1. 1 Hnin Thandar Nwe, 2 Latt Latt Win, 3 Min Wun & 1 Khine Kyi Han
“In Vitro and In Vivo Bioequivalence Studies on Three Brands of Levofloxacin in Myanmar Market”
1 Hnin Thandar Nwe, 2 Latt Latt Win, 3 Min Wun & 1 Khine Kyi Han
1 Department of Pharmacology, University of Pharmacy, Yangon
2 Department of Pharmacology, University of Medicine, Taung-gyi 3 Department of Food and Drug Administration, Yangon
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2. INTRODUCTION Bioequivalence
absence of a significant difference in bioavailability between two or more pharmaceutically equivalent products under similar conditions in appropriate study design
Pharmaceutically equivalence
same amount of the active ingredient in the same dosage form and administered by the same route1
1Southern African Development Community (SADC). Guideline for Bioavailability and Bioequivalence. 2007
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3. INTRODUCTION (Cont.)
one of the biggest public health challenges in the world
range of bacteria resistance to at least one of the most commonly used antibiotics ranged tremendously between different countries from zero to 82% (quinolone resistance 8% to 65%) 2
2 million people get antibiotic resistance in the US and 23,000 people die in each year3
2World Health Organization (WHO). “High levels of antibiotic resistance found worldwide, new data shows,” accessed January, 2018
3Centers for Disease Control and Prevention (CDC). “CDC 24/7: Saving Lives. Protecting People,”
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4. INTRODUCTION (Cont.)
One of the problems of drug resistance: counterfeit and substandard drugs
Many pharmaceuticals including antibiotics are manufactured by various pharmaceutical companies from different countries
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5. INTRODUCTION (Cont.)
Fluoroquinolones: main classes of antibiotics (from UTIs to infections of almost all body compartments in 800 million patients)4
Levofloxacin
now used increasingly among fluoroquinolone class in clinical setting
should have comparable quality, efficacy, and safety to the originator drug although it is manufactured by different pharmaceutical companies
4Bambeke, F.V., Michot, J.M., Eldere, J. V. and Tulkens, P. M. Quinolones in 2005: an update. Clin Microbiol Infect ; 11, pp. 256–280.
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6. INTRODUCTION (Cont.)
To combat drug resistance problems and enormous production and consumption of generic drugs, many bioequivalence studies are increasingly demanded in developing countries
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7. OBJECTIVES
To determine the pharmaceutical equivalence of three brands of levofloxacin by pharmacopoeial and non-pharmacopoeial tests
To determine the pharmacokinetic parameters of three brands of levofloxacin after single oral dose administration in Myanmar healthy volunteers
To compare the bioequivalence of two cheaper brands with reference brand according to ASEAN bioequivalence guidelines, 2004
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8. MATERIALS Reference drug
LEVO-DENK (Levofloxacin 500 mg) Film-coated tablet
Manufacturer - Denk Pharma, Germany
Myanmar Registration No. - R1812AA6773
Lot No
Manufacturing Date - 09/2014
Expiry Date - 08/2017
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9. MATERIALS (Cont.) Test drug (1)
LVZ (Levofloxacin 500 mg) Film-coated tablet
Manufacturer - Zifam Pharma, India
Myanmar Registration No. - R1902AA1208
Lot No. - FT197
Manufacturing Date - 09/2014
Expiry Date - 08/2017
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10. MATERIALS (Cont.) Test drug (2)
LIVOX (Levofloxacin 500 mg) Film-coated tablet
Manufacturer - Zee Laboratory, India
Myanmar Registration No. - R1608A8571
Lot No. - ZET0628
Manufacturing Date - 09/2014
Expiry Date - 08/2017
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11. MATERIALS (Cont.) Chemicals
Levofloxacin standard powder (SM Chemicals Ltd, Bangkok)
Copper sulfate pentahydrate (AnalaR), Merck, Mumbai
L- isoleucine (AnalaR), BDH chemicals Ltd., England
Methanol (HPLC Grade), Fisher Scientific, UK
Deionized water
Potssium phosphate monobasic solution (AnalaR), Merck, Mumbai
Sodium phosphate dibasic solution (AnalaR), Merck, Mumbai
Dichloromethane (AnalaR), Merck, Mumbai
Hydrochloric acid (AnalaR), BDH chemicals Ltd., England
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12. METHODS Study design  Cross-over study design
Study Period  From February 2016 to December 2016
Place of study  Pharmaceutical Toxicology Research Division, DMR (Yangon)
 Tablet Section, Pharmaceutical Research Department
Sample size  Eight healthy volunteers
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13. METHODS (Cont.) Inclusion criteria Healthy volunteers
age between 18 to 55 years
either sex
with normal serum creatinine and urine RE results
who did not take any medication within one week
who had given written informed consent after full explanation of experimental procedure
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14. METHODS (Cont.) Exclusion criteria Withdrawal criteria
Subjects with history of
Subjects who
allergy to levofloxacin and any drug
experienced any adverse effects of levofloxacin
renal diseases or alcoholism
with subject’s request
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15. Flow Chart for In Vitro Bioequivalence Procedure
Identification of levofloxacin tablets by UV-Vis Spectrophotometers and FTIR 1
Uniformity of Weight according to USP 1
Assay of active indregredients by UV-Vis Spectrophotometers 1
Tablet Length, Width and Thickeness by Caliper 1
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16. Flow Chart for In Vitro Bioequivalence Procedure (Cont.)
Tablet Hardness by Monsanto hardness tester 1
Disintegration By Tablet Disintegration Apparatus 1
Dissolution by Paddly Type Dissolution Apparatus 1
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1 The United State Pharmacopoeia. The National Formulary 29. The United State Pharmacopoeial Conention. Rockelle; 2011.

17. Flow Chart for In Vivo Bioequivalence Procedure (Bioavailability)
Explaining procedure of study to healthy volunteers
No consent
Informed consent
Exclude
History taking, basic clinical examination
& laboratory investigations
Selection of Eight healthy volunteers according to inclusion criteria
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18. Flow Chart for In Vivo Bioequivalence Procedure (Bioavailability) (Cont.)
After overnight fasting, 500 mg Levo-Denk was given with 200 mL water
to healthy volunteers
Blood sampling at 0 hr, 0.5 hr, 1 hr, 1.5 hr, 2 hr, 6 hr, 24 hr
Pharmacokinetic parameters of Levo-Denk were determined
Two weeks (wash-out period)
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19. Flow Chart for In Vivo Bioequivalence Procedure (Bioavailability) (Cont.)
After overnight fasting, 500 mg LVZ was given again with 200 mL water to healthy volunteers
Blood sampling at 0 hr, 0.5 hr, 1 hr, 1.5 hr, 2 hr, 6 hr, 24 hrs
Pharmacokinetic parameters of LVZ were determined
Two weeks (wash-out period)
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20. Flow Chart for In Vivo Bioequivalence Procedure (Bioavailability) (Cont.)
After overnight fasting, 500mg Livox was given with 200 mL water to healthy volunteers
Blood sampling at 0 hr, 0.5 hr, 1 hr, 1.5 hr, 2 hr, 6 hr, 24 hrs
Pharmacokinetic parameters of Livox were determined
Compare the pharmacokinetic parameters of three brands of Levofloxacin tablets and assess the bioequivalent range
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21. Flow diagram of plasma extraction procedure of Levofloxacin
0.25 mLof plasma sample μL phosphate buffer + 4 mL dichlormethane 5
Vortex for 30 sec
Centrifuge at 4000 rpm x 15 mins
Filter supernatant through 0.45 μm Nylon syringe filter
Evaporated under nitrogen
Reconstitute in 100 μL of mobile phase and inject 20 μL into HPLC column
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22. METHODS (Cont.)
HPLC
Mobile phase: 87.5% [Copper II sulphate (5mM) + L- isoleucine (10mM)] % Methanol
Stationary phase: reverse phase C18 silica column (150 x 4.6 mm i.d, 5 μm)
Flow rate: 1.0 mL/min
Wavelength: 236 nm
Sample injection volume: 20 μL
Mobile phase was filtered by using a 0.45 μm nylon membrane filter and sonicated for 20 mins by ultrasonic cleaner
5 Wong, F.A., Juzwin, S.J. and Flor, S.C. Rapid stereospecific high-performance liquid chromatographic determination of levofloxacin in human plasma and urine. Journal of Pharmaceutical and Biomedical Analysis. 1997; 15, pp
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23. METHODS (Cont.) Statistical analysis Data analysis
Plasma concentrations of levofloxacin were calculated by Microsoft Office Excel 2010
Pharmacokinetic parameters (Cmax, AUC, T ½ab, Kab, T½el, Kel, Vd and CL) were calculated
Statistical analysis
done by using SPSS (version 22.0) statistical software
Key pharmacokinetic parameters among three brands were analyzed by using ANOVA Test
Statistically significant level was p < 0.05 at 95% confidence interval
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24. METHODS (Cont.) Ethical considerations
This study was approved by Ethical and Research Committee of University of Pharmacy, Yangon
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25. RESULTS
Idenfication by FIIR
Idenfication by FIIR
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26. TABLE (1) Weight, Width, Length, Thickness and Hardness of three different brands of Levofloxacin Tablets
Sample
Weight (mg) (Mean ± SD)
Width (mm)
(Mean ± SD)
Length (mm)
Thickness
(mm)
Hardness (Kg)
Levo-Denk
730 ± 0.01
9.11 ± 0.01
± 0.01
5.26 ± 0.03
± 1.33
LVZ
660 ± 0.01
8.80 ± 0.01
± 0.03
5.07 ± 0.23
± 1.52
Livox
970 ± 0.02
9.30 ± 0.02
± 0.04
5.43 ± 0.05
17.57 ± 2.09
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27. Disintegration (mins)
TABLE (2) Disintegration, Dissolution and Assay content of three different brands of levofloxacin tablets
Sample
Disintegration (mins)
Dissolution (%)
Assay Content
(% in mg)
Levo-Denk
± 6.43
± 2.07
± 0.27
LVZ
2.16 ± 0.40
97.83 ± 2.25
± 0.40
Livox
7.66 ± 1.21
± 0.31
± 0.37
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28. Plasma concentration-time curve of three brands of Levofloxacin
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29. TABLE (3) Pharmacokinetic Parameters Levo-Denk, LVZ and Livox
(mean ± SD)
LVZ
Livox
C0 (µg/mL)
15.68 ± 6.59
12.66 ± 3.93
15.16 ± 4.85
Cmax (µg/mL)
8.32 ± 2.60
7.18 ± 1.55
8.60 ± 2.25
Tmax (hr)
1.31 ± 0.35
1.44 ± 0.18
1.25 ± 0.38
Kab (hr-1)
1.83 ± 0.73
1.63 ± 0.67
1.28 ± 0.78
T1/2ab (hr)
0.43 ± 0.16
0.50 ± 0.20
0.66 ± 0.24
Kel (hr-1)
0.10 ± 0.03
0.09 ± 0.03
T1/2el (hr)
7.84 ± 3.19
8.39 ± 2.49
7.62 ± 1.79
AUC0-25 (µg/mL.hr)
84.72 ± 25.24
90.72 ± 39.54
89.93 ± 29.08
AUC0-α (µg/mL.hr)
85.98 ± 26.09
93.74 ± 43.00
91.41 ± 29.93
Vd (L)
69.25 ± 25.36
59.58 ± 18.93
66.75 ± 28.16
CL (L/hr)
5.82 ± 2.85
5.33 ± 2.77
5.47 ± 1.95
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30. Comparison of Cmax of three brands of Levofloxacin (Levo-Denk, LVZ & Livox)
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31. Comparison of Tmax of three brands of Levofloxacin (Levo-Denk, LVZ & Livox)
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32. Comparison of AUC0-25 of three brands of Levofloxacin (Levo-Denk, LVZ & Livox)
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33. TABLE (4) Bioequivalent ratio of two brands of levofloxacin to reference brand
Test A
AUC0-25
AUC0-α
Cmax
Tmax
Levo-Denk
84.72
85.98
8.32
1.31
LVZ
90.72
93.74
7.18
1.44
Point estimate of
difference of the mean
1.07
1.09
0.86
Acceptance range
Conclusion
Bioequivalent
Test B
Livox
89.93
91.41
8.6
1.25
1.06
1.03
0.95
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34. DISCUSSION In vitro pharmaceutical equivalence study
In weight variation test, all tablets were complied with USP limits which were not more than ± 5% deviations and there was no tablet by more than ± 10%
Disintegration occurred within 30 minutes
In dissolution test, released their ingredients not less than 80% within 30 minutes
Assay results were within 90% ‐ 110%
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35. DISCUSSION (Cont.) In vivo bioequivalence study
Key pharmacokinetic parameters (Cmax, Tmax and AUC0-t) of Levo-Denk, LVZ and Livox - not statistically significant
All are within the acceptable bioequivalence range of
6Association of Southeast Asian Nations (ASEAN), Guidelines for The Conduct of The Bioavailability and Bioequivalence Study. 2004; pp.5.
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36. CONCLUSION
According to this study, the quality of a drug is not always directly proportional to their price in spite of huge fluctuation with different brands
These brands are pharmaceutically equivalent as well as biologically equivalent and can be used interchangeably with each other
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37. CONCLUSION (Cont.) The results of this study
could provide the useful information to the prescribers to choose the products with acceptable quality and affordable price because price differential between generics does not necessarily mean poor quality for the cheaper brand
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38. ACKNOWLEDGEMENT I would like to express my sincere thanks to
Prof. Dr. Latt Latt Win, Professor and Head, Department of Pharmacology, UM (TG) for her valuable guidance and close supervision of research throughout the study
Prof. Dr. Aye Soe, Professor and Head, Department of Pharmacology, UOP (Ygn) for her precious suggestions
Dr. Min Wun, Deputy Director and Head , FDA, Yangon for his expert opinion on pharmacokinetics
Dr. Khine Kyi Han, Assistant Lecturer, Department of Pharmacology, UOP (Ygn)
Last, I also like to thank all the volunteers for their kind participation in this study
Without them, the study would not be possible
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