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Chapter 4 Results
Pharmaceutical Medicine 61 Jamia Hamdard
4.0 RESULTS
The study results are divided into five parts:
1. Clinical Study Results
2. Bioanalytical Method Development
3. Bioanalytical Method Validation Results
4. Analysis of Clinical Study Samples
5. Pharmacokinetic and Statistical Results
4.1 CLINICAL STUDY RESULTS
The clinical study was conducted according to the protocol and informed
consent form (ICF), designed for this study, which were reviewed and approved
by the Jamia Hamdard Institutional Review Board (JH-IRB). All the subjects
provided the written informed consent, after attending an oral presentation of
ICF and thoroughly reading the same.
4.1.1 Study Population
The study was conducted in an Asian male population. Eighteen (18) healthy,
adult, human, male subjects, fulfilling the inclusion and exclusion criteria as
mentioned in the protocol, were enrolled in the study.
All subjects were similar with respect to age, weight, and height distribution
(Table 4.1.1). The mean age, weight and height of the study subjects were 26.72
years (range 19 to 41 years), 53.39 kg (range 47 to 71 kg) and 167 cm (range
159 to 178 cm) respectively (Table 4.1.2).
All subjects completed both periods of the study.
4.1.2 Dropouts and Withdrawal
There was no dropout or withdrawal in this study.
4.1.3 Clinical procedures and observations
4.1.3.1 Dose administration
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The dosing was done between 0900-0916 hrs on 14th November 2009 and on
21st November 2009 for period I and II respectively. The study drug was
administered with 240 mL of water at an ambient temperature, as described in
the methodology in each period after an overnight fast of at least 10 hours
under the supervision of trained study personnel.
4.1.3.2 Duration of treatment
The duration of treatment was one time, single dose in each period. Each
subject completing the study was dosed with test (T) or reference (R) product
once in each period as per the randomization schedule, separated by a washout
period of seven (07) days.
4.1.3.3 Medications
The subjects did not take any prescription and /or OTC medications for at least
thirty days prior to the study initiation and during the study.
4.1.3.4 Diet
All subjects abstained from alcoholic products for 48 hours prior to the dosing
and during in-house stay in each period. They also abstained from any xanthine
containing food and beverages during in-house stay in each period.
4.1.3.5 Activity
All subjects were dosed while seated and were asked to remain seated or
ambulatory for the first two hours following drug administration in each period.
Thereafter the subjects were allowed to engage in the normal activities avoiding
severe physical exertion until discharge in each period.
4.1.4 Clinical Safety Measurements
4.1.4.1 Vital Signs Recording
Vital signs of oral temperature, sitting blood pressure and radial pulse were
found to be normal for all the subjects during the course of the study in both
the periods. No major/significant fluctuations were observed in the sitting blood
pressure, pulse rate and oral temperature in any of the subjects until the
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completion of the study. The clinical examinations of all subjects were found
out to be normal in both the periods of the study.
4.1.4.2 Adverse Events
The subjects were monitored throughout the study period for adverse events.
Subjects were specifically asked about any adverse event at admission, prior to
dosing and approximately at 2, 6 and 24 hours (within 2 hour of the scheduled
time) post dose in each period. No adverse event was reported during this
study.
4.1.4.3 Clinical Laboratory Evaluation
4.1.4.3.1 Evaluation of Each Laboratory Parameter
Laboratory tests to ensure subject safety included haematology and
biochemistry parameters both at the time of screening and at the end of the
study, urinalysis and serology at the time of screening. Out of eighteen (18)
subjects, three (03) subjects (subject numbers 04, 08 and 15) had out of
acceptable range laboratory test results at the end of the study. Upon follow-up,
laboratory test of subject numbers 04 and 08 came under acceptable limits of
laboratory parameters as defined in the protocol. Laboratory test result of
subject number 15 was not within the acceptable limit after first follow up. After
repeated attempts to contact the subject, he refused to come for further follow-
up and was considered as “Lost to follow-up”.
„Out-of-range laboratory parameters at the End of Study Safety Assessment ‟ is
given in Table 4.1.3.
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Table 4.1.1 Subject demographics
Subject
No.
Age
(Yrs.)
Height
(cm)
Weight
(Kg) Diet* Gender Race
Smokers#
(Yes/No)
1 19 175 53 2 Male Asian No
2 30 165 48 2 Male Asian No
3 29 163 48 2 Male Asian No
4 34 167 52 2 Male Asian No
5 25 159 48 2 Male Asian No
6 41 178 57 2 Male Asian No
7 21 168 61 2 Male Asian No
8 26 163 47 2 Male Asian No
9 22 168 48 2 Male Asian No
10 35 161 50 2 Male Asian No
11 24 168 54 2 Male Asian No
12 20 166 53 2 Male Asian No
13 19 172 57 2 Male Asian Yes
14 20 170 56 2 Male Asian No
15 30 159 54 2 Male Asian No
16 33 168 71 2 Male Asian No
17 33 167 57 2 Male Asian No
18 20 169 47 2 Male Asian Yes
Mean 26.72 167 53.39
SD 6.65 5.02 6.07
CV% 24.88 3.00 11.37
N 18 18 18
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Table 4.1.2 Summary of subject demographic
Subjects enrolled in the study
(N = 18)
Age (years)
Mean ± SD
26.72 ± 6.65
Range
19-41
Groups
< 18
0 %
18 – 40
17 (94.44 %)
41-64
01 (5.56 %)
65-75
0 %
>75
0 %
Sex
Female
0 %
Male
18 (100%)
Race
Asian
18 (100%)
Black
0 %
Caucasian
0 %
Hispanic
0 %
Other
0%
Height (cm)
Mean ± SD 167 ± 5.02
Range 159-178
Weight (kg)
Mean ± SD
53.39 ± 6.07
Range
47-71
Smokers
Yes
02 (11.11 %)
No
16 (88.89 %)
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Table 4.1.3 Out Of Range Laboratory Parameters at the End of study
safety assessment :
Subject No. Parameter Reference
range Screening
At the end
of the
Study
(22.11.09)
Follow up
1
04 Eosinophils
(%) 0-6 7.4 10.9
7.2 (07.12.09)
08 Eosinophils
(%) 0-6 7.7 9.4
7.5
(27.11.09)
15* Eosinophils
(%) 0-6 7.1 11.3
14.0
(03.12.09)
* Subject was asked to come for follow-up but he refused to come and therefore he was
considered as “Lost to follow-up”.
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4.2 BIOANALYTICAL METHOD DEVELOPMENT RESULTS
The analytical method for the determination of Esomeprazole in human CPDA
plasma using Pantoprazole as internal standard was developed and validated
at the Department of Clinical Pharmacology and Pharmacokinetics, Ranbaxy
Research Laboratories.
Table 4.2.1 Chromatographic Conditions
Chromatographic Conditions
Column Discovery C18, 50 X 4.6mm, 5μ
Mobile Phase Acetonitrile :Buffer-2 :: 90:10, v/v
Flow Rate 0.7 mL/minute
Column Oven Temperature 35ºC ± 1.0ºC
Sample Cooler Temperature
10ºC ± 1.0ºC
Injection Volume 10 μL
Retention Time Esomeprazole: 0.6 to 1.4 minutes
Ion source Turbo-ion spray in Positive Ion mode
Rinsing solution Acetonitrile: HPLC grade water (80:20,
v/v).
Mass Spectrometric Settings
Curtain gas (CUR) 40#
Collision gas (CAD) 3#
Ion Source Gas-1 (GS1) 50#
Ion Source Gas-2 (GS2) 50#
Ion Spray Voltage (IS) 5500 V
Temperature (TEM) 5500C
Declustering Potential- DP (V) Esomeprazole : 25V
Pantoprazole : 33V
Entrance Potential - EP (V) Esomeprazole : 7V
Pantoprazole : 7V
Collision Energy -CE (V) Esomeprazole : 18V
Pantoprazole : 15V
Exit Potential -CXP (V) Esomeprazole : 3V
Pantoprazole : 3V
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4.3 BIOANALYTICAL METHOD VALIDATION RESULTS
A specific LC-MS/MS method to determine Esomeprazole concentrations in
human plasma was developed and validated at Ranbaxy Laboratories Ltd.
according to the applicable guidelines (FDA, CDER).
The validation of this procedure was performed in order to evaluate the method
in terms of selectivity, sensitivity, linearity of response, accuracy, precision,
recovery, stability, dilution integrity, matrix effect, matrix factor and ruggedness
in human plasma for Esomeprazole. The sensitivity, linearity, precision and
accuracy evaluations were performed on three batches of spiked samples. Each
batch of spiked plasma samples included one complete calibration curve
(consisting of two blank plasma, two blank plasma with internal standard and
eight different non-zero concentrations) and six replicate of quality control
samples at LOQQC,LQC, MQC and HQC levels
4.3.1 System Suitability Test
System suitability test was performed and it reflects that the % CV of the ratio of
drug/internal standard was under acceptable range (<4%), also % CV for
retention time is less than 10%.
4.3.2 Selectivity
The proposed method was found to be very selective for plasma samples as
there was negligible / no interference at retention times of esomeprazole (2.55-
6.46%) and Internal Standard (0.01-0.2%) against ≤ 20 % and ≤5 % respectively
for acceptance criteria. Selectivity evaluation was within the acceptance criteria
as described in methodology. Selectivity data are given in Table 4.3.1.
4.3.3 Sensitivity
The limit of quantitation was 9.78 ng/mL for Esomeprazole. The between batch
precision and accuracy at LOQQC concentration for Esomeprazole using
internal standard ratio method was 9.7% and 101.2%, respectively. This was
within the acceptance criteria of ≤20% and 80-120% for between batch precision
and accuracy, respectively at LOQQC concentration.
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4.3.4 Linearity of standard curve
The linearity of the method was determined by weighted least square
regression analysis of standard plot associated with eight point standard curve
(1/amount2). The results of linear regression analysis showed that the
correlation coefficients of all three standard curves were 0.9992 or greater
during the course of validation.
The mean of slopes/intercepts/correlation coefficients (± SD) are reported in the
Table 4.3.2.
4.3.5 Precision and accuracy
The chromatographic system was validated for within and between batch
precision & accuracy exercises. The results of within batch and between batch
precision and accuracy are shown in Table 4.3.3 and 4.3.4 respectively. All
estimates were from the six replicate analyses of four quality control samples
(LOQQC, Low, Middle and High QC samples) based on back calculation from
fresh calibrates. Within batch precision and accuracy ranged from 0.3 – 13.3 %
& 96.3-110.0% respectively. Between batches precision and accuracy ranged
from 4.1-9.7% and 101.2-104.4 % respectively. These results indicated that the
method was reliable, reproducible and accurate as all the parameters were
within the acceptance limit of ≤ 15% and ± 15% for precision and accuracy
respectively.
4.3.6 Recovery
Recovery of Esomeprazole and Pantoprazole (ISTD)
Upon analysis of six replicates of extracted quality control samples at low,
middle and high concentration levels, the overall recovery of Esomeprazole at
LQC, MQC and HQC levels were 64.8%, 57.2% and 69.0%, respectively. The
C.V. of recovery across the low, middle and high quality control concentrations
was 9.4%. The C.V. was within the acceptance criteria of ≤ 20% for mean
recovery between low, middle and high QC concentrations.
The recovery of Pantoprazole (ISTD) at MQC level was 66.4%.
A summary of recovery studies are presented in Table 4.3.5 (A and B).
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4.3.7 Ruggedness
The ruggedness of the extraction procedure and chromatographic method was
evaluated by analyzing a batch of six sets of quality control samples and a set of
calibration curve standards using a different column (same type) and by a
different analyst. Within-batch precision (% CV) and within-batch accuracy (%
Nominal) for ruggedness were 1.4% to 18.5% and 98.6% to 107.4%
respectively. The results indicated that the batch met the acceptance criteria for
ruggedness evaluation.
4.3.8 Stability and Integrity Evaluation
4.3.8.1 Freeze-Thaw Stability
The stability of spiked plasma samples was determined after three freeze thaw
cycles. The comparative stability ranged from 98.5% to 100.0% and its precision
ranged from 1.8% to 3.8% for Esomeprazole stability samples. These were
within the acceptance criteria of ± 15% for stability and ≤ 15% for precision at
low & high QC concentrations (Table 4.3.6).
4.3.8.2 Bench Top Stability
The bench top stability evaluation involved analysis of four replicates of low and
high QC stability samples, which had been kept at room temperature for 6.60
hours against freshly spiked quality control samples (comparison samples)
using freshly spiked calibration curve. The comparative stability ranged from
97.7% to 101.4% and its precision ranged from 1.9% to 2.0% for Esomeprazole
stability samples. These were within the acceptance criteria of ± 15% for
stability and ≤ 15% for precision at low & high QC concentrations (Table 4. 3.
7).
4.3.8.3 In-Injector Stability
The comparative stability ranged from 93.4% to 100.8% and precision ranged
from 0.3% to 2.2% for Esomeprazole stability samples. These were within the
acceptance criteria of ± 15% for stability and ≤ 15% for precision at low & high
QC concentrations. Refer Table 4.3.8 for In-Injector Stability of Esomeprazole
in Human Plasma.
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4.3.8.4 Stock Solution Stability
The stock solution of Esomeprazole and Pantoprazole (ISTD) was found to be
stable for seven days when stored at refrigerated temperature (between 1–10ºC,
protected from light). The stability of the stock solution for Esomeprazole and
Pantoprazole (ISTD) were 87.9% and 102.0% respectively. The mean response
of stability samples was within the acceptance criteria of 85% - 115% of the
mean response of comparison samples.
4.3.8.5 Short-Term Stability
The stability of the stock dilutions for Esomeprazole and Pantoprazole (ISTD)
were 93.6% and 101.2%, respectively. The precision for stability and
comparison stock dilution was found to be 0.8% and 3.6%, respectively for
Esomeprazole and 0.7% and 0.2%, respectively for Pantoprazole (ISTD). The
duration of stability was 7.15 hours and 7.12 hours for Esomeprazole and
Pantoprazole (stored at room temperature, protected from light) respectively.
The stability of the reference solution of Esomeprazole was 101.0%. The
precision for stability and comparison stock dilution was 1.2% and 0.6%,
respectively. The mean responses of stability samples were within the
acceptance criteria of 85% - 115% of the mean response of comparison
samples.The results of short term stability of Esomeprazole and Pantoprazole
(ISTD) are shown in Table 4.3.9 (A and B) respectively.
4.3.8.6 Dilution Integrity
Dilution integrity was determined by spiking about 1.70 times ULOQ
concentration for Esomeprazole. These were then diluted by factor of 2 and 4
with drug free plasma.The precision for Dilution Integrity samples at two times
and four times dilution were 2.4% and 2.7%, respectively. The accuracy for
Dilution Integrity samples at two times and four times dilution were 105.1% and
108.2%, respectively. Dilution Integrity evaluation was within the acceptance
criteria as described in methodology. Refer Table 4.3.10 for Dilution Integrity of
Esomeprazole.
4.3.9 Matrix Effect
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Matrix effect was performed by using six different batches of accepted blank
matrix. The precision for matrix effect samples at LQC and HQC level were
4.2%% and 1.7%, respectively. The accuracy for matrix effect samples at LQC
and HQC level were 97.7 % and 97.1%, respectively. The %C.V. of matrix factor
between LQC, MQC and HQC level was 1.5% and was within the acceptance
criteria (Table
4.3.11( A and B).
4.3. 10 Extended Precision and Accuracy Batch
Thirty (30) sets of QC samples were run for extended batch against a single
calibration curve.The within batch precision ranged from 3.8% to 6.0% and
within batch accuracy ranged from 93.0% to 98.7%.