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Journal of Bioequivalence & Bioavailability
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In vitro Bioequivalence Studies in Tablet Formulation Containing 625 mg of Colesevelam Hydrochlorides

Gulcin Tok*, Ediz Yildirim and Ali Turkyilmaz

Research and Development Center, Sanovel Pharmaceuticals, Istanbul, Turkey

*Corresponding Author:
Gulcin Tok
Research and Development Center
Sanovel Pharmaceuticals
Istanbul, Turkey
Tel: +90 212 746 52 52/3825
Fax: +90 212 746 54 30
E-mail: gulcintok@sanovel.com.tr

Received Date: January 17, 2017; Accepted Date: January 30, 2017; Published Date: February 06, 2017

Citation: Tok G, Yildirim E, Turkyilmaz A (2017) In vitro Bioequivalence Studies in Tablet Formulation Containing 625 mg of Colesevelam Hydrochloride. J Bioequiv Availab 9:346-352. doi: 10.4172/jbb.1000323

Copyright: © 2017 Tok G, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Colesevelam hydrochloride is a second generation bile acid sequestrant that is used principally for treatment of elevated LDL cholesterol. Additionally, it is indicated to improve glycemic control in adults with type 2 diabetes mellitus. Colesevelam hydrochloride is an insoluble, non-absorbed polymer that binds bile acids in the intestine, impeding their reabsorption. Because the drug is not absorbed into the systemic circulation, pharmacokinetic information is not available. The present abstract focuses on the study of in vitro BE in tablet formulation containing 625 mg of Colesevelam hydrochloride. Bile acid salts of Glycocholic Acid (GC), Glycochenodeoxycholic Acid (GCDC) and Taurodeoxycholic Acid (TDC) were used in vitro BE studies. The binding capacity HPLC method was developed and validated for these bile acid salts. The equilibrium binding study that is the pivotal BE study and in vitro kinetic binding study that is the support the pivotal equilibrium binding study was repeated twelve times. In the in vitro equilibrium binding studies, the Langmuir binding constants k1 (affinity) and k2 (capacity) were calculated for the three salts, individually and combined (GC+GCDC+TDC) using both nonlinear Langmuir equation and linear Langmuir equation for the test and reference products. The calculated capacity constant (k2), the more important parameter, were obtained very similar between test and reference products in the 90% confidence interval and acceptance criteria of 80% to 120%. The test/reference ratio for capacity constant, k2, was obtained 0.918 and 0.922 using equation 1 and 2 for total bile acid salts from the without acid pre-treatment equilibrium binding study and 0.948, 0.953 using equation 1 and 2 for total bile acid salts from the with acid pre-treatment equilibrium binding study.

Keywords

Colesevelam hydrochloride; In vitro bioequivalence; Bile acid sequestrant

Introduction

Colesevelam hydrochloride is a novel non-absorbed, lipid lowering polymer that binds bile acids in the intestine, impeding their reabsorption and it is used principally for treatment of elevated LDL cholesterol. The mechanism of action for the activity of colesevelam has been evaluated in several in vitro and in vivo studies. These studies have demonstrated that colesevelam binds bile acids, including glycocholic acid, the major bile acid in humans. Cholesterol is the sole precursor of bile acids. During normal digestion, bile acids are secreted into the intestine. A major portion of bile acids is then absorbed from the intestinal tract and returned to the liver via the enterohepatic circulation [1]. Colesevelam binds with bile acids in the intestine to form an insoluble complex that is eliminated in feces. This increased excretion of bile acids results in an increased oxidation of cholesterol to bile acid and a lowering of the serum cholesterol [2]. Additionally, Colesevelam hydrochloride is indicated to improve glycemic control in adults with type 2 diabetes mellitus [3,4].

Colesevelam hydrochloride is a second generation bile acid sequestrant. Second generation bile acid sequestrants have higher bile acid binding capacity then first generation bile acid sequestrants. Accordingly colesevelam hydrochloride is administered at lower doses than first-generation agents [5]. The in vivo studies in hamsters and rats confirmed that colesevelam is effective in enhancing the faecal excretion of bile acids. Both colesevelam hydrochloride and cholestyramine (first generation) cause a dose dependent increase in bile acid sequestration in these two rodent species. Colesevelam was at least 2-fold more potent and efficacious in increasing faecal bile acid excretion in both models than was cholestyramine. Additionally colesevelam hydrochloride is a substance of low acute toxicity. However, the safety margin in humans on repeated dosing for long periods is rather low [1].

The recommended dose of 625 mg colesevelam hydrochloride tablet is 3 tablets taken twice per day with meals or 6 tablets once per day with a meal and should be taken with a liquid [3]. When a meal is eaten, cholecystokinin is released from the endocrine cells of the intestinal mucosa. The hormone acts on vagal afferents or the nerve ganglia innervating the gallbladder and induces gallbladder contraction. At the same time it acts on the nerves innervating the sphincter of Oddi, causing it to relax. The end result is that gallbladder bile is delivered to the duodenum [6]. The affinity, capacity, and kinetics of colesevelam hydrochloride binding for GCDC, TDC, and GC were not significantly altered after suspension in water, carbonated water, Coca-Cola, Sprite, grape juice, orange juice, tomato juice, or Gatorade. The amount of bile acid sodium salt bound as a function of time was unchanged by pre-treatment with any beverage tested. The in vitro binding characteristics of colesevelam hydrochloride are unchanged by suspension in common beverages [7].

Chemical structure, molecular formula and chemical name of colesevelam hydrochloride are provided (Figure 1). It is poly (allylamine hydrochloride), cross-linked with epichlorohydrin and alkylated with (6-bromohexyl) trimethylammonium bromide and 1-bromodecane [8].

bioequivalence-bioavailability-colesevelam-hydrochloride

Figure 1: Chemical structure, molecular formula and chemical name of colesevelam hydrochloride.

Pharmacology

Traditional absorption, distribution, metabolism, and excretion studies were not conducted with colesevelam hydrochloride, as absorption was expected to be negligible. The results of the excretion balance studies in rat and dog (and in human) showed that the bulk of radioactivity was excreted in the faeces and no radioactivity beyond minimal quantities in any tissues, plasma or urine after a 72-h or 96-h period in dog and rat, respectively, and were consistent with the non-absorbable nature of colesevelam hydrochloride [1].

Pharmacokinetics

Colesevelam is a hydrophilic, water-insoluble polymer that is not hydrolyzed by digestive enzymes and is not absorbed. Because the drug is not absorbed into the systemic circulation, pharmacokinetic information is not available [1].

Pharmacodynamics

In the pharmacodynamic in vitro study, colesevelam, colestipol and cholestyramine demonstrated similar overall bile acid binding capacity when evaluated in mixed bile acid solutions. At all free ligand concentrations, colesevelam was found to bind Glycocholic Acid (GC) significantly more tightly than did cholestyramine, which in turn was more effective than colestipol. This is an important finding since GC is the major bile acid in humans. In contrast to the clear distinction for GC, the bile acid binding capacity of taurodeoxycholic acid, taurocholicdeoxycholic acid, glycodeoxycholic acid, glycocholicdeoxycholic acid and taurocholic acid were very similar for colesevelam and cholestyramine.

Colesevelam hydrochloride is a polymer that is not likely to be absorbed from the gastrointestinal tract due its particle size. The absence of specific safety pharmacology studies and secondary pharmacodynamic studies is justified by the insolubility of colesevelam and by the observation that colesevelam is not absorbed [1].

Objective

To compare in vitro binding characteristics of colesevelam hydrocholoride between generic product and reference product under identical experimental conditions.

Colesevelam hydrochloride 625 mg Film Tablet which is brand name Zander, Sanovel Pharmaceuticals used for test product and Cholestagel 625 mg Film Tablet, Genzyme used for reference product in in vitro bioequivalence studies.

In vitro Bioequivalence Studies

The clinical efficacy of colesevelam depends on its binding capacity to intestinal bile acids.

Each bile salt-containing incubation medium should contain Glycocholic Acid (GC), Glycochenodeoxycholic Acid (GCDC) and Taurodeoxycholic Acid (TDC) [9-11].

Study Design

image

Equilibrium binding studies

An equilibrium binding study is conducted under conditions of constant time and varying concentrations of bile acid salts. This study should be conducted by incubating the test and reference products with at least eight different concentrations of total bile salts, with and without acid pre-treatment. The equilibrium binding study is considered the pivotal bioequivalence study [9,10].

Kinetic binding studies

A kinetic binding study is conducted under constant concentration of bile acid salts with varying times of observation. This study should be conducted by incubating the test and reference products for at least eight different lengths of time, with two different constant total bile salt concentrations, without acid pre-treatment. The kinetic binding study should be used to support the pivotal equilibrium binding study [9,10].

Data Treatment and Analysis

The amount of bile acid salt bound to colesevelam hydrochloride is obtained from the difference between the initial concentration of bile acid salt introduced into the system and the concentration present in the filtrate at the end of the study. The monomolecular adsorption of bile acid salt molecules from solution, at constant temperature, on to colesevelam hydrochloride is described by the following Langmuir-type nonlinear Equation 1:

image (1)

Upon rearranging, linear Equation 2 is obtained:

image (2)

where:

Ceq=Concentration of the bile acid salt remaining in the solution at equilibrium;

x=The amount of bile acid salt bound to the colesevelam hydrochloride;

m=The amount of colesevelam hydrochloride used.

The constant, k1, is defined as the adsorption coefficient or affinity constant and is related to the magnitude of the forces involved in the binding process. The Langmuir-capacity constant, k2, indicates the apparent maximum amount of bile acid salt that can be adsorbed per unit weight of colesevelam hydrochloride.

For the in vitro equilibrium binding study, the Langmuir binding constants k1 and k2 should be determined based on total bile salt binding (GC+GCDC+TDC). The test/reference ratio should be calculated for k1. The 90% confidence interval (CI) should be calculated for k2 with the acceptance criteria of 80% to 120%. For the in vitro kinetic binding study, the test/reference bound bile acid salt ratios at the various times should be compared but not subjected to the 90% CI criteria [10].

Bioequivalence Based on 90% CI

The Langmuir binding constant k2 from the equilibrium binding study [9-13].

Experimental

The entire equilibrium test procedure with and without acid pre-treatment and entire kinetic test procedure with 0.3 mM and 3.0 mM aqueous solution in the presence of 0.1 M sodium chloride solution were repeated twelve times each for the reference and test tablets.

For each study, both reference and test product tablets, a tablet composite sample was prepared by grinding 10 tablets and weighing out an amount equivalent to 10 mg of active ingredient based on the average tablet weight.

Equilibrium binding studies

For stock bile acid salts solution, 40 mM bile acid salts solutions in Simulated Intestinal Fluid (SIF) without enzyme was prepared contain 3:3:1 molar proportion of Glycocholic Acid (GC), 17.14 mM, Glycochenodeoxycholic Acid (GCDC), 17.14 mM and Taurodeoxycholic Acid (TDC), 5.72 mM.

In without pre-treatment study, eight incubation flasks of the test product and eight incubation flasks of the reference product, each containing the equivalent of 10 mg colesevelam hydrochloride, were set up and soaked with SIF each incubation flask at room temperature overnight. The following day, requisite volumes of stock bile acid salts solution with the target concentrations of bile acid salts covering the ranges of 0.1-30 mM were added to each incubation flasks. Incubated all flasks at 37oC for 24 h and filtered.

In with acid pre-treatment study, eight incubation flasks of the test product and eight incubation flasks of the reference product, each containing the equivalent of 10 mg colesevelam hydrochloride, were set up and incubated in 0.1 N hydrochloric acid at 37oC for 1 h and centrifuged. The supernatant was aspirated; the pellet was resuspended with SIF until pH 6.8 was attained. The acid pre-treated colesevelam hydrochloride product was soaked in SIF at room temperature overnight. The following day, requisite volumes of stock bile acid salts solution with the target concentrations of bile acid salts covering the ranges of 0.1-30 mM were added to each incubation flask. Incubated all flasks at 37oC for 24 h and filtered.

Kinetic binding studies

For stock bile acid salts solution, 40 mM bile acid salts solution in water was prepared contain 3:3:1 molar proportion of GC, 17.14 mM, GCDC, 17.14 mM TDC, 5.72 mM.

Twelve incubation flasks of the test product and twelve incubation flasks of the reference product, each containing the equivalent of 10 mg colesevelam hydrochloride, were set up and soaked with 0.1 M sodium chloride solution each incubation flask at room temperature overnight. The following day, requisite volumes of stock bile acid salts solution with the target concentrations, 0.3 mM or 3.0 mM, added to each incubation flasks. Incubate flasks at 37oC for twelve different time points covering 0.05-24 h (0.05, 0.1, 0.15, 0.2, 0.25, 0.50, 1, 2, 4, 8, 16, 24 h) and filtered.

Analytical method and validation

An isocratic HPLC method was developed suitable for use in bioequivalence studies and validated for GC, GCDC and TDC bile acid salts. The study parameters for both equilibrium and kinetic binding experiments were optimized and finalized for in vitro bioequivalence (Figure 2).

bioequivalence-bioavailability-unbound-bile

Figure 2: Chromatogram of unbound bile acids in the filtrate.

Unbound bile acids in the filtrate were determined by UV-detector (210 nm). Bile acids were separated from each other using an Inertsil ODS-3V (250 × 4.6 mm) column and the mobile phase of phosphate buffer acetonitrile mixture.

The method was accurate with peak area %RSD ≤ 2.0% and resolution ≥ 1.5 for each component. The assay solution concentration range was validated between LOQ concentration for each bile acid salt to 40 mM total bile acid salts for equilibrium study and LOQ concentration for each bile acid salt to 4 mM total bile acid salts for kinetic study. The validation consists of the following experiments: specificity, linearity of individual bile acid salts, LOQ confirmation, accuracy/recovery of samples, precision/repeatability, intermediate precision, standard and sample solution stability, and filter evaluation.

Results

Equilibrium binding studies

Without acid pre-treatment: As indicated in the Tables 1 and 2; Figure 3, both the equation 1 and the equation 2 results in 90% CI for individual bile acid and total bile acid were within the 80-120% interval used in the approval of ANDA [11].

Langmuir constant Bile acid salts Test (T) Estimate ± SE Reference (R) Estimate ± SE T/R 90% CI for Ratio
Affinity constant (k1) GC 2.871 ± 0.025 2.965 ± 0.023 0.968 NA
GCDC 4.695 ± 0.023 5.028 ± 0.115 0.934 NA
TDC 16.401 ± 0.123 18.314 ± 0.709 0.896 NA
GC+GCDC+TDC 0.867 ± 0.003 0.866 ± 0.008 1.001 NA
Capacity constant (k2) GC 1.059 ± 0.006 1.167 ± 0.002 0.908 89.83–91.75
GCDC 3.622 ±0.009 3.937 ± 0.004 0.920 91.56–92.41
TDC 1.529 ± 0.004 1.655 ± 0.001 0.924 91.95–92.77
GC+GCDC+TDC 6.376 ± 0.021 6.946 ± 0.006 0.918 91.24–92.34

Table 1: Point estimation results of Langmuir constants calculated using Langmuir nonlinear equation (equation 1) from the without acid pre-treatment equilibrium studies of colesevelam hydrochloride 625 mg film tablet test and reference products (mean of twelve determination).

Bile acid salts Test Reference T/R
k1 k2 r2 k1 k2 r2 k1 k2
GC 8.451 10.300 0.9877 0.953 1.040 0.9942 0.820 0.917
GCDC 8.009 8.144 0.9983 3.452 3.741 0.9984 0.984 0.923
TDC 22.609 26.915 0.9981 1.515 1.634 0.9986 0.840 0.928
GC+GCDC+TDC 1.395 1.418 0.9955 6.031 6.543 0.9963 0.984 0.922

Table 2: Mean values of affinity (k1), capacity (k2) and linear regression (r2) for GC, GCDC, TDC and total bile acid salts calculated using Langmuir linear equation (equation 2) from the equilibrium studies without acid pre-treatment of colesevelam hydrochloride 625 mg film tablet test and reference products (mean of twelve determination).

bioequivalence-bioavailability-reference-result

Figure 3: Mean test/mean reference result of capacity constant, k2, calculated using (a) Langmuir nonlinear equation (equation 1) and (b) Langmuir linear equation (equation 2) for total bile salts at without acid pre-treatment equilibrium studies (mean of twelve determination).

The Langmuir constants k1 (GC+GCDC+TDC) and k2 (GC+GCDC+TDC) for test and reference was comparable ± 20%.

With acid pre-treatment: As indicated in the Tables 3 and 4; Figure 4, both the Equation 1 and the Equation 2 results the 90% CI for individual bile acid and total bile acid was within the 80-120%.

Langmuir constant Bile acid salts Test (T) Estimate ± SE Reference (R) Estimate ± SE T/R 90% CI for Ratio
Affinity constant (k1) GC 3.202 ± 0.120 2.596 ± 0.079 1.233 NA
GCDC 6.700 ± 0.149 5.966 ± 0.071 1.173 NA
TDC 21.892 ± 0.440 19.140 ± 0.297 1.144 NA
GC+GCDC+TDC 1.118 ± 0.021 1.001 ± 0.013 1.117 NA
Capacity constant (k2) GC 1.242 ± 0.004 1.349 ± 0.005 0.921 91.29–92.83
GCDC 3.513 ±0.007 3.695 ± 0.005 0.951 94.69–95.49
TDC 1.506 ± 0.003 1.561 ± 0.002 0.965 96.04–96.95
GC+GCDC+TDC 6.419 ± 0.016 6.770 ± 0.010 0.948 94.35–95.30

Table 3: Point estimation results of Langmuir constants calculated using Langmuir nonlinear equation (equation 1) from the equilibrium studies with acid pre-treatment of colesevelam hydrochloride 625 mg film tablet test and reference products (mean of twelve determination).

Bile acid salts Test Reference T/R
k1 k2 r2 k1 k2 r2 k1 k2
GC 6.962 1.159 0.9926 6.281 1.245 0.9933 1.108 0.931
GCDC 16.995 3.315 0.9985 10.667 3.485 0.9987 1.593 0.951
TDC 32.368 1.493 0.9981 29.288 1.535 0.9985 1.105 0.973
GC+GCDC+TDC 2.010 6.058 0.9969 1.801 6.358 0.9967 1.116 0.953

Table 4: Mean values of affinity (k1), capacity (k2) and linear regression (r2) for GC, GCDC, TDC and total bile acid salts calculated using Langmuir linear equation (equation 2) from the equilibrium studies with acid pre-treatment of Colesevelam hydrochloride 625 mg film tablet test and reference products (mean of twelve determination).

bioequivalence-bioavailability-Langmuir-nonlinear

Figure 4: Mean test/mean reference result of capacity constant, k2, calculated using (a) Langmuir nonlinear equation (equation 1) and (b) Langmuir linear equation (equation 2) for total bile salts at with acid pre-treatment equilibrium studies (mean of twelve determination).

The results indicate that the percent binding and the binding capacity of both test and reference products at several initial concentrations (Table 5) (ranging from 0.1-30 mM) are very similar and there was no significant difference in the binding performances of test and reference products in both with and without pre-treatment equilibrium studies (Figure 5).

Concentration (mM, Total bile acids) Without acid pre-treatment With acid pre-treatment
Test     Av. x/m Reference Av. x/m T/R Test Av. x/m Reference Av. x/m T/R
0.1 0.0969 0.0974 0.99 0.0974 0.0965 1.01
0.3 0.2791 0.2800 1.00 0.2787 0.2833 0.98
1 0.7920 0.8086 0.98 0.8497 0.8314 1.02
3 2.1713 2.1874 0.99 2.2028 2.1877 1.01
7 4.6125 4.8006 0.96 4.8168 4.8790 0.99
10 5.5473 5.8279 0.95 5.7754 5.9750 0.97
20 5.8537 6.2884 0.93 5.8643 6.0935 0.96
30 5.8882 6.3056 0.93 5.9513 6.2066 0.96

Table 5: Results of binding capacity of the equilibrium studies. Average micromoles total bile acid salts bound per 10 mg colesevelam hydrochloride (x/m) (mean of twelve determinations).

bioequivalence-bioavailability-binding-studies

Figure 5: Results of the percent binding versus concentration for total bile acid salts at with and without acid pre-treatment equilibrium binding studies (mean of twelve determinations).

Kinetic binding studies

Comparison of kinetic data of binding of 0.3 mM and 3.0 mM total bile acid salts concentration.

As indicated in the Figure 6 each product achieved equilibrium between 15-30 min for 0.3 mM and 3.0 mM kinetic studies (Tables 6 and 7).

bioequivalence-bioavailability-acid-salts

Figure 6: Results of the percent binding vs. time for total bile acid salts at 0.3 mM and 3.0 mM kinetic studies (mean of twelve determinations).

Test Reference T/R
Time (Hour) Av. x/m %CV Av. %Bound Av. x/m %CV Av. %Bound
0.05 0.2488 4.1831 83.4540 0.2499 3.9379 83.8365 1.00
0.10 0.2661 2.2006 89.2541 0.2623 1.5726 88.0054 1.01
0.15 0.2675 2.0025 89.7242 0.2704 1.1614 90.7026 0.99
0.20 0.2709 0.7342 90.8805 0.2738 1.2664 91.8330 0.99
0.25 0.2761 2.0225 92.6123 0.2791 1.2786 93.6126 0.99
0.50 0.2800 0.8312 93.9397 0.2822 1.4577 94.6763 0.99
1 0.2861 0.6182 95.9863 0.2834 0.5315 95.0535 1.01
2 0.2863 0.8390 96.0314 0.2859 1.0938 95.9071 1.00
4 0.2865 1.0939 96.0926 0.2873 0.9346 96.3837 1.00
8 0.2861 0.6812 95.9741 0.2870 1.2848 96.2779 1.00
16 0.2881 0.7881 96.6353 0.2859 0.8168 95.9028 1.01
24 0.2879 0.6789 96.5614 0.2843 0.5938 95.3714 1.01

Table 6: Results of the kinetics study of the binding of the total bile acid salts to Colesevelam hydrochloride in 0.3 mM aqueous bile salt solution in the presence of added 0.1 M NaCl. Average micromoles total bile acid salts bound per 10 mg colesevelam hydrochloride (x/m) and average % bound (mean of twelve determinations).

Test Reference T/R
Time (Hour) Av. x/m %CV Av. %Bound Av. x/m %CV Av. %Bound
0.05 1.9467 0.0217 65.1983 2.0017 0.0165 67.0610 0.97
0.10 2.1832 0.0054 73.1706 2.2602 0.0172 75.7207 0.97
0.15 2.2945 0.0096 76.7145 2.3680 0.0176 79.3315 0.97
0.20 2.3779 0.0029 79.6140 2.3836 0.0027 79.8556 1.00
0.25 2.3906 0.0082 80.1770 2.4090 0.0096 80.7057 0.99
0.50 2.4410 0.0035 81.8262 2.4096 0.0070 80.7239 1.01
1 2.4248 0.0125 81.0222 2.4302 0.0070 81.4162 1.00
2 2.4513 0.0025 82.1018 2.4408 0.0047 81.7721 1.00
4 2.4285 0.0069 81.2411 2.4211 0.0017 81.1124 1.00
8 2.4440 0.0073 81.8772 2.4338 0.0018 81.5368 1.00
16 2.4498 0.0048 82.0151 2.4260 0.0037 81.2737 1.01
24 2.4366 0.0048 81.6263 2.4340 0.0025 81.5424 1.00

Table 7: Results of the kinetics study of the binding of the total bile acid salts to Colesevelam hydrochloride in 3.0 mM aqueous bile salt solution in the presence of added 0.1 M NaCl. Average micromoles total bile acid salts bound per 10 mg colesevelam hydrochloride (x/m) and average % bound (mean of twelve determinations).

Discussion

Because the colesevelam hydrochloride is not absorbed into the systemic circulation, in vivo bioequivalence is not available. Thence, equivalence to the reference product was compared in vitro condition.

Mean test/mean reference (T/R) of twelve determination result of capacity constant, k2, was obtained 0.918 (91.24-92.34% CI) using equation 1 and 0.922 (91.88-92.47% CI) equation 2 for total bile salts at without acid pre-treatment equilibrium studies. Equation 1 and equation 2 results were very close to each other and both results calculated with equations 1 and equation 2 were obtained very similar between test and reference products in the 90% confidence interval and acceptance criteria of 80-120%.

These parameter indicated in the approval of ANDA to pivotal bioequivalence study [11]. The results obtained from without acid pre-treatment equilibrium binding studies meet the acceptance criteria [9-11].

Additionally T/R ratio of twelve determination result of capacity constant, k2, was obtained 0.948 (94.35-95.30% CI) using equation 1 and 0.953 (94.88-95.68%) equation 2 for total bile salts at with acid pre-treatment equilibrium binding studies. These results are also in the acceptance criteria.

Based on these data generic Colesevelam 625 mg Film Tablet (Zander) is comparable to Cholestagel 625 mg Film Tablet in the equilibrium study of bile acid salt binding in SIF with and without acid pre-treatment.

In the kinetic binding study, the binding at the initial both 0.3 mM and 3.0 mM concentration was extremely rapid for both test and reference product. The results for both concentrations were extremely reproducible.

Additionally, the kinetic binding of 0.3 mM and 3.0 mM bile salts for both test and reference product was nearly identical. T/R ratio was obtained between 0.99–1.01 for 0.3 mM concentration and 0.97–1.01 for 3.0 mM concentration. Therefore, based on these data test and reference products are comparable in terms of its in vitro binding characteristics.

Concentration of bile acid in the intestine varies according to fasting state [6]. For this reason, in the equilibrium studies, the binding characteristics of the test product to the reference product at 8 different concentrations in the concentration range of 0.1-30 mM were compared and it was found that the test product was comparable to the reference product at all concentrations. In addition, results of the kinetic studies shown that maximum binding to bile acids achieved in 15-30 min. When taken tablets with a meal, it takes much longer than 30 min to pass through the intestine [14]. Therefore, the tablets perform maximum binding pass through the intestine. The results of binding capacity shown in Table 5 indicate that test and reference products at 8 concentrations ranging from 0.1 to 30 mM are comparable. T/R ratio of twelve determination result of binding capacity was obtained ranging from 0.93-1.00 for without acid pre-treatment equilibrium study and 0.96-1.02 with acid pre-treatment equilibrium study.

An important parameter that affects the variation between repetitions is the waiting time for soaked with SIF each incubation flask at room temperature overnight. It should be repeated in the same way for less variation between repetitions.

Conclusion

All results obtained from equilibrium binding studies proved that generic Colesevelam 625 mg Film Tablet (Zander) bioequivalent to reference product Cholestagel 625 mg Film Tablet. In addition to the equilibrium binding results, the results of kinetic binding studies supported to bioequivalence.

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