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Bioavailability of carbamazepine:β-cyclodextrin complex in beagle dogs from hydroxypropylmethylcellulose matrix tablets
European Journal of Pharmaceutical Sciences 22 (2004) 201–207
Bioavailability of carbamazepine:-cyclodextrin complex in beagle dogs from hydroxypropylmethylcellulose matrix tablets Let´ıcia S. Koester a , Juliana B. Bertuol a , Kátia R. Groch b , Clarissa R. Xavier a , Roseli Moellerke b , Paulo Mayorga a , Teresa Dalla Costa a , Valquiria L. Bassani a,∗ a
Programa de Pós-Graduação em Ciˆencias Farmacˆeuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil b Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil Received 3 September 2003; received in revised form 19 February 2004; accepted 2 March 2004
Abstract The bioavailability of a carbamazepine:-cyclodextrin (CBZ:CD) complex from hydroxypropylmethylcellulose (HPMC) matrix tablets was evaluated in beagle dogs. A solubility study demonstrated the improvement of CBZ aqueous solubility by adding increasing amounts of CD. The 1:1 CBZ:CD molar ratio was chosen to produce the complex, which was obtained by spray-drying. Matrix tablets were prepared by direct compressing either a CBZ:CD complex or a physical mixture of both substances with HPMC. Both matrix formulations displayed a controlled-release profile when compared to the reference formulation (Tegretol CR 200® ). CBZ presented a significantly higher bioavailability from matrix tablets containing the CBZ:CD complex than that obtained from Tegretol CR 200® . Although a high inter-subject variability was observed, the results pointed to the feasibility of using CD in order to modulate CBZ release and absorption, as well as to reduce the drug dosage maintaining the same plasma levels. © 2004 Elsevier B.V. All rights reserved. Keywords: Carbamazepine; -Cyclodextrin; Hydroxypropylmethylcellulose; Matrix tablets; Spray-drying; Bioavailability
1. Introduction Carbamazepine (CBZ) is a widely used anticonvulsant drug, whose absorption is generally slow and irregular when immediate-release tablets are administered. Time to peak concentration after oral administration varies from 4 to 8 h or longer due to the very low water solubility (<200 g/ml) of this drug and its dissolution rate-limited absorption (Levy et al., 1992). The drug also presents a decrease in the half-life during chronic dosing due to metabolism autoinduction (USP DI, 1997). The improvement of the aqueous solubility of CBZ by using cyclodextrins (CDs) has been reported by Brewster et al. (1991, 1997), Choudhury and Nelson (1992), Betlach et al. (1993), Löscher et al. (1995), El-Nahhas (1996), El-Zein et al. (1998) and El-Gindy et al. (2002). Betlach ∗ Corresponding author. Tel.: +55-51-3316-5090; fax: +55-51-3316-5437. E-mail address: [email protected] (V.L. Bassani).
0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2004.03.010
et al. (1993) and Brewster et al. (1997) have also reported an improvement in CBZ bioavailability in dogs using hydroxypropyl--cyclodextrin (HPCD) as complexation agent. In both studies, an oral dosing of CBZ:HPCD in solution yielded improved values of areas under the plasma concentration–time curves (AUC) compared to that obtained for immediate-release tablets. Choudhury and Nelson (1992) also observed a greater AUC for a CBZ:HPCD lyophilized solid complex, as compared with that of the pure drug, when administered as suspensions to rats. No reports regarding the evaluation of the bioavailability of CBZ:CD complexes from tablets have been found, not even their well known advantages. Sustained release formulations of CBZ have been introduced into drug therapy with a twofold purpose: to reduce the number of single doses during the day, and to decrease the fluctuations of serum levels in view to obtain better therapeutic efficacy and diminished toxicity (Wolf et al., 1992). In order to extend CBZ release, hydroxypropylmethylcellulose (HPMC) has been successfully employed
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(Giunchedi et al., 1991; Ikinci et al., 1999; Qadan and Süss, 2000). In this context, the development of tablets combining the benefits of a controlled-release dosage form with an improvement in CBZ solubility reveals evident advantages. In our previous work (Koester et al., 2003), we demonstrated that the incorporation of CBZ previously complexed with CD in a HPMC matrix tablet influenced its in vitro dissolution profile. The complexation of CBZ with CD was indicated by differential scanning calorimetry and infrared spectroscopy. The present study was designed to evaluate the influence of CD on CBZ bioavailability from HPMC matrix tablets in beagle dogs and the need of previous CBZ complexation with CD rather than simple mixture. All tablet formulations were compared to a commercially available CBZ controlled-release tablet.
2. Materials and methods 2.1. Materials Carbamazepine (molar weight = 236.27 g/mol) was purchased from Henrifarma (São Paulo, Brazil), -cyclodextrin (molar weight = 1135 g/mol) was supplied by Roquette (France) and Blanver (São Paulo, Brazil) provided the HPMC (Methocel K100LV® , DOW Chemical Company). Methanol and acetonitrile were of HPLC quality and all other reagents were of analytical grade. 2.2. Phase solubility study A solubility study (Higuchi and Connors, 1965) was carried out by adding an excess amount of CBZ (16 mM) to 50 ml of aqueous media containing increasing amounts of CD (4, 8 and 16 mM). The resulting suspensions, which presented CBZ:CD molar ratios of 1:0.25, 1:0.5 and 1:1, were stirred for 24 h at 25 ◦ C. After this period the suspensions were filtered and the solutions were assayed by a spectrophotometric method at 286 nm. Each molar ratio was prepared in triplicate. 2.3. Preparation of CBZ:βCD solid complex and CBZ:βCD physical mixture A complex of CBZ and CD in the solid state was prepared by spray-drying 5 l of an aqueous solution containing the complex in a Niro-Production Minor® spray dryer. The liquid complex was prepared as described above for a 1:1 CBZ:CD molar ratio, which in fact produces a solution containing approximately 1.2 mg of CBZ complexed with CD. After spray-drying, CBZ content in the resulting powder was assayed by spectrophotometric method at 286 nm and was found to be 9.7%. In this way, the actual stoichiometry ratio of the complex, after all steps, was ca. 1:2 CBZ:CD. In parallel, a mixture containing the same ratio of CBZ and CD was prepared in a rotating-shell blender.
Table 1 Formula composition (%, w/w)a Composition
Matrix-C
Matrix-M
CBZ:CD complex CBZ + CD HPMC Magnesium stearate
74 – 25 1
– 74 25 1
a Matrix-C: matrix tablets containing a complex of CBZ with CD; Matrix-M: matrix tablets containing CBZ and CD simply mixed.
2.4. Preparation of matrix tablets Tablets were prepared by directly compressing a mixture containing either pre-complexed CBZ:CD powder or a simple mixture of CBZ and CD in a similar proportion. Table 1 presents the formula composition. Both tablet formulations contained 80 mg of CBZ, and the same hardness value (6.24 ± 0.16 Kp). A single punch Korsch EK-0 machine, equipped with flat 15.0 mm punches was employed. The quantitative composition of the reference formulation (Tegretol CR 200® ) is not disclosed, but the following excipients are listed: colloidal silicon dioxide, ethylcellulose, microcrystalline cellulose, co-polymers of acrylic and methacrylic esters, magnesium stearate, sodium croscarmellose, talc, hydroxypropylmethylcellulose, polyoxyethylene sorbitan monooleate, red iron oxide, yellow iron oxide and titanium dioxide. 2.5. In vitro dissolution testing Drug release determination from both matrix tablet formulations, as well as from Tegretol CR 200® , was performed in a Pharma Test dissolution tester coupled to a Hewlett-Packard 8452A spectrophotometer. The following conditions were employed: paddle speed of 75 rpm, 900 ml of water containing 1% of sodium lauryl sulfate as dissolution medium and temperature of 37 ± 0.5 ◦ C. Matrix tablets were placed in baskets (mesh diameter 1 mm) in order to avoid floating. Samples (three replicates) were automatically collected each 30 min and filtered through 0.45 m. CBZ was assayed by spectrophotometric method at 286 nm. Similarity factor (f2 ) and difference factor (f1 ), were used to compare the dissolution profiles (Shah et al., 1998). For curves to be considered similar, f1 values should be between 0 and 15, while f2 values should be in the range of 50–100. 2.6. Animal experiments The study was approved by the Ethics Committee of Porto Alegre Clinical Hospital (HCPA; Porto Alegre, Brazil). Six healthy 2–3-year-old female beagle dogs weighing 9.7 ± 0.7 kg were employed in a Latin square crossover design, where the columns correspond to periods and the lines, to the formulations. Each dog was fasted for 14–16 h prior to each study day, but water was allowed ad libitum. On each occasion, dogs received orally the following formulations
L.S. Koester et al. / European Journal of Pharmaceutical Sciences 22 (2004) 201–207
2.7. Analysis of CBZ concentration in the plasma CBZ was assayed using an HPLC method, which was validated following ICH guidelines (ICH, 1996). Plasma samples (200 l) were mixed with 400 l of acetonitrile containing internal standard (alprazolam, 4 g/ml) and vortexed for 30 s. The samples were centrifuged for 10 min at 10,000 rpm and 500 l of the supernatant were removed by aspiration into centrifuge tubes. These aliquots were evaporated under vacuum at 43 ◦ C in a SC 110A Speed Vac® Plus Thermo Savant centrifuge and reconstituted in 100 l of mobile phase. The samples were analyzed on a Shim-pack CLC-ODS (M) RP18 5 m, 250 mm × 4 mm column. The chromatographic equipment consisted of a Waters 600 pump and a Waters 2487 dual λ absorbance detector. The HPLC conditions were as follows: mobile phase constituted of phosphate buffer (0.05 M):acetonitrile:methanol, 56:28:16 (pH 4.0); flow rate of 1.0 ml/min; detection wavelength at 286 nm; injection volume of 50 l. Under these conditions, CBZ eluted at approximately 13 min, and alprazolam at 21 min. Standard curves were linear (r2 > 0.999) over the examined concentration ranges of CBZ: 0.125–8.0 g/ml. The coefficient of variation (CV) was <8.5 and <8.7% for the repeatability and for the intermediate precision, respectively. The limit of quantitation was 0.125 g/ml. CBZ recoveries were in the range of 97.7–117.6% for three different concentrations (0.150, 1.5 and 6.4 g/ml). 2.8. Pharmacokinetic and statistical analyses The maximum plasma concentration (Cmax ) and the time to reach peak concentration (Tmax ) were obtained directly from the concentration–time data of each dog. The area under the curve (AUC) was determined until infinity using the trapezoidal rule plus extrapolation for the terminal part of the curve. The elimination constant (Ke ) was estimated from the elimination segment of the curve, as the slope of the plot of
logarithm of concentration versus time, while the half-time (t1/2 ) was calculated as 0.693/Ke . The relative bioavailability (frel ) was calculated by [(AUCtest × Dref )/(AUCref × Dtest )] × 100, where D is the dose and “test” and “ref” correspond to the matrix tablets and commercial formulations, respectively. Dogs were excluded from the pharmacokinetic analysis when the individual AUC value was higher or lower than one standard deviation of the mean value (n = 6 dogs) in two out of three formulations tested. Based on this, two animals were excluded from the analysis. The significance of the differences observed for the mean pharmacokinetic parameters after tests and reference tablets when administered to dogs was evaluated using analysis of variance (ANOVA) at a significance level of P ≤ 0.05. Subsequent analysis was performed using Tukey’s test (P ≤ 0.05). A 95% confidence interval for the ratio of the mean test formulation AUC to that of the reference (ln-transformed data) was calculated using the statistical software Bioeqv 3.4. For each formulation, mean percent absorbed–time plots were obtained by deconvolution of the corresponding mean concentration versus time plots using the Wagner–Nelson method. These plots allowed the construction of percent unabsorbed–time plots, which were used for the evaluation of the absorption rate constants (Gibaldi and Perrier, 1982).
3. Results and discussion The CBZ:CD solubility curve is presented in Fig. 1. As can be observed, there is a linear relationship between the increase in CBZ solubility and the increase in CD concentration, which characterizes the complexation of the drug. A water solubility increase of up to seven-fold could be observed when a concentration of 16 mM of CD was employed. Therefore, this theoretical molar ratio (1:1 CBZ:CD) was chosen to prepare a CBZ:CD solid complex for Matrix-C formulation which, in fact, presents a 1:2 CBZ:CD molar ratio. The curve can be classified as the AL type, according to Higuchi and Connors (1965). Ks value was found to be 636 mol−1 . Some complexation conditions were slightly changed with respect to our previous work (Koester et al., 2003). Moreover, in that study we
CBZ concentration (µg/ml)
with 40 ml of water: a controlled-release commercial tablet (Tegretol CR 200® ), a matrix tablet containing CBZ:CD complex (Matrix-C), and a matrix tablet containing a mixture of CBZ and CD (Matrix-M). In the treatment with Tegretol CR 200® , doses of 200 mg were given. In the treatment with Matrix-C and Matrix-M, doses of 160 mg of carbamazepine (two 80 mg tablets) were administered. A washout period of at least 5 days was allowed between successive dosing. The dog’s legs were shaven and cannulated through the cephalic vein using a 22-gauge catheter. Blood samples (2 ml) were taken prior to drug administration and at 10, 20, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420 and 480 min after dosing into heparinized microcentrifuge tubes. For Tegretol CR 200® , further blood samples were taken at 45, 75, 105, 135, 165 and 210 min. Plasma was separated by centrifugation (3000 rpm, 15 min) and stored at −20 ◦ C until analyzed.
203
1200 900 600 300 0 0
4
8
12
16
BCD concentration (mM)
Fig. 1. Phase solubility diagram of CBZ:CD.
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CBZ released (%)
100 80 60 40 20 0 0
60
M atrix-C
120 180 240 300 360 420 480 540 600
Time (min) Matrix-M
Tegretol CR 200
Fig. 2. In vitro dissolution profile of carbamazepine released from Matrix-C, Matrix-M and Tegretol CR 200® .
used a laboratory scale Büchi 190 spray dryer in order to obtain the solid complex. In the present study, a production spray dryer was used. The scaling-up is possible and was investigated in a separate paper (Koester et al., 2004). Fig. 2 shows the release profiles of CBZ from both matrix tablet formulations as well as from the commercial formulation, Tegretol CR 200® . In the case of the HPMC matrices, tablets underwent gelling and very slow matrix erosion. One can clearly observe that both matrix tablets displayed dissolution profiles compatible with zero-order kinetic in the interval 0–300 min, which is suitable for controlled-release formulations. CBZ release from Tegretol CR 200® was faster compared to the matrices, and showed distinct kinetic behavior. The comparison of the dissolution profiles by applying f1 and f2 confirmed that the curves of Matrix-C and Matrix-M could be considered similar (f2 = 65.2; f1 = 9.3 when Matrix-C is set as reference). Nevertheless, CBZ release profiles from both formulations can be considered different from the profile observed for the reference formulation (f2 = 26.9 for Matrix-C versus Tegretol CR 200® ; f2 = 23.6 for Matrix-M versus Tegretol CR 200® ; f1 = 36.7 for Matrix-C versus Tegretol CR 200® ; f1 = 42.0 for Matrix-M versus Tegretol CR 200® ). In order to evaluate the influence the complexation (Matrix-C) or mixture (Matrix-M) with CD on CBZ
bioavailability, an in vivo study was conducted in beagle dogs. Tegretol CR 200® was used as reference. A high inter-subject variability was observed in the concentration–time curves obtained in this study. According to the literature, carbamazepine is known as a highly variable drug and, for this reason, many authors have suggested widening the bioequivalence limits from 80–125 to 70–143% (Hoffmann et al., 1997; Wangemann et al., 1998; Mayer et al., 1999). To allow the comparison of the different formulation profiles obtained in this study, a criterion of exclusion was adopted. Applying the criterion described in Section 2.8, two dogs were excluded from the study: one dog failed the criterion for both Matrix-M and Tegretol CR 200® , while the other failed for Matrix-M and Matrix-C. The mean plasma concentration–time curves after oral administration of the three formulations to beagle dogs are presented in Fig. 3. Different plasma profiles can be observed for each formulation. Although two dogs have been excluded, the coefficients of variation observed for the drug concentrations after the administration of Matrix-C and Matrix-M were higher than that for Tegretol CR 200® . This fact may be mainly attributed to the administration of two matrix tablets rather than one. Adhesion of the matrices to the gastrointestinal tract is possible once HPMC is known as a polymer that presents bioadhesive properties (Minghetti et al., 1998; Miyazaki et al., 2000). The mean CBZ pharmacokinetic parameters determined from the individual profiles after oral administration of the formulations are summarized in Table 2. Mean CBZ plasma peak levels of 1.5, 0.81 and 0.49 g/ml were observed for Matrix-C, Matrix-M and Tegretol CR 200® , respectively (P < 0.05). For Matrix-M, the plasma profile presented a more pronounced plateau. For the matrix tablets, CBZ concentrations could be quantified up to 300 min while from Tegretol CR 200® it was possible to determine only up to 135 min due to the analytical method sensitivity. CBZ concentrations decreased with an average t1/2 , for all formulations, of approximately 50 min (P > 0.05). The average t1/2 agrees with values reported after oral administration of solutions containing CBZ:HPCD (Betlach et al., 1993; Löscher et al., 1995). According to Löscher et al. (1995), the t1/2 of CBZ is shorter in dogs than in humans, which could explain the relatively low peak plasma levels obtained with oral administration in this specie.
Table 2 Pharmacokinetic (PK) parameters (mean ± S.D.) of CBZ after oral administration of the following formulations: matrix tablets containing a complex of CBZ with CD (Matrix-C), matrix tablets containing CBZ and CD simply mixed (Matrix-M), and commercial controlled-release tablets (Tegretol CR 200® ) to beagle dogs (n = 4) PK parameter
Matrix-C
Cmax (g/ml) Tmax (min) Ke (min−1 ) t1/2 (min) AUC0–␣ (g h/ml) frel (%)
1.5 128 0.017 42 228 594
± ± ± ± ±
Matrix-M 0.42 29 0.0007 1.9 80
0.81 105 0.012 61 160 417
± ± ± ± ±
0.44 39 0.003 16 111
Tegretol CR 200® 0.49 58 0.016 46 48 –
± ± ± ± ±
0.09 26 0.005 17 16
L.S. Koester et al. / European Journal of Pharmaceutical Sciences 22 (2004) 201–207
Fig. 3. Mean (±S.D.) plasma concentration–time profiles of CBZ after oral administration of: (A) Matrix-C (160 mg); (B) Matrix-M (160 mg); (C) Tegretol CR 200® (200 mg).
CBZ presented a significantly higher AUC for Matrix-C formulation than for Tegretol CR 200® (P < 0.05). The AUC for Tegretol CR 200® was smaller than that observed for an immediate-release Tegretol formulation by Brewster
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et al. (1997). Nevertheless, the results may not be directly compared once the formulations are different and the dose of CBZ administered, in that study, was higher (20 mg/kg, approximately 400 mg). The relative bioavailability of CBZ from Matrix-C was ca. six times higher than the bioavailability of the commercial reference. This six-fold increase in CBZ bioavailability from the matrix containing the complex seems to be related to the seven-fold increase in CBZ water solubility when CBZ:CD are complexed using a 1:1 molar ratio. The relative bioavailability of CBZ from Matrix-M was ca. four times higher than the bioavailability of Tegretol CR 200® using ANOVA. Nevertheless, due to the high variability of the data, the AUC of Matrix-M could not be considered significantly different from neither Matrix-C nor Tegretol CR 200® . It is generally accepted by most regulatory agencies a bioequivalence criterion based on the calculation of a 90 or 95% confidence interval for the ratio of the test product true population mean bioavailability to that of the reference. Two formulations are bioequivalent when this interval lies entirely between 80 and 125%, using ln-transformed data. The 95% confidence interval for the ratio of the Matrix-M mean AUC to that of the reference formulation was 80.6–982.7%, demonstrating the lack of bioequivalence of these tablets. According to this criterion, Matrix-C and Matrix-M cannot be considered bioequivalent either (IC95% = 29.9–122.5%, when Matrix-C is set as reference). Nor are bioequivalent Matrix-C and Tegretol CR 200® (IC95% = 176.2–1227.5%). As demonstrated by Betlach et al. (1993), the dissolution of CBZ from tablets is the rate-limiting step for the absorption of the drug in dogs. In the mentioned study, the bioavailability of a solution containing a CBZ:HPCD complex was compared with that of immediate-release commercial tablets (Tegretol CR 200® ) and the authors verified a 5.6-fold improvement in the bioavailability of the solution. In the present study, we demonstrated a similar increase in the bioavailability using a less soluble, but much less expensive cyclodextrin and, additionally, from a solid pharmaceutical dosage form. Based on the bioequivalence criterion described above, the extent of absorption of CBZ from Matrix-C and Matrix-M are different, suggesting that previous complexation may be compulsory for the modulation of CBZ release and dissolution from HPMC matrices. Moreover, the coefficient of variation observed for the drug plasma concentrations after the administration of Matrix-C was smaller than that for Matrix-M, which corroborates to the hypothesis that previous complexation may be a necessary step in order to smooth out CBZ dissolution after drug release. Finally, an evaluation of the absorption kinetic was performed. Fig. 4 presents a plot of percent CBZ not absorbed versus time after oral administration of the formulations, where a linear relationship can be observed (r 2 > 0.97) between percent unabsorbed and time on rectilinear coordinates, which suggests apparent zero-order absorption. This
L.S. Koester et al. / European Journal of Pharmaceutical Sciences 22 (2004) 201–207
Percent unabsorbed
206
120
Acknowledgements
100
We thank the Brazilian government (CAPES, CNPq and FAPERGS) for the financial support of this research. We also would like to thank Mr. Luis C. Batista and Mr. Daniel Mendes for assistance in this experiment.
80 60 40
References
20 0 0
30
60
90
120
Time (min) M atrix-C
M atrix-M
Tegretol CR 200
Fig. 4. Percent of CBZ unabsorbed vs. time after oral administration of Matrix-C, Matrix-M and Tegretol CR 200® .
kinetic is usually related to controlled-release profile of the drug from the dosage form. The apparent zero-order absorption rate constants (K0 ) were estimated from the slope of the curves and were found to be approximately 0.7% CBZ dose/min for both Matrix-C and Matrix-M, and 1.3% CBZ dose/min for Tegretol CR 200® . These results suggest that the absorption rate of the drug from the reference formulation is faster than that from the matrix tablets. Furthermore, previous complexation does not seem to influence the absorption rate of CBZ from the matrix tablets. Hence, complexation influences the extent of CBZ but not the rate of absorption.
4. Conclusions CBZ aqueous solubility was increased to up to seven times by its complexation with CD. In vitro dissolution revealed that HPMC matrix tablets presented a controlled-release profile when compared to the controlled-release reference formulation (Tegretol CR 200® ). Different plasma concentration profiles were observed for the three formulations after oral administration to beagle dogs. The relative bioavailabilities of CBZ from the matrix tablets containing the complex and the physical mixture of CBZ and CD were approximately six and four times higher, respectively, than the bioavailability of the reference formulation suggesting that the CBZ dose could be reduced in this dosage forms in order to obtain the same plasma levels. The high inter-subject variability raised difficulties on the interpretation of the results. Taken together, the results suggest that previous complexation may be important for the modulation of CBZ release and dissolution from matrix tablets. Further animal studies or even human studies could be undertaken with a larger number of subjects in order to confirm these results, which were found very positive in terms of obtaining a controlled-release formulation for CBZ that may present less adverse-effects.
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Bioavailability of carbamazepine:-cyclodextrin complex in beagle dogs from hydroxypropylmethylcellulose matrix tablets Let´ıcia S. Koester a , Juliana B. Bertuol a , Kátia R. Groch b , Clarissa R. Xavier a , Roseli Moellerke b , Paulo Mayorga a , Teresa Dalla Costa a , Valquiria L. Bassani a,∗ a
Programa de Pós-Graduação em Ciˆencias Farmacˆeuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil b Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil Received 3 September 2003; received in revised form 19 February 2004; accepted 2 March 2004
Abstract The bioavailability of a carbamazepine:-cyclodextrin (CBZ:CD) complex from hydroxypropylmethylcellulose (HPMC) matrix tablets was evaluated in beagle dogs. A solubility study demonstrated the improvement of CBZ aqueous solubility by adding increasing amounts of CD. The 1:1 CBZ:CD molar ratio was chosen to produce the complex, which was obtained by spray-drying. Matrix tablets were prepared by direct compressing either a CBZ:CD complex or a physical mixture of both substances with HPMC. Both matrix formulations displayed a controlled-release profile when compared to the reference formulation (Tegretol CR 200® ). CBZ presented a significantly higher bioavailability from matrix tablets containing the CBZ:CD complex than that obtained from Tegretol CR 200® . Although a high inter-subject variability was observed, the results pointed to the feasibility of using CD in order to modulate CBZ release and absorption, as well as to reduce the drug dosage maintaining the same plasma levels. © 2004 Elsevier B.V. All rights reserved. Keywords: Carbamazepine; -Cyclodextrin; Hydroxypropylmethylcellulose; Matrix tablets; Spray-drying; Bioavailability
1. Introduction Carbamazepine (CBZ) is a widely used anticonvulsant drug, whose absorption is generally slow and irregular when immediate-release tablets are administered. Time to peak concentration after oral administration varies from 4 to 8 h or longer due to the very low water solubility (<200 g/ml) of this drug and its dissolution rate-limited absorption (Levy et al., 1992). The drug also presents a decrease in the half-life during chronic dosing due to metabolism autoinduction (USP DI, 1997). The improvement of the aqueous solubility of CBZ by using cyclodextrins (CDs) has been reported by Brewster et al. (1991, 1997), Choudhury and Nelson (1992), Betlach et al. (1993), Löscher et al. (1995), El-Nahhas (1996), El-Zein et al. (1998) and El-Gindy et al. (2002). Betlach ∗ Corresponding author. Tel.: +55-51-3316-5090; fax: +55-51-3316-5437. E-mail address: [email protected] (V.L. Bassani).
0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2004.03.010
et al. (1993) and Brewster et al. (1997) have also reported an improvement in CBZ bioavailability in dogs using hydroxypropyl--cyclodextrin (HPCD) as complexation agent. In both studies, an oral dosing of CBZ:HPCD in solution yielded improved values of areas under the plasma concentration–time curves (AUC) compared to that obtained for immediate-release tablets. Choudhury and Nelson (1992) also observed a greater AUC for a CBZ:HPCD lyophilized solid complex, as compared with that of the pure drug, when administered as suspensions to rats. No reports regarding the evaluation of the bioavailability of CBZ:CD complexes from tablets have been found, not even their well known advantages. Sustained release formulations of CBZ have been introduced into drug therapy with a twofold purpose: to reduce the number of single doses during the day, and to decrease the fluctuations of serum levels in view to obtain better therapeutic efficacy and diminished toxicity (Wolf et al., 1992). In order to extend CBZ release, hydroxypropylmethylcellulose (HPMC) has been successfully employed
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(Giunchedi et al., 1991; Ikinci et al., 1999; Qadan and Süss, 2000). In this context, the development of tablets combining the benefits of a controlled-release dosage form with an improvement in CBZ solubility reveals evident advantages. In our previous work (Koester et al., 2003), we demonstrated that the incorporation of CBZ previously complexed with CD in a HPMC matrix tablet influenced its in vitro dissolution profile. The complexation of CBZ with CD was indicated by differential scanning calorimetry and infrared spectroscopy. The present study was designed to evaluate the influence of CD on CBZ bioavailability from HPMC matrix tablets in beagle dogs and the need of previous CBZ complexation with CD rather than simple mixture. All tablet formulations were compared to a commercially available CBZ controlled-release tablet.
2. Materials and methods 2.1. Materials Carbamazepine (molar weight = 236.27 g/mol) was purchased from Henrifarma (São Paulo, Brazil), -cyclodextrin (molar weight = 1135 g/mol) was supplied by Roquette (France) and Blanver (São Paulo, Brazil) provided the HPMC (Methocel K100LV® , DOW Chemical Company). Methanol and acetonitrile were of HPLC quality and all other reagents were of analytical grade. 2.2. Phase solubility study A solubility study (Higuchi and Connors, 1965) was carried out by adding an excess amount of CBZ (16 mM) to 50 ml of aqueous media containing increasing amounts of CD (4, 8 and 16 mM). The resulting suspensions, which presented CBZ:CD molar ratios of 1:0.25, 1:0.5 and 1:1, were stirred for 24 h at 25 ◦ C. After this period the suspensions were filtered and the solutions were assayed by a spectrophotometric method at 286 nm. Each molar ratio was prepared in triplicate. 2.3. Preparation of CBZ:βCD solid complex and CBZ:βCD physical mixture A complex of CBZ and CD in the solid state was prepared by spray-drying 5 l of an aqueous solution containing the complex in a Niro-Production Minor® spray dryer. The liquid complex was prepared as described above for a 1:1 CBZ:CD molar ratio, which in fact produces a solution containing approximately 1.2 mg of CBZ complexed with CD. After spray-drying, CBZ content in the resulting powder was assayed by spectrophotometric method at 286 nm and was found to be 9.7%. In this way, the actual stoichiometry ratio of the complex, after all steps, was ca. 1:2 CBZ:CD. In parallel, a mixture containing the same ratio of CBZ and CD was prepared in a rotating-shell blender.
Table 1 Formula composition (%, w/w)a Composition
Matrix-C
Matrix-M
CBZ:CD complex CBZ + CD HPMC Magnesium stearate
74 – 25 1
– 74 25 1
a Matrix-C: matrix tablets containing a complex of CBZ with CD; Matrix-M: matrix tablets containing CBZ and CD simply mixed.
2.4. Preparation of matrix tablets Tablets were prepared by directly compressing a mixture containing either pre-complexed CBZ:CD powder or a simple mixture of CBZ and CD in a similar proportion. Table 1 presents the formula composition. Both tablet formulations contained 80 mg of CBZ, and the same hardness value (6.24 ± 0.16 Kp). A single punch Korsch EK-0 machine, equipped with flat 15.0 mm punches was employed. The quantitative composition of the reference formulation (Tegretol CR 200® ) is not disclosed, but the following excipients are listed: colloidal silicon dioxide, ethylcellulose, microcrystalline cellulose, co-polymers of acrylic and methacrylic esters, magnesium stearate, sodium croscarmellose, talc, hydroxypropylmethylcellulose, polyoxyethylene sorbitan monooleate, red iron oxide, yellow iron oxide and titanium dioxide. 2.5. In vitro dissolution testing Drug release determination from both matrix tablet formulations, as well as from Tegretol CR 200® , was performed in a Pharma Test dissolution tester coupled to a Hewlett-Packard 8452A spectrophotometer. The following conditions were employed: paddle speed of 75 rpm, 900 ml of water containing 1% of sodium lauryl sulfate as dissolution medium and temperature of 37 ± 0.5 ◦ C. Matrix tablets were placed in baskets (mesh diameter 1 mm) in order to avoid floating. Samples (three replicates) were automatically collected each 30 min and filtered through 0.45 m. CBZ was assayed by spectrophotometric method at 286 nm. Similarity factor (f2 ) and difference factor (f1 ), were used to compare the dissolution profiles (Shah et al., 1998). For curves to be considered similar, f1 values should be between 0 and 15, while f2 values should be in the range of 50–100. 2.6. Animal experiments The study was approved by the Ethics Committee of Porto Alegre Clinical Hospital (HCPA; Porto Alegre, Brazil). Six healthy 2–3-year-old female beagle dogs weighing 9.7 ± 0.7 kg were employed in a Latin square crossover design, where the columns correspond to periods and the lines, to the formulations. Each dog was fasted for 14–16 h prior to each study day, but water was allowed ad libitum. On each occasion, dogs received orally the following formulations
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2.7. Analysis of CBZ concentration in the plasma CBZ was assayed using an HPLC method, which was validated following ICH guidelines (ICH, 1996). Plasma samples (200 l) were mixed with 400 l of acetonitrile containing internal standard (alprazolam, 4 g/ml) and vortexed for 30 s. The samples were centrifuged for 10 min at 10,000 rpm and 500 l of the supernatant were removed by aspiration into centrifuge tubes. These aliquots were evaporated under vacuum at 43 ◦ C in a SC 110A Speed Vac® Plus Thermo Savant centrifuge and reconstituted in 100 l of mobile phase. The samples were analyzed on a Shim-pack CLC-ODS (M) RP18 5 m, 250 mm × 4 mm column. The chromatographic equipment consisted of a Waters 600 pump and a Waters 2487 dual λ absorbance detector. The HPLC conditions were as follows: mobile phase constituted of phosphate buffer (0.05 M):acetonitrile:methanol, 56:28:16 (pH 4.0); flow rate of 1.0 ml/min; detection wavelength at 286 nm; injection volume of 50 l. Under these conditions, CBZ eluted at approximately 13 min, and alprazolam at 21 min. Standard curves were linear (r2 > 0.999) over the examined concentration ranges of CBZ: 0.125–8.0 g/ml. The coefficient of variation (CV) was <8.5 and <8.7% for the repeatability and for the intermediate precision, respectively. The limit of quantitation was 0.125 g/ml. CBZ recoveries were in the range of 97.7–117.6% for three different concentrations (0.150, 1.5 and 6.4 g/ml). 2.8. Pharmacokinetic and statistical analyses The maximum plasma concentration (Cmax ) and the time to reach peak concentration (Tmax ) were obtained directly from the concentration–time data of each dog. The area under the curve (AUC) was determined until infinity using the trapezoidal rule plus extrapolation for the terminal part of the curve. The elimination constant (Ke ) was estimated from the elimination segment of the curve, as the slope of the plot of
logarithm of concentration versus time, while the half-time (t1/2 ) was calculated as 0.693/Ke . The relative bioavailability (frel ) was calculated by [(AUCtest × Dref )/(AUCref × Dtest )] × 100, where D is the dose and “test” and “ref” correspond to the matrix tablets and commercial formulations, respectively. Dogs were excluded from the pharmacokinetic analysis when the individual AUC value was higher or lower than one standard deviation of the mean value (n = 6 dogs) in two out of three formulations tested. Based on this, two animals were excluded from the analysis. The significance of the differences observed for the mean pharmacokinetic parameters after tests and reference tablets when administered to dogs was evaluated using analysis of variance (ANOVA) at a significance level of P ≤ 0.05. Subsequent analysis was performed using Tukey’s test (P ≤ 0.05). A 95% confidence interval for the ratio of the mean test formulation AUC to that of the reference (ln-transformed data) was calculated using the statistical software Bioeqv 3.4. For each formulation, mean percent absorbed–time plots were obtained by deconvolution of the corresponding mean concentration versus time plots using the Wagner–Nelson method. These plots allowed the construction of percent unabsorbed–time plots, which were used for the evaluation of the absorption rate constants (Gibaldi and Perrier, 1982).
3. Results and discussion The CBZ:CD solubility curve is presented in Fig. 1. As can be observed, there is a linear relationship between the increase in CBZ solubility and the increase in CD concentration, which characterizes the complexation of the drug. A water solubility increase of up to seven-fold could be observed when a concentration of 16 mM of CD was employed. Therefore, this theoretical molar ratio (1:1 CBZ:CD) was chosen to prepare a CBZ:CD solid complex for Matrix-C formulation which, in fact, presents a 1:2 CBZ:CD molar ratio. The curve can be classified as the AL type, according to Higuchi and Connors (1965). Ks value was found to be 636 mol−1 . Some complexation conditions were slightly changed with respect to our previous work (Koester et al., 2003). Moreover, in that study we
CBZ concentration (µg/ml)
with 40 ml of water: a controlled-release commercial tablet (Tegretol CR 200® ), a matrix tablet containing CBZ:CD complex (Matrix-C), and a matrix tablet containing a mixture of CBZ and CD (Matrix-M). In the treatment with Tegretol CR 200® , doses of 200 mg were given. In the treatment with Matrix-C and Matrix-M, doses of 160 mg of carbamazepine (two 80 mg tablets) were administered. A washout period of at least 5 days was allowed between successive dosing. The dog’s legs were shaven and cannulated through the cephalic vein using a 22-gauge catheter. Blood samples (2 ml) were taken prior to drug administration and at 10, 20, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420 and 480 min after dosing into heparinized microcentrifuge tubes. For Tegretol CR 200® , further blood samples were taken at 45, 75, 105, 135, 165 and 210 min. Plasma was separated by centrifugation (3000 rpm, 15 min) and stored at −20 ◦ C until analyzed.
203
1200 900 600 300 0 0
4
8
12
16
BCD concentration (mM)
Fig. 1. Phase solubility diagram of CBZ:CD.
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CBZ released (%)
100 80 60 40 20 0 0
60
M atrix-C
120 180 240 300 360 420 480 540 600
Time (min) Matrix-M
Tegretol CR 200
Fig. 2. In vitro dissolution profile of carbamazepine released from Matrix-C, Matrix-M and Tegretol CR 200® .
used a laboratory scale Büchi 190 spray dryer in order to obtain the solid complex. In the present study, a production spray dryer was used. The scaling-up is possible and was investigated in a separate paper (Koester et al., 2004). Fig. 2 shows the release profiles of CBZ from both matrix tablet formulations as well as from the commercial formulation, Tegretol CR 200® . In the case of the HPMC matrices, tablets underwent gelling and very slow matrix erosion. One can clearly observe that both matrix tablets displayed dissolution profiles compatible with zero-order kinetic in the interval 0–300 min, which is suitable for controlled-release formulations. CBZ release from Tegretol CR 200® was faster compared to the matrices, and showed distinct kinetic behavior. The comparison of the dissolution profiles by applying f1 and f2 confirmed that the curves of Matrix-C and Matrix-M could be considered similar (f2 = 65.2; f1 = 9.3 when Matrix-C is set as reference). Nevertheless, CBZ release profiles from both formulations can be considered different from the profile observed for the reference formulation (f2 = 26.9 for Matrix-C versus Tegretol CR 200® ; f2 = 23.6 for Matrix-M versus Tegretol CR 200® ; f1 = 36.7 for Matrix-C versus Tegretol CR 200® ; f1 = 42.0 for Matrix-M versus Tegretol CR 200® ). In order to evaluate the influence the complexation (Matrix-C) or mixture (Matrix-M) with CD on CBZ
bioavailability, an in vivo study was conducted in beagle dogs. Tegretol CR 200® was used as reference. A high inter-subject variability was observed in the concentration–time curves obtained in this study. According to the literature, carbamazepine is known as a highly variable drug and, for this reason, many authors have suggested widening the bioequivalence limits from 80–125 to 70–143% (Hoffmann et al., 1997; Wangemann et al., 1998; Mayer et al., 1999). To allow the comparison of the different formulation profiles obtained in this study, a criterion of exclusion was adopted. Applying the criterion described in Section 2.8, two dogs were excluded from the study: one dog failed the criterion for both Matrix-M and Tegretol CR 200® , while the other failed for Matrix-M and Matrix-C. The mean plasma concentration–time curves after oral administration of the three formulations to beagle dogs are presented in Fig. 3. Different plasma profiles can be observed for each formulation. Although two dogs have been excluded, the coefficients of variation observed for the drug concentrations after the administration of Matrix-C and Matrix-M were higher than that for Tegretol CR 200® . This fact may be mainly attributed to the administration of two matrix tablets rather than one. Adhesion of the matrices to the gastrointestinal tract is possible once HPMC is known as a polymer that presents bioadhesive properties (Minghetti et al., 1998; Miyazaki et al., 2000). The mean CBZ pharmacokinetic parameters determined from the individual profiles after oral administration of the formulations are summarized in Table 2. Mean CBZ plasma peak levels of 1.5, 0.81 and 0.49 g/ml were observed for Matrix-C, Matrix-M and Tegretol CR 200® , respectively (P < 0.05). For Matrix-M, the plasma profile presented a more pronounced plateau. For the matrix tablets, CBZ concentrations could be quantified up to 300 min while from Tegretol CR 200® it was possible to determine only up to 135 min due to the analytical method sensitivity. CBZ concentrations decreased with an average t1/2 , for all formulations, of approximately 50 min (P > 0.05). The average t1/2 agrees with values reported after oral administration of solutions containing CBZ:HPCD (Betlach et al., 1993; Löscher et al., 1995). According to Löscher et al. (1995), the t1/2 of CBZ is shorter in dogs than in humans, which could explain the relatively low peak plasma levels obtained with oral administration in this specie.
Table 2 Pharmacokinetic (PK) parameters (mean ± S.D.) of CBZ after oral administration of the following formulations: matrix tablets containing a complex of CBZ with CD (Matrix-C), matrix tablets containing CBZ and CD simply mixed (Matrix-M), and commercial controlled-release tablets (Tegretol CR 200® ) to beagle dogs (n = 4) PK parameter
Matrix-C
Cmax (g/ml) Tmax (min) Ke (min−1 ) t1/2 (min) AUC0–␣ (g h/ml) frel (%)
1.5 128 0.017 42 228 594
± ± ± ± ±
Matrix-M 0.42 29 0.0007 1.9 80
0.81 105 0.012 61 160 417
± ± ± ± ±
0.44 39 0.003 16 111
Tegretol CR 200® 0.49 58 0.016 46 48 –
± ± ± ± ±
0.09 26 0.005 17 16
L.S. Koester et al. / European Journal of Pharmaceutical Sciences 22 (2004) 201–207
Fig. 3. Mean (±S.D.) plasma concentration–time profiles of CBZ after oral administration of: (A) Matrix-C (160 mg); (B) Matrix-M (160 mg); (C) Tegretol CR 200® (200 mg).
CBZ presented a significantly higher AUC for Matrix-C formulation than for Tegretol CR 200® (P < 0.05). The AUC for Tegretol CR 200® was smaller than that observed for an immediate-release Tegretol formulation by Brewster
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et al. (1997). Nevertheless, the results may not be directly compared once the formulations are different and the dose of CBZ administered, in that study, was higher (20 mg/kg, approximately 400 mg). The relative bioavailability of CBZ from Matrix-C was ca. six times higher than the bioavailability of the commercial reference. This six-fold increase in CBZ bioavailability from the matrix containing the complex seems to be related to the seven-fold increase in CBZ water solubility when CBZ:CD are complexed using a 1:1 molar ratio. The relative bioavailability of CBZ from Matrix-M was ca. four times higher than the bioavailability of Tegretol CR 200® using ANOVA. Nevertheless, due to the high variability of the data, the AUC of Matrix-M could not be considered significantly different from neither Matrix-C nor Tegretol CR 200® . It is generally accepted by most regulatory agencies a bioequivalence criterion based on the calculation of a 90 or 95% confidence interval for the ratio of the test product true population mean bioavailability to that of the reference. Two formulations are bioequivalent when this interval lies entirely between 80 and 125%, using ln-transformed data. The 95% confidence interval for the ratio of the Matrix-M mean AUC to that of the reference formulation was 80.6–982.7%, demonstrating the lack of bioequivalence of these tablets. According to this criterion, Matrix-C and Matrix-M cannot be considered bioequivalent either (IC95% = 29.9–122.5%, when Matrix-C is set as reference). Nor are bioequivalent Matrix-C and Tegretol CR 200® (IC95% = 176.2–1227.5%). As demonstrated by Betlach et al. (1993), the dissolution of CBZ from tablets is the rate-limiting step for the absorption of the drug in dogs. In the mentioned study, the bioavailability of a solution containing a CBZ:HPCD complex was compared with that of immediate-release commercial tablets (Tegretol CR 200® ) and the authors verified a 5.6-fold improvement in the bioavailability of the solution. In the present study, we demonstrated a similar increase in the bioavailability using a less soluble, but much less expensive cyclodextrin and, additionally, from a solid pharmaceutical dosage form. Based on the bioequivalence criterion described above, the extent of absorption of CBZ from Matrix-C and Matrix-M are different, suggesting that previous complexation may be compulsory for the modulation of CBZ release and dissolution from HPMC matrices. Moreover, the coefficient of variation observed for the drug plasma concentrations after the administration of Matrix-C was smaller than that for Matrix-M, which corroborates to the hypothesis that previous complexation may be a necessary step in order to smooth out CBZ dissolution after drug release. Finally, an evaluation of the absorption kinetic was performed. Fig. 4 presents a plot of percent CBZ not absorbed versus time after oral administration of the formulations, where a linear relationship can be observed (r 2 > 0.97) between percent unabsorbed and time on rectilinear coordinates, which suggests apparent zero-order absorption. This
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Percent unabsorbed
206
120
Acknowledgements
100
We thank the Brazilian government (CAPES, CNPq and FAPERGS) for the financial support of this research. We also would like to thank Mr. Luis C. Batista and Mr. Daniel Mendes for assistance in this experiment.
80 60 40
References
20 0 0
30
60
90
120
Time (min) M atrix-C
M atrix-M
Tegretol CR 200
Fig. 4. Percent of CBZ unabsorbed vs. time after oral administration of Matrix-C, Matrix-M and Tegretol CR 200® .
kinetic is usually related to controlled-release profile of the drug from the dosage form. The apparent zero-order absorption rate constants (K0 ) were estimated from the slope of the curves and were found to be approximately 0.7% CBZ dose/min for both Matrix-C and Matrix-M, and 1.3% CBZ dose/min for Tegretol CR 200® . These results suggest that the absorption rate of the drug from the reference formulation is faster than that from the matrix tablets. Furthermore, previous complexation does not seem to influence the absorption rate of CBZ from the matrix tablets. Hence, complexation influences the extent of CBZ but not the rate of absorption.
4. Conclusions CBZ aqueous solubility was increased to up to seven times by its complexation with CD. In vitro dissolution revealed that HPMC matrix tablets presented a controlled-release profile when compared to the controlled-release reference formulation (Tegretol CR 200® ). Different plasma concentration profiles were observed for the three formulations after oral administration to beagle dogs. The relative bioavailabilities of CBZ from the matrix tablets containing the complex and the physical mixture of CBZ and CD were approximately six and four times higher, respectively, than the bioavailability of the reference formulation suggesting that the CBZ dose could be reduced in this dosage forms in order to obtain the same plasma levels. The high inter-subject variability raised difficulties on the interpretation of the results. Taken together, the results suggest that previous complexation may be important for the modulation of CBZ release and dissolution from matrix tablets. Further animal studies or even human studies could be undertaken with a larger number of subjects in order to confirm these results, which were found very positive in terms of obtaining a controlled-release formulation for CBZ that may present less adverse-effects.
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