International Journal of Pharmaceutical and Phytopharmacological Research (eIJPPR)

International Journal of Pharmaceutical and Phytopharmacological Research (eIJPPR)
ISSN (Online) 2249-6084 (Print) 2250-1029


                                            International Journal of Pharmaceutical and
                                              Phytopharmacological Research (eIJPPR)
                                                            [Impact Factor – 0.7826]


Research Article
     COMPARATIVE BIOEQUIVALENCE STUDY OF DIFFERENT
    BRANDS OF TELMISARTAN TABLETS MARKETED IN INDIA
  BY DISSOLUTION MODELING AND QUALITY CONTROL TESTS
            TAPAS KUMAR PAL1*, USASHI GHOSH1, MOUMITA PANDA1
        1
          NSHM KNOWLEDGE CAMPUS, KOLKATA – GROUP OF INSTITUTIONS
                 NSHM COLLEGE OF PHARMACEUTICAL TECHNOLOGY,
                      124 B.L. SAHA ROAD, KOLKATA – 700053, INDIA
                     *Corresponding author’s email: tapas.pal@nshm.com
ABSTRACT:
The marketing of multisource therapeutically equivalent branded drug products with variable
pricing poses a great problem to common patient as well as to the prescribing physician with
respect to their quality, safety, bioavailability and interchangeability. Considering that only in
vitro bioequivalence studies can predict in vivo bioequivalence of all therapeutically
equivalent branded & generic versions of the same API formulations; the present study had
been performed to explore & justify interchangeability of branded generic drug products by
comparing the multipoint in vitro dissolution profile of SIX different brands of Telmisartan
20 mg tablets available in Kolkata, Indian market along with routine quality control tests like
assay of drug content, weight variation, hardness, friability tests, disintegration tests,
dissolution tests. For assessing the bioequivalence and the possibility of interchangeability of
different brands, the multipoint in vitro dissolution profiles were compared through various
statistical methods recommended by FDA such as Model-independent methods including the
dissimilarity factor (f1) and the similarity factor (f2), Model-dependent methods like Zero-
order, First-order, Higuchi model, Hixson-Crowell model, and Korsmeyer-Pepas model.
Dissolution efficiency (DE%) values of these brands had also been compared by using
Analysis of Variance (ANOVA) to determine whether all the formulations used were
equivalent or significantly different. The result demonstrated that all the tested brands except
Brand B released more than 85% in 30 minutes time as per USP specification. However, the
assay result claimed that all brands except Brand A contained more than 96-98% of the
labeled amount. The experimental data showed that ANOVA-based and model-dependent
approaches are more discriminative than the f -factors, which are easier to be interpreted by a
single value to describe the closeness of the two dissolution profiles. Hence the experimental
findings call for more post marketing surveillance so as to ensure that only safe, effective
bioequivalent drug products with economic benefit reaches the common man and the
prescribing physicians should develop awareness regarding interchangeability of multisource
branded generic drug products.

KEYWORDS: ANNOVA, Biowaiver, Bioequivalent, Branded Generic, Dissolution
Efficiency, Dissolution Profile, Therapeutically Equivalent,

                                                1
1. INTRODUCTION:

Bioequivalence (BE) studies focus on the drug release from the formulation and subsequent
absorption into the systemic blood circulation which consist of both invivo and invitro
studies. Until recent years, bioequivalence was determined only by invivo tests. However,
there are many reports that have been utilized invitro bioequivalence studies instead of invivo
bioequivalence tests for immediate release solid oral dosage forms of highly soluble BCS
class I and III drugs. According to US Pharmacopeia, necessary in vitro tests are assay,
content uniformity and dissolution studies. The dissolution profile comparison is more
precise than others to characterize the drug product [1]. The similarity in in vitro dissolution
behavior of oral dosage forms has long been sought from the perspective of both
bioavailability and quality control considerations as key pre-requisite to be systemically
effective. Variable therapeutic responses to therapeutically equivalent drug products have
been reported with so called branded generics and batch-to-batch inconsistencies have also
been reported. Different products with the same amount of API have shown distinct
differences in their therapeutic effects [2, 3]. The reasons may be either due to the differences
in rate and extent of absorption, or difference between the purity of active ingredients, type of
excipients, proportion between them and the manufacturing variables such as the influence of
mixing method and granulation procedure as well as coating parameters [4,5]. Therefore
there are serious issues to the prescribing physicians that various branded generic
substitutions may have different bioavailability and couldn’t be used interchangeably.

Dissolution is extremely important for all conventional solid oral dosage forms and can be the
rate limiting step for the absorption of drugs administered orally especially for Class II drugs
in BCS classification i.e. low solubility and high permeability. Three categories of dissolution
test specification for immediate release products are described in the guidance provided by
the Center for Drug Evaluation and Research at the Food and Drug Administration, USA: (a)
single point specifications, (b) two-point specifications, and (c) dissolution profile
comparison. The dissolution profile comparison is more precise than others to characterize
the drug product [6]. On the other side, in vitro dissolution profile is the most frequently
used in vitro variable parameter to generate IVIVC [7]. Furthermore, Dissolution efficiency
(DE%) is defined as the area under the dissolution curve up to a certain time ‘t’, expressed as
a percentage of the area under the rectangle described by 100% dissolution in the same time
[8]. The comparison of DE% of several formulations simultaneously can be theoretically
related to the comparison among mean plasma concentration-time curve obtained after
deconvolution of the in vivo data [9-11]. The similarity factor (f2) is also used to determine
whether any selected branded formulation under test is similar to the therapeutically
equivalent reference product (usually called an Innovator) with same API to allow
interchangeability between the therapeutically equivalent products. An f2 value higher than
50% means that the average difference between both dissolution profiles is less than 10% at
all sampling points indicating similarity of the two branded therapeutically equivalent
products [12].

Telmisartan is an angiotensin II receptor antagonist (ARB) used in the management of
hypertension. The solubility of Telmisartan in water is very low i.e. 0.078 mg/ml in water and
is strongly pH-dependent, with maximum solubility observed at high and low pH. However,
in the range of pH 3–9, it is only poorly soluble. Telmisartan belongs to Class II drug in BCS
classification i.e. low solubility and high permeability. One of the major problems with this
drug is its low solubility in biological fluids, which results into poor bioavailability after oral
administration. Absolute bioavailability of the Telmisartan was 42-58% and biological half-

                                                2
life is only 24 hours. Poor solubility of Telmisartan leads to poor dissolution and variation in
dose dependant bioavailability. Pharmacokinetics of orally administered Telmisartan is
nonlinear over the dose range 20-160 mg, with greater than proportional increases of plasma
concentrations (Cmax and AUC) with increasing doses.

Conventional Telmisartan tablet formulations of different brands have different types and/or
amount of diluents, binders, disintegrants, lubricants, or other excipients. They may be also
subjected to different formulation variables which affect the hardness, disintegration and
dissolution rate of a given branded formulation. Manufacturing methods and the excipients
used in the production processes could also contribute to the quality and release skillfulness
of API. So in order to ensure the products’ quality, the manufacturers perform in-process
quality control tests regularly throughout the production process and the products’ shelf-life.
Besides, feedback from few prescription audits reveal that some Telmisartan brands need to
be given more than the recommended once daily dose or that daily doses higher than the
recommended 40 mg are required to produce the desirable clinical effects. This has
necessitated the present study to assure & justify interchangeability of these therapeutically
equivalent Telmisartan brands by comparing the dissolution profiles and other quality
parameters of Telmisartan 20 mg tablets of six different brands marketed in Kolkata, Indian
Market.


   2. MATERIALS AND METHODS:

Telmisartan, chemically is 4-((2-n-propyl-4-methyl-6-(1- methylbenzimidazol – 2 – yl) –
benzimidazol – 1 - yl) methyl) biphenyl-2- Carboxylic acid being an angiotensin II receptor
antagonist (ARB) used in the management of hypertension bind to the angiotensin II type 1
(AT1) receptors with high affinity, causing inhibition of the action of angiotensin II on
vascular smooth muscle, ultimately leading to a reduction in arterial blood pressure.




                             Chemical structure of Telmisartan


Immediate release Telmisartan 20 mg tablets of six different brands were purchased from
different local retail pharmacies at Kolkata local market, India. Products were codified as
Brand A, Brand B, Brand C, Brand D, Brand E and Brand F. The following commercial
tablets of Telmisartan 20 mg were taken for the study:




                                               3
Table 1: Drug product Information of Six Brands of telmisartan Tablets (20mg)

 Sl Brand                          Mfg.        Exp.      Price/10
                    Batch No.                                              Manufacturer
 no. Code                          date        date       units
                                   Sep-        Aug-
    1       C       48003034                             Rs.42.50           USV Limited
                                   2013        2015
                                   Aug-         Jul-
    2       F      E3ALM021                              Rs.14.50     Mankind Pharma Limited
                                   2013        2015
                                   Mar-        Feb-                  Alembic Pharmaceuticals
    3       B      1301000377                            Rs.38.15
                                   2013        2015                           Limited
                                   Jun-        May-                    Unichem Laboratories
    4       D      BTEL13004                             Rs.41.50
                                   2013        2015                           Limited
                                   Nov-        Oct-                  Aristo pharmaceuticals pvt
    5       E       B324l023                             Rs.25.30
                                   2013        2015                             Ltd
                                   Nov-        Oct-
    6       A           --                              Rs. 32.10    IPCA Laboratories Limited
                                   2013        2015

In order to evaluate bioequivalence & brand interchangeability, similarity in dissolution and
dissolution efficiency for telmisartan immediate release tablets, various assessments were
conducted and six different commercial brands were compared, using Brand C as reference
control brand and Brand A, Brand B, Brand D, Brand E and Brand F as test formulations.

TELMISARTAN bulk drug was obtained as a gift sample from M/S AUCTUS PHARMA
LIMITED (UNIT-11). All other chemicals and solvents used were of analytical grade. The
reagents used were methanol (GR grade), sodium hydroxide, potassium dihydrogen
phosphate. Freshly prepared glass distilled water was used throughout the work.


2.2 Dissolution Testing Parameters

Dissolution Apparatus:                   Lab India Dissolution Test Apparatus, DS 8000
                                         USP Type II (Paddle Type)
Volume of Dissolution medium:            900 ml Phosphate Buffer (pH 7.5)
Temperature:                             37 ± 0.5 0 C
RPM:                                     75
Dissolution Time:                        60min
Time interval for each sample:           5 min up to 30 min; then 15 min up to 1 hr
Sample withdrawn:                        10ml each time point

2.3 Apparatus/Instruments:

       UV Visible Spectrophotometer:    Shimadzu UV-1560
       Digital Balance:                 Mettler Toledo (ML204/A01)
       Roche Friabilator:               Digital Roche Friabilator (Model 902)
       Monsanto Hardness Tester:        Model: EI 66 Expo
       Disintegration Test Apparatus:   Digital Tablet Disintegration Test Apparatus
                                         Microprocessor Based, VEEGO, (VTD-DV)

2.4 Visual Inspection:


                                                 4
The shape and color of the different brands of tablets were examined visually. The diameter
and thickness of 10 tablets from each brand were measured and the average was taken and
standard deviation was calculated.

2.5 Friability Test:
Ten tablets of each brand were weighed and subjected to abrasion using a Roche friabilator at
100 revolutions for 4 min. The tablets were dedusted and weighed again then percent of
weight loss was recorded. The friability of the tablets was then calculated using the
expression: % Friability = [(Initial weight – Final weight) / Initial weight] × 100

2.6 Hardness Test:
The crushing strength of the tablets was determined using Monsanto hardness tester. Ten
sample tablets of each brand were taken and the pressure required to break the tablet was then
read off the machine and recorded.

2.7 Uniformity of Weight:
Twenty tablets of each brand were weighed individually using a digital analytical balance.
The average weight was determined and the percentage (%) deviation of the individual
tablets from the mean was determined.

2.8 Disintegration Test:
The disintegration test was performed using the Microprocessor Based Digital Tablet
Disintegration Test Apparatus (VEEGO). Six tablets of each brand were selected randomly
and the disintegration time was recorded using freshly prepared distilled water as medium at
37 ± 2 °C. The time when no granule of any of the tablets was left on the mesh was noted as
the disintegration time.


2.9 ASSAY USING UV-VISIBLE SPECTROPHOTOMETER:

2.9.1 Preparation of standard drug solution:
For preparation of Standard drug solution of Telmisartan, 44.0 mg of Telmisartan RS was
accurately weighed and transferred to a 100ml volumetric flask in which 1.0 ml of 0.1(M) of
sodium hydroxide (NaOH) solution was added. Finally methanol was added up to 100ml to
form a clear drug solution with a concentration of 0.44 mg/ml. (4.4 μg/ml)

2.9.2 Preparation of calibration curve:
Aliquots, containing 1 to 4 ml portion of standard drug solution, were transferred to four nos.
individual 50 ml volumetric flask and volume was made up to mark with methanol in each
volumetric flask. Solutions were scanned in the range of 200-400 nm against blank. The
absorption maxima were found to be at 296nm against blank. The calibration curve was
plotted.

2.9.3 Assay of Telmisartan contents in different brands:
For performing the assay of the Telmisartan tablets, the following method was applied. Ten
tablets from each brand were separately weighed and finely powdered. The powdered tablets
were passed through 60# sieve. An accurately weighed quantity of each lot of sieved powder
equivalent to 50mg of Telmisartan was taken in individual 100ml volumetric flask to which
70ml methanol and 1ml of 0.1(M) NaOH were added. The mixture was sonicated for 15-30
minutes. The volume in each flask had been made up to 100ml with methanol. The solution

                                              5
was then filtered through Whattman filter paper. The filtrate was then diluted suitably with
methanol to get the solution of concentration range of 8 - 35 μg/ml. The absorbance of each
assay preparations arising out of each of SIX Brands of Telmisartan 20 mg Tablets was
measured against methanol as blank solution. The drug content of each branded preparation
was calculated using standard calibration curve. The absorbances of the standard drug
solution and branded preparation were concomitantly determined at λmax 296nm with
Shimadzu UV-1800 Spectrophotometer using methanol as a blank.

2.9.4 Dissolution Rate Determination:
Dissolution test had been performed on nine tablets of each brand using the USP monograph
method with 900 ml phosphate buffer as dissolution medium (pH 7.5) in USP type II
apparatus (Paddle Type) [Lab India Dissolution Test Apparatus, DS 8000]. Temperature of
the medium was maintained at 37 ± 0.5 °C. The paddle was rotated at 75 rpm for 1 hour.
During this process 10 milliliters of the samples were collected each time with a 10ml syringe
at a regular interval of 5min up to 30 minutes and then at 15 min interval up to 1 hour ( i.e. at
5, 10, 15, 20, 25, 30, 45, 60 minutes). Also, at the same time 10ml of fresh phosphate buffer
solution was replenished in the medium in order to maintain the volume at 900 ml. Each of
the withdrawn samples was filtered through Whatman filter paper (No 41). The absorbance
was then measured at λmax 296nm using UV-Visible spectrophotometer. The concentration
was determined against standard drug solution having a known concentration of Telmisartan
RS in the same medium. The percentage of drug released is calculated using the given
formula. From the absorbance values obtained at each time interval percentage release of the
drug was calculated using standard graph and graph was plotted by taking % of drug release
on y-axis and time on x-axis for each tablet. Dissolution efficiency was calculated for each
tablet from the graph by using the following formula. Same experiment was repeated for
remaining brands.

2.10 DISSOLUTION EFFICIENCY:
The dissolution efficiency (DE%) of a pharmaceutical dosage form is defined as the area
under the dissolution curve up to a certain time, t, expressed as a percentage of the area of the
rectangle described by 100% dissolution in the same time. This concept was proposed by
khan and Rhodes [13] and is calculated by the equation:
                ∫
       DE% =           *100%
Where y is the percent drug release as the function of time, t. y100 is 100% drug release and t
is the total time of drug release.

2.11 MODEL-DEPENDENT METHODS:
Model dependent methods are designed on different mathematical functions, which interpret
the dissolution profile. The quantitative interpretation of the values obtained in dissolution
testing is easier using mathematical equations, which express the API release profile in
function of some critical parameters related with the pharmaceutical dosage forms. Some of
the most relevant and more commonly used mathematical models describing multipoint
dissolution profile are shown below.

2.11.1 Zero-Order Model: Drug dissolution from dosage forms that do not disaggregate &
release the drug slowly can be represented by the equation:
                      Qt = Q0 + K0 t
where, Qt is the amount of drug dissolved in time t, Q0 is the initial amount of drug in the
solution (most times, Q0 = 0) and K0 is the zero order release constant expressed in units of

                                               6
concentration/time. To study the release kinetics, data obtained from in vitro drug release
studies were plotted as cumulative amount of drug released versus time.

Application: This relationship can be used to describe the drug dissolution of several types of
modified release pharmaceutical dosage forms, as in the case of some transdermal systems, as
well as matrix tablets with low soluble drugs in coated forms, osmotic systems, etc, coated
forms, osmotic systems.

2.11.2 First –Order Model: This model has been used to describe absorption/elimination of
some drugs, although it is difficult to conceptualize this mechanism on a theoretical basis.
The release of the drug which followed first order kinetics can be expressed by the equation:
                        dC/dt = -KC
where, K is first order rate constant expressed in units of time-1.

This equation can also be expressed as:                log C = log C0 – Kt / 2.303
where, C0 is the initial concentration of drug, K is the first order rate constant, and t is the
time. The data obtained are plotted as log cumulative percentage of drug remaining vs. time
which would yield a straight line with a slope of - K/2.303.

Application: This relationship can be used to describe the drug dissolution in pharmaceutical
dosage forms such as those containing water-soluble drugs in porous matrices.

2.11.3 Higuchi Model: The first example of a mathematical model aimed to describe drug
release from a matrix system was proposed by Higuchi in 1961. Initially conceived for planar
systems, it was extended to different geometrics and porous systems.

This model is based on the hypotheses that
(i) Initial drug concentration in the matrix is much higher than drug solubility;
(ii) drug diffusion takes place only in one dimension (edge effect must be negligible);
(iii) drug particles are much smaller than system thickness;
(iv) matrix swelling and dissolution are negligible;
(v) drug diffusivity is constant; and
(vi) perfect sink conditions always attained in the release environment

Accordingly, model expression is given by the equation:
        ft = Q = A √ [D (2C - Cs)*Cs t]
where Q is the amount of drug released in time t per unit area A, C is the drug initial concn,
Cs is the drug solubility in the matrix media and D is the diffusivity of the drug molecules
(diffusion coefficient) in the matrix base. In a general way, it is possible to simplify the
Higuchi model as (generally known as The Simplified Higuchi Model):
                               ft = Q = KH * t 1/2
where, KH is the Higuchi dissolution constant. The data obtained were plotted as cumulative
percentage drug release versus square root of time.

Application: This relationship can be used to describe the drug dissolution from several
types of modified release pharmaceutical dosage forms, as in the case of some transdermal
systems and matrix tablets with water soluble drugs




                                                7
2.11.4 Hixson And Crowell Model: Hixson and Crowell (1931) recognized that the
Particle’s regular area is proportional to the cube root of its volume. They derived the
equation:
                                W01/3 - Wt1/3 = k t
Where, W0 is the initial amount of drug in the pharmaceutical dosage form, Wt is the
remaining amount of drug in the pharmaceutical dosage form at time t, and k (kappa) is a
constant incorporating the surface - volume relation. The equation describes the release from
systems where there is a change in surface area and diameter of particles or tablets. To study
the release kinetics, data obtained from in vitro drug release studies were plotted as cube root
of drug percentage remaining in matrix (Wt1/3) versus time (t).

Application: This expression applies to pharmaceutical dosage form such as tablets, where
the dissolution occurs in planes that are parallel to the drug surface if the tablet dimensions
diminish proportionally, in such a manner that the initial geometrical form keeps constant all
the time

2.11.5 Korsmeyer-Peppas Model: Korsmeyer et al. (1983) derived a simple semi-emperical
relationship which relates exponentially drug release from a polymeric system with respect to
relapsed time. To find out the mechanism of drug release, first 60% drug release data were
fitted in Korsmeyer - Peppas model .
                               Mt / M ∞ = K * t n
where Mt / M∞ is a fraction of drug released at time t, K is the release rate constant and n is
the release exponent. The n value is used to characterize different release for cylindrical
shaped matrices. This above equation can also be expressed as:
                       log Mt – log M∞ = n log t + log K
To find out the exponent of n, the portion of the release curve, where Mt / M∞ < 0.6 should
only be used. To study the release kinetics, data obtained from in vitro drug release studies
were plotted as log cumulative percentage drug release versus log time. This model is
generally used to analyze the release of polymeric dosage form, when the release mechanism
is not well known or when more than one type of release phenomenon is involved

2.12 MODEL-INDEPENDENT METHODS:
The description of the in vitro dissolution profiles by using model-independent methods
includes the calculation of mean dissolution time (MDT) from the dissolution profile, mean
residence time (MRT) from the residence profile, or Area Under Dissolution Curve (AUC).
The concept of Fit Factors or similarity indices was introduced by Moor and Flanner [11] in
1996. A simple model independent approach uses a dissimilarity factor (f1) and a similarity
factor (f2) to compare dissolution profiles [12]. For comparison of in vitro dissolution
profiles, similarity and dissimilarity factors are emphasized by US FDA.

2.12.1 Dissimilarity factor (f1):
The dissimilarity factor is the percent difference between the two curves at each time point
and is a measurement of the relative error between the two curves. The percent error is zero
when the test and drug reference profiles are identical and increase proportionally with the
dissimilarity between the two dissolution profiles. It is expressed as:
                       f1 = {[∑ t=1n |Rt -Tt|] / [∑t=1n Rt]} x100
where, n is the number of dissolution sample time points, Rt and Tt are the cumulative mean
percentage dissolved at each of the selected n time points of the reference and test product
respectively. .


                                               8
2.12.2 Similarity factor (f2):
The similarity factor 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 the
two curves. This model independent method is most suitable for dissolution profile
comparison when three to four or more dissolution time points are available. It represents
closeness of two comparative formulations. Generally similarity factor in the range of 50-100
is acceptable according to US FDA. It is expressed as:
                      f2 = 50xlog {[1+ (1/n) ∑ t=1n (Rt -Tt) 2] -0.5 x100}
Where, n is the number of dissolution sample times, where Rt and Tt are the cumulative
percentage dissolved at each of the selected n time points of the reference and test product
respectively. The similarity factor should range between 0 and 100. It is 100 when two
comparative groups of reference and test are identical and approaches 0 as the dissimilarity
increases. Among several methods investigated for dissolution profile comparison, f2 is the simplest.
Moore and Flanner proposed a model independent mathematical approach to compare the dissolution
profile using two factors, f1 and f2 (10).

2.13 STATISTICAL METHODS:
The dissolution profile data can also be illustrated graphically by plotting the mean
dissolution profile data for each formulation with error bars extending to two standard errors
at each dissolution time point. Then, the data of the dissolution profiles are summarized
numerically and 95% confidence intervals for the differences in the mean dissolution profiles
at each dissolution time point are evaluated (11) The statistical methods assess the difference
between the means of two drug release data sets in single time point dissolution (ANOVA or
t-student test).


    3. RESULTS & DISCUSSION:

Test sample quantification was based on a previously constructed calibration curve with a
standard reference sample of Telmisartan RS and submitted to a linear regression analysis.
Linear relations with reference standard Telmisartan bulk drug were obtained with the
concentration interval of 8 -35 μg/mL (R2 = 0.9999), according to the equation Abs = 0.0445
x C (where, C = μg/mL). Nowadays, the study of dissolution in vitro is considered a
fundamental requirement in the pharmaceutical industry in order to assure the quality of solid
pharmaceutical dosage forms for oral use, guarantee the quality from Batch to Batch,
orientate the development of new formulations and secure the uniformity in quality and
performance of the drug even after modifications. On a parallel basis, this allows formulation
optimization in the development phase and, in the same way, it allows stability studies,
manufacturing process monitoring, and the establishment of in vivo/in vitro correlations [14-
16]. During the development of pharmaceutical formulations - innovator or generic - was
expected that chemically and physically stable products would be formulated, in such a way
that they were bioavailable with similar in vitro dissolution profile.


The pharmaceutical industry and the registration authorities do focus, nowadays, on drug
dissolution studies. In vitro dissolution has been recognized as an important element in drug
development. Under certain conditions it can be used as a surrogate for the assessment of
Bioequivalence. The kind of drug, its polymorphic form, crystallinity, particle size, solubility
and amount in the pharmaceutical dosage form can influence the release kinetics. A water-
soluble drug incorporated in a matrix is mainly released by diffusion, while for a low water-

                                                 9
soluble drug the self-erosion of the matrix will be principal release mechanism. To
accomplish these studies the cumulative profiles of the dissolved drug are more commonly
used. To compare dissolution profiles between two drug products model dependent (curve
fitting), statistic and model independent methods can be used. In recent years, FDA has
placed more emphasis on a dissolution profile comparison in the area of post-approval
changes and biowaivers. Under appropriate test conditions, a dissolution profile can
characterize the product more precisely than a single point dissolution test. A dissolution
profile comparison between pre-change and post-change products for SUPAC related
changes, or with different strengths, helps assure similarity in product performance and
signals bioinequivalence.

Once a tablet is ingested it undergoes disintegration and dissolution before being absorbed.
The rate and extent of the drug when it enters into the systemic circulation determines its
bioavailability. Assuming the solubility/dissolution behavior of the drug is key determinant of
its oral bioavailability and absorption from the gastro-intestinal tract being the rate-limiting
step, the experimental results indicate that Brand A, Brand D, Brand E, Brand F are
bioequivalent and interchangeable to Brand C (Reference product) as in all cases f2 value
exceeds 50 and f1 value approaches 1. However in case of Brand B, the f2 value of 9.99
indicates that it is not bioequivalent to reference product Brand C. Models have been
established to describe the relationship between drug dissolution and geometry on drug
release patterns mathematically. The release models are shown in the Fig.9 to Fig.13 with
major appliance and best describing drug release phenomena such as Zero order model, First
order model, the Higuchi model, Hixon-Crowell model and Korsmeyer–Peppas model.

Table 2: Standard Calibration data of Telmisartan RS (concentration range 8–35 μg/ml)

                                                      Absorbance at 296 nm
                 SL.NO. Concentration (μg/ml)
                                                      (± Standard deviation)
                    1                  0                        0
                    2                 8.8                   0.392±0.008
                    3                13.2                   0.588±0.006
                    4                17.6                   0.785±0.016
                    5                22.0                   0.970±0.011
                    6                26.4                   1.180±0.005
                    7                35.2                   1.566±0.013




                                              10
STANDARD CALIBRATION CURVE OF TELMISARTAN

                                      1.8
                                      1.6                                               y = 0.0445x
                                                                                        R² = 0.9999
                                      1.4
               Absorbance at 296 nm



                                      1.2
                                       1
                                      0.8
                                      0.6
                                      0.4
                                      0.2
                                       0
                                            0      5           10        15        20         25      30       35   40
                                                                        Concentration in mcg/ml


                                            Fig 1: Standard calibration curve of Telmisartan RS



Table 3: Quality control parameters of different Brands of Telmisartan 20 mg Tablets
(data showing avg. ± standard deviation; an = 20, bn = 10, cn = 10, dn = 6, en = 10)

           a                                                              c               d                e
           Uniformity of                               b                  Hardness         DT             Drug Content
Brand                                                      Friability
           Weight (mg) ±                                                 (Kg/cm2) ±     (Min &         Amount
Code                                                          %                                                  Percentage
               S.D                                                           S.D          Sec)          (mg)
                                                                           14.14 ±      14 min
  A        119.44 ± 1.56                                     0.01                                     16.87±0.036   84.35%
                                                                            1.27         35 sec
                                                                           7.46 ±        8 min
  B        141.39 ± 1.87                                     0.04                                     23.66±0.883   118.30%
                                                                            1.38         20 sec
                                                                           12.56±        4 min
  C        131.46 ± 1.24                                     0.02                                     22.26±0.514   111.30%
                                                                            2.29         43 sec
                                                                           11.62 ±       4 min
  D        166.28 ± 4.13                                     0.02                                     20.59±0.620   102.95%
                                                                            0.70         25 sec
                                                                           12.42 ±       5 min
  E        125.43 ± 2.77                                     0.01                                     20.92±1.134   104.60%
                                                                            1.92         35 sec
                                                                                         4 min
  F        190.73 ± 1.71                                     0.01        6.52 ± 1.96                  22.21±0.323   111.05%
                                                                                         40 sec




                                                                              11
Table 4: Percent mean cumulative dissolved amount of Drug from different brands of
       Telmisartan 20 mg tablets at different time points (Each data is mean of 9 samples)

                       % Mean CDR from different Brands
 TIME                                                                       Rt = Cumulative
             Rt        Tt1        Tt2         Tt3        Tt4        Tt5     percentage
 (min)
                                                                            dissolved Qty. of
          brand C    brand F    brand B    brand D    brand E    brand A    Reference product
                                                                            at time t;
                                                                            Tt = Cumulative
   5       62.23      47.42      18.54      55.70      41.59       31.97
                                                                            percentage
                                                                            dissolved Qty. of
   10      94.25      92.15      26.87      87.19      74.09       55.03    Test product at
                                                                            time t
   15     103.82     107.48      35.08      100.95      96.62      73.85
   20     103.98     109.50      37.86      103.66     106.89      87.32
   25     103.47     108.68      40.32      103.22     108.30      95.50
   30     102.04     109.24      41.93      102.67     107.91      96.92
   45     101.26     108.45      41.90      102.30     107.40      96.65
   60      98.24     107.51      42.33      100.93     106.51      98.29
                                                                            No. of Time
   N         7          7          0          7           6          5      points, where
                                                                            % CDR ≥ 85%

The dissolution results as the means of percents cumulative dissolved amount of Drug from
different brands of Telmisartan 20 mg tablets versus time are estimated and given in Table 3
and results of descriptive analyses for routine quality parameters of commercially available
tablets of all brands of Telmisartan 20 mg tablets are given in Table 2. The in vitro
dissolution profiles of the different tablets individual brands tablets are shown in Fig. 2 to Fig
7. Each data point had represented a mean of nine measurements for each branded product.
All branded drug products except brand B had complied with the dissolution specification,
Q, stated in the US Pharmacopeia, as cumulative dissolution not less than 85 % of the labeled
amount of Telmisartan within 30 minutes in all brands (brand C, F, D, E & A).




                                               12
Table 5: Area under the dissolution curve of % CDR from different brands of Telmisartan 20
mg Tablets at different time points (Each data is mean of 9 samples)


                                 Area under the Curve (AUC) of %CDR vs Time

 TIME (min)                             AUC = 0.5*{(CDR1+CDR2)*(t2-t1)}

                    brand C      brand F        brand B      brand D         brand E   brand A
        5            391.20       348.91         113.53       357.24         289.21     217.50
        10           495.18        499.06        154.88       470.37          426.79    322.19
        15           519.51        542.43        182.35       511.52          508.78    402.91
        20           518.64        545.45        195.44       517.18          537.97    457.04
        25           513.78        544.81        205.61       514.71          540.52    481.04
        30          1524.75       1632.70        628.72      1537.23         1614.83   1451.71
        45          1496.27       1619.72        631.76      1524.17         1604.29   1462.05
        60
        ∑           5459.32       5733.08       2112.29      5432.41         5522.39   4794.45



Table 6: Percent dissolution efficiency of different brands of Telmisartan 20 mg Tablets at
two different time points (Each data is mean of 9 samples)

                                             % Dissolution Efficiency

%DEmax                                    DEtmax = AUC /(Ymax*tmax)

                 brand C       brand F        brand B       brand D      brand E       brand A

  DE30            81.28         82.69          28.39         79.03           76.78      62.69
  DE45            88.07         91.41          32.90         86.85           87.07      74.05


Table 7: Fit Factors showing similarity & dissimilarity within different brands of Telmisartan
20 mg tablets with respect to Ref. product [Brand C] (Each data is mean of 9 samples)


   f %                                % Similarity & Dissimilarity Factors

               brand C        brand F        brand B       brand D       brand E       brand A
   f1                          5.08           62.34          0.87            2.94       4.85
             Ref. Product
   f2                         60.27            9.99         74.41            60.41      52.59


                                                 13
Table 8: Comparison among different brands of Telmisartan 20 mg tablets with respect to
Bioequivalency, similarity, dissolution efficiency and variance from dissolution profile

Telmisartan 20 mg                                                   Variance                Variance
                           f2              f1        DE30                         DE45
     Tablets                                                         at t30                  at t45

     brand C           Reference Product             81.28           28.85        88.07       30.6
     brand F             60.27            5.08       82.69            4.98        91.41       4.13
     brand B              9.99            62.34      28.39           10.41        32.9       27.81
     brand D             74.41            0.87       79.03            4.31        86.85       3.71
     brand E             60.41            2.94       76.78            8.34        87.07       6.35
     brand A             52.59            4.85       62.69           81.53        74.05      73.31

According to FDA’s guides for industry (FDA, 1995, 1997a, b), generally, f1 values up to 15
(0–15) and f2 values greater than 50 (50–100) ensure similarity or equivalence of the two
curves. The values of f1 and f2 factors for test products versus reference were calculated from
the means of percent dissolved at each time point (Table 7). From the above Table 8, it might
be forecasted with respect to similarity factor (f2) being more than 50 value; brand F, D, E
& A of Telmisartan 20 mg tablets are bioequivalent to the Reference Product (Brand C)
and these findings can also be correlated with dissimilarity factor (f1) values of the
corresponding brands. Brand B is proven as outlier in this study and not bioequivalent with
other brands due to having very low value of f2 and very low dissolution efficiency (both
DE30 as well as DE45) as found in Table 6 and has not further been considered in subsequent
ANNOVA – Single factor calculation. However, the result

Table 9: Calculation of ANNOVA- SINGLE FACTOR and VARIANCE within different
brands of Telmisartan 20 mg tablets (Brand B being outlier, not taken into calculation)

                       Sample
     BRANDS
                       Count
                                          Sum     Avg (Mean) % CDR at t30 Variance within brand
  Test Brand - A         9               872.24               96.92                       81.53
  Test brand - E         9               971.22              107.91                        8.34
  Test brand - D         9               924.00              102.67                        4.31
Reference brand - C      9               918.33              102.04                       28.85
  Test brand - F         9               983.17              109.24                        4.98

 Source of                       df =       MS =
                  SS                                  Fcalculated              P-value            FCV
 Variation                      (N -1)      SS/df
 Between
                884.74            4         221.18        8.64       0.0000400747759798713        2.61
  brands
  Within
                1024.06          40         25.60
  brand
   Total
                1908.80          44
 Variation



                                                     14
To find the critical value from an F distribution, one must know the numerator (MSTR) and
denominator (MSE) degrees of freedom, along with the significance level. FCV has df1 and
df2 degrees of freedom, where df1 is the numerator degrees of freedom equal to (C – 1) and
df2 is the denominator degrees of freedom equal to (N – C).
In our study, C = no of brands (5) & N = no of samples within the Brand (9).
Therefore, df1 = (5 – 1) = 4 and df2 = (9*5 – 5) = 40 and calculated F value is 8.64
Hence, we need to find FCV [4,40] corresponding to α = 5% using the F tables and the value
obtained is: FCV [4,40] = 2.61

Decision Rule
We reject the null hypothesis if: Fcalculated (observed value) > FCV (critical value).

In our findings, we get 8.64 > 2.61, so we reject the null hypothesis that there are significant
similarity of Bioavailability within the brands (A,C,D,E & F) of Telmisartan 20 mg tablets
based on the cumulative drug release as evident from multipoint dissolution profile data. The
results of ANOVA also showed that the drug products were significantly different in terms of
percent dissolved at each time point (P
% CDR From brand F tablets
                                               120
                                                                                                                        TAB 1
                % CDR from different tablets

                                               100
                                                                                                                        TAB 2
                                                80                                                                      TAB 3

                                                60                                                                      TAB 4
                                                                                                                        TAB 5
                                                40
                                                                                                                        TAB 6
                                                20
                                                                                                                        TAB 7
                                                 0                                                                      TAB 8
                                                     5   10     15      20      25        30        45        60
                                                                                                                        TAB 9
                                                                      Time in minutes


Fig 3: Percent Cumulative release of Telmisartan from Brand (F) of Telmisartan 20 mg
        tablets at different time points (n = 9)



                                                               % CDR From brand B tablets
                                                60

                                                                                                                         TAB 1
                                                50
       % CDR from different tablets




                                                                                                                         TAB 2
                                                40                                                                       TAB 3
                                                                                                                         TAB 4
                                                30
                                                                                                                         TAB 5
                                                20                                                                       TAB 6
                                                                                                                         TAB 7
                                                10
                                                                                                                         TAB 8
                                                 0                                                                       TAB 9
                                                     5    10     15        20     25           30        45        60
                                                                        Time in minutes

Fig 4: Percent Cumulative release of Telmisartan from Brand (B) of Telmisartan 20 mg
        tablets at different time points (n = 9)




                                                                               16
% CDR From brand D tablets                  TAB 1
                                       120
                                                                                                  TAB 2
                                       100
        % CDR from different tablets


                                                                                                  TAB 3

                                        80                                                        TAB 4

                                        60                                                        TAB 5

                                                                                                  TAB 6
                                        40
                                                                                                  TAB 7
                                        20
                                                                                                  TAB 8

                                         0                                                        TAB 9
                                             5   10    15      20      25       30    45    60
                                                             Time in minutes


Fig 5: Percent Cumulative release of Telmisartan from Brand (D) of Telmisartan 20 mg
        tablets at different time points (n = 9)




                                                      % CDR from brand E tablets
                                       120                                                        TAB 1
        % CDR from different tablets




                                       100                                                        TAB 2

                                        80                                                        TAB 3

                                                                                                  TAB 4
                                        60
                                                                                                  TAB 5
                                        40
                                                                                                  TAB 6
                                        20
                                                                                                  TAB 7
                                         0                                                        TAB 8
                                             5   10     15      20      25       30    45    60
                                                              Time in minutes                     TAB 9




Fig 6: Percent Cumulative release of Telmisartan from Brand (E) of Telmisartan 20 mg
       tablets at different time points (n = 9)




                                                                      17
% CDR from brand A tablets                                     TAB 1
                                         140
                                                                                                                               TAB 2
                                         120
                                                                                                                               TAB 3
            % CDR of different tablets




                                         100
                                                                                                                               TAB 4

                                          80
                                                                                                                               TAB 5

                                          60                                                                                   TAB 6

                                          40                                                                                   TAB 7

                                          20                                                                                   TAB 8

                                           0                                                                                   TAB 9
                                                     5    10        15        20    25      30        45        60
                                                                         Time in minutes

Fig 7: Percent Cumulative release of Telmisartan from Brand (A) of Telmisartan 20 mg
       tablets at different time points (n = 9)




                                                     % Cumulative release profile of diff. Brands of
                                  140.00                     Telmisartan 20 mg tablets
       % cumulative dissolved amount of




                                  120.00

                                  100.00
                 Temisartan




                                         80.00

                                         60.00

                                         40.00

                                         20.00

                                          0.00
                                                 0         10            20         30           40        50           60        70
                                                                                   Time in Minutes
                                                          brand C                     brand F                        brand B
                                                          brand D                     brand E                        brand A



Fig 8: Mean Percent Cumulative Release pattern from SIX different Brands of Telmisartan
       20 mg tablets vs different time points in minutes ( n = 9)




                                                                                   18
Zero order plots of different brands
                                                      25.00
         Qty of telmisartan dissolved (mg)
                                                      20.00


                                                      15.00


                                                      10.00


                                                                        5.00


                                                                        0.00
                                                                                 5        10       15      20       25     30       45        60
                                                                                                        Time in minutes
                                                                                         brand C                 brand F                 brand B
                                                                                         brand D                 brand E                 brand A


                                                                          Fig 9 : Qty. of Telmisartan dissolved at different time point 't'




                                                                                      First order plots of different brands
                                                                         1.50
                                 Log of qty remaining to be dissolved




                                                                         1.00

                                                                         0.50

                                                                         0.00
                                                                                  5       10       15      20       25     30      45         60
                                                                        -0.50

                                                                        -1.00

                                                                        -1.50

                                                                        -2.00

                                                                        -2.50
                                                                                                         Time in minutes
                                                                                         brand C                brand F             brand B
                                                                                         brand D                brand E             brand A


Fig 10: Log of Qty. of Telmisartan remaining undissolved at different time point 't'




                                                                                                         19
Simplified Higuchi plots of different brands
         Qty of telmisartan dissolved per unit area            25.00

                                                               20.00

                                                               15.00

                                                               10.00

                                                                5.00

                                                                0.00
                                                                           2.24       3.16      3.87    4.47      5.00    5.48      6.71    7.75
                                                                                                       Sq. Root of Time
                                                                            brand C                    brand F                   brand B
                                                                            brand D                    brand E                   brand A


              Fig 11: Qty. of Telmisartan dissolved per unit area at different time point 't'




                                                                             Hixson and Crowell plots of different brands
                                                                       3
                 Cube Root of Qty of Telmisartan undissolved




                                                                   2.5

                                                                       2

                                                                   1.5

                                                                       1

                                                                   0.5

                                                                       0
                                                                              5         10        15      20      25       30        45         60
                                                                                                       Time in Minutes
                                                                                      brand C                  brand F                brand B
                                                                                      brand D                  brand E                brand A



Fig 12: Cube root of Telmisartan Qty remaining at different time point 't'




                                                                                                        20
Korsmeyer - Peppas plots of different brands


            Log of Fraction of Telmisartan dissolved
                                                       0.20


                                                       0.00
                                                               5      10       15     20      25      30    45       60
                                                       -0.20


                                                       -0.40


                                                       -0.60


                                                       -0.80                        Time in minutes
                                                                     brand C             brand F           brand B
                                                                     brand D             brand E           brand A


     Fig 13: Log of fractional Qty of Telmisartan released at time't' from different brands

    4. CONCLUSION:
In the present study, the f1 and f2 values were found within the range of 0.87 – 5.08 and
52.59 – 74.41 respectively with the deviation in case of Brand B. This states that the release
of Telmisartan from all formulations were similar with reference. On the other hand, for all
products except formulation B the cumulative drug delivery was satisfactory since at least
85% had been dissolved in 30 min. Therefore results confirm the presence of bioequivalence
and interchangeability between the analyzed brands and reference product with the exception
of brand B. This study verifies serious need for constant post marketing monitoring of the
marketed products with the view to bioequivalence and agreement with pharmacopoeia
standards.

In conclusion, each method described here for the comparison of dissolution profiles seems to
be applicable. In general, it was observed that ANOVA-based and model-dependent methods
have narrower & stringent limits and are more discriminative than the f-factors. For ready
interpretation f-factors are easier to use; only one value is obtained to describe equivalency of
the two dissolution profiles. [16-20].

However, the last point for dissolution has to be determined in order to emphasize the
difference between two profiles, since the values of the f-factors depend on this point. In the
model-dependent methods, for the formation of a non-linear regression model, it is important
that the assumption of least squares be continually checked (Rawlings, 1988). The application
and evaluation of model-dependent methods are more complicated and these methods present
an acceptable model approach to the true relationship between the dependent and independent
variables. ANOVA-based methods including post hoc procedures provide a possibility for the
comparison of dissolution data on the basis of point-by-point and for finding the sources of
differences among the variables. It is possible to obtain detailed information about dissolution
data which can be useful also in formulation development to match release of reference
product. Consequently, ANOVA-based methods would seem to be more informative than the
other comparison methods.



                                                                                    21
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                                               22
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