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PHARMACOKINETIC EVALUATION OF TWO BRANDS OF LINEZOLID TABLETS IN HEALTHY HUMAN VOLUNTEERS

METHODS AND MATERIALS:

These studies were performed in accordance with good clinicalpractice guidelines. The study protocols were reviewed and approvedby the institutional review board of the participating institution(Nizam's Institute of Medical Sciences, Hyderabad), and all subjectsprovided written informed consent prior to participation (Slatter, J. G et al., 2001).

 

Study design

A randomized, balanced, two treatment, two-period, two-sequence, single-dose, crossover pilot bioavailability and bioequivalence study of Linezolid (Linezolid 600mg tablets, Dr.Reddy's laboratories Ltd) with Linezolid (Zyvox ® 600mg tablets, Pharmacia &Upjohn Company, USA) (Grunder G et al., 2006).

 

Drug administration:

In each of the two study periods, single oral dose of assigned formulation were orally administrated with 240ml of water in the morning after an overnight fasting of at least 10 hrs. (Slatter, J. G et al., 2001).

 

Number of subjects

 

Twelve healthy, adult, male, human subjects were included in the study. The selection was based on the inclusion and exclusion criteria as for the protocol. All eligible participants were healthy non-smokerswilling to abstain from vigorous exercise and to follow a controlleddiet. Subjects were excluded for the following: a history ofclinically significant cardiovascular, renal, hepatic, pulmonary,gastrointestinal, endocrine, haematological, vascular or collagendiseases; a history of nervous system or muscle disease, seizuredisorder or a psychiatric disorder that might hinder compliancewith the study. The following precautions were incorporated in to the study to minimize the bias: 1) subjects were sequentially assigned to randomly ordered treatment 2) subject enrolment was dependent on satisfactory fulfillment of given list of inclusion criteria. 3) If individual subject were withdrawn prior to completion of the study were specified. 4) The analysis was blinded to the randomization code (Von Eiff, C.et al., 1999).

Blood sampling

The pre dose blood samples (7 ml) were collected with in 1 hr prior to dosing. The post dose serial blood samples (7ml) were collected at time intervals (Hrs) like 0.00, 0.33, 0.67, 1.0, 1.33, 1.67, 2.00, 2.50, 3.00, 4.0, 6.0, 8.0, 12.0, 16.0, and 24.00. All the blood samples were drawn by direct venipuncture or indwellingcatheter into a K3-EDTA Vacutainer. The blood samples were centrifuged at 3000 RPM at 100C for 10 minutes. The plasma was separated and stored at -200C of the test and reference product (Grunder G et al., 2006).

 

Bio Analysis

using sensitiveand selective high-performance liquid chromatography methods,with ultraviolet detection (251 nm) and PNU-101145 as the internalstandard. In brief, plasma (0.5 mL) was prepared using solid-phase extraction. Each sample waseluted with methanol. Upon evaporation of the organic material,the residue was reconstituted in acetonitrile: water and transferredto injection vials. 0.060 mL aliquots for plasma samples were injected onto the chromatographysystem.  Using a reverse phase column(Zorbax RX-8, MAC-MOD Analytical, Chadds Ford, PA, USA) anda mobile phase comprising trifluoroacetic acid:tetrahydrofuran:methanol:water(0.1:1.2:25:73.7, v/v/v/v). Detection was by UV absorbance at251 nm. Retention times of linezolid and the internal standard were 7 and 10 min, respectively. In plasma, mean recoveriesfor linezolid and internal standard were 95.4% and 95.8%,respectively

Pharmacokinetic assessments

(Cmax): maximum measured plasma concentration over the time span specified under steady state

(Tmax): time of the maximum measures plasma concentration.

(AUC0-t): the area under the plasma concentration versus time curve from time zero to last measurable concentration

(AUCO-α): the area under the plasma concentration versus time curve from time zero to infinity. This is calculated as the sum of the AUC 0-t plus the ratio of the last measurable concentration, to the elimination rate constant. (Ballow, C.H et at., 2002)

Statistical Analysis:

Statistical analysis was performed using SAS software and manual calculations, the following summary statistics for the pharmacokinetic parameters were calculated for both the Test and Reference products: Arithmetic mean, Standard Deviation (SD), percentage of coefficient of variation (CV%) were calculated. The analysis of variance (ANOVA) model included sequence, formulation (treatment) and period as fixed effects and subject nested within the sequence as a random effect. The sequence effect was tested at the 0.10 level of significance using mean square of subjects nested with in sequence from the ANOVA as the error term; all other main effects were tested at the 0.05 level of significance using residual error from the ANOVA. The ratio of the Test and Reference product averages (Least Square Mean) was estimated for the differences in the Least Square Mean (LSM) of the Log- transformed data then taking the anti-log for the estimates. 90% confidence interval for the ratio of test and reference was estimated using the t value at mean square error degrees of freedom (df), and the stranded error of estimate. The standard error of estimate was calculated using the mean square error, and number of reference subjects from the ANOVA model.

Safety assessments

Adverse events, laboratory assays (e.g. haematology, includinghaematocrit, haemoglobin, reticulocytes, platelets and othermeasures, clinical chemistries and urinalysis), vital signs,12-lead electrocardiogram and cardiac telemetry were performedfor each subject on selected study days to assess the tolerabilityof linezolid. Adverse event data were obtained voluntarily fromsubjects and by daily monitoring and questioning of subjectsby study personnel. Adverse events were assessed for seriousness,intensity, potential relation to study medication, clinicaloutcome and effect on study treatment.

 

Subject dropout:

Total 12 volunteers were dosed once during the study. All the volunteers were included in the safety evaluation. Of the 12 subjects enrolled, 12 subjects received both the test and reference products and completed the study, but subject no 11 and volunteer code K was withdrawn due to adverse events.

 

 

RESULTS

 

The present bioavailability and bioequivalence study was conducted in 12 healthy volunteers in the age range 18-32 years. The final evaluation was carried out on data obtained from 11 volunteers who completed the study according to protocol. The individual and mean plasma concentrations of Linezolid test and reference products data had shown in Tables 1& 2. Linear (untransformed) and logarithmic (log transformed) scales are shown in Figures 1 &2.The mean Cmax for test was 12.592 µg/ml and for the reference products was 12.882 µg/ml, the mean (+/-SD) of Cmax test  was 2.4822 µg/ml and for the reference was 2.7383 µg/ml,  the mean Tmax  for test and reference were 1.02 hr and 1.36 hr, the mean (+/-SD) of  Tmax  for test and reference were 0.7328 hr and 0.7799 hr, the mean  AUC 0-t for test was 95.88 µg.hr / ml and for reference was 93.753 µg.hr / ml. the mean (+/-SD) of  AUC 0-t test and reference were 26.1986 µg.hr / ml and 25.7797 µg.hr / ml. The mean AUC 0-α for test was 103.502 µg.hr/ml and for reference was                  102.024 µg.hr / ml,the mean (+/-SD) of AUC 0-α test and reference were 29.2290 µg.hr / ml and 29.4988 µg.hr / ml, the mean  Kel for test was 0.151 hr -1 and for reference 0.145 hr -1, the mean (+/-SD) of Kel test and reference were 0.0525 hr -1 and 0.0423 hr -1.The mean t ½ for test was 5.07 hrs and for reference for 5.20 hrs and the mean (+/-SD) of t ½ for test and reference were 1.61hrs and 1.68 hrs, respectively,(Table - 4).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NDIVIDUAL AND MEAN PLASMA LINEZOLID CONCENTRATION (µg/mL) FOR PRODUCT ‘T ‘(TEST)

TABLE -1

subject

Volunteer

code

sequence

0.00

hrs

0.33

hrs

0.67

hrs

1.00

hrs

1.33

hrs

1.67

hrs

2.00

hrs

2.50

hrs

3.00

hrs

4.00

hrs

6.00

hrs

8.00

Hrs

12.00

hrs

16.00

hrs

24.00

hrs

1

A

ST

0.000

0.507

9.835

11.195

10.687

10.155

9.879

8.997

8.866

7.950

6.559

4.819

2.957

1.928

1.119

2

B

TS

0.000

14.838

15.435

15.865

14.781

14.393

14.480

13.629

13.151

12.176

9.226

7.012

4.262

2.642

0.831

3

C

TS

0.000

6.291

11.481

11.127

10.955

10.621

10.446

10.339

9.625

8.368

6.517

4.074

1.820

0.952

0.000

4

D

ST

0.000

4.261

6.355

6.914

7.220

7.465

8.372

7.766

7.170

6.150

4.177

2.700

1.073

0.519

0.000

5

E

TS

0.000

2.667

15.839

13.318

11.566

12.155

11.586

10.891

9.946

8.725

6.079

4.073

1.855

0.907

0.000

6

F

ST

0.000

2.953

3.390

9.152

15.744

12.647

11.392

9.988

9.308

8.335

6.631

4.188

2.646

1.611

0.750

7

G

TS

0.000

9.084

9.723

10.554

10.776

10.953

10.010

10.670

9.534

8.452

6.275

4.201

1.926

1.072

0.000

8

H

ST

0.000

0.000

14.733

11.671

11.562

11.088

10.995

10.085

9.434

8.715

6.925

5.027

2.875

1.917

0.909

9

I

ST

0.000

0.000

4.929

7.617

7.801

8.068

8.473

8.155

7.809

7.002

5.880

4.275

2.225

1.124

0.000

10

J

TS

0.000

12.332

13.046

12.492

12.182

11.453

10.983

10.558

9.746

8.903

7.072

5.133

2.558

1.470

0.000

12

L

TS

0.000

0.000

2.357

7.735

9.718

10.198

10.381

11.731

11.752

11.093

8.741

7.254

5.039

3.714

1.898

N

 

 

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

Mean

 

 

0.000

4.812

9.738

10.695

11.181

10.836

10.818

10.255

9.667

8.715

6.735

4.796

2.658

1.623

0.501

SD

 

 

0.000

5.233

4.878

2.704

2.545

1.951

1.688

1.628

1.646

1.676

1.324

1.325

1.139

0.914

0.646

Min

 

 

0.000

0.000

2.357

6.914

7.220

7.465

8.372

7.766

7.170

6.150

4.177

2.700

1.073

0.519

0.000

Med

 

 

0.000

2.953

9.835

11.127

10.955

10.953

10.983

10.339

9.534

8.452

6.559

4.275

2.558

1.470

0.000

Max

 

 

0.000

14.838

15.839

15.865

15.744

14.393

14.480

13.629

13.151

12.176

9.226

7.254

5.039

3.714

1.898

CV%

 

 

---

108.75

50.09

25.29

22.77

18.01

15.61

15.88

17.03

19.23

20.13

27.62

42.85

56.29

129.09

 

 

 

 

 

 

 

 

 

 

 

 

INDIVIDUAL AND MEAN PLASMA LINEZOLID CONCENTRATION (µg/mL) FOR PRODUCT ‘R' (REFERENCE)

TABLE -2

subject

Volunteer

code

sequence

0.00

hrs

0.33

hrs

0.67

hrs

1.00

hrs

1.33

hrs

1.67

hrs

2.00

hrs

2.50

hrs

3.00

hrs

4.00

hrs

6.00

hrs

8.00

hrs

12.00

hrs

16.00

hrs

24.00

hrs

1

A

ST

0.000

0.900

11.415

11.467

11.323

10.059

10.161

9.847

9.143

8.321

7.118

5.261

3.555

2.204

1.164

2

B

TS

0.000

14.366

15.462

15.631

15.504

14.380

13.995

13.450

10.933

9.665

8.580

5.811

3.451

1.980

0.778

3

C

TS

0.000

0.000

2.749

3.918

8.227

12.241

11.068

10.644

9.888

9.580

7.223

5.129

2.567

1.409

0.000

4

D

ST

0.000

11.764

11.065

9.623

8.503

8.320

8.190

7.815

6.961

5.268

3.827

2.422

0.824

0.000

0.000

5

E

TS

0.000

0.000

14.039

13.839

12.514

12.229

11.318

9.688

9.678

8.130

6.170

4.268

1.847

0.845

0.000

6

F

ST

0.000

0.440

3.466

4.551

5.741

9.007

10.422

10.180

9.978

9.254

7.545

5.104

3.205

1.948

0.769

7

G

TS

0.000

2.949

12.954

12.783

12.606

12.188

12.654

10.143

10.302

9.706

8.942

7.272

4.348

3.440

1.503

8

H

ST

0.000

3.438

14.698

13.671

12.616

12.301

11.883

11.067

10.188

9.642

7.505

5.691

3.314

1.759

0.698

9

I

ST

0.000

0.000

1.303

4.383

6.077

7.278

7.371

7.680

7.615

7.334

5.718

4.073

1.996

1.036

0.000

10

J

TS

0.000

15.422

13.123

11.398

10.520

10.082

9.668

9.338

8.227

7.705

5.454

4.058

1.773

0.809

0.000

12

L

TS

0.000

15.379

13.860

12.926

11.627

12.592

12.198

11.970

11.309

10.330

7.048

5.960

4.051

2.424

1.187

N

 

 

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

Mean

 

 

0.000

5.878

10.412

10.381

10.478

10.971

10.722

10.166

9.475

8.630

6.830

5.004

2.812

1.623

0.554

SD

 

 

0.000

6.786

5.184

4.208

3.022

2.164

1.863

1.667

1.364

1.470

1.461

1.274

1.093

0.939

0.576

Min

 

 

0.000

0.000

1.303

3.918

5.741

7.278

7.371

7.680

6.961

5.268

3.827

2.422

0.824

0.000

0.000

Med

 

 

0.000

2.949

12.954

11.467

11.323

12.188

11.068

10.143

9.888

9.254

7.118

5.129

3.205

1.759

0.698

Max

 

 

0.000

15.422

15.462

15.631

15.504

14.380

13.995

13.450

11.309

10.330

8.942

7.272

4.348

3.440

1.503

CV%

 

 

---

115.46

49.78

40.54

28.84

19.73

17.38

16.40

14.39

17.04

21.40

25.47

38.88

57.88

104.00

 

 

Summary statistics of Pharmacokinetic Parameters for

LINEZOLID in 11 healthy male subjects TABLE-3

Products/statistics

Cmax

AUC0-t

AUC 0-∞

Tmax

Ref.product

Arithametic mean

SD

CV%

N

 

12.882

2.4822

19.27

11

 

95.8892

26.1986

27.32

11

 

103.5029

29.2290

28.24

11

 

1.02

0.7328

72.16

11

Test product

Arithametic mean

SD

CV%

N

 

12.592

2.7383

21.75

11

 

93.7531

25.7797

27.50

11

 

102.0243

29.4988

28.91

11

 

1.36

0.7799

57.16

11

Least Squere mean

S

T

 

12.743

12.518

 

95.0593

92.8581

 

102.5796

101.0475

 

--

--

Least Squere mean

Ratio S/T%

98.23

97.68

98.51

--

90%Confidence interval

S vsT

Lower limit

Upper limit

 

 

87.23

102.24

 

 

86.20

109.17

 

 

85.48

111.54

 

 

--

--

p-value(ANOVA)

Sequence

Sub(seq)

Period

Form

 

0.0133

0.0844

0.3560

0.7751

 

0.0111

0.0084

0.9326

0.7202

 

0.0186

0.0153

0.9520

0.8382

 

--

--

--

--

Power%

84.40

81.19

70.30

--

Fig-1 Mean plasma concentration of Linezolid test and       Reference Vs time profile. (For untransformed data)

 

Fig-2 Mean plasma concentration of Linezolid test and Reference Vs time profile (log transformed data)

 

(Series 1 = test, series 2 =Reference)

Comparison of mean Pharmacokinetic Parameters of reference and test

TABLE-4

Parmeters

Statistics

Reference

Product (S)

Test

prodct (T)

T max   (hrs)

Arithmaticmean

S.D

1.02

0.7328

1.36

0.7799

C max (mcg/ml)

Arithmaticmean

S.D

12.88

2.4822

12.592

2.7383

AUC0-t

(µg.hr /ml)

Arithmaticmean

S.D

95.88

26.1986

93.75

25.7797

AUC 0-α

(µg.hr /ml)

Arithmaticmean

S.D

103.50

29.2290

102.02

29.4988

Kel hr -1

Arithmaticmean

S.D

0.151

0.0525

0.145

0.0423

t ½ hr

Arithmaticmean

S.D

5.07

1.61

5.20

1.68

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DISCUSSION

The main objective of the study was to assess the bioavailability and bioequivalence of the Linezolid 600 mg tablets of Dr. Reddy's Laboratories Ltd. In healthy human adult male volunteers, under fasting condition after single dose and to evaluate safety of both the products. The drug concentration level of Linezolid in plasma were determined by a validated HPLC method, sampling was done up to 24 hr after dosing on day 7 such that the plasma concentration could be measured for adequately profiling the pharmacokinetics of the product and study periods were separated by a washout period of 8 days for complete elimination of the product   substance. The pharmacokinetic and statistical analyses were done on 11 subjects excluding subject no. 11 and volunteer code no. K withdrawn due to adverse events in period-I

The results of pharmacokinetic analysis of Linezolid test product were comparable to the reference product. Analysis of variance for log transformed pharmacokinetics parameters revealed that there was no significant effect of variation due to period and formulation for all the pharmacokinetic parameters at 0.05 level of significance. Based on the results obtained in the study, the Linezolid 600 mg tablets of Dr. Reddy's Laboratories Ltd and Zyvox ® (Linezolid) 600mg tablets, Pharmacia & Upjohn Company, USA are bioequivalent in healthy human adult male subjects under fast conditions. Both the products were well tolerated. Plasma levels may be used as surrogate parameters for clinical activity. Therefore, the results of this study suggest comparable clinical efficacy of the products.

 

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