples had the best cost/benefit ratio by providing a significant increase in
the precision (and minor improvement in bias) of both PK and PD parameters
compared with no PK sampling.
Grosset et al. (2005) reported a simple therapeutic regimen for Parkinson’s

disease. Patients will comply much more readily to with fewer applications than to
treatments with more frequent drug administration. A prolonged-action drug permits
the steady release of the active ingredient over 24 hours preventing the active
concentration from continuously surging on and surging off as commonly
encountered with multiple applications of the substance. An unvarying, efficient
level by continuous release over 24 hours will help avoid losses during the course of
23
the day. Pramipexole, the dopamine agonist that had been a non-depot formula so far,
which patients had to take t.i.d (3x) daily, could be turned into a controlled-release
drug to be administered once daily. This paper gives an overview regarding the
development and pharmacology of the extended-release form of Pramipexole, and it
summarizes the available clinical data on depot Pramipexole in the treatment of early
and advanced Parkinson’s disease.
Hauser et al. (2009) reported the clinical study on extended-release

Pramipexole in the early stage of PD using 259 patients who had been ill for about a
year. The study was carried on for 18 weeks with an initial titration period of 7
weeks). This controlled double-blind trial (extended-release Pramipexole:
immediate-release Pramipexole: placebo in the ratio of 2 : 2 : 1) also focused on the
effect by employing UPDRS II and III plus CGI-I and PGI-I. An improvement by 7.5
points was found in the group treated with immediate-release Pramipexole: the group
taking extended-release Pramipexole scored 7.4 points and the placebo group 2.7
points. Response rates were determined by GCI-I and PGI-I and were indicative of
improvements in week 18 and 33 for extended-release Pramipexole in 37/35.6%. The
result was 48/23.8% under immediate-release Pramipexole and 18/12% under
placebo. The authors concluded that both Pramipexole formulas were safe and
superior to placebo, aside from being well tolerated. The efficacy of extended-release
Pramipexole was said to be similar to immediate-release Pramipexole with
comparable adverse effects.
Hauser et al. (2010) reported the randomized, double-blind, multicenter

evaluation of Pramipexole extended release once daily in early Parkinson's disease.
The objective of this study was to evaluate the efficacy and safety of Pramipexole
extended release (ER) administered once daily in early Parkinson's disease (PD).
Pramipexole immediate release (IR) administered three times daily (t.i.d) is an
efficacious and generally well-tolerated treatment for PD. A Pramipexole ER
formulation is now available. We performed a randomized, double-blind, placebo
and active comparator-controlled trial in subjects with early PD. The primary
efficacy and safety evaluation of Pramipexole ER compared with placebo took place
at week 18. Two hundred fifty-nine subjects were randomized 2:2:1 to treatment with
Pramipexole ER once daily, Pramipexole IR t.i.d, or placebo. Levodopa rescue was
24
required by 7 subjects in the placebo group (14%), 3 subjects in the Pramipexole ER
group (2.9%, P = 0.0160), and 1 subject in the Pramipexole IR group (1.0%,
P = 0.0017). Adjusted mean [standard error (SE)] change in Unified Parkinson
Disease Rating Scale (UPDRS) II [activities of daily living (ADL)] + III (motor)
scores from baseline to week 18, including post-levodopa rescue evaluations, was -
5.1 (1.3) in the placebo group, -8.1 (1.1) in the Pramipexole ER group (P = 0.0282),
and -8.4 (1.1) in the Pramipexole IR group (P = 0.0153). Adjusted mean (SE) change
in UPDRS ADL + motor scores, censoring post-levodopa rescue data, was -2.7 (1.3)
in the placebo group, -7.4 (1.1) in the Pramipexole ER group (P = 0.0010), and -7.5
(1.1) in the Pramipexole IR group (P = 0.0006). Adverse events more common with
Pramipexole ER than placebo included somnolence, nausea, constipation, and fatigue.
Pramipexole ER administered once daily was demonstrated to be efficacious
compared with placebo and provided similar efficacy and tolerability as Pramipexole
IR administered t.i.d.
Jantratid et al. (2009) reported an application of bio-relevant dissolution tests

to the prediction of in-vivo performance of an oral modified-release (MR) dosage
form. In vitro dissolution of MR diclofenac sodium pellets containing 100 mg active
ingredient was evaluated under simulated pre- and postprandial conditions using
USP Apparatus 3 (reciprocating cylinder, Bio-Dis) and 4 (flow-through cell) and
results compared with compendial methods using USP Apparatus 1 (basket) and 2
(paddle). In-vivo, the effects of food on the absorption of diclofenac sodium from the
pellet dosage form was investigated by administering the product to 16 healthy
volunteers pre- and postprandial in a crossover-design study. The in-vitro results
were compared with the in-vivo data by means of Level A in-vitro and in-vivo
correlation (IVIVC) and Weibull distribution analysis. The compendial dissolution
tests were not able to predict food effects. The bio relevant dissolution tests predicted
correctly that the release (and hence absorption) of diclofenac sodium would be
slower in the fed state than in the fasted state. No significant differences in extent of
absorption due to changes in extent of release were predicted or observed. The
results demonstrate good correlations between in-vitro drug release and in-vivo drug
absorption in both pre and post-prandial states using the bio relevant dissolution test
methods.
25
Jenner et al. (2009) reported a clinical phase I study for Pramipexole matrix
tablets. In a crossover study with in healthy male subjects, the pharmacokinetic
properties were investigated in the steady state. An optimal formula for further
clinical development was supposed to be identified based on the a maximum plasma
concentration, in the steady state (day 4 of each extended-release Pramipexole
therapy), that does not exceed the one obtained after immediate-release Pramipexole
t.i.d., and a minimum plasma concentration not less than the one observed after the
immediate-release formula applied t.i.d., a peak/trough fluctuation (PTF) over
24 hours that is smaller than or comparable to the one following immediate-release
Pramipexole,a bioavailability not less than ≥75% in the steady state that compares
with the immediate-release formula, no incidence of irregular release (“dose-
dumping” defined as a not more than 80% dose absorption within 4 hours after
application). One matrix tablet met all of the above criteria and was chosen for
further clinical development. It consists of an innovative hydrogel formula. With this
kind of tablet, the drug substance is homogeneously embedded in a polymeric matrix.
The slow down (= extended release) of the agent is accomplished by combining three
polymers hypromellose, corn starch, and carbomer. The interaction of these three
polymers warrants the uniform and long-lasting release of the agent over 24 hours.
Upon contact with digestive juices, the drug substance is first dissolved at the surface.
The matrix then starts swelling, forming a viscous jelly that releases Pramipexole
consistently over 24 hours. Because of Pramipexole’s good solubility independent
from pH-value, the drug substance is dissolved from the matrix also in deeper
intestinal segments and is ready for absorption. In patients with Parkinson's disease,
once-daily use of an ER formulation may improve the convenience of treatment
relative to the IR formulation taken 3 times daily and thus increase compliance.
Jose david et al. (2013) reported the permutation test (PT) and tolerated
difference test (TDT for statistical comparison of dissolution profiles. The most
popular way of comparing oral solid forms of drug formulations from different
batches or manufacturers is through dissolution profile comparison. Usually, a
similarity factor known as (f2) is employed; however, the level of confidence
associated with this method is uncertain and its statistical power is low. In addition,
f2 lacks the flexibility needed to perform in special scenarios. In this study two new
26
statistical tests based on non-parametrical Permutation Test theory are described, the
Permutation Test (PT), which is very restrictive to confer similarity, and the
Tolerated Difference Test (TDT), which has flexible restrictedness to confer
similarity, are described and compared to f2. The statistical power and robustness of
the tests were analyzed by simulation using the Higuchi, Korsmayer, Peppas and
Weibull dissolution models. Several batches of oral solid forms were simulated while
varying the velocity of dissolution (from 30 min to 300 min to dissolve 85% of the
total content) and the variability within each batch (CV 2–30%). For levels of
variability below 10% the new tests exhibited better statistical power than f2 and
equal or better robustness than f2. TDT can also be modified to distinguish different
levels of similarity and can be employed to obtain customized comparisons for
specific drugs. In conclusion, two new methods, more versatile and with a stronger
statistical basis than f2, are described and proposed as viable alternatives to that
method. Additionally, an optimized time sampling strategy and an experimental
design-driven strategy for performing dissolution profile comparisons are described.
Jost (2010) reported the clinical studies of Pramipexole Immediate-release and

extended-release tablets. The substance itself is unchanged, meaning there is an
identical receptor profile, identical efficacy, and identical receptor binding. The half-
life of the agent is also the same, but the continuous release from the depot tablet
results in an overall prolonged plasma half-life. Any of the accepted statements
linked to immediate-release Pramipexole also apply to extended-release
Pramipexole-except for the daily doses required. Extended-release Pramipexole can
be used in both early and late Parkinson’s disease. A switch can take place overnight,
the ratio being 1:1, for example, immediate-release Pramipexole mg (base) are
consistent with 2.1 mg extended-release Pramipexole. In most cases, the patient is
not going to be affected by this switch. More efficaciousness, increased dyskinesias
or undesired effects are not to be expected. In a few cases, the effect may be less,
which would require a dose adjustment. Some patients appreciate the stimulating
effect of immediate-release Pramipexole with a rapid afflux and higher peak and for
this reason, they prefer fast-release Pramipexole. So far, there have been no insights
regarding any increased individual side effect owing to the use of extended-release
Pramipexole. Reduction of plasma peaks might even result in decreased side effects.
27
Since the introduction of extended-release Pramipexole, and in consideration of
pharmacologic and clinical aspects, it has been recommended putting the patient on
extended-release Pramipexole in a new approach. All patients who have been under
immediate-release Pramipexole may furthermore be switched to extended-release
Pramipexole overnight. The inauguration of extended release Pramipexole is to be
regarded as another important option in the drug treatment of Parkinson’s disease.
Kasawar and Farooqui (2010) reported a simple, precise, specific, and accurate
reverse phase HPLC method for the determination of Pregabalin in capsule dosage
form. The chromatography was set on Hypersil BDS, C8, 150 × 4.6 mm, 5 μm
column using photodiode array detector. The mobile phase consisting of phosphate
buffer pH 6.9 and acetonitrile in the ratio of 95:05 with flow rate of1 ml/min. The
method was validated according to ICH guidelines with respect to specificity,
linearity, accuracy, precision and robustness. Lower limit of quantification is 0.6
mg/l. The Pregabalin sample solution was found to be stable at room temperature for
about 26 hour. The influence of fluctuating temperature and humidity conditions that
might occur during transportation of drug products can be estimated using stability
analysis of a drug. The assay is calibrated over the range of 500 μg/ml to 1500 μg/ml
and without derivatisation of analyte also the proposed method can quantify (LOQ)
at least0.61 μg/ml and can detect (LOD) at least 0.23 μg/ml. The developed method
has been validated showing the method accuracy, linearity and reproducibility.
Validation procedure was mainly based on the ICH guideline.
Koenen-Bergmann et al. (2008) reported a level A in-vitro and in-vivo

correlation (IVIVC) with the chosen formula was established in the course that was
able to predict a complete plasma concentration profile in humans by in-vitro
solubility charts with adequate precision. This IVIVC also showed that Pramipexole
of the extended-release formula was steadily absorbed all through the entire intestine
including the colon. The variability among other individuals in this process was
rather low and not influenced by other factors food for instance.
Kortejarvi et al. (2002) reported the different levels of correlation between in-
vitro release and in-vivo absorption rate for four modified-release levosimendan
capsule formulations. Differences and similarities in the in-vitro dissolution curves
28
were compared with pharmacokinetic parameters describing absorption rate.
Formulations F, G, H and I differed in the amounts of the delaying excipients alginic
acid and HPMC. In vitro release rate was studied by the USP basket method using
the following conditions: pH 5.8 or 7.4 and a rotation speed of 50 or 100 rpm. In-vivo
bioavailability was tested in nine healthy male volunteers and the fractions absorbed
were calculated by the Wagner–Nelson method. Dissolution conditions pH 5.8 and a
rotation speed of 100 rpm predicted best the similarities and differences in absorption
rates among different formulations, and levels C and B correlation coefficients were
0.85 and 0.97, respectively. For formulation Hlevel A correlation (r=0.997) was
found when in-vitro lag time was 0.2 h and time scale factor 1.9. This study indicated
that dissolution tests developed can be used as a surrogate for human bioequivalence
studies, for development processes of final commercial products, to ensure batch to
batch bioequivalence and in the future possible scale-up and post approval change
cases for modified release levosimendan formulation H.
Lake et al. (1999) have reported a dissolution test method for carbamazepine
immediate release tablets, giving the best in-vitro-in-vivo correlations and to
determine the potential of this method as an estimate for bioequivalence testing. Four
200 mg carbamazepine products which are sold on the Dutch market, covering the
innovator and three generic products were selected. They had been tested in a
randomised, four way cross-over bioavailability study in healthy volunteers. Their
dissolution rate behavior in-vitro was investigated using 1% sodium lauryl sulphate
in water and 0.1 M/l Hydrochloric acid in water. In the bioavailability study these
products had shown no large differences in the extent of absorption but large
differences in absorption rate. The products now also showed large differences in
dissolution rate in-vitro in both dissolution media, the rank order being the same as
for the absorption rate. It was concluded that the absorption rate in-vivo depends on
the dissolution rate in-vivo. Level C' IVIVC according to the USP were optimized by
plotting percentages dissolved on selected time points (D values) or their reciprocals
(1/D values), against several pharmacokinetic parameters primarily related to the
absorption phase and against AUC. In this way for each IVIVC the optimum D or
1/D value, was calculated. For both media no meaningful IVIVC were obtained with
AUC, but favorable IVIVC were obtained with the parameters primarily related to
29
the absorption phase. In the bioavailability study, among the pharmacokinetic
characteristics primarily related to the absorption phase, Cmax was the most promising
in expressing rate of absorption in bioequivalence testing in single dose studies with
carbamazepine immediate release tablets.
Malte Selch Larsen et al. (2015) reported an in-vivo and in-vitro Evaluations of
Intestinal Gabapentin. Gabapentin exhibits saturable absorption kinetics, however, it
remains unclear which transporters that are involved in the intestinal transport of
gabapentin. Thus, the aim of the current study was to explore the mechanistic
influence of transporters on the intestinal absorption of Gabapentin by both in-vivo
and in-vitro investigations. Gabapentin showed dose-dependent oral absorption
kinetics and dose-independent disposition kinetics. Co-application of BCH inhibited
intestinal absorption in-vivo and apical uptake in-vitro, whereas no effect was
observed following co-application of L-lysine. The present study shows for the first
time that BCH was capable of inhibiting intestinal absorption of gabapentin in-vivo.
Furthermore, in Caco-2 cell experiments BCH inhibited apical uptake of gabapentin.
These findings may imply that a BCH-sensitive transport-system was involved in the
apical and possibly the basolateral transport of gabapentin across the intestinal wall.
Meihua Rose Feng et al. (2001) reported a brain microdialysis and PK/PD
correlation of Pregabalin in rats. In this report, blood-brain barrier (BBB) influx and
efflux of PGB were investigated with microdialysis at efficacious doses in rats. BBB
influx (CLin) and efflux (CLout) permeability for Pregabalin were 4.8 and
37.2 μL/min/g brain, respectively, following an intravenous infusion to rats. The
results indicate that PGB is brain pentrable, supporting its anti-epilepsy and other
CNS pharmacology. Significant anticonvulsant action of PGB was detected between
2 and 8 hr post oral dose, which is lag behind ECF drug concentrations lees.
A PK/PD link model was used to describe the counter-clockwise hysteresis
relationship between Pregabalin brain ECF concentration and the anticonvulsant
effect in rats. The resulting Ce (concentration in effect compartment) versus effect
profile exhibits a sigmoidal curve and the calculated ECe50 and Keo values were
95.3 ng/mL and 0.0092/min, respectively. The small Keo value suggests that the
effect is not directly proportional to the amount of Pregabalin in the ECF
compartment possibly due to inherent delay.
30
Pathare et al. (2006) reported a validated chiral liquid chromatographic method
for the enantiomeric separation of Pramipexole dihydrochloride monohydrate. A
chiral liquid chromatographic method was developed for the enantiomeric resolution
of Pramipexole dihydrochloride monohydrate. The enantiomers of Pramipexole
dihydrochloride monohydrate were resolved on a Chiralpak AD (250 mm × 4.6 mm,
10 μm) column using a mobile phase system containing
n-hexane:ethanol:diethylamine (70:30:0.1, v/v/v). The resolution between the
enantiomers was found not less than eight. The presence of diethylamine in the
mobile phase has played an important role in enhancing chromatographic efficiency
and resolution between the enantiomers. The developed method was extensively
validated and proved to be robust. The limit of detection and limit of quantification
of (R)-enantiomer were found to be 300 and 900 ng/ml, respectively for 20 μl
injection volume. The percentage recovery of (R)-enantiomer was ranged from 97.3
to 102.0 in bulk drug samples of Pramipexole dihydrochloride monohydrate.
Pramipexole dihydrochloride monohydrate sample solution and mobile phase were
found to be stable for at least 48 h. The proposed method was found to be suitable
and accurate for the quantitative determination of (R)-enantiomer in bulk drugs.
Peter Jenner et al. (2009) investigated the pharmacokinetic properties of a

variety of prototypes for a once daily extended release (ER) formulation of
Pramipexole and to further characterize the prototype whose pharmacokinetics best
matched those of the IR formulation. Three Phase I studies were conducted, all in
2
healthy adult men aged ≤ 50 years with a body mass index of 18.5 to 29.9 kg/m . In
the first study, 7 prototypes of a once-daily ER formulation with various release
properties, including rate and pH dependence, were compared with the IR
formulation taken 3 times daily to identify the optimal pharmacokinetic resemblance
based on predefined criteria derived from plasma AUC0−24h, Cmax, and Cmin. In the
second study, a level A in-vitro/in-vivo correlation (IVIVC) suitable for predicting an
entire in-vivo bioavailability time course based on in-vitro dissolution was
established and validated, and the single-dose pharmacokinetics of the optimal ER
formulation identified in the first study were analyzed for food effect. In the third
study, steady-state pharmacokinetics of the optimal ER formulation were assessed
across a range of Pramipexole doses (0.375–4.5 mg/d), including investigation of the
31
food effect at steady state for the highest dose. Tolerability was assessed throughout
all studies based on physical examinations, laboratory measurements, and adverse
events (AEs). In these studies in healthy male volunteers, an ER Pramipexole
formulation was identified that resembled the IR formulation in terms of both
pharmacokinetics and tolerability. In patients with Parkinson's disease, once-daily
use of an ER formulation may improve the convenience of treatment relative to the
IR formulation taken 3 times daily and thus increase compliance.
Poewe et al. (2009) reported the effectiveness of extended-release Pramipexole

in the early stage of disease. this work group scrutinized 539 patients who had had
symptoms for averagely 12 months and treated them with immediate-release
Pramipexole by 3-arm design: depot Pramipexole: placebo in a ratio of 2 : 2 : 1 for 26
weeks. The effectiveness was checked upon after 18 weeks, the (non-inferiority)
after 33 weeks. UPDRS II and III besides CGI-I (Clinical Global Impression
Improvement) and PGI-I (Patient Global Impression Improvement) served as
parameters. The two goals of the study were attained, that is, proof of the superiority
versus placebo in week 18 and proof of the non-inferiority of extended-release
Pramipexole in week 33 (−8.6 versus −8.8 scores). Relevant differences in respect of
CGI-I and PGI-I were not seen (46.1/33.3 versus 43.3/34.4%), the incidence and
severity of side effects did not differ either.
Pui Shan Chana et al. (2014) reported an in-vitro transport assay of rufinamide,
Pregabalin, and zonisamide by human P-glycoprotein. P-glycoprotein (Pgp) export
may contribute to antiepileptic drug (AED) resistance. Concentration equilibrium
transport assays (CETA) measure permeable-drug transport. Zonisamide, Pregabalin,
and rufinamide are not Pgp substrates in CETA. Epilepsy is resistant to treatment
with antiepileptic drugs (AEDs) in about one third of epilepsy patients. AED export
by P-glycoprotein (Pgp) overexpressed in the blood–brain barrier may contribute to
AED resistance. The Pgp transport status of many of the recently approved AEDs
remains unknown. We investigated whether several new AEDs - zonisamide (ZNS),
Pregabalin (PGB), and rufinamide (RFM) - are human Pgp substrates. MDCKII and
LLC-PK1 cells transfected with the human MDR1 gene, which encodes the Pgp
protein, were used in concentration equilibrium transport assays (CETA) to
determine the substrate status of ZNS, PGB, and RFM. For each drug, an equal
32
concentration was added to apical and basal chambers, and the concentration in both
chambers was measured up to 4 hours. RFM, ZNS, and PGB were not transported by
MDR1-transfected cells from basolateral to apical sides in CETA. ZNS, RFM, and
PGB are not substrates of human Pgp. These data suggest that resistance to these
drugs may not be attributed to increased Pgp activity in resistant patients.
Quinones et al. (2010) reported a comparative bioavailability study of two

formulations of Pregabalin in healthy Chilean volunteers. The aim of this study was
to compare the pharmacokinetic parameters between two brands of Pregabalin in
healthy Chilean volunteers. A randomized, single-dose, two-period, two-sequence,
crossover study design with a 2-week washout period was conducted in healthy
Chilean males. Plasma samples were collected over a 12-hour period after
administration of 150 mg Pregabalin in each period. A validated ultra-performance
liquid chromatography with positive ionization mass spectrometric detection method
was used to analyze Pregabalin concentration in plasma. Pharmacokinetic parameters
were determined using a non-compartmental method. Bioequivalence between the
test and reference products was determined when the ratio for the 90% confidence
intervals (CIs) of the difference in the means of the log-transformed area under the
curve (AUC0-t, AUC0-∞, and maximum concentration Cmax) of the two products were
within 0.80 and 1.25. These results suggest that both products are bioequivalent and
can be used as interchangeable options in the clinical setting.
Rascol et al. (2009) investigated the extent of switching a patient from

immediate-release Pramipexole to extended-release Pramipexole. This was a
double-blind study,156 patients were switched who had received invariably more
than 1.05 mg (base) Pramipexole for at least 4 weeks (on the average 1.9 mg (base)
immediate-release Pramipexole). Changes up to 15% in the UPDRS (Unified
Parkinson Disease Rating Scale) II and III were rated noninferior. 84.5% were
successfully changed overnight (87 out of 103 patients). The proof on noninferiority
failed although extended-release Pramipexole turned out even better per UPDRS and
CGI-I (Clinical Global Impression Improvement), responder rate (87. 4 versus
78.8%). There was no difference in side effects.
33
Rascol et al. (2010) reported the efficacy, safety, and tolerability of overnight
switching from immediate to once daily extended release Pramipexole in early
Parkinson's disease. Non-fluctuating patients on Pramipexole IR three times daily,
alone or with levodopa, for early PD were randomly switched overnight to double
blind IR three times daily (N = 52) or ER once-daily (N = 104) at initially unchanged
daily dosage. Successful switching (defined as no worsening >15% of baseline
UPDRS II+III score and no drug-related adverse event withdrawal) was assessed at 9
weeks, after optional dosage adjustments (primary endpoint), and at 4 weeks, before
adjustment. Other secondary endpoints included adjusted mean changes from
baseline in UPDRS scores and proportion of responders based on Clinical or Patient
Global Impression (CGI/PGI). Absolute difference between percentage of successful
switch to ER versus IR was tested for ER noninferiority, defined as a 95%
confidence-interval lower bound not exceeding -15%. At 9 weeks, 84.5% of the ER
group had been successfully switched, versus 94.2% for IR. Noninferiority was not
demonstrated, with a difference of -9.76% (95% CI: [-18.81%, +1.66%]). At 4 weeks,
81.6 % of the ER group had been successfully switched, versus 92.3% for IR, a
difference of -10.75 % (95% CI: [-20.51%, +1.48%]). UPDRS changes and CGI/PGI
analyses showed no differences between the groups. Both formulations were safe and
well tolerated. Pramipexole ER was not equivalent to IR, but the difference was
marginal. The fact that >80% of the patients successfully switched overnight at
unchanged dosage shows that this practice was feasible in most patients.
Reza Ahmadkhaniha et al. (2014) reported a validated HPLC method for

quantification of Pregabalin in human plasma using 1-fluoro-2,4-dinitrobenzene as
derivatization agent. In this study, a sensitive, simple, and reliable HPLC method for
quantification of Pregabalin in human plasma was developed and validated. 1-
Fluoro-2,4-dinitrobenzene was used as pre-column derivatization agent. For
chromatography, an analytical reversed phase (C18) column and a mixture of
Na2HPO4 10 mM (pH 8.0)—methanol (35 : 65 v/v) were used as stationary and
mobile phase, respectively. Detection was performed using a UV detector tuned at
360 nm. The linearity of the method was tested over the concentration range
1–4500 ng/mL in 500 μL of human plasma and satisfactory results were obtained
(r2 >0.999). The method showed good precision and accuracy in terms of within
34
between days relative standard deviations and percent deviation from nominated
values (in the range of 4.3–12.7% and 2.6–8.0%, resp.). The limit of quantification of
the method was found to be 1 ng/mL which is better than previously reported method
and indicates its potential application for sensitive bio-analysis.
Robert Lee et al. (2008) reported the possible heart failure exacerbation
associated with Pregabalin. Regabalin is an analog of the neurotransmitter γ-
aminobutyric acid that exhibits analgesic, anticonvulsant, and anxiolytic properties.
Owing to its pharmacologic properties, the drug has been used worldwide in the
management of diabetic peripheral neuropathy, post therapeutic neuralgia,
generalized anxiety disorder, and social anxiety disorder. Although central nervous
system disturbances account for the majority of Pregabalin's side effects, dose-
dependent peripheral edema and weight gain have also been reported. Recently, three
case reports have been published documenting a possible association between
Pregabalin administration and chronic heart failure decompensation. They presented
three additional cases of possible heart failure exacerbation in patients with clinically
stable heart failure who received Pregabalin for neuropathic pain. Additionally,
literature was reviewed addressing the nature and possible etiology for this adverse
effect.
Rossi et al. (2007) have reported a dissolution procedure for Ritonavir soft
gelatin capsules (Norvir) based on in-vivo data. Several conditions such as medium
composition, pH, surfactant concentration and rotation speed were evaluated. The
method was carried out using the same batch of Norvir used in a bioequivalence
study and the in-vivo data were used to select the best dissolution test conditions
based on in-vitro-in-vivo correlation (IVIVC). The dissolution test was validated
using a high-performance liquid chromatographic method (HPLC). For this
formulation, the best dissolution conditions were achieved using paddle, 900 ml of
medium containing water with 0.7% (w/v) of sodium lauryl sulfate at a rotation
speed of 25 rpm. Under these conditions a significant linear relationship between
fraction of ritonavir absorbed and dissolved was obtained (R(2)=0.993) and a level A
IVIVC was established. In the HPLC method a relative standard deviation for intra-
day precision was <1.6% and for inter-day precision was <1.4%. Accuracy was from
98.5% to 101.6% over the concentration range required for the dissolution test
35
(4.0-124.0 μg/ml). Both the HPLC method and the dissolution test are validated and
could be used to evaluate the dissolution profile of ritonavir soft gelatin capsules.
Salin et al. (2009) reported the 3 arm study using 101 patients for 33 weeks.
84 patients were evaluated at the end of the study, 35 of them being on extended-
release Pramipexole, 31 under immediate-release Pramipexole and 18 under placebo.
Changes of UPDRS II and III were compared with week 18 to 33. The group taking
extended-release Pramipexole scored higher by a change of 0.3 (−11.8 versus −11.5
points). The group treated with immediate-release Pramipexole presented with a
change of −11.9 points. The change in the placebo group amounted to −4.2 points up
to week 18 and −2.7 points to week 33. This means that in both drug groups the
effect was maintained between week 18 and 33 whereas a decline of 1.5 points in
was recorded in the placebo group. Parallel data on response were also collected.
PGI interestingly enough revealed a slight superiority of the extended-release
formula compared to immediate-release Pramipexole in week 18 and 33 (45.7/42.9
versus 35.5/41.9%), evaluation of CGI disclosed a superiority of the immediate-
release formula in week 18 and 33 (64.5/51.6 versus 46.9/40.6%).
Schapira et al. (2009) reported the Effectiveness of Extended-Release

Pramipexole in Advanced Parkinson’s Disease. This examination included 517
patients, who had been diseased for about 6 years, treated already by approximately
600 mg levodopa. Used in a 3-arm design were immediate-release Pramipexole:
extended-release pramipexol: placebo in a ratio of 1 : 1 : 1. The superiority of
Pramipexole was studied after 18 weeks by UPDRS part II and III (Figure 6) and by
off periods. The effectiveness was rechecked then 33 weeks later. Extended-release
Pramipexole proved superior after 18 weeks, reflected by 4.9 points (−11.0 versus
6.1) in UPDRS and 0.7 (−2.1 versus 1.4) hours off time as opposed to placebo. There
were no relevant differences between immediate-release Pramipexole and extended-
release Pramipexole. The great effect of placebo was astonishing. Giddiness and
vomiting occurred less under extended-relapse Pramipexole than under immediate-
release Pramipexole.
Soto et al. (2010) reported the population in-vitro and in-vivo correlation model
for Pramipexole slow-release oral formulations. The IVIVC was developed based on
36
data from an immediate-release (IR) and three slow-release (SR) formulations of
Pramipexole; a fourth SR formulation was used for validation purposes. In vitro
dissolution profiles were obtained from all SR formulations. Fifteen volunteers
received all Pramipexole formulations in a randomized cross-over trial. Data were
analyzed using the population modelling approach as implemented in NONMEM VI.
Dissolution profiles of the SR formulations were described by the Weibull model.
The pharmacokinetics of the IR formulation were described by a two-compartment
disposition model with first-order absorption. Difference between the in-vivo and in-
vitro estimates of the release rate constants (k(d)) from the Weibull function suggests
the release process occurs faster in-vivo. Pharmacokinetic profiles for SR
formulations were described based on the in-vitro release model with k(d) increased
in0.058/h and the population pharmacokinetic model developed from the IR
formulation. A level A IVIVC was established and evaluated for the Pramipexole SR
formulations, which can be used in the future as a surrogate to avoid certain
bioequivalence studies.
Thrasivoulos et al. (2008) reported the efficacy of Pregabalin and gabapentin

for neuropathic pain in spinal-cord injury. Spinal-cord injury (SCI) is a leading cause
of neuropathic pain (NP). Current pharmaceutical treatments for NP in SCI patients
are not effective. Two promising options are gabapentin (GP) and Pregabalin (PB).
Their predominant mechanism of action is believed to be the inhibition of calcium
currents, leading in turn to reduced neurotransmitter release and attenuation of
postsynaptic excitability. This could explain much of their efficacy in the treatment
of both seizure disorders and pain syndromes. However, evidence for their efficacy
in attenuating NP of SCI is still controversial. There is a lack of studies comparing
GP and PB in treating NP in SCI. This systematic review indicates the possible
efficacy of PB and GP in NP of SCI. Recommendations for future research to inform
clinical practice should include cost-effectiveness studies and dose-response analysis
in order to determine the schema employed and the duration of treatment.
Trond Kvernmo et al. (2006) reported a dopamine agonists (DAs), which can
be categorized as ergot derived and non-ergot derived, are used in the treatment of
Parkinson's disease. Relevant articles were identified through a search of MEDLINE
using the terms dopamine agonists (or each individual drug name) and
37
pharmacokinetics, metabolism, drug-drug interaction, interactions, CYP450, fibrosis,
valvular heart disease, tremor, clinical trials, reviews, and meta-analyses. Abstracts
from recent sessions of the International Congress of Parkinson's Disease and
Movement Disorders were also examined. Clinical studies with <20 patients overall
or <10 patients per treatment group in the final analysis were excluded. All DAs that
were graded at least possibly useful with respect to at least 3 of 4 items connected to
the treatment/prevention of motor symptoms/complications in the most recent
evidence-based medical review update were included. This resulted in a focus on the
ergot-derived DAs Bromocriptine, Cabergoline, and Pergolide, and the non-ergot-
derived DAs Pramipexole and Ropinirole. As reflected in the results of the clinical
trials included in this review, dyskinesia associated with DA therapy may be linked
to stimulation of the D1 receptor. Fibrosis (including VHD) seemed to be a class
effect of the ergot-derived DAs. Each of the DAs except Pramipexole has the
potential to interact with other drugs via the CYP enzyme system.
Wolfram Eisenreich et al. (2010) reported a novel treatment option in

Parkinson's disease using Pramipexole extended release formulations. Based on
numerous clinical data and vast experiences, efficacy and safety profiles of this non-
ergoline dopamine agonist are well characterized. Since October 2009, an extended-
release formulation of Pramipexole has been available for symptomatic treatment of
Parkinson's disease. Pramipexole administration can be cut down from three times to
once a day due to the newly developed extended-release formulation. This is
considerable progress in regard to minimizing pill burden and enhancing compliance.
Moreover, the 24 h continuous drug release of the once-daily extended-release
formulation results in fewer fluctuations in plasma concentrations over time
compared to immediate-release Pramipexole, given three times daily. The present
study summarizes pharmacokinetics and all essential pharmacological and clinical
characteristics of the extended-release formulation. In addition, it provides all study
data, available so far, with regard to transition and de-novo administration of
extended-release formulation for patients with Parkinson's disease. It further
compares efficacy and safety data of immediate-release Pramipexole with the
extended-release formulation of Pramipexole.
38
Yi Lau Yau et al. (1996) reported a sensitive and selective high-performance
liquid chromatographic (HPLC) method was developed for the determination of
Pramipexole in human plasma and urine. Plasma/urine is made alkaline before
Pramipexole and BHT-920 (internal standard) are extracted by ethyl ether and back-
extracted with a solution that contains heptanes sulfonic acid. Separation is achieved
by ion-pair chromatography on a Zorbax Rx C8 column with electrochemical
detection at 0.6 V for plasma and ultraviolet detection at 286 nm for urine. The
retention times of Pramipexole and internal standard are approximately 14.4 and 10.7
min, respectively. The assay is linear in concentration ranges of 50 to 15 000 pg/ml
(plasma) and 10 to 10 000 ng/ml (urine). The correlation coefficients are greater than
0.9992 for all curves. For the plasma method, the analysis of pooled quality controls
(300, 3000, and 10 000 pg/ml) demonstrates excellent precision with relative
standard deviations (R.S.D.) (n=18) of 1.1%, 2.3%, and 6.8%, respectively. For the
urine method, quality control pools prepared at 30, 300, and 3000 ng/ml had R.S.D.
values (n=18) of 2.9%, 1.7%, and 3.0%, respectively. The plasma and urine controls
were stable for more than nine and three months, respectively. The mean recoveries
for Pramipexole and internal standard from plasma were 97.7% and 98.2%,
respectively. The mean recoveries for Pramipexole and internal standard from urine
were 89.8% and 95.1%, respectively. The method is accurate with all intra-day (n=6)
and overall (n=18) mean values for the quality control samples being less than 6.4
and 5.8% from theoretical for plasma and urine, respectively.
39
CHAPTER 3
MATERIALS AND METHODS
3.1 Reagents, Chemicals and Instruments
3.1.1 Reagents and Chemicals used
Methanol, Acetonitrile and Water of HPLC grade from Merck, Ammonium

Formate, Trichloro acetic acid, Dichloromethane, n-Hexane, Diethyl ether and
Formic Acid were AR Grade, Pregabalin, and Tramadol obtained from Aarti Drugs
Limited, Pramipexole dihydrochloride monohydrate from Orchid Chemicals and
Pharmaceuticals Ltd, India. Quetiapine Fumarate obtained from Lupin Laboratories
Limited, Maharashtra, India.
3.1.2 Instruments used
i. Agilent 6460 Triple Quad LC-MS/MS

ii. AB Sciex API 5500 Triple Quad LC-MS/MS
iii. Sartorius single pan digital balance (R200D & 1702)
iv. Systronics pH meter,  pH system 361.
v. Shimadzu LC 2010A HT HPLC system with following
configurations;
vi. Electrolab USP Type I and II dissolution testing apparatus
vii. Shimadzu 160A UV-VIS spectrophotometer
viii. Perkin-Elmer FT IR 1600 series
ix. Ultra Sonicator
x. Solid phase Extractor
40
3.2 Bioequivalence study
This section describes the study design, products of evaluation, subject

selection, inclusion and exclusion criteria, safely and ethics review procedure, drugs
administration, meals and food restrictions, blood collection and extraction,
evaluation of pharmacokinetic parameters and statistical analysis involved in the
bioequivalence studies.
3.2.1 Study Design
A single dose, randomized, two treatment, two period, two sequence, single dose,
crossover bioequivalence study for the immediate release formulation and modified
release formulation in twenty four healthy, adult, male, human subjects under fasting
conditions.
3.2.2 Product for Evaluation
In each dosing session, volunteers received either immediate release

formulation or modified release formulation. A wash out period of seven days was
allowed between dose administrations.
3.2.3 Subjects
The subjects for the bioavailability study were selected from the panel of
volunteers enrolled with the Centre of Bioequivalence, Roxaane Research Private
Limited, Chennai. Seven days prior to the commencement of the study, volunteers
were screened for the following for inclusion in the study;
 Healthy males, 20 - 35 years of age
 Not more than ±15% from ideal weight for subjects height and elbow
breadth
 General good health as determined by medical history and physical

examination within 30 days prior to the start of the study (without a history
of clinically significant organ system disorders, or ongoing infectious
diseases, history of benign prostatic hypertrophy, prostate infections, or
urinary retention, history of asthma and drug allergy history of peripheral
41
neuropathy, history of alcohol abuse or drug addiction requiring treatment
within the last 12 months)
 No prescription drugs within 14 days, or OTC preparations, herbal remedies,

or nutritional supplements (excluding vitamins) within 7 days prior to drug
administration and
 Subjects with systolic blood pressure 90-140 mmHg, diastolic pressure

50-90 mmHg and pulse rate within 50- 100 bpm
On the basis of this preliminary screening, 24 volunteers were selected and

their liver and renal functions and hematological parameters such as hemoglobin
content, RBC and WBC counts, blood sugar, cholesterol, bilirubin and ECG were
examined by standard clinical and biochemical investigations. No grapefruit juice
or grapefruit containing products for at least 72 h and caffeine or xanthine
consumption for at least 12 h prior to drug administration was allowed in each
period. No concomitant medication (other than the study drug) was allowed during
the study phase. Volunteers were also instructed to refrain from consuming alcohol,
smoking or other stimulant drinks during this period.
3.2.4 Ethics Review Procedure
The protocol of the study was then submitted to the Institutional Human
Ethical Committee and the approval for conducting the same was obtained. Prior to
the commencement of the study, each subject was provided with an information
sheet giving details of the investigational drugs, procedure and potential risk
involved and a written consent was obtained. They were instructed that they are free
to withdraw their consent and to discontinue their participation in the study at any
time without prejudice.
3.2.5 Drug Administration
All the volunteers were made to assemble in the Bioequivalence Centre, 12

hours prior to the initiation of the study. After overnight fasting, the volunteers were
given code numbers and allocated to the treatment in accordance with the
randomized code. Their pulse rates and blood pressures were recorded and a sterile
intravenous cannula introduced with strict aseptic precautions for blood collection.
42
Volunteers received either immediate release formulation or modified release
formulation according to their code numbers with 240 ml of water. The order of
treatment administration was randomized in two sequences (AB and BA) in blocks
of two.
Blood samples (4 ml) were collected using disposable syringes in

pre-heparinised centrifugal tubes at 0 (before drug administration), 0.5, 1.0, 1.5, 2.0,
3.0, 4.0, 6.0, 8.0, 12.0, 18.0 and 24.0 hours post dosing. The samples were
centrifuged at 3500 rpm for 10 minutes to separate plasma. They were transferred
into airtight containers and stored at deep freeze condition until starting of analysis.
A similar procedure adopting cross-over design in drug treatment was repeated after
7 days of wash out period.
3.2.6 Subject Monitoring
The study was monitored by a physician and a clinical pharmacologist. In

addition, a staff nurse and a technician were present throughout the study for blood
collection and plasma separation. The blood pressure and pulse rate were measured
at 0.5, 1, 3, 6, 12 and 24 hours post dosing. The volunteers were monitored for
abnormal symptoms during the study period and for one week after the study period
and if noticed, the details were entered in the case report sheets and tabulated at the
end of the study.
3.2.7 Meals and Food Restrictions
Standard breakfast was provided after 3 h post dosing. Subjects were

instructed to eat their entire breakfast in 30 minutes. Lunch and dinner, consisting
of caffeine-free, xanthine-free, and grapefruit-free foods and beverages, were served
after 7 and 12 hours post dosing during the in house portion of the study.
3.2.8 Extraction of drugs from plasma
All the plasma samples were extracted and their drug levels were quantified
using LC-MS/MS technique. Detailed procedures involved in extraction and
quantification are discussed under section 3.3.8.
43
3.2.9 Estimation of Pharmacokinetic Parameters
Pharmacokinetic parameters namely, the peak height concentration (Cmax),

the time of peak concentration (Tmax), and elimination rate constant (Keli) were
determined for individual drug treatments from the observed plasma concentration-
time data. The area under the plasma concentration-time curves (AUC) were
calculated by trapezoidal rule from time zero to the last observed concentration.
3.3 Analytical Method Development
Method development involves evaluation and optimization of the various

stages of sample preparation, chromatographic separation, detection and
quantification. Optimization of various parameters were tried in order to develop a
selective and sensitive method for the analysis of Pregabalin and Pramipexole in
plasma samples.
3.3.1 Selection of Molecular Ions
LC-MS/MS instrument was calibrated with Reserpine standard in positive

and negative ionization mode. LC-MS/MS detector at unit resolution in the multiple
reaction monitoring (MRM) mode was preferred. 100 ng/ml of Pregabalin,
Pramipexole, Quetiapine Fumarate and Tramadol solutions were infused and mass
spectrums were recorded. Using the mass spectra of the infused solutions, parent and
product ions were selected for analyte and internal standards.
The transition of the protonated molecular parent ions were m/z 160.2, m/z
264.2, m/z 212.30, m/z 384.80 and the product ions m/z 55.1, m/z 58.0, m/z 153.30,
m/z 220.30 for the Pregabalin, Tramadol, Pramipexole and Quetiapine Fumarate,
respectively.
3.3.2 Selection of Source Parameters
The source parameters namely Gas temperature, Gas flow, Nebulizer, Sheath
gas temperatures, Sheath gas flow, Capillary voltage, Charging voltage were
optimized for Pregabalin and Tramadol. The optimized source parameters for
Pregabalin and Tramadol is presented in Table 3.2.
44
Table 3.1 Source Parameters for Pregabalin
Source parameters Value

o
Gas temperature ( C) 300
Gas flow (lml/min) 10
Nebulizer (psi) 45
o
Sheath gas temperature ( C) 400
Sheath gas flow (lml/min) 11
Capillary voltage (V) 3500
Charging voltage (V) 500
The optimized source parameters for Pramipexole and Quetiapine is presented in

Table 3.2.
Table 3.2 Source Parameters for Pramipexole
Source parameters Value

o
Gas temperature ( C) 500
Gas flow (l/min) 10
Nebulizer (psi) 45
o
Sheath gas temperature ( C) 400
Sheath gas flow (l/min) 11
Capillary voltage (V) 5500
Charging voltage (V) 500
45
3.3.3 Column Selection
Reversed phase Hypersil, Symmetry and Phenomenox columns of different

sizes and different particle sizes (C8 (100 x 4.6 mm, 5 µm), C18 (100 x 4.6 mm, 5 µm),
C18 (50 x 4.6 mm, 5 µm), C18 (50 x 4.6, 10 µm) and C18 (3 x 30 mm, 3.5 µm)) were
tried. Based on better resolution and reproducibility, Kromasil C18 (3x 30 mm,
3.5µm) was selected for the present study.
3.3.4 Selection of Mobile Phase
Different mobile phases, such as acetonitrile and 2 mM ammonium formate

in different ratios ie. 80:20 (v/v), 70:30 (v/v), 65:35 (v/v) and 60:40 (v/v), 0.5 %
formic acid and acetonitrile in different ratios ie. 90:10 (v/v), 85:15 (v/v), 80:20 (v/v)
and 70:30 (v/v) were tried.
Based on the peak shape and separation, acetonitrile: 0.5% formic acid
(80:20) was selected as mobile phase for Pregabalin and Acetonitrile: 5mM
Ammonium Formate (65:35) was selected as mobile phase for Pramipexole analysis.
3.3.5 Effect of Injection Volume
The process of quantification at very low concentrations is a competition

between signal and noise. In order to keep the instrument clean the amount of sample
reaching into the mass spectrometer should be kept as low as possible. A comparison
was made between injecting 5 µl and 2 µl of reconstituted extracts of Pramipexole
and Pregabalinfrom 50 ng/ml of plasma samples. At 5 µl injection volume, signal to
noise ratio of Pramipexole peak was 15, whereas at 2 µl injection volume, signal to
noise ratio of Pregabalin was 15, hence, 5 µl of injection volume for Pregabalin and
2 µl of injection volume for Pramipexole was selected for the present analysis.
3.3.6 Effect of Flow Rate
Effect of flow rate on chromatogram was studied at a flow rate of 0.5, 0.75
and 1.0 ml/min of mobile phase. Pregabalin and internal standard was eluted at 1.6
and 1.4 minutes respectively at 1ml/min flow rate. Pramipexole and internal standard
46
was eluted at 1.9 and 1.81 minutes respectively at 0.5 ml/min flow rate. Flow rate of
1.0 ml/min for Pregabalin and 0.5 ml/min for Pramipexole was selected for the
present study.
3.3.7 Selection of Internal standard
Different compounds such as Tramadol, Verapamil, Paracetamol,

Itraconazole, Quetiapine, Atorvastatin etc. were tried for internal standards. Based on
the peak response with good elution and recovery Tramadol was selected as internal
standard for Pregabalin and Quetiapine was selected as internal standard for
Pramipixole.
3.3.8 Selection of Extraction Procedure
Extraction was tried with Liquid-Liquid Extraction (LLE). Aliquots of 500 µl

of spiked plasma in a glass tube was added with 50 µl of 500 ng/ml Pregabalin and
tried with different extraction solvents. The ratio of the solvents and percentage
recovery obtained is shown in Table 3.3.
Table 3.3 Liquid –Liquid Extraction of Pregabalin
% Recovery
Solvents (%) Volume
Analyte IS
Methyl Tertiary butyl

3ml 25 48
Ether(MTBE)
MTBE:
3ml 45 16
Dichloromethane(DCM)
n-Hexane 3ml 20 18
Diethyl ether :
3ml 38 28
DCM(80:20)
47
The recovery of LLE method was very low, hence Solid Phase Extraction
procedure was attempted with various cartridges. Strata-X 33 µm cartridge was
selected for the present study.
The Solid phase extraction procedure for the extraction of Pregabalin from
plasma samples as follows,
 The solid phase cartridges were conditioned using 1 ml of 100%

methanol.
 It was equilibrated and acidified with 1 ml of 1% Formic acid.
 550 µl plasma sample and 200 µl of internal standard was added.
 Washed twice with 1 ml of 1% Formic Acid.
 Pregabalin was eluted with 500 µl of mobile phase (Acetonitrile: 0.5%
Formic Acid). Eluted samples were dried under Nitrogen
 Reconstituted the samples with 300 µl of Mobile phase, vortexed for 1
minute and 2 µL of the sample was injected.
The Solid phase extraction procedure for the extraction of Pramipexole from
plasma samples as follows,
 The solid phase cartridges were conditioned using 1ml of 100% methanol.
 It was equilibrated and acidified with 1 ml of 1% Formic acid.
 550 µl plasma sample and 200 µl of internal standard were added.
 Washed twice with 1 ml of 20% methanol in water.
 Pramipixole was eluted with 500 µl of methanol.
 Eluted samples were dried under Nitrogen
 Reconstituted the samples with 300 µl of Mobile phase, vortexed for 1
minute and 2 µL of the sample was injected.
48
3.4 Method Validation
3.4.1 System Suitability
Un-extracted standard was prepared equivalent to upper level of calibration

curve concentration and internal standard. It was injected for six times and the
chromatograms were recorded as per developed bioanalytical method. If the system
suitability did not pass with six injections, six more injections were performed until
the system suitability test passes. The retention times and responses of the analyte
and internal standard were recorded. The system suitability was evaluated by
manually calculating the mean, standard deviation and coefficient of variation for the
retention time and area.
The %CV of area ratio of drug and internal standard is ≤3% for single analyte
and for ≤5% for multiple analyte. The %CV of retention time of drug and internal
standard is 2%. If the results did not comply with the acceptance criteria, then the
system was checked for its malfunction and suitable remedial actions were taken and
the system suitability should be performed again.
Following parameters were assessed during validation
 Selectivity
 Specificity
 Ruggedness
 Recovery
 Linearity
 Precision
 Accuracy
 Dilution integrity
 Matrix effect and
 Stability
49
3.4.2 Linearity
The linearity of the selected range was determined by preparing calibration curve
consists of a blank sample (matrix sample processed without internal standard),
blank with internal standard and 8 non-zero standards covering the expected range
(coded as CS1 to CS8) were analysed.
3.4.3 Accuracy and Precision
Accuracy and precision of the method were determined by analyzing six

replicates of Lower limit of quantification (LLOQ), Lower quality control (LQC),
Middle quality control (MQC) and Higher Quality control ( HQC) in different
occasions.
Within Run accuracy and precision
Within run accuracy and precision evaluations were performed by analyzing

replicate concentration of Pregabalin in Human plasma. The run consisted of a
calibration curve standard plus a total of 24 spiked samples, 6 replicates of each of
lower limit of quantitation (LLOQ), low (LQC), medium (MQC) and high (HQC)
quality control samples.
Between run accuracy and precision
The between run accuracy and precision evaluations were assessed by the
repeated analysis of human plasma samples containing different concentrations of
Pregabalin by changing analyst to analyst and by changing the column. A single run
consisted of a calibration curve standards plus 6 replicates of LLOQ, LQC, MQC and
HQC samples.
3.4.4 Recovery
Recovery of the developed method can be evaluated by analyzing six

replicates of analyte along with internal standard by comparing the analytical results
for extracted samples at three concentrations equivalent to LQC, MQC and HQC
50
with unextracted samples that represent 100% recovery. The percentage recovery of
analyte and internal standard were calculated using appropriate chromatographic
conditions. For the internal standard, mean internal standard responses of eighteen
processed samples were compared to the mean internal standard responses of
eighteen appropriately diluted pure internal standard solutions.
3.4.5 Selectivity
Selectivity was assessed by analyzing blank plasma samples obtained from

six different sources with six samples at LLOQ concentrations spiked using the
biological matrix of any one source. Randomly selected blank human plasma sources
were taken to determine the extent to which endogenous human plasma interfere
with the analyte or the internal standard.
3.4.6 Sensitivity
Sensitivity was determined by limit of quantification by analyzing six

replicates of lower limit of quantification (LLOQ) that can be measured with
acceptable accuracy and precision.
3.4.7 Matrix Effect
It had been noted that co eluting, undetected endogenous matrix components

might reduce the ion intensity of the analyte and adversely affect the reproducibility
and accuracy of the LCMS/MS assay. In order to determine whether this effect
(matrix effect) was present or not, 6 different plasma pools were extracted and then
spiked with standard solution concentration equal to LQC (post extracted spiked
sample). Samples were prepared at low quality control level (LQC) in different
human plasma sources analysed with 3 replicates of comparison samples in a single
run. Percentage nominal concentrations were calculated for each matrix.
3.4.8 Carry over Test
Carry over is calculated as the percentage peak area observed in a processed

plasma blank injected immediately after a processed Upper limit of quantification
51
(ULOQ) calibration standard. No significant carry over was observed for analyte and
internal Standard.
3.4.9 Stability
The stabilities were assessed under varying storage and handling conditions
and determined by calculating the percentage nominal of LQC and HQC samples
against freshly prepared calibration curve standards and compared with bulk spiked
comparison samples (CS). As a part of method validation bench-top, in-injector,
stock solution, freeze thaw, dry extract, wet extract, short and long term stabilities
were also evaluated. Samples were considered to be stable if the assay values were
within the acceptable limits of accuracy (± 15% SD) and precision (± 15% RSD)
Freeze and Thaw Stability
Samples were prepared at low (LQC) and higher (HQC) quality control levels,
0
aliquoted and frozen at (-70 C), some of the aliquots of quality control samples were
subjected to three freeze-thaw cycles (stability samples). Six replicates of each LQC,
o
MQC and HQC stored at (-70 C) were thawed completely unassisted at room
0
temperature and refrozen immediately to (-70 C). This cycle was repeated for three
times with 12 hour intervals and the samples were extracted and analysed with
freshly prepared calibration curve standards and comparison samples. A calibration
curve and quality control samples were freshly prepared and processed with 6
replicates of stability samples and analyzed in a single run. At the time of analysis,
the samples were removed from deep freezer and kept in the room temperature and
allowed to thaw.
Bench Top stability
The stability of samples on the bench i.e., when kept outside the freezer were
studied to know the stability of samples at room temperature. Six replicates of LQC
& HQC were kept at room temperature for 6 hours, these samples were processed
and analysed with a freshly prepared calibration curve standards and comparison
samples.
52
3.5 Estimation of Pregabalin in Plasma Samples
3.5.1 Preparation of standard Stock Solution
10 mg of Pregabalin was transferred into a 10 mL volumetric flask. Add 5 ml

of methanol and water mixture (1:1) was added and made up the volume with same
to produce a solution of 1mg/mL strength.
3.5.2 Preparation of Calibration Curve Samples Stock Dilutions
Standard stock solution prepared ranging from 1.0 ng/mL to 200.0 ng mL

using 50:50 methanol and water mixture (1:1) and is presented in Table 3.5 and 3.6.
3.5.3 Internal standard stock solution
10 mg of internal standard was transferred to a 10 ml volumetric flask to

which 5 ml of methanol was added to dissolve. The volume was then made up to 10
ml using methanol (stock solution A).
100 µl of stock solution A was taken in a volumetric flask and the volume
was made upto 10 ml using mobile phase (stock solution B).
500 µl of stock solution B was taken in a volumetric flask and the volume
was made upto 10 ml using mobile phase (stock solution C).
53
Table 3.4 Preparation of PregabalinStandards for Calibration Curve (Dilution I)
Stock Volume
Stock Spiking
Solution Volume of Final Final conc.
Solution solution
concentration taken(mL) diluent volume(mL) (ng/mL)
ID ID
(ng/mL) (mL)
C 10160.755 2.460 7.540 10.000 2500.000 CS1
C 10160.755 4.921 5.079 10.000 5000.000 CS2
B 101607.551 1.845 8.155 10.000 18750.000 CS3
B 101607.551 3.691 6.309 10.000 37500.000 CS4
A 1016075.50 1.015 8.985 10.000 103200.000 CS5
A 1016075.50 2.031 7.969 10.000 206400.000 CS6
A 1016075.50 2.986 7.014 10.000 303500.000 CS7
A 1016075.50 4.207 5.793 10.000 427500.000 CS8
A 1016075.50 4.920 5.080 10.000 500000.000 CS9
54
Table 3.5 Preparation of Pregabalin Standards for Calibration Curve (Dilution II)
Spiking
Spiking Final
Solution Volume Volume of Final CC
Solution conc.
Concentration taken(mL) diluent(mL) volume(mL) ID
ID (ng/mL)
(ng/ml)
CS1 2500.000 0.200 9.800 10.000 50.000 CS1
CS2 5000.000 0.200 9.800 10.000 100.000 CS2
CS3 18750.000 0.200 9.800 10.000 375.000 CS3
CS4 37500.000 0.200 9.800 10.000 750.000 CS4
CS5 103200.000 0.200 9.800 10.000 2064.000 CS5
CS6 206400.000 0.200 9.800 10.000 4128.000 CS6
CS7 303500.000 0.200 9.800 10.000 6070.000 CS7
CS8 427500.000 0.200 9.800 10.000 8550.000 CS8
CS9 500000.000 0.200 9.800 10.000 10000.000 CS9
3.5.4 Preparation of QC Samples
In this the spiked samples were taken and they were used to monitor the performance
of the method. For this the samples were taken from the stock solution and they were
prepared according to the procedures given in Table 3.7 and 3.8.
55
Table 3.6 Preparation of Pregabalin QC Samples (Dilution-I)
Final
Solution Volume Volume of Final
Solution concentra Spiking solution
concentratio taken diluent volume
ID tion. ID
n (ng/ml) (mL) (mL) (mL)
(ng/mL)
C 10160.75 2.460 7.540 10.000 2500.00 SS01-LLOQ
C 10160.75 6.292 3.708 10.000 6400.00 SS01-LQC
A 1016075.50 1.575 8.425 10.000 160000.00 SS01-MQC
A 1016075.50 3.937 6.063 10.000 400000.00 SS01-HQC
Table 3.7 Preparation of Pregabalin QC Samples (Dilution-II)
Spiking Solution Volume of Final Spiking

Volume Final
Solution concentration diluent conc. solution
taken(mL) volume(mL)
ID (ng/mL) (mL) (ng/mL) ID
SS01-
2500.000 0.200 9.800 10.000 50.000 LLOQ
LLOQ
SS01-
6400.000 0.200 9.800 10.000 128.000 LQC
LQC
SS01-
160000.000 0.200 9.800 10.000 3200.000 MQC
MQC
SS01-
400000.000 0.200 9.800 10.000 8000.000 HQC
HQC
56
injected with the optimised chromatographic conditions and the chromatograms
were recorded. The quantification of the chromatogram was performed using peak
area ratios (response factor) of the drug to internal standard. The calibration curves
are constructed routinely for spiked plasma containing drug and internal standard
during the process of pre-study validation and in-study validation.
3.6 Estimation of Pramipexole in Plasma Samples
3.6.1 Preparation of Pramipexole Calibration Curve Samples Stock Dilutions
Pramipexole standard calibration stock solutions ranging from 1.0 ng/mL

to 200.0 ng/mL was prepared using methanol and water mixture (1:1) as diluent as
shown in Table 3.9.
Table 3.8 Stock Dilution for Pramipexole Calibration Standard
Volume Volume
Initial Initial stock Volume of Final stock
solution conc. stock of of final conc.(ng/m Final
S.No.
solution
ID (ng/mL) taken(mL) Diluent solution L)
(mL) (mL)
1 Std I 19662.2 0.05 4.95 5.000 196.622 SS1
2 SS 1 196.622 3.75 1.25 5.000 147.466 SS2
3 SS 2 147.466 2.50 2.50 5.000 73.733 SS3
4 SS 3 73.733 2.50 2.50 5.000 36.867 SS4
5 SS 4 36.866 1.88 3.12 5.000 13.825 SS5
6 SS 5 13.825 1.52 3.48 5.000 4.2166 SS6
7 SS 6 4.2166 2.40 2.60 5.000 2.024 SS7
8 SS 7 2.024 2.50 2.50 5.000 1.012 SS8
57
3.6.2 Stock Dilution for Pramipexole Quality Control Sample
Pramipexole quality control stock solution ranging from 100.0 to 20000.0

ng/mL using methanol and water mixture (1:1) was prepared as shown in the
Table.3.10.
Table 3.9 Stock Dilution for Pramipexole Quality Control Samples
Volume Total
Initial Volume of of volume Final
Initial stock Final stock
S.No solution stock Diluent of final solution
conc.(ng/mL) conc.(ng/mL
ID taken(mL) added solution ID
(mL) mL
1 QCStd 1 9799.54 0.2 9.8 10 195.09 QC1
2 QC1 195.99 3.75 6.26 10 73.49 QC2
3 QC2 73.49 5 5 10 36.74 QC3
4 QC3 36.74 0.75 9.25 10 2.75 QC4
5 QC4 2.75 3.7 6.3 10 1.01 QC5
3.6.3 Preparation of Quetiapine Stock Solution

9.831 mg of Quetiapine was transferred into a 10mL volumetric flask. 5 ml of
Methanol was added, mixed the contents until Quetiapine was dissolved. The volume
was made up with methanol to produce a solution of 0.983.13 mg/mL concentration
of Quetiapine.
3.6.4 Preparation of Quetiapine Stock Dilutions

0.1ml of Quetiapine stock solution was transferred into 5 ml volumetric flask
and the final volume was made using methanol:water mixture (1:1) to produce
working concentration of 19622.72 ng/mL Quetiapine.
58
3.6.5 Pramipexole Calibration Standards in Human plasma
Pramipexole stock dilutions ranging from 20.0 to 4000.0 pg/mL was prepared
as per Table 3.10.
Table 3.10 Pramipexole Calibration standards in Human plasma
Total
Initial Volume of Volume
Initial stock Final Stock Final
S.No solution stock of Final
conc.(ng/mL) Conc.(pg/ml) Solution ID
ID taken(mL) Solution
(mL)
1 SS 1 196.62 0.020 1 3932.44 STD- 8
2 SS 2 147.46 0.020 1 2949.33 STD- 7
3 SS 3 73.73 0.020 1 1474.66 STD- 6
4 SS 4 36.86 0.020 1 737.33 STD- 5
5 SS 5 13.82 0.020 1 276.50 STD- 4
6 SS 6 4.21 0.020 1 84.33 STD- 3
7 SS 7 2.02 0.020 1 40.48 STD- 2
8 SS 8 1.01 0.020 1 20.24 STD- 1
59
3.6.6 Preparation of Quality Control Samples of Pramipexole
Quality control samples in Human K3EDTA plasma ranging from 20.0

to 1500.0 pg/ml was prepared as per Table 3.11.
Table 3.11 Pramipexole Quality Control samples in Human Plasma
Volume Total
Initial Volume of of volume Final
S. solution Initial stock Final stock solution
stock Diluent of final
No ID conc.(ng/mL) taken(mL) added solution conc(pg/mL) ID
(mL) (mL)
1 QC 1 7349.66 0.200 0.800 1 1469.9320 HQC
2 QC 2 3674.83 0.200 0.800 1 734.9660 MQC
3 QC 3 275.61 0.200 0.800 1 55.1220 LQC
4 QC 4 101.98 0.200 0.800 1 20.3960 LOQQC

injected with the optimised chromatographic conditions and the chromatograms
were recorded. The quantification of the chromatogram was performed using peak
area ratios (response factor) of the drug to internal standard. The calibration curves
are constructed routinely for spiked plasma containing drug and internal standard
during the process of pre-study validation and in-study validation.
3.7 Determination of Pharmacokinetic Parameters
The following Pharmacokinetic parameters were determined for individual

drug treatments from the observed plasma concentration-time data. The area under
the plasma concentration-time curves (AUC) were calculated by trapezoidal rule
from time zero to the last observed concentration.
60
 Cmax Maximum plasma concentration
 tmax Time of maximum plasma concentration
 AUC (0-t) Area under plasma concentrations time curve 0 to 24 hrs
 AUC (0- ∞) Area under plasma concentrations time curve 0 to  hrs
 t 1/2 Elimination half-life
 keli Elimination constant
The statistical analysis was carried out for Cmax, AUC(0-t) and AUC (0- ∞)
3.8 Development of in-vitro dissolution studies
The release characteristics of selected drugs in their formulations is carried out

using USP XXIII dissolution apparatus (type I, basket; type II, paddle), at different
rpm, with the media of pH 1.2, 4.5, 6.8, distilled water and 7.4 buffers maintained at
37±0.5˚C. Dissolution tests was performed on twelve tablets. Percentage drug release
at various time intervals was calculated and compared.
5 ml of the samples were withdrawn at 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0,
12.0, 18.0 and 24.0 hr time intervals for a period of 24 hours. Equal quantity of the
dissolution medium was replaced to the dissolution jar after each sampling.
The amount of the drug released was estimated. Percentage drug release at
various time intervals were calculated and compared.
3.9 In-vitro and in-vivo correlation
Difference factor (f1) and a similarity factor (f2) were calculated. The
difference factor calculates the percent difference between the two curves at each
time point and is a measurement of the relative error between the two curves. The
similarity factor is a logarithmic reciprocal square root transformation of the sum
squared error and is a measurement of the similarity in the percent dissolution
between the two curves. Generally, f1 values up to 15 (0-15) and f2 values greater
than 50 (50-100) ensure sameness or equivalence of the two curves.
61
The measured plasma concentrations were used to calculate the area under
the plasma concentration-time profile from time zero to the last concentration time
point (AUC(0-t)). The AUC(0-t) was determined by the trapezoidal method. AUC(0-ω)
was determined by the following equation:
AUC(0-ω) = AUC(0-t) + C(t)/ ke
kewas estimated by fitting the logarithm of the concentrations versus time to a

42
straight line over the observed exponential decline. The Wagner-Nelson method
was used to calculate the percentage of the selected drugs dose absorbed
F(t) = C(t) + ke AUC(0-t)
Where F(t) is the amount absorbed.
The percent absorbed is determined by dividing the amount absorbed at any

time by the plateau value, keAUC(0-ω) and multiplying this ratio by 100:
C(t) + ke AUC(0-t)
% dose absorbed = x 100
ke AUC(0-ω)
The percentage of drug dissolved was determined using the aforementioned

dissolution testing method and the fraction of drug absorbed was determined using
the method of Wagner-Nelson. Linear regression analysis was used to examine the
relationship between percentage of drug dissolved and the percentage of drug
absorbed. The percentage of drug un-absorbed was calculated from the percentage
absorbed. The slope of the best-fit line for the semi-log treatment of this data was
taken as the first order rate constant for absorption. The dissolution rate constants
were determined from percentage released vs the square root of time. Linear
regression analysis was applied to the in-vitro and in-vivo correlation plots and
2
coefficient of determination (r ), slope and intercept values were calculated.
62
3.9.1 Validation of IVIVC
dissolution data and mean in-vivo pharmacokinetics of the selected modified release
formulations. Briefly, the correlation of the mean in-vitro dissolution rate constants
was correlated to the mean absorption rate constants for the modified release
formulations. These two data points, along with the zero-zero intercept were used to
calculate the expected absorption rate constants.
The prediction of plasma concentration was accomplished using the

following curve fitting equation:
ka
-ket -kat
y = Const. X (Dose) X ------------ (e – e )
ka - ke
Where, y is predicted plasma concentration (mcg/ml);

Const. is the constant representing F / Vd (where F is the fraction
absorbed, and Vd is the volume of distribution);
ka is absorption rate constant;
keisoverall elimination rate constant.
The de-convolution was accomplished on a spread-sheet in Excel.
To further assess the predictability and the validity of the correlations, IVIVC
model-predicted Cmax and AUC values were determined for each formulation. The
percent prediction errors for Cmax and AUC were calculated as follows:
Cmax (obs) - Cmax (pred)

% PECmax = ------------------------------- X 100
Cmax (obs)
AUC (obs) - AUC (pred)

% PEAUC = ------------------------------- X 100
AUC (obs)
63
Where Cmax (obs) and Cmax (pred) are the observed and IVIVC model-
predicted maximum plasma concentrations, respectively;
AUC (obs) and AUC (pred) are the observed and IVIVC model-predicted
AUC for the plasma concentration profiles, respectively.
64
CHAPTER 4
RESULTS AND DISCUSSION
This chapter describes the experimental results obtained in the present

investigation in the form of Tables and Figures along with a detailed discussion on
results of bioequivalence study design and data handling, development of LC-
MS/MS methods for the estimation of Pregabalin and Pramipexole in plasma
samples, validation of the developed methods, estimation of the selected drugs in
plasma samples, determination of pharmacokinetic parameters, development of
in-vitro dissolution studies, in-vivo and in-vitro data analysis and in-vitro and in-vivo
correlation model development and validation.
The developed in-vitro and in-vivo correlation, may serve as surrogate for
additional bioequivalence studies as part of the formulation development, scale up
and pre and post approval changes.
4.1 Bioequivalence study design and data handling
Comparative bioavailability studies between immediate release formulation

(IR formulation) and modified release formulations (MR formulation) of selected
drugs were carried out in 24 healthy human subjects adopting a single dose,
randomized, complete, two ways, two periods cross over design.
After overnight fasting, the volunteers were given code numbers and were
allocated to the treatment in accordance with the randomisation code. The order of
treatment administration was randomised in two sequences (AB and BA) in blocks of
two. In each dosing session, volunteers received either immediate release
65
formulation or modified release formulation. A washout period of seven days was
allowed between dose administrations.
A total of 13 blood samples were collected at 0 hour (before drug

administration) 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 18.0 and 24.0 hours
post dosing. Blood Samples were collected through an indwelling cannula placed in
a forearm vein. The pre-dose blood sample was collected within a period of one hour
before dosing and post-dose samples were collected within 2-minutes of the schedule
time. The blood samples were collected in 6-ml vacutainers containing EDTA as the
anticoagulant. The Plasma was separated, divided into two portions while
transferring to labeled storage vials and promptly frozen at -70 C until analysis.
All subjects were on fast overnight for a period of 10 hours before

commencement of dosing. Drinking water was not allowed from one hour pre-dosing
to two hours post dosing except while administering the dose. Uniform and low fat
meals were provided to all the subjects. A standard breakfast and lunch were
provided at 4, 8, and 12 hours after the drug administration.
The samples were centrifuged and plasma was separated. There were no
serious adverse effects observed during the entire study. The total number of blood
samples drawn during the study was 28 and the total volume of blood drawn
including 15ml for screening was about 200 ml.
4.2 Development of LC-MS/MS Methods for the Estimation of Pregabalin and

Pramipexole in Plasma Samples
The LC MS/MS instrument was calibrated with polypropylene glycol

standard in positive and negative ion mode. Infusion was done using 500 ng/ml of
Pregabalin, Pramipexole and internal standards in mobile phase. Using the mass
spectra of this solution, mass spectrometer parameters were optimized.
The various LC-MS/MS conditions namely, source parameters, column

selection, volatile mobile phase, selective extraction procedure and sensitive
acquisition methods, pH of the mobile phase, solvent strength, solvent ratio, flow
rate, addition of peak modifiers in mobile phase, nature of the stationary phase,
66
injection volume, molecular ion and internal standard were optimized in order to
develop a selective and sensitive method for the analysis of Pregabalin and
Pramipexole in plasma samples.
The optimized method for the estimation of Pregabalin in plasma samples is,
Mass Spectrometer : Agilent 6460 QQQ

Ion source : ESI + Agilent Jet Stream
Polarity : Positive
Pregabalin Ion : 160.2 (parent), 55.1 (product)
Tramadol Ion : 264.2 (parent), 58.0 (product)
Column : Kromasil C18 (3.5µm, 3, 30 mm)
0
Column oven temperature : 35.0 C
0
Auto sampler temperature : 15 C
Mobile phase : Acetonitrile: 0.5% Formic Acid (80:20)
Flow rate : 1 ml/min
Volume of injection : 2µl
Pregabalin Retention Time : 1.042
Tramadol Retention Time : 1.120
Run time : 2 minutes
Dwell time : 200 ms
67
The optimized method for the estimation of Pramipexole in plasma samples is,
Mass Spectrometer : API 5500, AB Sciex

Ion source : ESI + Agilent Jet Stream
Polarity : Positive
Pramipexole Ion : 212.30 (parent), 153.30 (product)
Quetiapine Fumarate Ion : 384.80 (parent), 220.300 (product)
Column : Hypersil gold column (5 µm,50 x 4.6 mm)
0
Column oven temperature : 35.0 C
0
Auto sampler temperature : 15 C
Mobile phase : Acetonitrile :5mM Ammonium Formate
Mobile Phase Ratio : (65:35) Flow
rate : 0.5 mL/min
Volume of injection : 5µl
Pramipexole RT : 1.81 minutes
Quetiapine Fumarate RT : 1.30 minutes
Run time : 3 minutes
Dwell time : 200 ms
68
4.3 Method Validation for Pregabalin
The developed method was validated according to currently accepted FDA

guidelines of bio-analytical method validation.
4.3.1 Linearity
Calibration curves are found to be consistently accurate and precise, linear over
the range of 50 to 10000 ng/ml. The correlation coefficient (r) is equal to 0.9949.
Back calculations were made from the calibration curves to determine Pregabalin
concentrations of each calibration standard. Datas are presented in Table 4.1 and a
typical calibration curve is presented in Figure 4.1

replicates of lower limit of quantitation (LLOQ), low (LQC), medium (MQC) and
high (HQC) quality control samples in different occasions.
Intra-batch accuracy and precision evaluations were performed by analyzing

replicate concentration of Pregabalin in Human plasma. The run consisted of a
quality control samples. The percentage of nominal value should be within 15% of
the actual except at LLOQ, where it should not deviate by more than 20%. Each
concentration level should not exceed 15% of the coefficient of variation (CV)
except for the LLOQ, where it should not exceed 20% of the coefficient of variation
(CV).
Intra-batch coefficients of variation for limit of quantification of quality

control (LOQQC), low (LQC), medium (MQC), high (HQC) quality control for
Pregabalin samples were 8.48, 1.14, 3.67 and 4.57 %, respectively. Intra-batch
percentages of nominal concentrations for LOQQC, LQC, MQC and HQC were
105.07, 90.73, 108.81 and 106.20 %, respectively, and it is within the limit. The
results are shown in Table 4.2.
69
Inter-batchaccuracy and precision evaluations were assessed by the repeated
analysis of human plasma samples containing different concentrations of Pregabalin
by changing analyst to analyst and by changing the column. A single run consisted of
a calibration curve standards plus 6 replicates of lower limit of quantitation (LLOQ),
low (LQC), medium (MQC) and high (HQC) quality control samples. The
percentage of nominal value should be within 15% of the actual value except at
LLOQ, where it should not deviate by more than 20%. Each concentration level
should not exceed 15% of the coefficient of variation (CV) except for the LLOQ,
where it should not exceed 20% of the coefficient of variation (CV).
Inter-batch coefficients of variation for LOQQC, LQC, MQC, HQC samples

of Pregabalin were 4.88, 2.39, 3.35 and 4.75 % respectively. The between run
percentages of nominal concentrations for LOQQC, LQC, MQC and HQC were
103.06, 97.97, 102.42 and 103.08 % respectively and it is within the limit. The
results are shown in Table 4.3.
Recovery of the developed method can be evaluated by analyzing six

replicates of analyte along with internal standard by comparing the analytical results
for extracted samples at three concentrations (equivalent to LQC, MQC and HQC)
with un-extracted samples that represent 100 % recovery. The percentage recovery of
analyte and internal standard (IS) were calculated. For the internal standard, mean
internal standard responses of eighteen processed samples were compared to the
mean internal standard responses of eighteen appropriately diluted pure internal
standard solutions. Recovery of the analyte need not be 100%, but the extent of
recovery of an analyte and of the internal standard should be consistent, precise and
reproducible.
Mean recovery values are 88.92, 88.25 and 84.81 % at low, medium and high
quality control levels respectively. Mean recovery value for the internal standard
was 89.43% and it is within the limit. Results are presented in Table 4.4 and 4.5.
70
4.3.3 Matrix Selectivity
Selectivity was assessed by analyzing blank plasma samples obtained from six
different sources with six samples at LLOQ concentrations spiked using the
with the analyte or the internal standard. The Results are presented in Table 4.5.
No significant interference was observed in six different sources of human

plasma samples.
4.3.4 Sensitivity

acceptable accuracy and precision. A calibration curve standards and lower limit of
quantification samples (LLOQ) were processed and analysed in a single run. At the
time of analysis, the samples were removed from the deep freezer and kept in the
room temperature and allowed to thaw. Analyte peak (response) should be
identifiable, discrete and reproducible with a precision of 20% and accuracy of 80 to
120%. Results are presented in Table 4.6.
Lower limit of quantitation for Pregabalin coefficient of variation was 6.766

and a percentage of nominal concentration was 109.07 % which is within the limit.
4.3.5 Matrix effect
Undetected endogenous matrix components might reduce the ion intensity of

the analyte and adversely affect the reproducibility and accuracy of the LC-MS/MS
assay. In order to determine the matrix effect, 6 different plasma pools were
extracted and then spiked with standard solution concentration equal to LQC (post
extracted spiked sample). Samples were prepared at low quality control level (LQC)
in different human plasma sources analysed with 3 replicates of comparison samples
in a single run. Percentage nominal concentrations were calculated for each matrix.
71
The Results are presented in Table 4.7. The Matrix effect was found to be 104.84%
for Pregabalin.
Carry over test was calculated as the percentage peak area observed in a
processed plasma blank injected immediately after a processed ULOQ calibration
standard. No significant carry over was observed for Analyte and Internal Standard.
The Results are presented in Table 4.8.
4.3.7 Stability
o
Six replicates of each LQC, MQC and HQC stored at (-70 C) were thawed
0
completely unassisted at room temperature and refrozen immediately to (-70 C). This
cycle was repeated for three times with 12 hour intervals and the samples were
extracted and analysed with freshly prepared calibration curve standards and
comparison samples.
Samples were prepared at low (LQC) and higher (HQC) quality control and
0
frozen at (-70 C), some of the aliquots of quality control samples were subjected to
three freeze thaw cycles (stability samples). A calibration curve and quality control
samples were freshly prepared and processed with 6 replicates of stability samples
and analyzed in a single run. At the time of analysis, the samples were removed from
deep freezer and kept in the room temperature and allowed to thaw. The Results are
presented in Table 4.9.
The mean accuracy of Quality Control samples at each level was within ±15%
of the actual value.
Bench Top stability
Six replicates of LQC and HQC were kept at room temperature for 6 hours,
these samples were processed and analysed with a freshly prepared calibration curve
standards and comparison samples. The Results are presented in Table 4.10.
72
Table 4.1 Intercept, Slope and Correlation Coefficient Values for Pregabalin
Calibration Curve
Correlation
Curve No. Intercept Slope 2
coefficient(r )
1 0.275 0.0007 0.9974
2 0.981 0.0006 0.9958
3 1.024 0.0001 0.9941
4 0.289 0.0005 0.9953
5 0.265 0.0004 0.9962
Fig. 4.1: Calibration Curve of Pregabalin
73
Table 4.2 Intra-batch Accuracy and Precision of Pregabalin
QC ID LLOQ LQC MQC HQC

Actual
concentration 50.00 128.06 3201.42 8003.55
ng/mL
47.13 118.60 3436.57 7908.46
55.52 116.11 3459.41 8310.70
57.08 115.87 3428.68 8658.44
PA-01
55.15 115.34 3523.49 9141.63
55.91 116.87 3526.73 9039.95
56.42 114.31 3605.57 8973.44
Mean 54.53 116.18 3496.76 8672.11
SD 3.69 1.46 67.98 481.77
%CV 6.77 1.26 1.94 5.56
%Nominal 109.06 90.73 109.23 108.35
48.16 124.62 3126.52 7906.43
54.42 129.11 3359.42 7830.63
52.06 126.86 3228.43 8258.43
PA-02
49.14 120.12 3423.46 8141.64
53.98 125.89 3326.62 8890.95
51.41 126.11 3208.53 8473.24
Mean 51.53 125.45 3278.83 8250.22
SD 2.52 3.00 109.88 391.50
%CV 4.88 2.39 3.35 4.75
%Nominal 103.06 97.97 102.42 103.08
48.12 118.60 3436.54 7808.43
45.53 116.11 3359.41 8410.69
55.06 115.87 3628.48 8258.43
PA-03
52.14 115.34 3543.48 8941.62
57.92 116.87 3326.72 8803.93
56.43 114.31 3606.57 8773.43
Mean 52.53 116.18 3483.53 8499.42
SD 4.46 1.33 128.02 388.80
%CV 8.48 1.14 3.67 4.57
%Nominal 105.07 90.73 108.81 106.20
74
Table 4.3 Inter-batch Accuracy and Precision of Pregabalin
QC ID LLOQ LQC MQC HQC

Actual
concentration 50.00 128.05 3201.41 8003.54
ng/mL
54.53 116.18 3496.76 8672.11
55.52 116.10 3459.40 8410.69
57.08 115.86 3428.67 8658.43
PA-01
56.02 118.12 3302.63 8868.20
52.08 128.12 3102.61 8168.20
52.52 126.10 3159.40 8080.63
Mean 54.62 120.08 3324.91 8476.38
SD 1.98 5.54 164.73 310.12
%CV 3.62 4.61 4.95 3.65
% Nominal 109.25 93.77 103.85 105.90
48.12 118.60 3436.54 7808.42
45.52 116.10 3359.40 8410.68
53.23 126.16 3486.76 8682.12
PA-02
54.62 116.14 3259.42 8310.68
55.28 118.86 3628.67 8638.42
52.03 124.87 3428.62 8820.24
Mean 51.468 120.12 3433.24 8445.09
SD 3.857 4.36 123.89 363.01
% CV 7.49 3.63 3.61 4.29
% Nominal 102.94 96.19 107.24 105.52
48.16 124.62 3126.52 7906.42
54.42 129.11 3359.41 7830.62
52.06 126.86 3228.42 8258.42
PA-03
49.14 120.12 3423.46 8141.64
53.98 125.88 3326.62 8890.94
51.41 126.11 3208.52 8473.23
Mean 51.53 125.45 3278.82 8250.21
SD 2.519 3.003 109.88 391.50
% CV 4.88 2.39 3.35 4.75
% Nominal 103.06 97.97 102.42 103.08
75
Table 4.4 Recovery of Pregabalin
Extracted sample Un extracted

Sample Name % Recovery
Response sample Response
6912 7680
7765 8533
7903 8815
8181 9037
LQC 7508 8763 88.92 %
7949 9149
Mean 7703 8663
SD 446.44 527.81
%CV 0.602 6.092
250914 285486
283355 314839
MQC
234636 272928
280869 323712
264981 287898 88.25 %
265775 305946
Mean 263421 298468
SD 18410.72 19461.52
%CV 6.989 6.520
611211 712699
657090 782436
HQC 645259 799875
647679 731593
569744 702434 84.81 %
605876 676956
Mean 622809 734332
SD 33271.13 47738.47
%CV 5.342 6.500
76
Table 4.5 Recovery of Tramadol (lnternal Standard)
Extracted sample Un extracted

S. No % Recovery
Response sample Response
1 1128024 1261348
2 1015235 1135228
3 1088757 1217440
4 1151089 1287139
5 1152370 1289004
6 1061858 1187362
7 1523122 1703144
8 1177545 1316722
9 1073840 1200760
10 1083053 1211062
11 1020475 1141088 89.43 %
12 1109863 1241041
13 1140817 1275653
14 1126721 1259891
15 1188609 1329094
16 1055095 1179799
17 1147110 1282690
18 1019673 1140191
Mean 1125736 1258814
SD 112809.1 126148.4
%CV 10.020 10.021
77
Table 4.6 Matrix Selectivity of Pregabalin
Interference with
Matrix Identification Interference with IS
Analyte
MT-144/09 0.00 0.00
MT-146/09 0.00 0.00
MT-157/09 0.00 0.00
MT-159/09 0.00 0.00
Heamolysed plasma 0.00 0.00
Lipemic plasma 0.00 0.00
Table 4.7 Lower Limit of Quantitation (LLOQ)
Cal. concentration
S. No Accuracy
(2.001 ng/mL)
1 47.13 94.25
2 55.52 111.03
3 57.08 114.15
4 55.15 110.29
5 55.91 111.81
6 56.41 112.82
Mean 54.53
SD 3.68
%CV 6.76
% Nominal 109.07
78
Table 4.8 Matrix Effect of Pregabalin
LQC
Matrix ID
Response of Response of Post
Matrix factor
standard solution Extracted sample
9158 8733 104.87
MT-110/09 9346 8875 105.31
9417 9037 104.20
9170 8733 105.00
MT-114/09 9157 8875 103.18
9977 9037 110.40
8870 8733 101.57
MT-115/09 9284 8875 104.61
9551 9037 105.69
9293 8733 106.41
MT-124/09 9351 8875 105.36
10111 9037 111.88
9334 8733 106.88
MT-123/09 9600 8875 108.17
9922 9037 109.79
8863 8733 101.49
MT-125/09 9473 8875 106.74
9478 9037 104.88
Table 4.9 Carry over test of Pregabalin
Sample ID Analyte peak area IS peak area

Extracted blank 0 0
Extracted LLOQ+IS 1061858 1187362
Extracted ULOQ+IS 1188609 1329094
Extracted blank 0 0
% Carry over 0.00 0.00
79
Table 4.10 Freeze and Thaw Stability of Pregabalin
Fresh samples After 3 cycle

Sample
Cal. Con. Cal. Con.
Name S. No Accuracy Accuracy
(µg/mL) (µg/mL)
1 134.16 104.77 135.27 105.64
2 139.83 109.2 137.50 107.38
3 132.48 103.46 132.92 103.8
4 136.17 106.34 142.43 111.23
LQC 5 135.42 105.75 139.51 108.95
6 133.52 104.27 140.32 109.58
Mean 135.26 137.99
SD 2.59 3.48
%CV 1.91 2.52
% Stability 102.01
1 7014.30 87.64 8218.84 102.69
2 7328.84 91.57 8671.04 108.34
3 7105.55 88.78 8253.25 103.12
4 7456.10 93.16 7580.96 94.72
HQC 5 7874.69 98.39 8215.64 102.65
6 8528.58 106.56 8020.35 100.21
Mean 7551.34 8160.01
SD 566.61 355.30
%CV 7.50 4.35
% Stability 108.06
80
Table 4.11 Bench Top stability of Pregabalin
Fresh samples After 6 hours

Sample
Cal. Con. Cal. Con.
Name S. No Accuracy Accuracy
(µg/mL) (µg/mL)
1 137.32 107.24 127.89 99.87
2 143.33 111.93 141.61 110.59
3 140.76 109.92 121.87 95.17
4 127.89 99.87 133.89 104.56
5 134.75 105.23 115.00 89.81
6 145.05 113.27 136.47 106.57
LQC
Mean 138.18 129.45
SD 6.30 9.85
%CV 4.56 7.61
% Stability 93.68
1 7856.28 98.16 7010.30 87.59
2 7327.24 91.55 7143.16 89.25
3 7441.69 92.98 7752.23 96.86
4 6953.48 86.88 7666.59 95.79
5 7183.98 89.76 8027.55 100.30
6 6826.22 85.29 8378.11 104.68
HQC
Mean 7264.82 7662.99
SD 369.00 519.32
%CV 5.07 6.77
% Stability 105.48
81
4.4 Validation of Pramipexole
4.4.1 Linearity
Calibration curves are found to be consistently accurate and precise, linear

over the range of 200 to 4000 pg/mL. The correlation coefficient (r) is equal to
0.9928. Back calculations were made from the calibration curves to determine
Pramipexole concentrations of each calibration standard. Correlation coefficient
values are presented in Table 4.12 and a typical calibration curve is presented in
Figure 4.2.

replicates of LLOQ, LQC, MQC and HQC in different occasions.
Intra-batch accuracy and precision evaluations were performed by analyzing

replicate concentration of Pramipexole in Human plasma. The run consisted of a
quality control samples. Intra-batch coefficients of variation for limit of
quantification of quality control (LOQQC), low (LQC), medium (MQC), high
(HQC) quality control for Pramipexole samples were 11.37, 6.38, 3.01 and 5.92 %
respectively. Intra-batch percentages of nominal concentrations for LOQQC, LQC,
MQC and HQC were 116.80, 103.90, 103.07 and 97.59 % respectively and it is
within the limit. The results are shown in Table 4.13.
Inter-batchaccuracy and precision evaluations were assessed by the repeated

analysis of human plasma samples containing different concentrations of
Pramipexole by changing analyst to analyst and by changing the column. A single
run consisted of a calibration curve standards plus 6 replicates of lower limit of
quantitation (LLOQ), low (LQC), medium (MQC) and high (HQC) quality control
samples. Inter-batch coefficients of variation for LOQQC, LQC, MQC, HQC
samples of Pramipexole were 14.70, 8.56, 4.18 and 1.53% respectively. The
between run percentages of nominal concentrations for LOQQC, LQC, MQC and
82
HQC were 116.77, 96.85, 106.96 and 109.79% respectively and it is within the limit.
The results are shown in Table 4.14.
Recovery of the developed method can be evaluated by analyzing six replicates

of analyte along with internal standard by comparing the analytical results for
extracted samples at three concentrations (equivalent to LQC, MQC and HQC) with
un-extracted samples that represent 100% recovery. The percentage recovery of
analyte and internal standard were calculated using appropriate chromatographic
conditions. For the internal standard, mean internal standard responses of eighteen
processed samples were compared to the mean internal standard responses of
eighteen appropriately diluted pure internal standard solutions. Mean recovery
values were 74.03, 82.52 and 80.12% at low, medium and high quality control levels
respectively. Mean recovery value for the internal standard was 80.43 % and it is
within the limit. Results are presented in Table 4.15 and 4.16.
4.4.3 Matrix Selectivity
Selectivity was assessed by analyzing blank plasma samples obtained from six
different sources with six samples at LLOQ concentrations spiked using the
with the analyte or the internal standard. The Results are presented in Table 4.17.
4.4.4 Sensitivity

acceptable accuracy and precision. Results are presented in Table 4.17. Lower limit
of quantitation for Pramipexole coefficient of variation was 3.64 and a percentage of
nominal concentration was 98.07 % which is within the limit.
83
4.4.5 Matrix effect
It had been noted that co eluting, undetected endogenous matrix components

might reduce the ion intensity of the analyte and adversely affect the reproducibility
and accuracy of the LC-MS/MS method. In order to determine whether this effect
(matrix effect) was present or not, 6 different plasma pools were extracted and then
spiked with standard solution concentration equal to LQC (post extracted spiked
sample). Samples were prepared at low quality control level (LQC) in different
human plasma sources analysed with 3 replicates of comparison samples in a single
run. Percentage nominal concentrations were calculated for each matrix. The Results
are presented in Table 4.18. The Matrix effect was found to be 101.84 % for
Pramipexole.
Carry over is calculated as the percentage peak area observed in a processed

plasma blank injected immediately after a processed ULOQ calibration standard. No
significant carry over was observed for Analyte and Internal Standard. The Results
are presented in Table 4.19.
4.4.7 Stability
o
Six replicates of each LQC, MQC and HQC stored at (-70 C) were thawed
0
completely unassisted at room temperature and refrozen immediately to (-70 C). This
cycle was repeated for three times with 12 hour intervals and the samples were
extracted and analysed with freshly prepared calibration curve standards and
comparison samples. Samples were prepared at low (LQC) and high (HQC) quality
0
control levels and frozen at (-70 C), some of the aliquots of quality control samples
were subjected to three freeze-thaw cycles (stability samples). A calibration curve
and quality control samples were freshly prepared and processed with 6 replicates of
stability samples and analyzed in a single run. At the time of analysis, the samples
were removed from deep freezer and kept in the room temperature and allowed to
84
thaw. The Results are presented in Table 4.20. The mean accuracy of Quality Control
samples at each level was within ±15% of the actual value.
Bench Top stability
The stability of samples on the bench i.e., when kept outside the freezer were
studied to know the stability of samples at room temperature. Six replicates of LQC
& HQC were kept at room temperature for 6 hours these samples were processed and
analysed with a freshly prepared calibration curve standards and comparison
samples. The Results are presented in Table 4.21.
85
Table 4.12 Intercept, Slope and Correlation coefficient values for
Pramipexole Calibration Curve
Curve Correlation
Intercept Slope 2
No. coefficient(r )
1 0.265 0.0004 0.9962
2 0.971 0.0005 0.9948
3 0.289 0.0005 0.9953
4 1.024 0.0001 0.9941
5 0.275 0.0007 0.9974
Fig. 4.2: Calibration Curve of Pramipexole
86
Table 4.13 Intra-batch Accuracy and Precision of Pramipexole
QC ID LOQQC LQC MQC HQC

Actual
Concentration 20.40 55.12 1469.93 2939.86
pg/mL
20.13 47.49 1648.28 3284.71
22.42 51.90 1530.35 3282.22
24.09 57.63 1562.10 3162.53
PA-01
22.43 50.63 1510.47 3187.47
19.65 52.84 1659.87 3222.18
24.18 59.83 1522.64 3227.41
Mean 23.82 53.39 1572.28 3227.76
SD 3.50 4.57 65.72 49.23
%CV 14.70 8.56 4.18 1.53
%Nominal 116.77 96.85 106.96 109.79
24.16 50.12 1538.16 3315.38
23.02 57.41 1484.66 3100.34
21.80 48.30 1515.32 2934.57
PA-02 24.06 49.62 1537.21 2989.89
25.01 54.12 1554.41 2876.49
24.47 52.14 1560.85 3075.70
Mean 25.42 51.95 1531.77 3048.73
SD 4.43 3.37 28.00 155.34
%CV 17.44 6.38 1.83 5.10
%Nominal 124.63 94.25 104.21 103.70
28.48 45.47 1598.67 2886.47
23.25 49.67 1504.33 2983.14
23.73 99.58 1477.95 2958.73
PA-03 23.17 49.35 1534.46 2960.82
24.22 51.78 1485.63 2893.64
20.08 47.77 1489.11 2531.37
Mean 23.82 57.27 1515.03 2869.03
SD 2.71 20.84 45.60 169.95
% CV 11.37 6.38 3.01 5.92
% Nominal 116.80 103.90 103.07 97.59
87
Table 4.14 Inter-batch Accuracy and Precision of Pramipexole
QC ID LOQQC LQC MQC HQC

Actual
Concentration 20.40 55.12 1469.93 2939.86
pg/mL
24.16 50.12 1538.16 3315.38
23.02 57.41 1484.66 3100.34
21.80 48.30 1515.32 2934.57
PA-01
24.06 49.62 1537.21 2989.89
25.01 54.12 1554.41 2876.49
24.47 52.14 1560.85 3075.70
Mean 25.42 51.95 1531.77 3048.73
SD 4.43 3.37 28.00 155.34
% CV 17.44 6.48 1.83 5.10
% Nominal 124.63 94.25 104.21 103.70
28.48 45.47 1598.67 2886.47
23.25 49.67 1504.33 2983.14
23.73 54..5839 1477.95 2958.73
PA-02
23.17 49.35 1534.46 2960.82
24.22 51.78 1485.63 2893.64
20.08 47.77 1489.11 2531.37
Mean 23.82 57.27 1515.03 2869.03
SD 2.71 20.84 45.60 169.95
% CV 11.37 6.38 3.01 5.92
% Nominal 116.80 103.90 103.07 97.59
20.13 47.49 1648.28 3284.71
22.42 51.90 1530.35 3282.22
24.09 57.63 1562.10 3162.53
PA-03
22.43 50.63 1510.47 3187.47
19.65 52.84 1659.87 3222.18
24.18 59.83 1522.64 3227.41
Mean 23.82 53.39 1572.28 3227.76
SD 3.50 4.57 65.72 49.23
% CV 14.70 8.56 4.18 1.53
% Nominal 116.77 96.85 106.96 109.79
88
Table 4.15 Recovery of Pramipexole
Quality control sample Aqueous analyte Extracted Analyte

ID area Area
5827 2861
3560 3196
3641 2729
LQC
3489 2831
3396 3017
3757 2889
Mean 3945 2921
%Recovery 74.03
108254 91159
112579 9390
113729 92370
MQC
116525 95627
115943 95699
118523 97800
Mean 114259 94291
%Recovery 82.52
237576 200475
234150 190470
233862 180647
HQC
229446 178929
223307 175996
227618 183945
Mean 230993 185077
% Recovery 80.12
89
Table 4.16 Recovery of Quetiapine (Internal Standard)
Quality control Extracted Analyte

Aqueous analyte area
sample ID Area
286951 274785
182502 264753
248380 273006
LQC
304910 274887
311395 266484
319042 265670
362114 266398
365558 270301
359083 268334
MQC
363605 273828
365346 270992
367941 275798
366101 265758
367659 270033
366802 270595
HQC
366252 263057
361254 268961
361082 262875
Mean 334777 269251
% Recovery 80.43
90
Table 4.17 Matrix Selectivity of Pramipexole
Plasma Selectivity (spiked %inter-ference in

Specificity (Blank) Area Ratio SN Ratio
lot LLOQ) Blank
Analyte Analyte/ IS
IS peak
Analyte IS peak Analyte IS peak Analyte
(≤5%)
(≤20%)
MAT 916 0 0 1892 128972 0.000 0.000 0.0147 44.862
MAT 917 0 0 1876 125755 0.000 0.000 0.0149 45.794
MAT 918 0 0 1928 124231 0.000 0.000 0.0155 49.631
MAT 919 0 0 1863 122650 0.000 0.000 0.0152 51.504
MAT 920 0 0 1850 122876 0.000 0.000 0.0151 44.596
MAT 921 0 0 1720 122123 0.000 0.000 0.0141 37.892
MAT 0 0 1851 123922 0.000 0.000 0.0149 52.306
136(H*)
Mean 0.01491
SD 0.000441
%CV 2.96
91
(H)*- Haemolyzed Lot % of lots passed = 100.00%
91
Table 4.18 Lower Limit of Quantitation (LLOQ) of Pramipexole
Parameters LLOQ
20.240
20.993
19.407
Calculated concentration (pg mL)
20.082
19.600
19.161
Mean 19.848
SD 0.723
%CV 3.640
%Nominal 98.073
92
92
Table 4.19 Matrix Effect of Pramipexole
Aqueous sample Spiked sample Area ratio Matrix Factor

Analyte Analyte Aqueous Spiked Matrix
Lot No IS Area IS Area
Area Area sample sample Factor
MAT 916 4489 121787 4429 119724 0.0369 0.037 1.02
MAT 917 4586 122720 4116 120522 0.0374 0.0342 0.94
MAT 918 4542 124656 4401 119279 0.0364 0.0369 1.02
MAT 919 4339 122989 4858 120102 0.0353 0.0404 1.11
MAT 920 4402 122223 4405 120335 0.036 0.0366 1.01
MAT 921 4465 124470 4519 119748 0.0359 0.0377 1.04
MAT 4386 121450 0.0361 0.99

136(H*)
Mean 0.036 0.036 1.018
SD 0.051
%CV 5.04
(H)*- Haemolyzed Lot Matrix Effect = 101.84

93
93
Table 4.20 Carry over test of Pramipexole
Sample ID Analyte peak area IS peak area
Extracted blank 0 0
Extracted LLOQ+IS 4489 121787
Extracted ULOQ+IS 4586 122720
Extracted blank 0 0
%Carry over 0.00 0.00
Table 4.21 Freeze and Thaw stability of Pramipexole
LQC CS HQC CS LQC FT4 HQC FT4
55.122 2939.864 55.122 2939.864
Actual 58.822 2979.755 63.466 2982.360

concentration
61.391 2924.904 55.401 2572.076
(pg/mL)
60.429 2928.377 53.287 2898.413
Calculated
concentration 62.534 3014.649 54.065 2797.862
(pg/mL) 63.728 2852.733 51.702 2775.815
62.828 2942.260 55.189 2883.421
Mean 61.622 2940.446 55.519 2818.324
SD 1.790 55.0183 4.1217 141.720
%CV 2.91 1.87 7.42 5.03
%Nominal 111.79 100.02 100.72 95.87
% Nominal against 90.10 95.85

CS
94
Table 4.22 Bench Top stability of Pramipexole
0.hr 8hrs
LQC HQC
LQC CS HQC CS
stability stability
Actual concentration 55.122 2939.864 55.122 2939.864

(pg/mL)
55.921 2991.807 53.741 3002.031
54.392 3021.0761 52.296 3064.895

Calculated
62.302 2968.565 48.587 3033.745
concentration(pg/mL)
54.816 2989.871 50.670 2921.980
52.401 2963.158 52.930 3000.352
52.469 2875.439 62.163 3005.113
Mean 55.384 2968.316 53.398 3004.686
SD 3.656 49.905 4.668 47.614
%CV 6.60 1.68 8.74 1.58
%Nominal 100.48 100.97 96.87 102.20
% Nominal against CS 96.41 101.23
95
4.5 Estimation of Pregabalin and Pramipexole in plasma samples
Estimation of Pregabalin and Pramipexole in plasma samples from the

volunteers was carried out using optimized chromatographic conditions.
The mass spectrum of Pregabalin parent ion and product ions were given in
Fig. 4.3 and 4.4 and for Tramadol parent ion and product ions were given in Fig. 4.5
and 4.6. A typical chromatogram obtained from a processed blank human plasma
sample is presented in Figure 4.7 and representative chromatograms of the lower
limit of quantitation, lower quality control, middle quality control, higher quality
control and upper limit of quantitation samples were given in Fig.4.8, 4.9, 4.10, 4.11
and 4.12.
The mass spectrum of Pramipexole parent ion and product ions were given in
Figure 4.13 and 4.14 and for Quetiapine parent ion and product ions were given in
Figure 4.15 and 4.16. A typical chromatogram obtained from a processed blank
human plasma sample is presented in Figure 4.17 and representative chromatograms
of the lower limit of quantitation, lower quality control, middle quality control, high
quality control and upper limit of quantitation samples were given in Figures 4.18,
4.19, 4.20, 4.21 and 4.22. The calibration samples (CC samples), quality control
samples (QC samples) and plasma sample solutions were injected with the optimised
and validated chromatographic conditions and chromatograms were recorded. The
retention time of Pragabalin and internal standard were 1.0 and 1.1, respectively. The
retention time Pramipexole and internal standard were 1.3 and 1.8, respectively. The
quantification of the chromatogram was performed using peak area ratios (response
factor) of the drug to internal standard. The calibration curves were constructed
routinely during the process of pre-study validation and in-study validation. The
mobile phase used for the assay provided a well-defined separation between the drug,
internal standard and endogenous components. The zero h (pre-dose) samples of all
subjects showed no interference at retention time of both selected drugs and internal
standards. The concentration of the selected drugs present in the plasma samples
were calculated.
96
Fig. 4.3: Mass Spectrum of Pregabalin (Parent Ion)
x102 +ESI MRM:52 (0.020-0.340 min, 5 scans) Frag=80.0V

(160.1 -> …**)
2.5Pregabalin_PREGABALIN_26155.1
1.5
0.5
0
52.5 53 53.5 54 54.5 55 55.5 56 56.5 57
57.5
Counts vs. Mass-to-Charge (m/z)
Fig. 4.4: Mass Spectrum of Pregabalin (Product Ion)
97
Fig. 4.5: Mass Spectrum of Tramadol (Parent Ion)
Fig. 4.6: Mass Spectrum of Tramadol (Product Ion)
98
Fig. 4.7: Representative chromatogram of processed blank plasma
Fig. 4.8: Typical chromatogram obtained from LLOQ
99
Fig. 4.9: Typical chromatogram obtained from LQC
Fig. 4.10: Typical chromatogram obtained from MQC
100
Fig. 4.11: Typical chromatogram obtained from HQC
Fig. 4.12: Typical chromatogram obtained from ULOQ
101
Fig. 4.13: Mass Spectrum of Pramipexole (Parent Ion)
Fig. 4.14: Mass Spectrum of Pramipexole (Product Ion)
102
Fig. 4.15: Mass Spectrum of Quetiapine (Parent Ion)
Fig. 4.16: Mass Spectrum of Quetiapine (Product Ion)
103
Fig. 4.17: Representative chromatogram of processed blank plasma
Fig. 4.18: Typical chromatogram obtained from LLOQ
104
Fig. 4.19: Typical chromatogram obtained from LQC
Fig. 4.20: Typical chromatogram obtained from MQC
105
Fig. 4.21: Typical chromatogram obtained from HQC
Fig. 4.22: Typical chromatogram obtained from ULOQ
106
4.6 In-vitro and in-vivo correlations for Pregabalin
4.6.1 In-vivo data analysis
Comparative bioavailability studies for immediate and modified release

formulations of Pregabalin were carried out in healthy human subjects. The drug-
plasma levels were measured over 24 hours by validated LCMSMS method. The
plasma concentration of the individual subjects and Pharmacokinetic parameters such
as, Peak plasma concentration (Cmax), Time to peak Concentration (tmax), Area under
the plasma concentration time curve (AUC0-24 and AUC0-∞), elimination rate
constant (keli) and Elimination half-life (t1/2) were calculated and are presented in
Table 4.23 to 4.24. The mean plasma concentration of the immediate and modified
release formulation of Pregabalin is presented in Table 4.25 and the mean
concentration time curve is presented in Fig. 4.23. The mean pharmacokinetic
profiles of immediate and modified release formulation of Pregabalin are presented
in Table 4.26.
The In-transformed values of Cmax, AUC0-t and AUC0-∞, along with the factors
included in this statistical analysis were period, sequence, treatments and subjects.
The factor subject was random and others were fixed. A difference between the
treatments was including 95% confidence interval. The design statement indicates
that the subjects were nested within the sequences.
The statistical parameters for In-transformed values of Cmax, that is the sum of
square, degree of freedom, mean square, F, significance values for immediate and
modified release formulations of Pregabalin, between subjects effects are presented
in Table 4.27. From these values, it is seen that the period, sequence and treatment
effects are non-significant when immediate release formulation was compared with
modified release formulation. However, the significant differences were observed
when immediate release formulation was compared with modified release
formulation. The 95% confidence interval of the difference between the two In-
transformed Cmax values for individual subjects and mean percentage ratio are
presented in Table 4.30. The 95% confidence interval for immediate and modified
release formulations ranges from -0.1306 to -0.0292, while the mean differences for
107
immediate and modified release formulations of Pregabalin was -0.0791. Back-
transformed to regular units, this means that the mean Cmax-0.0791=1.008, while
95% confidence interval for immediate and modified release formulations ranges
from -0.1306= 1.139 to -0.0292=1.089. The mean percentage ratio between
immediate and modified release formulation of Pregabalin was 194.65. The
percentage confidence interval for immediate vs modified release formulations
ranges from 88.50 to 108.04.
The statistical parameters for In-transformed values of AUC0-24, that is the

sum of square, degree of freedom, mean square, F, significance values for immediate
and modified release formulations of Pregabalin, between subjects effects are
presented in Table 4.28. From these values, it is seen that the period, sequence and
treatment effects are non-significant when immediate release formulation was
compared with modified release formulations. However, the significant differences
were observed when immediate release formulation was compared with modified
release formulation. The 95% confidence interval of the difference between the two
In-transformed AUC0-24 values for individual subjects and mean percentage ratio are
immediate and modified release formulations of Pregabalin was -0.0967. Back-
transformed to regular units, this means that the mean AUC0-24 -0.0967= 1.101, while
the 95 % confidence interval for immediate Vs modified release formulations ranges
from -0.1277 = 1.136 to -0.0721 = 1.075. The mean percentage ratio between
immediate and modified release formulation was 151.42. The percentage confidence
interval for immediate vs modified release formulations ranges from 96.32 to 97.64
The statistical parameters for In-transformed values of AUC0-∞, that is the

and modified release formulations of Pregabalin, between subjects effects are
release formulation. The 95 % confidence interval of the difference between the two
108
In-transformed AUC0-∞ values for individual subjects and mean percentage ratio are
presented in Table 4.30. The 95 % confidence interval for immediate and modified
immediate and modified release formulations of Pregabalinwas -0.0951. Back-
transformed to regular units, this means that the mean
AUC0-∞ -0.0951= 1.0998, while the 95 % confidence interval for immediate Vs
modified release formulations ranges from -0.1232 = 1.132 to -0.0665= 1.068. The
mean percentage ratio between immediate and modified release formulation was of
Pregabalin was 146.79. The percentage confidence interval for immediate Vs
modified release formulations ranges from 97.16 to 97.11.
The in-vitro release characteristics of the immediate and modifies release

formulations of Pregabalin was determined. Cumulative percentage drug release at
various time intervals were calculated and are presented in Table 4.31 and 4.32 and
in Fig. 4.24 and 4.25. The similarity factor (f2) was calculated and presented in
Table 4.33.
4.6.3 In-vitro and in-vivo correlations
This section describes in-vivo and in-vitro data analysis, in-vitro and in-vivo
model ‘A’ correlation development and validation for immediate and modified
release formulations.
When dissolution tests were performed at pH 1.2 buffer, pH 4.5 buffer, water
and pH 7.4 buffer at 50 and 75 rpm, the release of the Pregabalin was found to be
almost indistinguishable between the immediate and modified release formulations.
The higher similarity factor (f2) values (more than 50) confirms that these dissolution
mediums are indistinguishable and ensures sameness or equivalence between the two
dissolution profiles and hence not considered for the present study.
The best discrimination was achieved at pH 6.8 buffer at 50 rpm as well as 75

rpm. The (f2) value for pH 6.8 buffer at 50 rpm and 75 rpm were 45.98 and 42.68,
respectively. The associated f2 metric, and f2 value below 50 suggests that the two
109
dissolution profiles are dissimilar and reveals pH 6.8 buffer at 50 and 75 rpm were
more discriminating dissolution mediums and hence selected for IVIVC model
development.
A Level A correlation was developed by a two-stage procedure,

deconvolution followed by comparison of the percent dissolved vs the percent
absorbed data for both immediate and modified release formulations. The in-vitro
and in-vivo correlation plot was constructed using percentage of drug dissolved at
pH 6.8 buffer dissolution media at both 50 and 75 rpm vs the percentage of drug
absorbed. The slope of the best-fit line was examined using linear regression
2
analysis and coefficient of correlation (r ). The slope and intercept values were
calculated and are presented in Table 4.34 and in Fig. 4.26 and 4.27. The correlation
2
coefficient (r ) of pH 6.8 buffer and at 50 and 75 rpm was 0.8098 and 0.8130,
respectively for modified release formulation, while 0.8960 and 0.9084, respectively
for immediate release formulation. A good linear regression relationship was thus
observed when the dissolution studies were carried out in pH 6.8 buffer at 75 rpm
and hence this was selected for further analysis.
The dissolution rate constants were determined from percentage drug release
versus the square root of time. The slope of the best-fit line for the semi-log
treatment of this data was taken as the first order rate constant for absorption. Linear
regression analysis was applied to the in-vitro and in- vivo correlation plots and
2
coefficient of correlation (r ), slope and intercept values were calculated and are
2
presented in Fig. 4.28 to 4.29. The correlation coefficient (r ) for pH 6.8 buffer at
75 rpm for modified release and immediate release formulation were 0.9251 and
0.9483, respectively. A good linear regression relationship was thus observed using
pH 6.8 buffer as dissolution medium at 75 rpm and hence this was selected at the
dissolution media of choice.
4.6.4 Internal validation
dissolution data and mean in-vivo pharmacokinetics of the immediate and modified
release formulations. The mean in-vitro dissolution rate constants were correlated to
110
the mean absorption rate constants for the modified release formulations. These two
data points, along with the zero–zero intercept were used to calculate the expected
absorption rate constants.
The prediction of plasma concentration was calculated. From this, percentage

prediction errors for Cmax and AUC were calculated and are presented in Table 4.35
and 4.36 and in Fig.4 30 and 4.31. The Cmax prediction errors for both the immediate
and modified release formulations were found to be 2.190 and -12.60, respectively.
The AUC prediction errors for both the immediate and modified release formulations
were found to be 9.39 and -2.76, respectively. These values were very close to the
observed mean values.
4.6.5 External validation
The in-vitro release characteristics of the immediate and modified release

formulations of Pregabalin were determined. Cumulative percentage drug release at
various time intervals and similarity factor (f2) were calculated. When dissolution
test was performed at pH 6.8 buffer at 75 rpm, best discrimination was achieved
between the immediate and modified release formulations of Pregabalin. The
associated f2 metric, and f2 value below 50 suggests that the two dissolution profiles
are dissimilar and reveals pH 6.8 buffer at 75 rpm was discriminating dissolution
medium.
An in-vitro and in-vivo correlation plot was constructed using percentage of

the drug dissolved at pH 6.8 buffer dissolution media at 75 rpm vs the percentage of
drug absorbed. The slope of the best fit line was examined using Linear regression
2
analysis and the coefficient of determination (r ), slope and intercept values
2
calculated are presented. The correlation coefficient (r ) value for immediate and
modified release formulations was 0.9084 and 0.813, respectively. A good linear
regression relationship was observed.
Vs the square root of time. The slope of the best fit line for the semi-log treatment of
this data was taken as the first order rate constant for absorption. Linear regression
analysis was applied to the in-vitro and in-vivo correlation plots and the coefficient of
111
2
correlation (r ), slope and intercept values calculated are presented in Table 4.37. The
2
correlation coefficients (r ) value for immediate and modified release formulations
was 0.9251 and 0.9483, respectively.
The prediction of plasma concentration for immediate and modified release

formulations of Pregabalin was calculated. From this, percentage prediction errors
for Cmax and AUC were calculated. The Cmax prediction errors for both the immediate
and modified release formulations were found to be 2.190 and -12.60, respectively.
The AUCprediction errors for both the immediate and modified release formulations
were found to be 9.39 and -2.76, respectively. The Cmax and AUC prediction error
was within the specified limit and hence, the IVIVC is considered as validated both
in terms of internal and external validation.
112
Table 4.23 Individual plasma concentrations (mcg/ml) and pharmacokinetic parameters for Pregabalin Immediate Release
Formulation
Time A B C D E F G H I J K L
0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.5 693.88 1233.89 1009.43 912.05 1079.76 1167.68 926.91 769.08 998.55 741.19 1247.71 1034.66
1 890.98 1223.04 982.75 1228.39 1169.44 1130.67 880.00 1027.01 962.66 1084.40 1221.97 1056.43
1.5 844.23 1140.27 924.60 1217.09 1102.79 1087.28 864.50 968.67 926.07 1021.00 1105.73 964.13
2 796.75 1068.28 875.72 1102.39 1008.13 1038.79 818.74 922.20 900.59 967.07 1025.43 972.38
3 686.76 958.20 768.81 1039.51 824.13 968.26 757.22 809.55 832.27 848.09 893.08 897.50
4 592.69 816.33 657.19 972.17 722.65 849.06 631.61 678.56 691.43 723.21 721.23 751.43
6 487.84 652.04 515.32 815.22 576.14 687.19 539.41 546.04 543.36 564.57 588.36 638.52
8 365.36 478.97 376.27 684.43 404.46 543.50 401.86 402.49 357.09 411.99 418.13 404.12
12 259.27 304.36 261.99 522.06 333.32 292.18 254.86 263.50 235.35 286.17 236.16 271.07
18 109.93 143.88 140.29 319.82 232.41 161.80 142.59 136.44 141.06 150.65 106.83 140.91
24 0.00 0.00 0.00 71.49 62.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Cmax 890.98 1233.89 1009.43 1228.39 1169.44 1167.68 926.91 1027.01 998.55 1084.40 1247.71 1056.43
Tmax 1.00 0.50 0.50 1.00 1.00 0.50 0.50 1.00 0.50 1.00 0.50 1.00
AUC 0-t 7643.85 9065.68 7338.02 12587.92 9110.02 9307.44 7225.74 7498.71 7326.70 7938.46 7941.69 8156.38
Keli 0.18 0.23 0.21 0.19 0.25 0.22 0.29 0.22 0.22 0.25 0.26 0.22
T-half 4.62 4.76 5.03 6.27 5.92 5.14 5.28 4.83 4.89 5.02 4.00 4.91
AUC0-∞ 7983.32 9652.60 8324.32 13188.42 9593.87 10075.77 7871.93 8043.61 7911.38 8609.06 8216.11 8742.40
113
113
Table 4.23 (continued). Individual plasma concentrations (mcg/ml) and pharmacokinetic parameters for Pregabalin Immediate
Release Formulation
Time M N O P Q R S T U V W X
0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.5 996.73 934.18 1054.32 1101.33 803.33 920.32 1056.98 981.88 618.44 987.43 870.56 714.31
1 1216.98 1097.34 1186.45 1259.43 874.59 926.47 1231.08 967.73 877.82 1041.59 998.78 1040.97
1.5 1324.57 1161.28 1133.55 1227.55 864.16 903.32 1014.38 922.72 848.47 965.44 971.43 1005.37
2 172.30 1086.90 1075.23 1103.03 797.07 888.29 983.37 859.45 811.10 857.05 915.38 956.77
3 1035.28 973.10 953.11 1030.98 712.50 777.12 874.11 751.10 734.47 731.07 837.22 861.49
4 847.05 731.57 829.19 964.90 591.36 665.05 707.54 698.09 635.31 588.00 732.48 766.77
6 668.94 605.85 650.54 759.19 400.86 476.29 438.81 490.01 4395.84 383.22 520.21 618.99
8 522.65 395.34 497.43 593.16 260.74 353.46 275.10 388.36 318.60 241.07 364.74 462.90
12 315.64 292.68 277.53 324.00 123.57 214.90 131.61 240.28 154.30 165.40 215.46 284.25
18 120.33 143.68 144.21 155.98 104.04 104.79 105.61 152.13 89.35 110.25 122.65 158.20
24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Cmax 1324.57 1161.28 1186.45 1259.43 874.59 926.47 1231.08 981.88 877.82 1041.59 998.78 1040.97
Tmax 1.50 1.50 1.00 1.00 1.00 0.50 1.00 0.50 1.00 1.00 1.00 1.00
AUC 0-t 9772.32 8927.36 9315.37 10455.0 6532.90 7033.67 6720.94 7141.27 6144.71 6063.23 7560.25 8608.18
Keli 0.27 0.19 0.21 0.19 0.26 0.20 0.26 0.24 0.22 0.21 0.22 0.18
T-half 4.69 5.17 5.30 5.40 4.53 4.89 4.30 5.44 4.37 4.61 5.07 5.83
AUC0-∞ 10268.6 10453.5 10050.0 11290.8 7175.97 7450.12 7089.49 7617.33 6425.08 6488.09 8114.36 9531.01
114
114
Table 4.24 Individual Plasma Concentrations (Mcg/Ml) and Pharmacokinetic Parameters for Pregabalin Modified Release
Formulation
0 0 0 0 0 0 0 0 0 0 0 0 0
0.5 88.43 78.1 78.98 0 0 0 0 0 0 0 0 0
1 259.77 174.08 173.79 263.77 217.43 207.33 252.08 240.66 284.556 187.35 184.88 119.55
1.5 357.83 296 251.51 268.43 314.87 296.33 329.07 417.54 384.85 366 216.56 169.98
2 478.79 376.84 362.26 353.47 402.32 360.11 510.23 554.67 476.98 456 383.97 359.74
3 623.97 463.29 438.55 549.11 673.05 583.77 549.97 486.36 673.07 585.06 615.67 583.07
4 547.41 518.11 363.98 463.09 555.95 514.74 524.98 452.39 614.07 487.46 535.93 514.09
6 489.59 449.89 333.77 435.07 526.79 485.66 459.92 355.01 485.05 376.38 475.09 447.23
8 450.07 364.83 258.1 387.54 392.05 389.89 365.71 295.6 391.9 309.17 389.98 378.91
12 231.69 216.78 226.8 238.95 226.44 225.96 247.43 245.67 233.99 245.97 233.58 241.28
18 156.15 152.57 163.88 156.7 153.94 144.96 157.95 137.71 160.86 135.27 153.24 156.38
24 84.97 88.58 86.89 85.86 81.99 83.38 91.97 70.43 83.52 68.09 85.99 86.57
Cmax 623.97 518.11 438.55 549.11 673.05 583.77 549.97 554.67 673.07 585.06 615.67 583.07
Tmax 3 4 3 3 3 3 3 2 3 3 3 3
AUC 0-t 5999.59 5219.26 4493.9 5373.99 5772.16 5442.23 5734.17 5212.13 5991.84 5449.93 5547.44 5378.11
Keli 0.14 0.11 0.06 0.2 0.11 0.18 0.11 0.17 0.12 0.13 0.11 0.11
T-half 6.12 6.62 7.42 6.5 5.76 6.54 6.73 7.15 6.21 6.5 6.03 6.44
115
AUC0-∞ 6530.8 5807.79 5216.36 5956.31 6317.07 5961.82 6361.18 5873.7 6500.2 6034.98 6097.42 5952.02
115
Table 4.24 (continued) Individual Plasma Concentrations (Mcg/Ml) and Pharmacokinetic Parameters for Pregabalin Modified
Release Product
0 0 0 0 0 0 0 0 0 0 0 0 0
0.5 0 145.09 90 90.44 0 0 0 0 0 99.06 0 98.1
1 162.89 273.67 185.88 167.88 196.98 174.77 117.07 231.44 175 173.9 206.96 174.87
1.5 392.59 344.41 364.48 389.36 389.25 261.23 250.91 327.11 298.91 348.53 287.99 376.27
2 426.38 378.97 410.03 565.74 423.05 395 522.53 394.99 415.94 475.49 400.43 414.79
3 583.68 441.77 540.77 519.7 477.67 440.32 626.95 531.7 500.04 531.14 508.23 468.75
4 545.97 500.23 491.07 481.23 394.05 505.89 554.07 448.05 444.08 464.07 453.42 509.08
6 424.96 498.38 351.64 371.78 413.53 445.05 494.99 374.37 381.97 354.51 380.55 407.37
8 311.39 377.65 279.68 349.65 252.98 387.79 414.56 299.26 334.48 339.65 314.2 334.7
12 245.81 247.81 235.97 226.89 206.58 248.56 220.97 246.96 247.44 231.78 240.81 224.99
18 147.28 143.32 142.71 133.04 137.99 162.24 155.28 146.11 135.51 140.38 132.65 126.86
24 69.11 64.56 67.22 71.11 67.43 86.99 90.14 67.76 67.67 70.48 67.85 59.71
Cmax 583.68 500.23 540.77 565.74 477.67 505.89 626.95 531.7 500.04 531.14 508.23 509.08
Tmax 3 4 3 2 3 4 3 3 3 3 3 4
AUC 0-t 5543.51 5645.58 5107.37 5350.42 4767.48 5418.24 5748.3 5176.75 5174.92 5257.47 5088.56 5174.36
Keli 0.15 0.12 0.13 0.1 0.14 0.21 0.11 0.13 0.1 0.14 0.1 0.15
T-half 6.03 6.53 6.89 6.21 7.21 7.21 6.11 7.83 6.91 7.11 7.21 7.03
AUC0-∞ 6142.33 6147.07 5715.09 5992.51 5402.92 5995.77 6337.85 5786.52 5788.68 5905.79 5699.04 5649.71
116
116
Table 4.25 Mean plasma concentrations (mcg/ml) for Pregabalin
Time Immediate Release Modified Release

Formulation formulation
(h)
Mean S.D Mean S.D
0.00
0.00 0.00 0.00 0.00
0.50
952.28 166.10 32.01 47.70
1.00
1065.71 129.36 200.27 44.85
1.50
1021.19 132.18 320.83 62.63
2.00
916.77 187.23 429.11 62.23
3.00
856.46 107.22 541.49 70.51
4.00
731.87 104.87 495.14 54.72
6.00
731.78 787.42 425.77 56.35
8.00
413.43 102.95 348.74 50.70
12.00
260.83 80.18 234.96 11.40
18.00
143.24 47.49 147.21 10.64
24.00
5.56 18.90 77.01 10.02
117
Fig.4.23: Mean Concentration Time Curve for Pregabalin
118
118
Table 4.26 Pharmacokinetic Profile of Pregabalin
Parameters Immediate release product Modified Release product
Mean S.D Mean S.D
Cmax 1081.07 135.50 555.38 58.96
Tmax 0.88 0.30 3.08 0.50
AUC 0-t 8142.33 1496.59 5377.82 350.39
K eli 0.26 0.03 0.13 0.03
Tmax 5.01 0.52 6.68 0.52
AUC 0-t 8756.98 1603.36 5965.54 317.87

119
119
Table 4.27 Statistical data for Pregabalin Immediate Release Vs Modified Release Formulation
Dependent variable: Cmax Tests of Between-Subjects Effect
Source Type III df Mean F Sig. Partial Eta Noncent. Observed

a
sum of square squared parameter power
squares
Intercept Hypothesis 90.564 1 90.564 12343.706 .000 .996 12343.706 1.000

b
Error .332 29.675 9.567E-03
SEQ Hypothesis 3.453E-05 1 2.667E-05 .004 .956 .000 .004 .121

C
Error .180 22 8.421E-3
PERIOD Hypothesis .000 0 . . . . . .
Error . . .
d
TREATMENT Hypothesis .000 0 . . . . . .
Error . . .
d
SUBJECT Hypothesis .743 23 1.754E-02 2.167 .043 .743 51.656 .977

c
Error .231 22 8.421E-03
a. Computed using alpha = .1
b. 5.266E-02 MS(SUBJECT) +.947 MS (Error)
120
c. MS (Error)
d. Cannot compute the appropriate error term using Satterthwaite’s method
120
Dependent variable: AUC 0-t Tests of Between-Subjects Effect

a
squares
Error 9.545E-02 27.067 3.543E-03b

C
Error 7.993E-02 22 3.665-03
Error . . .
d
Error . . .
d

c
Error 7.993E-02 22 3.872E-03
c. MS (Error)
121
121
Dependent variable: AUC 0-inf Tests of Between-Subjects Effect

a
squares
Intercept Hypothesis 186.443 1 186.443 69656.098 .000 1.000 69656.098 1.000

b
Error 7.554E-02 27.998 2.554E-03

C
Error 5.976E-02 22 2.997E-03
Error . . .
d
Error . . .
d

C
Error 5.987E-02 22 2.997E-03
c. MS (Error)
122
122
Table 4.30 Paired Sample Test for Pregabalin
Paired Differences
Std. Std. 95% Confidence
Mean Deviation Error Interval of the
Mean Difference t df Sig. (2-
tailed)
Lower Upper
Pair 1 C max -0.0791 .12434 .02667 -.1306 -.0292 -3.553 23 0.005
Pair 2 AUC 0-t -.0967 0.0734 .01765 -.1277 -.0721 -4.556 23 .000
Pair 3 AUC 0-inf -.0951 .07098 .01332 -.1232 -.0665 -6.556 23 .000
123
123
Table 4.31 Cumulative percentage dissolved at 50 rpm for Pregabalin formulations
Time Square pH 1.2 buffer pH 4.5 buffer pH 6.8 buffer Water pH 7.4 buffer
(h) root of Formulation Formulation Formulation Formulation Formulation
time
Immediate Modified Immediate Modified Immediate Modified Immediate Modified Immediate Modified
(h)
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.50 0.71 25.32 0.00 26.57 0.00 16.32 7.56 27.94 11.86 16.93 10.98
1.00 1.00 38.48 36.87 38.82 32.35 35.56 15.99 32.37 30.22 40.21 19.09
1.50 1.22 40.45 42.83 49.21 43.92 58.96 32.97 40.67 38.94 52.06 32.19
2.00 1.41 48.30 46.06 52.13 44.67 84.18 41.90 48.03 42.38 58.01 36.90
3.00 1.73 55.87 50.06 58.77 55.55 97.44 62.08 56.85 51.27 62.09 42.32
4.00 2.00 65.16 54.96 64.69 64.43 100.02 75.03 69.00 58.88 74.32 49.76
6.00 2.45 88.32 64.76 70.44 66.75 100.87 84.02 100.12 87.32 82.19 63.99
8.00 2.83 90.21 74.52 85.94 74.62 101.01 95.21 100.44 100.09 100.67 73.07
12.00 3.46 100.01 96.75 99.53 90.22 101.86 99.02 100.59 100.15 101.98 101.09
18.00 4.24 100.37 99.53 100.61 100.01 102.18 99.87 100.98 101.67 102.44 102.98
24.00 4.90 100.57 99.83 100.11 102.34 102.39 99.99 101.43 101.43 102.98 103.00
124
124
100
80
60
RELEASE (%)
CUMULATIVE
40
20
0
0 5 15 20
10 TIME 25
(HOURS)
Fig. 4.24: Cumulative Pregabalin Release Vs Time Profile for Immediate and
Modified Release Formulations at 50 RPM
125
125
Table 4.32 Cumulative percentage dissolved at 75 rpm for Pregabalin formulations
(h) root of Formulation Formulation Formulation Formulation Formulation
time
Immediate Modified Immediate Modified Immediate Modified Immediate Modified Immediate Modified
(h)
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.50 0.71 29.11 0.00 32.54 0.00 17.99 10.32 28.63 20.32 17.21 15.43
1.00 1.00 39.64 40.32 42.83 34.99 36.78 22.64 35.92 36.21 43.28 25.32
1.50 1.22 43.01 47.9 50.84 45.89 64.89 40.21 40.67 40.21 54.90 45.22
2.00 1.41 53.72 50.44 61.66 55.55 75.66 45.89 48.03 47.29 62.67 53.20
3.00 1.73 64.67 67.93 67.93 64.90 97.32 61.21 56.85 52.64 67.00 59.12
4.00 2.00 75.64 83.54 70.52 74.90 100.02 71.64 78.21 74.32 79.32 62.00
6.00 2.45 99.56 99.32 84.83 92.67 100.97 86.83 99.21 88.32 96.33 70.53
8.00 2.83 100.42 99.54 99.11 100.03 101.01 99.43 99.29 98.32 99.32 99.43
12.00 3.46 100.72 100.43 100.61 100.43 100.86 99.67 99.45 98.64 99.78 100.05
18.00 4.24 100.81 101.23 100.11 101.95 100.18 100.01 99.59 99.43 100.32 100.56
24.00 4.90 100.99 101.89 100.50 102.64 102.09 100.23 100.19 100.02 101.21 100.94
126
126
CHART
TITLE
100
80
60
RELEASE (%)
CUMULATIVE
40
20
0
0 5 10 15
20 25
TIME
(HOURS)
127
127
Table 4.33 Similarity factors for Pregabalin modified release dosage forms in
Various dissolution condition
S.No pH Condition Formulation Similarity factor (f2)
1 pH 1.2 buffer 50 rpm IR Vs MR 83.01
7 Distilled water 50 rpm IR Vs MR 73.64
8 Distilled water 75 rpm IR Vs MR 77.45

128
128
Table 4.34 IVIVC model regression of % absorbed vs. % dissolved for Pregabalin
Formulation using pH 6.8 at 50 and 75 rpm
Time Percentage dissolved Percentage dissolved Percentage absorbed

(Hours) 50 RPM 75 RPM
Immediate Modified Immediate Modified Immediate Modified
0.00 0.00 0.00 0.00 0.00 0 0
0.50 16.32 7.56 17.99 10.32 32.65 3.75
1.00 35.56 15.99 36.78 22.64 62.38 25.43
1.50 58.96 32.97 64.89 40.21 78.10 48.54
2.00 84.18 41.90 75.66 45.89 95.32 70.54
3.00 97.44 62.08 97.32 61.21 97.43 93.45
4.00 100.02 75.03 100.02 71.64 98.54 96.44
6.00 100.87 84.02 100.97 86.83 99.54 99.43
8.00 101.01 95.21 101.01 99.43 100.21 99.54
12.00 101.86 99.00 100.86 99.67 100.45 99.96
18.00 102.18 99.87 100.18 100.01 101.43 100.49
24.00 102.39 99.99 102.09 100.23 101.45 100.54

129
129
100
90
80
70
60
50
40
30
20
10
0
0 20 40 60 80
100
Fig. 4.26: IVIVC model Linear Regression Plot of % Absorbed Vs % Dissolved

for Immediate and Modified Release Pregabalin Formulations using pH 6.8
Buffer at 50 RPM
Fig.4.27: IVIVC model Linear Regression Plot of % Absorbed Vs % Dissolved

for Immediate and Modified Release Pregabalin Formulations using pH 6.8
Buffer at 75 RPM
130
100
80
60
40
20
0 y = 19.708x y = 23.123x +
+ 32.195 11.504
R² = 0.619 R² =
0.8364
Fig. 4.28: Cumulative Pregabalin Release Vs Square Root of Time Profile for
Immediate and Modified Release Pregabalin Formulations using pH 6.8 Buffer
at 50 RPM
100
80
60
40
20
y = 19.708x + y = 23.123x +
32.195 11.504
0 R² = R² =
0.619 0.8364
Fig. 4.29: Cumulative Pregabalin Release Vs Square Root of Time Profile for
Immediate and Modified Release Pregabalin Formulations using pH 6.8 Buffer
at 75 RPM
Table 4.35 Observed and IVIVC model predicted Cmax and AUC values for
Pregabalin
Time Immediate Modified
(Hours) Fraction Fraction Fraction Fraction
observed predicted observed predicted
0.00 0.00 0.00 0.00 0
0.50 952.28 768.45 32.01 16.89
1.00 1065.71 999.87 200.27 83.54
1.50 1021.19 1089.56 320.83 220.67
2.00 916.77 1000.22 429.11 326.84
3.00 856.46 934.39 541.49 480.89
4.00 731.87 890.43 495.14 463.94
6.00 731.78 700.59 425.77 375.47
8.00 413.43 563.32 348.74 269.05
12.00 260.83 308.90 234.96 132.98
18.00 143.24 178.32 147.21 26.97
24.00 5.56 32.01 77.01 13.98
Cmax 1065.71 1089.56 541.49 480.89
AUC 9453.99 10433.32 5995.34 5834.06
Table 4.36 Prediction errors (%) associated with Cmax and AUC for Pregabalin
Formulation Cmax AUC
Immediate 2.19 9.39
Modified -12.60 -2.76
Average -5.21 3.31

3.5
2.5
1.5
0.5
0
0 5 10 15 20
25
Fraction observed Fraction predicted
Fig. 4.30 Observed and Predicted Pregabalin Plasma Concentration for the
Immediate Release Pregabalin Formulations using IVIVC Model
3.5
2.5
1.5
0.5
0
0 5 10 15 20
25
Fig. 4.31 Observed and Predicted Pregabalin Plasma Concentration for the
Modified Release Pregabalin Formulations using IVIVC Model
Table 4.37 IVIVC model linear regression plots of % absorbed vs % dissolved
for Pregabalin tablets using pH 6.8, at 75 rpm
Time Percentage dissolved (pH 6.8) Percentage absorbed
(Hours) Immediate Modified Immediate Modified
0.00 0.00 0.00 0 0
0.50 17.99 10.32 32.65 3.75
1.00 36.78 22.64 62.38 25.43
1.50 64.89 40.21 78.10 48.54
2.00 75.66 45.89 95.32 70.54
3.00 97.32 61.21 97.43 93.45
4.00 100.02 71.64 98.54 96.44
6.00 100.97 86.83 99.54 99.43
8.00 101.01 99.43 100.21 99.54
12.00 100.86 99.67 100.45 99.96
18.00 100.18 100.01 101.43 100.49
24.00 102.09 100.23 101.45 100.54

4.7 In-vitro and in-vivo correlations for Pramipexole
Comparative bioavailability studies for immediate and modified release

formulations of Pramipexole were carried out in healthy human subjects. The drug-
plasma levels were measured over 24 hours by validated LCMSMS method. The
plasma concentration of the individual subjects and Pharmacokinetic parameters such
as, Peak plasma concentration (Cmax), Time to peak Concentration (tmax), Area under
the plasma concentration time curve (AUC0-24 and AUC0-∞), elimination rate
constant (keli) and Elimination half-life (t1/2) were calculated and are presented in
Table 4.38 to 4.39. The mean plasma concentration of the immediate and modified
release formulation of Pramipexole is presented in Table 4.40. The mean
concentration time curve is presented in Fig.4.32. The mean pharmacokinetic profiles
of immediate and modified release formulation of Pramipexole are presented in
Table 4.41.
The In-transformed values of Cmax, AUC0-t and AUC0-∞ ,along with the factors
included in this statistical analysis were period, sequence, treatments and subjects.
The factor subject was random and others were fixed. A difference between the
treatments was including 95% confidence interval. The design statement indicates
that the subjects were nested within the sequences.
The statistical parameters for In-transformed values of Cmax, that is the sum of
square, degree of freedom, mean square, F, significance values for immediate and
modified release formulations of Pramipexole, between subject effects are presented
in Table 4.42. From these values, it is seen that the period, sequence and treatment
effects are non-significant when immediate release formulation was compared with
modified release formulation. However, the significant differences were observed
when immediate release formulation was compared with modified release
formulation. The 95% confidence interval of the difference between the two In-
transformed Cmax values for individual subjects and mean percentage ratio are
release formulations ranges from -0.097 to 0.085, while the mean differences for
immediate and modified release formulations of Pramipexole was -0.005. Back-
transformed to regular units, this means that the mean Cmax 0.005= 1.0051, while
95% confidence interval for immediate and modified release formulations ranges
from 0.097= 1.102 to 0.085=1.089. The mean percentage ratio between immediate
and modified release formulation of Pramipexole was 73.94. The percentage
confidence interval for immediate vs modified release formulations ranges from
108.64 to 109.64.
The statistical parameters for In-transformed values of AUC0-24, that is the

and modified release formulations of Pramipexole, between subject effects are
In-transformed AUC0-24 values for individual subjects and mean percentage ratio are
transformed to regular units, this means that the mean AUC0-24 -0.092 = 1.097, while
the 95 % confidence interval for immediate vs modified release formulations ranges
from -0.160=1.174 to -0.024= 1.025. The mean percentage ratio between immediate
and modified release formulation was 57.84. The percentage confidence interval for
immediate vs modified release formulations ranges from 93.43 to 107.01.
The statistical parameters for In-transformed values of AUC0-∞, that is the

and modified release formulations of Pramipexole, between subject effects are
In-transformed AUC0-∞ values for individual subjects and mean percentage ratio are
transformed to regular units, this means that the mean AUC0-∞ -0.086= 1.090, while
the 95 % confidence interval for immediate vs modified release formulations ranges
from -0.153= 1.166 to -0.028= 1.029. The mean percentage ratio between immediate
and modified release formulation was of Pramipexole was 56.63. The percentage
confidence interval for immediate vs modified release formulations ranges from
94.40 to 106.97.
4.7.2 In-vitro data analysis
The in-vitro release characteristics of the immediate and modifies release

formulations of Pramipexole was determined. Cumulative percentage drug release at
various time intervals were calculated and are presented in Table 4.46 and 4.47 and
in Fig. 4.33 and4.34. The similarity factor (f2) was calculated and presented in Table
4.48.
4.7.3 In-vitro and in-vivo correlations
This section describes in-vivo and in-vitro data analysis, in-vitro and in-vivo
model ‘A’ correlation development and validation for immediate and modified
release formulations. When dissolution tests were performed at pH 6.8 buffer, pH 7.4
buffer and water at 50 and 75 rpm, the release of the Pramipexole was found to be
almost indistinguishable between the immediate and modified release formulations.
The higher similarity factor (f2) values (more than 50) confirms that these dissolution
mediums are indistinguishable and ensures sameness or equivalence between the two
dissolution profiles and hence not considered for the present study.
The best discrimination was achieved at pH 1.2 buffer and pH 4.5 buffer at
50 rpm as well as 75 rpm. The (f2) value for pH 1.2 buffer and pH 4.5 buffer 50 rpm
was 45.87 and 43.76, respectively, whereas at 75 rpm the (f2) value 47.34 and 42.91,
respectively. The associated f2 metric, and f2 value below 50 suggests that the two
dissolution profiles are dissimilar and reveals pH 1.2 buffer and pH 4.5 buffer at 50
137
ad 75 rpm were more discriminating dissolution mediums and hence selected for
IVIVC model development.
A Level A correlation was developed by a two-stage procedure,

deconvolution followed by comparison of the percent dissolved Vs the percent
absorbed data for both immediate and modified release formulations. The in-vitro
and in-vivo correlation plot was constructed using percentage of drug dissolved at pH
1.2 buffer and pH 4.5 buffer dissolution media at both 50 and 75 rpm Vs the
percentage of drug absorbed. The slope of the best-fit line was examined using
2
linear regression analysis and coefficient of correlation (r ). The slope and intercept
values were calculated and are presented in Table 4.49 and 4.50 and in Fig. 4.34 to
2
4.37. The correlation coefficient (r ) of pH 1.2 buffer and pH 4.5 buffer at 50 rpm
was 0.9397 and 0.8473, respectively for modified release formulation, while for pH
1.2 buffer and pH 4.5 buffer at 50 rpm was 0.8473 and 0.7826, respectively for
2
immediate release formulation. The correlation coefficient (r ) of pH 1.2 buffer and
pH 4.5 buffer at 75 rpm was 0.9316 and 0.8539, respectively for modified release
formulation, while for pH 1.2 buffer and pH 4.5 buffer at 75 rpm was 0.913 and
0.8945, respectively for immediate release formulation. A good linear regression
relationship was thus observed when the dissolution studies were carried out in pH
1.2 buffer at 50 and 75 rpm and hence this was selected for further analysis.
vs the square root of time. The slope of the best-fit line for the semi-log treatment of
analysis was applied to the in-vitro and in- vivo correlation plots and coefficient of
2
correlation (r ), slope and intercept values were calculated. The correlation
2
coefficient (r ) for pH 1.2 buffer at 50 rpm for modified release and immediate
release formulation were 0.9827 and 0.9542, respectively, while at 75 rpm rpm for
modified release and immediate release formulation were 0.9914 and 0.9815,
respectively. A good linear regression relationship was thus observed using pH 1.2
buffer as dissolution medium at 75 rpm and hence this was selected at the dissolution
media of choice.
138
4.7.4 Internal validation
dissolution data and mean in-vivo pharmacokinetics of the immediate and modified
release formulations. The mean in-vitro dissolution rate constants were correlated to
the mean absorption rate constants for the modified release formulations. These two
data points, along with the zero –zero intercept were used to calculate the expected
absorption rate constants. The prediction of plasma concentration was calculated.
From this, percentage prediction errors for Cmax and AUC were calculated and are
presented in Tables 4.51 and in Fig. 4.38 and 4.39. The Cmax prediction errors for
both the immediate and modified release formulations were found to be -2.724 and
1.205, respectively. The AUCprediction errors for both the immediate and modified
release formulations were found to be -0.610 and 2.252, respectively. These values
were very close to the observed mean values.
4.7.5 External validation
The in-vitro release characteristics of the immediate and modified release

formulations of Pramipexole were determined. Cumulative percentage drug release
at various time intervals were calculated. The similarity factor (f2) was calculated.
When dissolution test was performed at pH 1.2 buffer at 75 rpm, best discrimination
was achieved between the immediate and modified release formulations of
Pramipexole. The associated f2 metric, and f2 value below 50 suggests that the two
dissolution profiles are dissimilar and reveals pH 1.2 buffer at 75 rpm was
discriminating dissolution medium.
An in-vitro and in-vivo correlation plot was constructed using percentage of

the drug dissolved at pH 1.2 buffer dissolution media at 75 rpm vs the percentage of
drug absorbed. The slope of the best fit line was examined using Linear regression
2
analysis and the coefficient of determination (r ), slope and intercept values
2
calculated. The correlation coefficient (r ) value was 0.7843. A good linear
regression relationship was observed.
vs the square root of time. The slope of the best fit line for the semi-log treatment of
139
analysis was applied to the in-vitro and in-vivo correlation plots and the coefficient of
2 2
correlation (r ), slope and intercept values calculated. The correlation coefficients (r )
value was 0.7439.
The prediction of plasma concentration for immediate and modified release

formulations of Pramipexole was calculated. From this, percentage prediction errors
for Cmax and AUC were calculated. The Cmax prediction error for the immediate and
modified release formulations of Pramipexole was found to be -2.724 and 1.205,
respectively. These values were very close to the observed mean values. The AUC
prediction error was -0.610 and 2.252 for immediate and modified release
formulations of Pramipexole, respectively and is presented in Table 4.52. The Cmax
and AUC prediction error was within the specified limit and hence, the IVIVC is
considered as validated both in terms of internal and external validation.
140
Table 4.38 Individual plasma concentrations (mcg/ml) and pharmacokinetic parameters for Pramipexole Immediate Release
Formulation
0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
0.5 0.721 0.357 1.643 1.734 0.265 1.924 1.231 1.238 1.488 1.430 0.118 1.501
1 0.985 0.849 3.352 2.318 0.869 2.370 1.435 2.123 2.674 2.283 2.601 2.409
1.5 1.586 1.380 3.478 2.366 2.528 2.037 1.847 3.680 2.355 2.373 2.375 3.657
2 1.615 2.462 2.898 2.445 1.970 1.722 1.971 2.539 2.248 2.473 2.026 3.390
3 1.357 1.634 2.130 2.063 1.830 1.317 1.428 1.726 1.590 1.495 1.537 2.201
4 1.213 1.587 1.678 1.350 1.528 1.141 1.134 1.419 1.204 1.229 1.256 1.626
6 0.472 0.733 1.055 0.877 1.157 0.859 0.731 0.958 0.917 0.866 0.877 1.108
8 0.312 0.305 0.698 0.645 0.944 0.710 0.583 0.759 0.701 0.608 0.553 0.717
12 0.148 0.268 0.286 0.582 0.758 0.382 0.301 0.442 0.395 0.32 0.328 0.347
18 0.122 0.083 0.088 0.111 0.317 0.076 0.170 0.239 0.198 0.079 0.146 0.127
24 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
Cmax 1.615 2.462 3.478 2.445 2.528 2.370 1.971 3.680 2.674 2.473 2.601 3.390
Tmax 2 2 1.5 2 1.5 1 2 1.5 1 2 1 1.5
AUC 0-t 10.452 11.732 18.008 16.782 18.532 14.672 12.021 17.211 16.043 15.126 13.543 17.513
Keli 0.163 0.231 0.223 0.178 0.249 0.195 0.183 0.162 0.134 0.221 0.173 0.243
T-half 4.275 4.159 3.105 4.956 4.298 4.674 5.211 5.709 5.744 4.674 5.217 4.978
AUC0-∞
141
10.854 12.453 18.453 16.976 19.564 15.001 13.229 17.924 16.532 15.453 15.832 17.453
141
Table 4.38 (continued) Individual plasma concentrations (mcg/ml) and pharmacokinetic parameters for Pramipexole
Immediate Release Formulation
0.0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
0.5 0.521 0.854 1.134 1.018 0.058 0.387 1.629 1.386 1.837 0.411 0.828 0.620
1 1.908 1.376 1.863 1.890 0.492 1.767 3.705 2.548 2.170 1.065 1.694 2.482
1.5 1.971 1.432 1.906 1.926 0.521 1.789 3.720 2.573 2.128 1.093 1.672 2.532
2 2.100 2.678 3.617 2.204 1.837 2.926 5.449 3.814 2.100 1.295 2.637 3.297
3 1.816 1.712 2.320 2.208 1.779 2.754 2.278 3.348 2.049 1.242 1.848 1.783
4 1.231 0.846 1.148 0.878 1.272 1.390 0.982 1.489 0.878 0.832 0.897 0.827
6 1.231 0.846 1.148 0.878 1.272 1.390 0.982 1.489 0.878 0.832 0.897 0.827
8 1.127 0.601 0.820 0.589 0.893 0.729 0.578 0.915 0.595 0.601 0.714 0.643
12 0.806 0.345 0.193 0.331 0.523 0.533 0.464 0.553 0.307 0.332 0.401 0.380
18 0.099 0.095 0.142 0.111 0.212 0.123 0.078 0.193 0.191 0.129 0.175 0.093
24 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
Cmax 2.100 2.678 3.617 2.204 1.837 2.926 5.449 3.814 2.128 1.295 2.637 3.297
Tmax 1.5 2 2 1.5 1.5 2 2 2 1.5 2 2 1.5

AUC 0-t 20.439 14.045 18.901 15.554 17.453 20.991 21.323 19.564 16.003 12.655 15.563 17.444
Keli 0.228 0.163 0.223 0.167 0.156 0.148 0.224 0.184 0.221 0.19 0.185 0.178
T-half 4.967 3.519 4.158 4.554 5.887 4.668 3.527 4.921 4.334 5.322 4.894 4.807
142
AUC0-∞ 21.047 14.564 19.328 16.001 17.855 20.964 21.457 20.487 16.531 13.275 16.445 18.209
142
Table 4.39 Individual plasma concentrations (mcg/ml) and pharmacokinetic parameters for Pramipexole Modified Release
Formulation
0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
0.5 0.033 0.272 0.062 0.337 0.155 0.741 0.360 0.268 0.080 0.198 0.179 0.089
1 0.137 0.546 0.209 0.405 0.315 0.985 0.712 0.490 0.138 0.407 0.301 0.175
1.5 0.392 1.208 0.410 0.722 0.996 2.268 1.411 1.414 0.249 0.478 0.556 0.225
2 0.645 2.006 1.082 1.185 1.526 4.507 1.730 2.578 0.371 0.901 1.187 0.303
3 2.081 3.108 2.612 2.484 2.175 3.038 2.129 3.802 0.816 1.505 2.457 1.260
4 3.202 4.334 3.466 3.608 3.074 2.083 3.148 3.979 2.011 2.031 3.872 1.892
6 2.498 1.781 1.968 1.772 2.017 1.012 1.545 2.036 2.251 3.867 1.533 3.624
8 1.978 1.559 1.784 1.521 1.773 0.749 1.337 1.784 2.084 3.269 1.363 3.273
12 1.158 0.975 0.872 1.008 0.941 0.484 0.909 1.072 1.263 1.239 0.856 1.552
18 0.589 0.420 0.466 0.369 0.364 0.162 0.425 0.393 0.424 0.449 0.448 0.450
24 0.257 0.313 0.245 0.350 0.277 0.227 0.245 0.238 0.204 0.332 0.357 0.254
Cmax 3.202 4.334 3.466 3.608 3.074 4.507 3.148 3.979 2.251 3.867 3.872 3.624
Tmax 4 4 4 4 4 2 4 4 6 6 4 6
AUC 0-t 30.776 31.821 23.113 25.956 27.121 20.810 24.896 31.098 27.113 33.901 26.278 33.987
Keli 0.130 0.129 0.139 0.123 0.132 0.145 0.126 0.153 0.164 0.157 0.115 0.178
T-half 5.674 5.394 5.041 5.559 5.461 4.824 5.670 4.384 4.108 4.369 6.023 3.700
143
AUC0-∞ 31.352 31.326 28.197 28.932 28.242 21.737 26.377 32.447 26.107 35.613 28.140 34.592
143
Table 4.39 (continued) Individual plasma concentrations (mcg/ml) and pharmacokinetic parameters for Pramipexole modified
release reference product
0.0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
0.5 0.159 0.023 0.151 0.015 0.011 0.095 0.019 0.032 0.170 0.122 0.119 0.075
1 0.261 0.149 0.247 0.139 0.104 0.224 0.104 0.107 0.230 0.239 0.178 0.168
1.5 0.309 0.371 0.681 0.394 0.168 0.330 0.144 0.615 0.388 0.416 0.362 0.239
2 0.524 0.764 0.982 0.631 0.397 0.581 0.282 0.994 0.760 0.756 0.963 0.299
3 1.381 1.450 1.719 1.670 1.340 1.507 1.042 2.075 1.453 1.559 1.710 1.097
4 2.713 1.646 2.624 3.032 2.583 1.796 1.522 2.760 1.645 2.125 2.534 1.587
6 1.916 2.986 2.143 5.232 4.529 4.456 4.436 1.225 4.470 4.930 2.084 4.198
8 1.688 1.990 1.971 2.664 3.442 3.336 2.167 1.773 2.010 2.375 1.754 3.631
12 1.287 1.044 1.296 1.392 1.385 1.201 0.990 1.105 1.290 1.157 1.165 1.722
18 0.392 0.421 0.439 0.536 0.474 0.593 0.417 0.437 0.518 0.458 0.553 0.682
24 0.216 0.186 0.253 0.213 0.217 0.259 0.144 0.185 0.209 0.234 0.183 0.269
Cmax 2.713 2.986 2.624 5.232 4.529 4.456 4.436 2.760 4.470 4.930 2.534 4.198
Tmax 4 6 4 6 6 6 6 4 6 6 4 6
AUC 0-t 23.339 24.268 26.526 25.839 35.233 31.998 26.358 25.047 29.556 31.267 25.461 35.173
Keli 0.148 0.143 0.484 0.133 0.213 0.215 0.162 0.143 0.083 0.133 0.288 0.386
T-half 4.897 4.147 5.691 3.933 3.963 3.850 3.569 4.780 4.164 3.970 5.395 4.220
144
AUC0-∞ 24.375 25.289 28.204 36.974 36.400 32.901 27.023 26.102 30.731 32.535 26.723 36.294
144
Table 4.40 Plasma concentrations (mcg/ml) of Pramipexole
Time Immediate Release Modified Release

(h) Formulation Formulation
Mean S.D Mean S.D
0.00 0.000 0.000 0.000 0.000
0.50 1.014 0.578 0.157 0.159
1.00 1.968 0.788 0.290 0.213
1.50 2.205 0.814 0.614 0.507
2.00 2.571 0.882 1.081 0.924
3.00 1.894 0.480 1.895 0.729
4.00 1.210 0.266 2.636 0.826
6.00 0.970 0.227 2.855 1.324
8.00 0.681 0.181 2.136 0.755
12.00 0.405 0.157 1.140 0.254
18.00 0.142 0.060 0.453 0.099
24.00 0.000 0.000 0.244 0.053
145
3
2.5
1.5
Title
Title
Axis
Axis
1
0.5
Axis
Title
Immediate Release Formulation Modifed Release Formulation
Fig 4.32: Mean Concentration Time Curve for Pramipexole

146
146
Table 4.41 Pharmacokinetic profile of Pramipexole
Immediate release product Modified Release product

Parameters Mean S.D Mean S.D
Cmax 2.736 0.878 3.700 0.826
Tmax 1.688 0.355 4.833 1.167
AUC 0-t 16.315 2.945 28.206 4.111
Cmax 0.193 0.032 0.176 0.091
Tmax 4.690 0.696 4.699 0.756
AUC 0-t 16.912 2.823 29.859 4.200

147
147
Table 4.42 Statistical data for Pramipexole Immediate Release Vs Modified Release Formulation
Dependent variable: Cmax Tests of Between-Subjects Effect

a
squares

b
Error .798 30.987 2.554E-02
SEQ Hypothesis 8.109E-02 1 8.324E-02 3.765 .060 .159 3.432 .540

C
Error .600 22 2.006E-02

Error . . .
d
TREATME Hypothesis .000 0 . . . . . .

NT Error . . d
.
SUBJECT Hypothesis 2.543 23 9.549E-02 4.443 .000 .843 106.998 1.000
c
Error .432 22 2.087E-02
e. Computed using alpha = .1
f. 5.266E-02 MS(SUBJECT) +.870 MS (Error)
g. MS (Error)
h. Cannot compute the appropriate error term using Satterthwaite’s method
148
148
Dependent variable: AUC 0-t Tests of Between-Subjects Effect

a
squares

b
Error .508 28.654 1.421E-02

C
Error .342 22 1.293E-02

Error . . .
d

Error . . .
d

c
Error .421 22 1.332E-02
g. MS (Error)
149
149
Dependent variable: AUC 0-inf Tests of Between-Subjects Effect

a
squares

b
Error .443 29.654 1.676E-02

C
Error .232 22 1.67E-02

Error . . .
d
TREATME Hypothesis .000 0 . . . . . .

NT Error . . d
.
c
Error .321 22 1.556E-02
g. MS (Error)
150
150
Table 4.45 Paired Sample Test for Pramipexole
Paired Differences
Std. Std. 95% Confidence t df Sig. (2-

Error Interval of the tailed)
Mean Deviation Difference
Mean
Lower Upper
Pair 1 C max -.005 .217 .044 -.097 .085 -.109 23 .967
Pair 2 AUC 0-t -.092 .160 .032 -.160 -.024 -2.232 23 .013
Pair 3 AUC 0-inf -.086 .157 .031 -.153 -.028 -2.45 23 .01
151
151
Table 4.46 Cumulative percentage dissolved at 50 rpm for Pramipexole test formulations
(h) root of
Formulation Formulation Formulation Formulation Formulation
time (h)
MR IR MR IR MR IR MR IR MR IR
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.50 0.71 6.17 15.83 8.63 16.53 4.22 5.92 5.55 4.21 4.32 4.53
1.00 1.00 12.60 27.05 13.12 30.98 9.74 11.43 11.53 12.67 10.43 12.93
1.50 1.22 16.32 41.53 19.47 39.01 13.95 15.37 16.30 19.56 17.83 18.55
2.00 1.41 21.76 55.92 23.86 66.91 20.56 22.54 19.54 23.67 21.55 23.56
3.00 1.73 28.54 73.73 32.75 83.28 28.48 31.26 30.20 33.49 23.67 26.54
4.00 2.00 34.95 87.32 46.91 93.64 35.21 42.54 44.67 49.54 35.26 39.43
6.00 2.45 40.87 97.38 70.42 98.54 53.90 56.48 62.54 66.37 49.32 55.32
8.00 2.83 49.21 99.80 79.32 100.55 67.02 70.43 75.20 83.24 65.34 72.10
12.00 3.46 71.55 100.04 90.76 100.69 83.20 86.34 86.77 91.17 74.21 79.00
18.00 4.24 91.77 100.08 96.09 101.76 97.03 97.97 99.21 97.56 83.24 90.53
24.00 4.90 95.51 100.92 99.53 102.20 101.32 98.10 99.55 98.20 93.42 89.41
152
152
CHART
TITLE
100
80
60
TITLE
AXIS
40
20
0
0 5 10 15
20 25
AXIS
TITLE
153
Fig 4.33: Cumulative Pregabalin Release Vs Time Profile for Immediate and
153
Table 4.47 Cumulative percentage dissolved at 75 rpm for Pramipexole test formulations
(h) root of
Formulation Formulation Formulation Formulation Formulation
time (h)
MR IR MR IR MR IR MR IR MR IR
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.50 0.71 9.45 18.54 10.49 20.42 9.43 17.43 7.32 09.67 8.09 11.76
1.00 1.00 16.56 33.21 15.32 33.89 14.78 21.86 14.98 18.56 14.89 18.34
1.50 1.22 22.89 49.27 20.98 46.92 20.54 26.89 18.08 23.05 19.45 21.75
2.00 1.41 25.94 55.98 26.79 57.23 26.97 35.01 22.65 28.53 22.67 25.56
3.00 1.73 33.01 71.45 35.64 76.07 35.74 58.21 32.95 36.78 28.06 35.78
4.00 2.00 38.90 88.21 44.10 95.31 45.82 69.02 48.94 53.11 40.21 45.20
6.00 2.45 44.21 99.65 72.21 99.00 59.20 79.98 65.20 72.05 57.93 63.56
8.00 2.83 54.67 100.43 83.43 100.63 72.34 89.04 77.55 84.43 71.22 75.65
12.00 3.46 75.23 100.76 96.29 101.69 88.30 99.00 89.08 96.21 79.03 82.08
18.00 4.24 92.43 100.90 99.06 101.77 100.05 100.34 100.56 100.07 89.32 100.65
24.00 4.90 99.76 101.15 100.62 101.98 101.32 100.94 101.67 100.76 97.54 100.71
154
154
CHART
TITLE
100
80
60
TITLE
AXIS
40
20
0
0 5 10 15
20 25
AXIS
TITLE
155
155
Table 4.48 Similarity Factors for Pramipexole Modified Release Formulations
in Various Dissolution Conditions
Similarity factor
S.No pH Condition Formulation
(f2)
1 pH 1.2 buffer 50 rpm 45.87
2 pH 1.2 buffer 75 rpm 47.34
3 pH 4.5 buffer 50 rpm 43.76

Immediate
4 pH 4.5 buffer 75 rpm Release verses 42.91
Modified
5 pH 6.8 buffer 50 rpm 93.10
Release
6 pH 6.8 buffer 75 rpm 77.20
7 Distilled water 50 rpm 91.67
8 Distilled water 75 rpm 95.21
9 pH 7.4 buffer 50 rpm 87.45
10 pH 7.4 buffer 75 rpm 88.76
156
Table 4.49 IVIVC model regression of % absorbed vs. % dissolved for

(pH 1.2) (pH 4.5)
(Hours)
MR IR MR IR MR IR
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.50 6.17 15.83 8.63 16.53 2.10 3.10
1.00 12.60 27.05 13.12 30.98 4.53 11.72
1.50 16.32 41.53 19.47 39.01 10.76 23.03
2.00 21.76 55.92 23.86 66.91 20.54 51.04
00 28.54 73.73 32.75 83.28 47.98 73.00
00 34.95 87.32 46.91 93.64 82.84 88.21
00 40.87 97.38 70.42 98.54 94.21 95.49
00 49.21 99.80 79.32 100.55 96.04 95.98
12.00 71.55 100.04 90.76 100.69 96.31 96.39
18.00 91.77 100.08 96.09 101.76 99.01 97.78
24.00 95.51 100.92 99.53 102.20 99.41 98.12
157
100
90
80
70
60
50
40
30
20
10
0
0 20 40 60 80
100
Fig. 4.35: IVIVC model Linear Regression Plot of % Absorbed Vs % Dissolved for
Immediate and Modified Release Pramipexole Formulations using pH 1.2 Buffer at 50
RPM
100
90
80
70
60
50
40
30
20
10
0
0 20 40 60 80
100

for Immediate and Modified Release Pramipexole Formulations using pH 4.5
Buffer at 50 RPM
158
Table 4.50 IVIVC model regression of % absorbed vs. % dissolved for

(Hours) (pH 1.2) (pH 4.5)
MR IR MR IR MR IR
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.50 9.45 18.54 10.49 20.42 2.10 3.10
1.00 16.56 33.21 15.32 33.89 4.53 11.72
1.50 22.89 49.27 20.98 46.92 10.76 23.03
2.00 25.94 55.98 26.79 57.23 20.54 51.04
3.00 33.01 71.45 35.64 76.07 47.98 73.00
4.00 38.90 88.21 44.10 95.31 82.84 88.21
6.00 44.21 99.65 72.21 99.00 94.21 95.49
8.00 54.67 100.43 83.43 100.63 96.04 95.98
12.00 75.23 100.76 96.29 101.69 96.31 96.39
18.00 92.43 100.90 99.06 101.77 99.01 97.78
24.00 99.76 101.15 100.62 101.98 99.41 98.12

100
90
80
70
60
50
40
30
20
10
0
0 20 40 60 80
100
Fig. 4.37: IVIVC model Linear Regression Plot of % Absorbed Vs % Dissolved for
Immediate and Modified Release Pramipexole Formulations using pH 1.2 Buffer at 75
RPM
100
90
80
70
60
50
40
30
20
10
0
0 20 40 60 80
100

for Immediate and Modified Release Pramipexole Formulations using pH 4.5
Buffer at 75 RPM
Table 4.51 Observed and IVIVC model predicted Cmax and AUC values for
Pramipexole
Time IR formulation MR formulation
Fraction Fraction Fraction Fraction

(Hours)
observed predicted observed predicted
0.00 0.000 0.000 0.000 0.000
0.50 1.014 1.176 0.157 0.162
1.00 1.968 2.043 0.290 0.265
1.50 2.205 2.328 0.614 0.609
2.00 2.571 2.643 1.081 1.025
3.00 1.894 1.934 1.895 1.792
4.00 1.210 1.158 2.636 2.603
6.00 0.970 1.043 2.855 2.821
8.00 0.681 0.721 2.136 2.098
12.00 0.405 0.428 1.140 1.021
18.00 0.142 0.154 0.453 0.407
24.00 0.000 0.073 0.244 0.189
Cmax 2.571 2.643 2.855 2.821
AUC 17.432 17.539 27.654 27.045

Fig. 4.39: Observed and Predicted Pregabalin Plasma Concentration for the
Immediate Release Pregabalin Formulations using IVIVC Model
3.5
2.5
1.5
0.5
0
0 5 10 15
20 25
Fig. 4.40: Observed and Predicted Pregabalin Plasma Concentration for the
Modified Release Pregabalin Formulations using IVIVC Model
162
Table 4.52 Prediction errors (%) associated with Cmax and AUC for
Pramipexole
Formulation Cmax AUC
Immediate -2.724 -0.610
Modified 1.205 2.252
Average -0.759 0.821
163
CHAPTER 5
SUMMARY AND CONCLUSION
This thesis deals with the studies carried out by the writer for the past three
years on the “Development and Validation of in-vitro and in-vivo correlations for
Pregabalin and Pramipexole formulations”.
The first chapter begins with a brief account of the in-vitro and in-vivo
correlations, biopharmaceutical classification system, IVIVC models, in-vitro and in-
vivo dissolutions and estimation of drugs in biological medium. The methods used
for the IVIVC model development, validation, the steps involved in bio-analytical
method development, in-vitro dissolution methods and their importance have also
been discussed.
The second chapter briefs the review of literature on the LC-MS/MS methods
available for the estimation of the selected drugs in biological fluids, IVIVC model
development, validation, in-vitro dissolution methods.
The third chapter deals with the scope and object of the present investigation.
The merits of IVIVC in the development of dosage forms and how IVIVC model
development necessitates development of in-vitro dissolution methods, bio-analytical
method development and validation are discussed. The objectives of the present
study, namely, to optimize the chromatographic conditions, to develop and validate
the methods to estimate the selected drugs in the biological fluids LC-MS/MS,
development of in-vitro dissolution methods and IVIVC model development and
validation, have been described.
The fourth chapter deals with the experimental procedures adopted. It describes
in detail the procedures adopted for the bioequivalence study design and data
164
handling, optimization and validation of the chromatographic conditions for the
estimation of the drugs in plasma and selected MR formations, IVIVC model
development and validation.
In the fifth chapter, the results obtained are presented, supported by tables and
figures and discussed in detail.
The discussion includes,
 Bioequivalence study design and data handling

 Optimization and validation of the chromatographic conditions for the
estimation of the drugs in plasma and selected MR formulations
 Chromatograms obtained
 Accuracy
 Reproducibility (intraday and interday variations)
 Specificity
 Linearity and range
 LOD and LOQ
 Ruggedness and robustness
 Stability and
 System suitability studies
 in-vitro – in-vivo data analysis
 in-vitro – in-vivo correlation
 Model development
 Internal and external validation
165
The following are some of the salient features of the present study;
 A single dose, randomized, complete two way and two treatments cross
over study was conducted in healthy human subjects and plasma
concentrations were estimated by sensitive and validated LC-MS/MS
methods.
 The selected drug candidates Pramipexole and Pregabalin are water

soluble drugs that are predominantly ionized in gastrointestinal pH ranges
and are well absorbed after oral administration.
 The selected drugs can be categorized as high solubility/high permeability

drugs under the proposed Class I Biopharmaceutical Classification System
(BCS) and hence it becomes necessary to determine the in-vitro and in-
vivo correlations for these drugs.
 The target to find out a predictive in-vitro dissolution method was reached
gradually. The first step was taken by observing which in-vitro dissolution
method predicted best similarities and differences in bioavailability.
Apparatus I, pH 6.8 at 75 rpm was found to yield acceptable IVIVC for
Pregabalin, whereas, Apparatus I, pH 1.2 at 75 rpm was found to yield
acceptable IVIVC for Pramipexole.
 From a comparison of the differences in the in-vitro pharmacokinetic

parameters and the differences in the in-vitro dissolution curves, it may be
concluded that the developed dissolution method will discriminate batches
which are non-bioequivalent.
 Level A correlation was observed for the selected formulations at the in-
vitro dissolution conditions developed. These dissolution methods
predicted also the best absorption rate for the selected modified release
formulations.
166
 The validity of the correlation was also assessed by determining how well
the IVIVC model could predict the rate and extent of absorption as
characterized by Cmax and AUC. The percent prediction error of ≤ 10 % for
Cmax and AUC was obtained, which establishes the predictability of the
developed IVIVC model. It may, thus, be concluded that the developed
dissolution methods can surrogate for human bioequivalence studies.
In conclusion, it may be pointed out that the developed in-vitro dissolution

methods can replace absorption studies during the pre-approval processes to develop
a desirable formulation and to ensure batch-to-batch bioequivalence. It will also be
very useful in performing possible post-approval changes in the formulation scale-up
or changes in the drug substance or excipients supplier.
167
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LIST OF PUBLICATIONS
 Uma, G, Manimala, M, Vasudevan, M, Karpagam, S & Deecaraman, M 2012,

‘LC-MS-MS method for the determination of Pregabalin in human plasma’,
International Journal of Pharmacy and Pharmaceutical Sciences, vol. 4,
no. 3, pp. 108-112 (Scopus indexed)
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‘Development and Validation of LCMS method for the estimation of
Pramipexole in Human Plasma’, International Journal of Pharmacy and
Pharmaceutical Sciences, vol. 2, no. 1, pp. 10-11 (Scopus indexed)
 Manimala, M, Karpagam, S & Deecaraman, M 2013, ‘LC-MS-MS method

for the determination of Digoxin in human plasma’, International Journal of
Pharmacy and Pharmaceutical Sciences, vol. 5, no. 2, pp. 131-132 (Scopus
indexed)
 Manimala, M, Clement Atlee, W, Sheik Mohamed, AA & Purushoth Prabhu,

T 2014, ‘Effect of Coccinia grandis on ammonium chloride and ethylene
glycol induced urolithiasis in rats’, International Journal of Drug
Development and Research, vol. 6, no. 3, pp. 138-146 (Scopus indexed)
 Manimala, M, Karpagam, S, Deecaraman, M, Clement Atlee, W & Purushoth

Prabhu, T 2015, ‘Evaluation of nephroprotective and antioxidant activity of
ethanoic extracts of Momordica dioica leaves’, Scholars research Library ,
vol. 7, no. 4, pp. 153-156
174

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DEVELOPMENT AND VALIDATION OF IN-VITRO

AND IN-VIVO CORRELATIONS FOR

I declare that the thesis entitled “DEVELOPMENT AND VALIDATION

I have also published my papers in International Journals (Scopus rated) as

Signature of Research Scholar

Certified that the thesis entitled “DEVELOPMENT AND VALIDATION OF

In each dosing session, volunteers received either immediate or modified

The LC-MS/MS instrument was calibrated with polypropylene glycol standard

The standard stock solutions, standard solutions, Calibration curve samples

The release characteristics of selected drugs in their formulations is carried out

The Wagner-Nelson method was used to calculate the percentage of the

Chapter No. Title Page No.

1.7.2 Development of LC-MS/MS methods for the 14

3.4.1 System Suitability 49

4 RESULTS AND DISCUSSION 65

4.6 In-vitro and in-vivo correlations for Pregabalin 107

4.6.1 In-vivo data analysis 107

4.7.2 In-vitro data analysis 137

Table No. Title Page No.

3.1 Source Parameters for Pregabalin 45

4.12 Intercept, Slope and Correlation coefficient values for 86

4.49 IVIVC model regression of % absorbed vs. % dissolved for 157

Figure No. Title Page No.

4.1 Calibration Curve of Pregabalin 73

4.25 Cumulative Pregabalin Release Vs Time Profile for 127

4.36 IVIVC model Linear Regression Plot of % Absorbed Vs % 158

ANOVA : Analysis of variance

1.1 In-vitro and in-vivo Correlations

IVIVC can be used in the development of new pharmaceuticals to reduce the

The term correlation is frequently employed within the pharmaceutical and

IVIVC is a predictive mathematical model describing the relationship between

A successful IVIVC model can be developed, if in-vitro dissolution is the rate-

1.2 Bio-pharmaceutics Classification System (BCS)

Bio-pharmaceutics Classification System (BCS) is a fundamental guideline

Class II (Low solubility/High permeability) drugs have a high Absorption

Class III (High solubility/Low permeability) drugs exhibit a high variability in

IVIVC are usually expected for Class I and Class II drugs.

1.3 Bio-availability Studies for Development of IVIVC

A bioavailability study should be performed to characterize the plasma

Liquid chromatographic coupled with tandem mass spectrometer (LC-MS/MS)

LC-MS/MS instrument consists of three major components

 LC (to resolve a complex mixture of components)

Several approaches can be employed for determining the in-vivo absorption.

The Wagner Nelson method is less complicated than the LooRiegelman, as

De-convolution is a numerical method used to estimate the time course of drug

1.4 In-vitro dissolution

Drug absorption from a solid dosage form following oral administration

Modified release dosage forms typically require dissolution testing over

However, for the IVIVC perspective, dissolution is proposed to be a surrogate

The utilization of in-vitro dissolution data for predicting in-vivo performance

IVIVC models are developed to explore the relationships between in-vitro

IVIVC modeling involves three stages, which are,

Developing a predictable IVIVC depends upon the complexity of the delivery

1.5.1 Level A Correlation

This level of correlation is the highest category of correlation and represents a

1.5.2 Level B Correlation

A level B IVIVC utilizes the principles of statistical moment analysis. In this

1.5.3 Level C Correlation

1.5.4 Multiple-level C correlation

A multiple level C correlation relates one or several pharmacokinetic

1.5.5 Level D correlation

Level D correlation is a rank order and qualitative analysis and is not

1.5.6 IVIVC Model Development