Chapter 4 Results

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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%.