In Vitro Bioequivalence for three Products of Warfarin Sodium in Syrian Market

 

Roula Faek Naddour, Nisrin Ali Kaddar

Department of Pharmaceutics, Faculty of Pharmacy , Tishreen University, Latakia, Syria

*Corresponding Author E-mail: roula.naddour@hotmail.com

 

ABSTRACT:

Three generic warfarin sodium 5mg tablets from different manufacturers had been evaluated to assess their bioequivalence in vitro. Other general quality assessments of these tablets like weight variation, content uniformity, hardness, friability and disintegration time were carried out and all these generic tablets passed these specification tests. To compare the dissolution profiles of all the tablet formulation withthe innovator brand, a model independent approach of difference factor (f1) and similarity factor (f2) was employed with all points included in the in vitro dissolution studies. These results showedthat all generic warfarin sodium tablets included in thisinvestigation were not bioequivalent with the chosen innovator brand, but two generic tablets were bioequivalent between each other.

 

KEYWORDS: Warfarin sodium, in vitro bioequivalence, dissolution profile, similarity factor, difference factor.

 


1.      INTRODUCTION:

Aiming to reduce the cost of medicines, the World Health Organization (WHO) has supported the use of generic drug products, especially for the below poverty line group of developing countries[1]. In 1984, the Hatch-Waxman Act approved generic drugs and bioequivalence tests to switch from brand to generic or between generics each other. The estimate of the US generic drug industry after the Hatch-Waxman Act had offered several views of great benefit to other countries, wishing to promotion a native generic drug industry.[2] To assist in substitution of brand with generic drugs, or generic with generic and attain therapeutic efficacy, bioequivalence study became essential.[3]

 

Bioequivalence has been described as the absence of considerable difference in the rate and extent to which the active moiety in pharmaceutical equivalent or pharmaceutical alternative become available at the site of drug action when they are administrated at the equal molar dose under similar conditions in an expedient designed study.[4] Bioavailability studies involve both in-vivo and in-vitro studies. In vitro studies are sometimes better than in vivo studies in assessing bioequivalence of immediate release solid oral dosage forms, because in vitro studies reduce costs, more directly assess product performance and offer benefits in terms of ethical consideration.[5]

 

Based on drug solubility and permeability, the following biopharmaceutical classification system (BCS) is recommended in the literature

Class 1: high solubility – high permeability drugs

Class 2: low solubility – high permeability drugs

Class 3: high solubility – low permeability drugs

Class 4: low solubility – low permeability drugs[6]

 

With the introduction of biopharmaceutical classification system (BCS), in-vivo bioequivalence studies could be waived for immediate release solid oral dosage forms for class 1 (high solubility and high permeability) and some of class 3 (high solubility and low permeability)[5]. Therefore, only in-vitro testing may be utilized to determine bioequivalence for highly soluble and highly permeable drugs, as warfarin sodium.Dissolution testing can serve as a tool to distinguish between acceptable and unacceptable drug products. Dissolution testing is the primary tool for the evaluation of formulation changes. Thus, dissolution-based drug product release methods are put in place to ensure the consistency of manufacturing processes and sites for the different product batches. However, it is generally acknowledged that a direct link between traditional dissolution methods and in vivo bio availability is not always possible. When such a link exists and a robust in vitro-in vivo correlation (IVIVC) can be established, dissolution data become a direct surrogate of in vivo dissolution and under certain circumstances can be used to waive bioequivalence studies.[7] It is a surrogate marker for bioequivalence test, practical and economic approach in developing countries, where both technology and resources are limited for in-vivo studies.[8]Warfarin sodium tablet (crystalline warfarin sodium) is an anticoagulant, which acts by inhibiting vitamin k-dependent coagulation factors. Chemically, it is 3-(α-acetonylbenzyl)-4-hydroxy coumarine and is a racemic mixture of the enantiomers[9]. It is available in two dose in Syria, 2 and 5 mg of warfarin sodium tablets, but in this study we chose only the most prescribed dose (5mg)[10]

 

Warfarin sodium is a narrow therapeutic index (NTI) drug for which several investigations compared the effects of different products. General reviews of studies noted that brand name and most generic products has closely similar bioavailability and clinical outcomes[11, 12], but there is not any reviews about the generic forms manufactured in Syria.An excellent correlation has been showed between results obtained in vivo rate with the dissolution test, which in turn delineating the critical area for in vitro dissolution rates of warfarin sodium tablets. [13]

 

During the war in Syria, many manufactories were destructed. Because of economic sanctions, there was a lack of primary resource; therefore many medicine was missed from the local market. In consequent,doctors were obliged to interchange generic drugs of Warfarin. It was difficult to adjust goal values of INR when switching from generic drug to another or from one patch to another for the same manufacturer.

 

This study aimed to evaluate the bioequivalence in vitro for warfarin sodium tablets(5 mg) produced by three local companies, and compared with the Brand, based on dissolution test.Other general quality assessments of the tablets were also determined.

2.      MATERIALS AND METHODS:

Warfarin sodium was gifted from Unipharma laboratories. Three generic warfarin sodium tablets, manufactured by different manufacturer and the innovator brand product with labeled contents of 5 mg each, were obtained from local market.

 

Assay:

weighed and powdered 20 tablets from each patch of generic product and the innovator drug product. The powder equivalent to 5 mg of warfarin sodium was taken and transferred to 100 ml volumetric flask. Then the volume made up to 100 ml with 0.01N NaOH. Strong shaking was done to dissolve the powdered material. After diluted two times (10 ml in 100 ml), absorbance values were measured at the maximum wavelength 308 nm (which was detected to the stander simple by drawing its spectrum) by using UV-UNVIS spectrophotometer (Jasco v-530nm/Japan) against blank.

 

Weight Variation Determination:

20 tablets from each generic and innovator brand products were weighted individually using a weighing balance (Precisa XB220A/Germany). The average weights of the tablet as well as their percentage deviation were calculated.

 

Content Uniformity:

10 tablets from each generic and innovator brand products were powdered and dissolved in 100 ml of 0.01 N NaOH, after 15 minutes 10 ml was obtained and diluted by using the same solution of NaOH. The absorbance value was measured at wavelength 308 nm by usingUV-UNVIS spectrophotometer against blank. Then the acceptance value (AV) was calculated from:

 

AV= | M-X | + KS

 

X: mean of individual contents expressed as a percentage of the label claim, K: acceptability constant, if n= 10 then k=2.4, and if n=30 then k=2.0, S: sample stander deviation, M: reference value.

 

Hardness Testing:

hardness was determined using a tablet hardness tester (Erweka TBH200/Germany).

 

Friability Testing:

friability test was conducted by employing a friability tester (Logan instruments crop/Germany) at 25 rev/min for 4 minutes. Percent friability was determined by using the following formula:

Friability% = I-F*100/I

I= Initial weight and F= Weight after friability test

 

Disintegration Testing:

6 tablets from each generic and innovator brand product were employed for the disintegration test in water at 37±0.5ºC using a disintegration apparatus (Erweka ZT-52/Germany). The disintegration time was taken to be the time when no particle remained on the basket.

 

In-vitro Dissolution Tests:

In-vitro dissolution tests were carried out using a dissolution apparatus (basket type). The dissolution medium was 1000 ml of distilled water, PH= 7, at 37ºC. In all dissolution points, 5 ml of dissolution sample were withdrawn and replaced with equal volume fresh dissolution medium at regular intervals. Collected dissolution samples were used for determination of released warfarin sodium concentrations by using a UV-UNVIS spectrophotometer, (Erweka DT600) against a blankat wavelength 308 nm.

 

3.      RESULTS AND DISCUSSION:

All the generic and innovator brand warfarin sodium 5mg tablets used in this study were within their shelf life. All tablets obtained from local market were subjected to a number of tests in order to assess their in vitro bioequivalence along with other parameters like assay, weight uniformity, content uniformity, friability, hardness and disintegration time.

 

All tablets (both generic and the brand) were approximate in hardness value. They were in a range of 5.522±0.562 to 9.522±1.661 even in case of the brand (table 1). This test showed the ability of tablets to stand handling without fracturing.

 

Friability test is used to evaluate the tablets resistance to scrape. The reference values of friability for tablets are less than 1% w/w according to USP and BP[14, 15]. The friability of all generic and brand tablets was within the range of 0.01 to 0.66% (table 1)

 

Table 1: Hardness and friability determination of all generic and innovator brand warfarin sodium tablets Where A, B, C are names of companies and 1, 2, 3 are patch numbers

Tablets

Hardness (kg/cm2)

Friability

Innovator brand

7.601±0.788

0.58

A1

9.522±1.661

0.05

A2

7.302±0.856

0.01

A3

6.225±0.904

0.03

B1

7.425±1.43

0.17

B2

5.609±1.143

0.66

B3

5.522±0.562

0.17

C1

5.95±0.232

0.29

C2

5.95±0.397

0.19

 

According to the USP, the uncoated tablets should disintegrate within 30 minutes. This mainly affects the drug released from the tablet to the site of absorption, and this depends on the drug dissolution. All the generic tablets disintegrated within the range of 2.82±0.061 to 9.07±1.1611, whereas in case of the brand, it was 17.83±2.401. (table 2)

 

Table 2: disintegration profiles of all generic and innovator brand of warfarin sodium tablets:

Tablets

Disintegration time (min)

Innovator brand

17.83±2.401

A1

8.78±0.432

A2

8.92±0.3936

A3

8.83±0.1619

B1

2.82±0.0616

B2

3.29±0.3594

B3

2.57±0.047

C1

9.07±1.1611

C2

9±1.012

 

The weight uniformity of all the tablets from each patch of generic and brand showed compliance within the official specifications (USP, BP), as none of the product deviated by up to 7.5% from their average weight (table 3)

 

The acceptance value was calculated for each patch of generic and innovator brand, and all of it was smaller than 15. (table 3)

 

Table 3: weight variation and content uniformity (AV) results of all generic and innovator brand of warfarin sodium tablets:

Tablets

Weight variation)mg)

AV

Innovator brand

221.7±2.5

8.264

A1

137.3±1.4

0.16

A2

139.1±2.6

8.03

A3

138.4±1.2

13.1

B1

202.6±6.4

13.4

B2

199.7±5.7

13.6

B3

198.9±2.1

13.41

C1

102.5±1

3.6

C2

110.3±1.8

13.92

 

According to the FDA guidance for industry, for the dissolution testing of immediate release solid oral dosage form, the BCS suggests that for class 1 and few of class 3 drugs, 85% w/w dissolution of the labeled content in appropriate medium (which referred in USP, BP or other pharmacopeias) within 15 minutes ensure that the bioavailability of the drug is not limited by dissolution. [8]

 

The amount released by all generic warfarin sodium tablets were over 85% within 15 min, whereas the brand released only 32% of the active ingredient within 15 minutes. (table 4)

 

Table 4: the amount released by all generic and innovator brand warfarin sodium tablets:

Tablets

Dissolution(%) at 15 min

Innovator brand

33±0.398

A1

108.16±0.313

A2

104.1±0.334

A3

103.16±0.126

B1

104.1±0.601

B2

88.06±0.126

B3

99.02±1.152

C1

110.1±0.655

C2

107.2±0.164

 

 

FIG 1: Dissolution profiles of all generic and innovator brand warfarin sodium tablets

 

To compare the dissolution profiles of all the generic tablets and the brand, a model independent approach of difference factor (f1) and similarity factor (f2) was employed with all points included in the in vitro dissolution studies. Difference factor (f1) is the percentage difference between two curves at each time point and is a measurement of the relative error between the two curves:

 

Where, n is a number of time points, Rt is the dissolution value of reference product at time t, and Tt is the dissolution value for the test product at time t.

 

Similarity factor (f2) is a logarithmic reciprocal square root transformation of the sum of squared error and is a measurement of the similarity in the percent (%) dissolution between these two curves:

 

For two dissolution profiles to be considered similar and bioequivalent, f1 should be between 0 to 15, while f2 should be between 50 and 100[8]. The calculated f1 and f2 values are shown in table 5. In other interpretation we chose the better batch of generic product to be the reference for other batches of different generic. F1 and were within the acceptable range for some generic tablets between each other (table6), but no one of the generic was at the acceptable range compared with the brand (table 5).This variation in dissolution time can be explained by the difference in the used excipients. The brand company states that it contains magnesium stearate, which in turn delays the time of dissolution [16]. Unfortunately we could not have any information about excipients used in local product. But  according the value of F1 and F2, we can estimate that they may use the same excipient in the local companies.

 

Table 5: calculated difference factor (f1) and similarity factor (f2) of all generic warfarin sodium tablets with its innovator brand

Tablets

Difference factor (f1)

Similarity factor (f2)

A1

87

19

A2

82

19

A3

87

19

B1

137

12

B2

116

15

B3

93

19

C1

103

16

C2

106

15

 

Table 6: calculated difference factor (f1) and similarity factor (f2) of all generic warfarin sodium tablets in comparisonwith generic patch (A2)

Tablets

Difference factor (f1)

Similarity factor (f2)

A1

5

61

A3

3

63

B1

33

24

B2

25

30

B3

32

31

C1

14

42

C2

19

35

 

4.      CONCLUSION:

In conclusion, our results showed that, for the quality control tests, all the generic and brand tablets were similar to each other, but in the dissolution test, there was not a bioequivalence between all the tablets. while national companies do not refer to excipients used, We should refer that brand is not available for treatment in Syria, and doctors depend on generic drugs only, so we viewed that changeability betweenbrand and generic is not a good choice, and will not give a similar effect to the patients, but between generics each other it is possible for two companies.

 

5.      REFERENCES:

1.     http://apps.who.int/medicinedocs/pdf/s5416e/s5416e.pdf, 2004.

2.     Boehm, G., et al., Development of the generic drug industry in the US after the Hatch-Waxman Act of 1984. Acta Pharmaceutica Sinica B, 2013. 3(5): p. 297-311.

3.     DP, c., conner dp. use of point estimates to demonstrate be: background, fda survey and recommendation. Presentation to pharmaceutical science advisory committee meeting. http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/AdvisoryCommitteeforPharmaceuticalScience and ClinicalPharmacology/UCM210796.pdf. FDA, 2010.

4.     Kusakabe, K., et al., Discovery of imidazo[1,2-b]pyridazine derivatives: selective and orally available Mps1 (TTK) kinase inhibitors exhibiting remarkable antiproliferative activity. J Med Chem, 2015. 58(4): p. 1760-75.

5.     Polli, J.E., In vitro studies are sometimes better than conventional human pharmacokinetic in vivo studies in assessing bioequivalence of immediate-release solid oral dosage forms. AAPS J, 2008. 10(2): p. 289-99.

6.     Amidon, G.L., et al., A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability. Pharmaceutical Research, 1995. 12(3): p. 413-420.

7.     Lu, Y., S. Kim, and K. Park, In vitro-in vivo correlation: perspectives on model development. Int J Pharm, 2011. 418(1): p. 142-8.

8.     guidance for industry; dissolution testing for immediate release solid oral dosage forms. http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm070237.pdf. aug 1997.

9.     Pryce, R., et al., The Use of Fish Oil with Warfarin Does Not Significantly Affect either the International Normalised Ratio or Incidence of Adverse Events in Patients with Atrial Fibrillation and Deep Vein Thrombosis: A Retrospective Study. Nutrients, 2016. 8(9).

10.   SDR, Syrian Drug Reference. 2013.

11.   Dentali, F., et al., Brand name versus generic warfarin: a systematic review of the literature. Pharmacotherapy, 2011. 31(4): p. 386-93.

12.   Vercaigne, L.M. and G.G. Zhanel, Clinical significance of bioequivalence and interchangeability of narrow therapeutic range drugs: focus on warfarins. J Pharm Pharm Sci, 1998. 1(3): p. 92-4.

13.   Wagner, J.G., et al., In vivo and in vitro availability of commercial warfarin tablets. Journal of Pharmaceutical Sciences, 1971. 60(5): p. 666-677.

14.   Niang, A., et al., [The primary pulmonary MALT lymphoma: a rare lung tumor]. Rev Pneumol Clin, 2014. 70(5): p. 293-7.

15.   Ferrando, M.L., et al., Carbohydrate availability regulates virulence gene expression in Streptococcus suis. PLoS One, 2014. 9(3): p. e89334.

16.   Iranloye, T.A. and E.L. Parrott, Effects of compression force, particle size, and lubricants on dissolution rate. Journal of Pharmaceutical Sciences, 1978. 67(4): p. 535-539.

 

 

 

 

 

 

Received on 11.06.2017          Modified on 18.07.2017

Accepted on 24.09.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2):532-536.

DOI: 10.5958/0974-360X.2018.00099.9