Original Paper Pharmacology 2014;94:170–178 DOI: 10.1159/000368084
Received: August 28, 2014 Accepted: September 2, 2014 Published online: October 22, 2014
Pharmacokinetic Evaluation of a Novel Benzopyridooxathiazepine Derivative as a Potential Anticancer Agent Florence Bourdon Marie Lecoeur Nicolas Lebègue Bernard Gressier Michel Luyckx Pascal Odou Thierry Dine Jean-François Goossens Nicolas Kambia UDSL, EA 4481, UFR Pharmacie, Université Lille Nord de France, Lille, France
Abstract Background/Aims: The in vivo metabolic profile of a benzopyridooxathiazepine (BPT) derivative, a potent tubulin polymerization inhibitor with a promising in vitro activity, was investigated. Methods: The quantification of the BPT derivative and the identification of metabolites in the plasma of Wistar rats after i.p. and oral administration of 10 mg/kg were performed by the HPLC-mass spectrometry method. Results: Following a single i.p. dose of the BPT derivative, the plasma concentrations showed a biexponential decay (with a rapid decline) followed by a slow decay with a terminal half-life of 77.90 min. The area under the concentration-time curve from time 0 to infinity (AUC0–∞) was 18.90 μg/ml · min. After oral administration, the plasmatic concentrations reached a peak of 0.06 μg/ml at 35 min and then decayed with a half-life of 108 min. The AUC0–∞ was 10.25 μg/ml · min, representing 54.2% of the relative bioavailability. The compound was well distributed in the body, and its elimination seemed to be fast, regardless of the administration route. The major metabolic pathways were demethylation and hy
© 2014 S. Karger AG, Basel 0031–7012/14/0944–0170$39.50/0 E-Mail [email protected] www.karger.com/pha
droxylation reactions, both followed by conjugation with glucuronic acid. Conclusion: In rats, the BPT derivative is well distributed and undergoes extensive metabolism, leading to several metabolites. With promising in vitro activity and very good oral bioavailability, this compound seems to be an attractive candidate for further development as an anticancer agent. © 2014 S. Karger AG, Basel
Introduction
Mitosis is a highly regulated process during cell division. Drugs that disrupt the mitotic progression process are extensively used to treat a wide variety of cancers. There are two major groups of these antimitotic agents: microtubule stabilizers such as paclitaxel and microtubule destabilizers such as vinca alkaloids, chalcones and chalcone derivatives, colchicine, combretastatin A-4, and sulfonamide E7010 [1–6]. However, these agents which target microtubules have high toxicity, and their potency is limited by the development of multidrug resistance [7, 8]. Therefore, there has been great interest in developing novel microtubule inhibitors that overcome various modes of resistance and exhibit improved pharmacologiNicolas Kambia Department of Pharmacology, Pharmacokinetics and Clinical Pharmacy Université Lille Nord de France, 3 rue du Professeur Laguesse FR–59006 Lille (France) E-Mail nicolas.kambia @ univ-lille2.fr
Downloaded by: Selçuk Universitesi 193.255.248.150 - 1/22/2015 12:36:49 PM
Key Words Benzopyridooxathiazepine derivative · Tubulin polymerization inhibitor · Pharmacokinetics · Rat · HPLC-MS
Pharmacokinetic Evaluation of a Novel BPT Derivative
O
O S O
O
O S
N
O
N N
O
N
O
a
b
Fig. 1. Chemical structures of the BPT derivative (a) and the IS (b).
to evaluate the pharmacokinetic profile of the compound. Then, high-resolution mass spectrometry (MS) was used to characterize the metabolites obtained and to propose the major metabolic pathway of the parent drug.
Materials and Methods Chemicals and Drug 1-(4-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (>98% purity) and 1-(4methoxyphenylethyl)-9-methyl-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (>98% purity), the internal standard (IS) (fig. 1), were synthesized according to the procedure described by Gallet et al. [12]. Dimethyl sulfoxide (DMSO) and sodium bicarbonate (NaHCO3) were purchased from Sigma Aldrich (Steinheim, Germany); formic acid (HCOOH) and ammonium hydroxide (NH4OH; 28 wt%) were obtained from Prolabo (Val de Fontenay, France). Methanol and acetonitrile (HPLC grade) were purchased from VWR (Val de Fontenay, France). Gum arabic was purchased from Cooper (Melun, France). NaCl 0.9% was obtained from Macopharma Laboratories (Mouvaux, France). Xylazine and ketamine were purchased from Merial Laboratory (Lyon, France). Ultrapure 18-MΩ water was supplied from a MilliQ system (Millipore, Saint-Quentin-en-Yvelines, France). Animals, Drug Administration, and Plasma Sample Collection Adult female Wistar rats, weighing 140–160 g, were obtained from Janvier Laboratories (Saint-Berthevin, France). They were specific pathogen free and were housed under controlled environmental conditions: temperature 20 ± 2 ° C, relative humidity 50 ± 5%, and 12-hour light-dark cycles in accordance with our institutional animal care guidelines. Throughout the study period, pelleted rat food and tap water were available ad libitum. Upon arrival, the animals were allowed to acclimatize for 7 days before being randomly assigned to the different groups. The animals were fasted overnight and water was allowed ad libitum. They were randomly divided into two groups (i.p. and oral
Pharmacology 2014;94:170–178 DOI: 10.1159/000368084
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cal profiles. In recent years, much effort has been devoted to the identification of new ligands binding to the colchicine site of tubulin, derived not only from natural sources but also by screening of compound libraries in combination with traditional medicinal chemistry [9, 10]. Quinoline and benzopyridooxathiazepine (BPT) derivatives are a pharmacologically active class of heterocyclic compounds that have been reported to inhibit tubulin polymerization, leading to cell cycle arrest and apoptosis [8, 11, 12]. Among the series of synthesized products, 2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,3dihydro-1H-indol-7-yl]acetamide was a novel class of quinoline derivative identified as a potent microtubule inhibitor acting through the colchicine-binding site of tubulin [8]. 1-(4-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide is a BPT derivative. Its pharmacological activity in vitro over a panel of six tumor cell lines (L1210, DU145, HT29, A549, KB-3-1, and KB-A-1) is in the nanomolar range (10 < IC50 < 13 nmol/l) [12]. This interesting profile, resulting from the inhibition of tubulin polymerization, encouraged us to perform in vivo biotransformation studies. Because Caco-2 cells derived from human colonic adenocarcinomas accurately predict the intestinal absorption of drugs, a preliminary study, conducted to assess the intestinal absorption of this compound, showed almost total permeability across these cells [12, 13]. Similarly, the chemical stability investigated under various stress conditions showed that the molecule was stable [14]. These results were encouraging for the development of a BPT derivative as a drug candidate. Likewise, other compounds derived from the oxathiazepine ring, whose chemical stability has been improved, were studied for their antiproliferative activity as well as their inhibition of tubulin polymerization [15]. Despite significant pharmacological activity, in vivo pharmacokinetic studies of this compound have not been reported. Because freshly isolated hepatocytes can be useful for predicting the in vivo clearance of various drugs [16, 17], the first approach with rat liver microsomes and hepatocytes was investigated as an in vitro drug-metabolizing system to predict both the route and rate of metabolism of the studied compound [18]. The aim of the present study was to examine the in vivo pharmacokinetics and bioavailability of the BPT derivative in Wistar rats by focusing on the most relevant pharmacokinetic parameters after single i.p. and oral administration. The first step consisted of the development and validation of a sensitive and selective HPLC-UV method
Toxicity Test and Dose Selection In order to administer a nontoxic dose of the BPT derivative to the animals during the pharmacokinetic study, test dosing was performed. Thus, the rats were randomly divided into two groups: a control and a treatment group. Both were deprived of food for 18 h before the experiments but were allowed access to water. In the oral administration group, the control group (n = 3) received 0.5 ml/100 g body weight of the mixture DMSO/Tween 80/gum arabic/NaCl 0.9% (3:4:18:75%) by gavage, whereas the treatment group (n = 3) received the BPT derivative dissolved in the abovementioned vehicles at a dose of 50 mg/kg. In the i.p. administration group, the control and treatment groups received the same doses as listed above, without gum arabic, at a ratio of 3:4:93%. Neither food nor water was given up to 2 h after administration. All animals were observed individually for 7 days to record any signs of ill-health or behavioral changes such as loss of body weight and a reduction in the quantity of food and tap water consumed. Intraperitoneal Administration After the toxicity test, a suitable dose was chosen (10 mg/kg) to perform the pharmacokinetic studies. Twenty-eight Wistar rats were randomly assigned to four treatment groups and received a single i.p. administration of 10 mg/kg body weight of the BPT derivative (dissolved in DMSO/Tween 80/NaCl 0.9% at a ratio of 3: 4:93%). Blood samples (1 sample per rat) were collected before drug administration and at 8, 10, 15, 30, 60, 120, and 180 min after drug administration in heparinized tubes after animal sacrifice by section of the carotid artery under deep xylazine-ketamine (1:0.5) anesthesia. Plasma was immediately isolated from the blood samples by centrifugation (3,000 g for 10 min) and then stored at –20 ° C until additional extraction and analysis.
Oral Administration As described above, a fixed dose of 10 mg/kg body weight of the BPT derivative was dissolved in a mixture of DMSO/Tween 80/ gum arabic/NaCl 0.9% (3:4:18:75%) and was administered by oral gavage to four groups of 9 rats. Blood samples were collected before drug administration and at 10, 15, 30, 45, 60, 90, 120, 150, and 180 min after drug administration (4 animals per time point), using the same protocol as presented above. Moreover, 5 drug-free rats were anesthetized as previously described, and blood samples were collected; the plasma obtained was pooled and subsequently used for preparing blank and standard curves. Instrumentation and Analytic Conditions HPLC-UV/MS Conditions The chromatographic equipment used to conduct the analyses of the BPT derivative and the IS was the same as that previously used [18]. Separations were carried out on a reversed-phase Symmetry C18 column (150 × 4.6 mm i.d., 5 μm) from Waters preceded by a LiChrospher C18 guard column (4 × 4 mm i.d., 5 μm) from Merck (Fontenay-sous-Bois, France), both kept at 25 ° C. The analytes were eluted under isocratic conditions using a mobile phase consisting of acetonitrile/water/formic acid (60: 40: 0.1,
172
Pharmacology 2014;94:170–178 DOI: 10.1159/000368084
v/v/v) delivered at a flow rate of 1 ml/min. All compounds were detected at 318 nm. An UPLC-ESI-MS system, including an Accela Autosampler, an Accela LC pump, and an Exactive FTMS mass spectrometer (Thermo Fisher Scientific, San Jose, Calif., USA), was used for the characterization of the metabolites [18]. All data were processed using the same software, which derives accurate masses from raw data. In addition, the chemical formula calculator was used to provide chemical formulas and saturation values (double bond equivalents). The full-scan mass spectrum of fragment ions of rat plasma after both i.p. and oral administration of the BPT derivative was compared with that of the blank plasma samples to find out the possible metabolites. Method Validation Stock solutions of the BPT derivative (1.91 mg/ml) and IS (7.94 mg/ml) were prepared in ACN/H2O (60: 40, v/v) and ACN/H2O (20:80, v/v), respectively. Calibration curves were constructed on rat plasma samples (800 μl) obtained from untreated rats. Five levels of calibration standards were prepared at 19.1, 38.2, 95.5, 191, and 382 ng/ml. Before HPLC-UV analysis, a solid-phase extraction was performed according to the procedure described by Bourdon et al. [18]. The linear regression analysis was performed by plotting the peak area ratio (y) against the analyte concentration (x) in nanograms per milliliter. The linearity of the relationship between peak area ratio and concentration was demonstrated by the correlation coefficient (r) obtained for the linear regression. The relative SD was calculated for all the standards. The intra- and interday assays of the method were evaluated by quintuplicate analyses of 3 quality control samples. The calibration standards were analyzed on 3 different days in order to determine intra- and interday precision and accuracy. The accepted criteria were that the relative SD and accuracy should not exceed 15%. Pharmacokinetic Data Analysis Pharmacokinetic parameters related to the absorption, distribution, and elimination of the BPT derivative were calculated using Kinetica 4.4.1 software (Thermo Fisher Scientific, Waltham, Mass., USA). Akaike’s information criterion was applied to select the compartment model to be used for data analysis [19]. Based on linear pharmacokinetics, the plasma disposition of the BPT derivative was best described by a one- or two-compartment open body model after oral and i.p. administration, respectively. The maximum plasma concentration (Cmax) and the time to reach the maximum concentration (Tmax) were directly determined from the plasma concentration-time curves. The other parameters, including the distribution volume of the central compartment (Vd/F), the elimination rate constant, and the microconstants, were estimated. Drug clearance (Cl/F) was determined directly from the plasma drug concentration-time curve. The area under the concentration-time curves from time 0 to infinity (AUC0–∞) were calculated from the model-derived parameters, and the elimination half-lives were calculated from the slope of the terminal elimination phase. The mean residence time (MRT) was calculated as the area under the moment curve extrapolated to infinity (AUMC0–∞)/AUC0–∞. The relative bioavailability was estimated from the ratios of the AUC0–∞ values for oral and i.p. administration. All values are expressed as means.
Bourdon/Lecoeur/Lebègue/Gressier/ Luyckx/Odou/Dine/Goossens/Kambia
Downloaded by: Selçuk Universitesi 193.255.248.150 - 1/22/2015 12:36:49 PM
administration) for the pharmacokinetic studies. All animal care and experimental procedures were approved by the institutional Experimental Animal Ethics Committee (EAEC-75 Nord-Pas de Calais) of INSERM U1011 (Pasteur Institute, Lille, France).
ŽŶĐĞŶƚƌĂƟŽŶƐ (μg/ml)
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100 Time (min)
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ConcentraƟŽŶƐ (μg/ml)
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0.01 b
Results
pharmacokinetic studies of the compound were characterized after both i.p. and oral administration in Wistar rats using 5 times less than the test dose, i.e. 10 mg/kg. The good sensitivity of our analytical method allows the detection of plasmatic concentrations.
Toxicity Test and Dose Selection Before carrying out the pharmacokinetic study of the BPT derivative, it was important to find the nontoxic dose. After a single i.p. or oral administration of the compound (50 mg/kg), the treatment was well tolerated without loss of body weight or decrease in the amount of food and tap water consumed during the study period (i.e. 7 days) in comparison with the untreated group. Moreover, there were no treatment-associated deaths. Thus, pilot
Method Validation The analytical methods described above were validated to simultaneously determine the parent drug and its metabolites with respect to linearity, accuracy, and precision. The HPLC-UV method has high selectivity,
Pharmacokinetic Evaluation of a Novel BPT Derivative
Pharmacology 2014;94:170–178 DOI: 10.1159/000368084
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Fig. 2. Mean plasma concentration-time profiles of the BPT derivative in rats after i.p. (a) and oral administration (b) of a dose of 10 mg/kg. The points represent the mean concentrations of 4 rats.
0.1
sensitivity, and specificity, which enables the determination of the BPT derivative in rat plasma with a limit of quantification of 11.45 ng/ml based on a signal-tonoise ratio of 10. Under the described chromatographic conditions, the compound, the IS, and the metabolites were eluted without an interference peak from the blank rat plasma. The standard curve obtained from the detection of plasma containing known amounts of the BPT derivative was linear over the concentration ranges from 19.1 to 382 ng/ml. The calibration curve was found to be linear and could be described by the regression equation y = 0.00248x – 0.0255 (with r ≥0.998). This sensitivity has proven useful in the analysis of pharmacokinetic data on rats treated both intraperitoneally and orally. The intra- and interday precision and the accuracy based on peak area ratios were determined. The overall intraday precision was
Received: August 28, 2014 Accepted: September 2, 2014 Published online: October 22, 2014
Pharmacokinetic Evaluation of a Novel Benzopyridooxathiazepine Derivative as a Potential Anticancer Agent Florence Bourdon Marie Lecoeur Nicolas Lebègue Bernard Gressier Michel Luyckx Pascal Odou Thierry Dine Jean-François Goossens Nicolas Kambia UDSL, EA 4481, UFR Pharmacie, Université Lille Nord de France, Lille, France
Abstract Background/Aims: The in vivo metabolic profile of a benzopyridooxathiazepine (BPT) derivative, a potent tubulin polymerization inhibitor with a promising in vitro activity, was investigated. Methods: The quantification of the BPT derivative and the identification of metabolites in the plasma of Wistar rats after i.p. and oral administration of 10 mg/kg were performed by the HPLC-mass spectrometry method. Results: Following a single i.p. dose of the BPT derivative, the plasma concentrations showed a biexponential decay (with a rapid decline) followed by a slow decay with a terminal half-life of 77.90 min. The area under the concentration-time curve from time 0 to infinity (AUC0–∞) was 18.90 μg/ml · min. After oral administration, the plasmatic concentrations reached a peak of 0.06 μg/ml at 35 min and then decayed with a half-life of 108 min. The AUC0–∞ was 10.25 μg/ml · min, representing 54.2% of the relative bioavailability. The compound was well distributed in the body, and its elimination seemed to be fast, regardless of the administration route. The major metabolic pathways were demethylation and hy
© 2014 S. Karger AG, Basel 0031–7012/14/0944–0170$39.50/0 E-Mail [email protected] www.karger.com/pha
droxylation reactions, both followed by conjugation with glucuronic acid. Conclusion: In rats, the BPT derivative is well distributed and undergoes extensive metabolism, leading to several metabolites. With promising in vitro activity and very good oral bioavailability, this compound seems to be an attractive candidate for further development as an anticancer agent. © 2014 S. Karger AG, Basel
Introduction
Mitosis is a highly regulated process during cell division. Drugs that disrupt the mitotic progression process are extensively used to treat a wide variety of cancers. There are two major groups of these antimitotic agents: microtubule stabilizers such as paclitaxel and microtubule destabilizers such as vinca alkaloids, chalcones and chalcone derivatives, colchicine, combretastatin A-4, and sulfonamide E7010 [1–6]. However, these agents which target microtubules have high toxicity, and their potency is limited by the development of multidrug resistance [7, 8]. Therefore, there has been great interest in developing novel microtubule inhibitors that overcome various modes of resistance and exhibit improved pharmacologiNicolas Kambia Department of Pharmacology, Pharmacokinetics and Clinical Pharmacy Université Lille Nord de France, 3 rue du Professeur Laguesse FR–59006 Lille (France) E-Mail nicolas.kambia @ univ-lille2.fr
Downloaded by: Selçuk Universitesi 193.255.248.150 - 1/22/2015 12:36:49 PM
Key Words Benzopyridooxathiazepine derivative · Tubulin polymerization inhibitor · Pharmacokinetics · Rat · HPLC-MS
Pharmacokinetic Evaluation of a Novel BPT Derivative
O
O S O
O
O S
N
O
N N
O
N
O
a
b
Fig. 1. Chemical structures of the BPT derivative (a) and the IS (b).
to evaluate the pharmacokinetic profile of the compound. Then, high-resolution mass spectrometry (MS) was used to characterize the metabolites obtained and to propose the major metabolic pathway of the parent drug.
Materials and Methods Chemicals and Drug 1-(4-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (>98% purity) and 1-(4methoxyphenylethyl)-9-methyl-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (>98% purity), the internal standard (IS) (fig. 1), were synthesized according to the procedure described by Gallet et al. [12]. Dimethyl sulfoxide (DMSO) and sodium bicarbonate (NaHCO3) were purchased from Sigma Aldrich (Steinheim, Germany); formic acid (HCOOH) and ammonium hydroxide (NH4OH; 28 wt%) were obtained from Prolabo (Val de Fontenay, France). Methanol and acetonitrile (HPLC grade) were purchased from VWR (Val de Fontenay, France). Gum arabic was purchased from Cooper (Melun, France). NaCl 0.9% was obtained from Macopharma Laboratories (Mouvaux, France). Xylazine and ketamine were purchased from Merial Laboratory (Lyon, France). Ultrapure 18-MΩ water was supplied from a MilliQ system (Millipore, Saint-Quentin-en-Yvelines, France). Animals, Drug Administration, and Plasma Sample Collection Adult female Wistar rats, weighing 140–160 g, were obtained from Janvier Laboratories (Saint-Berthevin, France). They were specific pathogen free and were housed under controlled environmental conditions: temperature 20 ± 2 ° C, relative humidity 50 ± 5%, and 12-hour light-dark cycles in accordance with our institutional animal care guidelines. Throughout the study period, pelleted rat food and tap water were available ad libitum. Upon arrival, the animals were allowed to acclimatize for 7 days before being randomly assigned to the different groups. The animals were fasted overnight and water was allowed ad libitum. They were randomly divided into two groups (i.p. and oral
Pharmacology 2014;94:170–178 DOI: 10.1159/000368084
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cal profiles. In recent years, much effort has been devoted to the identification of new ligands binding to the colchicine site of tubulin, derived not only from natural sources but also by screening of compound libraries in combination with traditional medicinal chemistry [9, 10]. Quinoline and benzopyridooxathiazepine (BPT) derivatives are a pharmacologically active class of heterocyclic compounds that have been reported to inhibit tubulin polymerization, leading to cell cycle arrest and apoptosis [8, 11, 12]. Among the series of synthesized products, 2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,3dihydro-1H-indol-7-yl]acetamide was a novel class of quinoline derivative identified as a potent microtubule inhibitor acting through the colchicine-binding site of tubulin [8]. 1-(4-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide is a BPT derivative. Its pharmacological activity in vitro over a panel of six tumor cell lines (L1210, DU145, HT29, A549, KB-3-1, and KB-A-1) is in the nanomolar range (10 < IC50 < 13 nmol/l) [12]. This interesting profile, resulting from the inhibition of tubulin polymerization, encouraged us to perform in vivo biotransformation studies. Because Caco-2 cells derived from human colonic adenocarcinomas accurately predict the intestinal absorption of drugs, a preliminary study, conducted to assess the intestinal absorption of this compound, showed almost total permeability across these cells [12, 13]. Similarly, the chemical stability investigated under various stress conditions showed that the molecule was stable [14]. These results were encouraging for the development of a BPT derivative as a drug candidate. Likewise, other compounds derived from the oxathiazepine ring, whose chemical stability has been improved, were studied for their antiproliferative activity as well as their inhibition of tubulin polymerization [15]. Despite significant pharmacological activity, in vivo pharmacokinetic studies of this compound have not been reported. Because freshly isolated hepatocytes can be useful for predicting the in vivo clearance of various drugs [16, 17], the first approach with rat liver microsomes and hepatocytes was investigated as an in vitro drug-metabolizing system to predict both the route and rate of metabolism of the studied compound [18]. The aim of the present study was to examine the in vivo pharmacokinetics and bioavailability of the BPT derivative in Wistar rats by focusing on the most relevant pharmacokinetic parameters after single i.p. and oral administration. The first step consisted of the development and validation of a sensitive and selective HPLC-UV method
Toxicity Test and Dose Selection In order to administer a nontoxic dose of the BPT derivative to the animals during the pharmacokinetic study, test dosing was performed. Thus, the rats were randomly divided into two groups: a control and a treatment group. Both were deprived of food for 18 h before the experiments but were allowed access to water. In the oral administration group, the control group (n = 3) received 0.5 ml/100 g body weight of the mixture DMSO/Tween 80/gum arabic/NaCl 0.9% (3:4:18:75%) by gavage, whereas the treatment group (n = 3) received the BPT derivative dissolved in the abovementioned vehicles at a dose of 50 mg/kg. In the i.p. administration group, the control and treatment groups received the same doses as listed above, without gum arabic, at a ratio of 3:4:93%. Neither food nor water was given up to 2 h after administration. All animals were observed individually for 7 days to record any signs of ill-health or behavioral changes such as loss of body weight and a reduction in the quantity of food and tap water consumed. Intraperitoneal Administration After the toxicity test, a suitable dose was chosen (10 mg/kg) to perform the pharmacokinetic studies. Twenty-eight Wistar rats were randomly assigned to four treatment groups and received a single i.p. administration of 10 mg/kg body weight of the BPT derivative (dissolved in DMSO/Tween 80/NaCl 0.9% at a ratio of 3: 4:93%). Blood samples (1 sample per rat) were collected before drug administration and at 8, 10, 15, 30, 60, 120, and 180 min after drug administration in heparinized tubes after animal sacrifice by section of the carotid artery under deep xylazine-ketamine (1:0.5) anesthesia. Plasma was immediately isolated from the blood samples by centrifugation (3,000 g for 10 min) and then stored at –20 ° C until additional extraction and analysis.
Oral Administration As described above, a fixed dose of 10 mg/kg body weight of the BPT derivative was dissolved in a mixture of DMSO/Tween 80/ gum arabic/NaCl 0.9% (3:4:18:75%) and was administered by oral gavage to four groups of 9 rats. Blood samples were collected before drug administration and at 10, 15, 30, 45, 60, 90, 120, 150, and 180 min after drug administration (4 animals per time point), using the same protocol as presented above. Moreover, 5 drug-free rats were anesthetized as previously described, and blood samples were collected; the plasma obtained was pooled and subsequently used for preparing blank and standard curves. Instrumentation and Analytic Conditions HPLC-UV/MS Conditions The chromatographic equipment used to conduct the analyses of the BPT derivative and the IS was the same as that previously used [18]. Separations were carried out on a reversed-phase Symmetry C18 column (150 × 4.6 mm i.d., 5 μm) from Waters preceded by a LiChrospher C18 guard column (4 × 4 mm i.d., 5 μm) from Merck (Fontenay-sous-Bois, France), both kept at 25 ° C. The analytes were eluted under isocratic conditions using a mobile phase consisting of acetonitrile/water/formic acid (60: 40: 0.1,
172
Pharmacology 2014;94:170–178 DOI: 10.1159/000368084
v/v/v) delivered at a flow rate of 1 ml/min. All compounds were detected at 318 nm. An UPLC-ESI-MS system, including an Accela Autosampler, an Accela LC pump, and an Exactive FTMS mass spectrometer (Thermo Fisher Scientific, San Jose, Calif., USA), was used for the characterization of the metabolites [18]. All data were processed using the same software, which derives accurate masses from raw data. In addition, the chemical formula calculator was used to provide chemical formulas and saturation values (double bond equivalents). The full-scan mass spectrum of fragment ions of rat plasma after both i.p. and oral administration of the BPT derivative was compared with that of the blank plasma samples to find out the possible metabolites. Method Validation Stock solutions of the BPT derivative (1.91 mg/ml) and IS (7.94 mg/ml) were prepared in ACN/H2O (60: 40, v/v) and ACN/H2O (20:80, v/v), respectively. Calibration curves were constructed on rat plasma samples (800 μl) obtained from untreated rats. Five levels of calibration standards were prepared at 19.1, 38.2, 95.5, 191, and 382 ng/ml. Before HPLC-UV analysis, a solid-phase extraction was performed according to the procedure described by Bourdon et al. [18]. The linear regression analysis was performed by plotting the peak area ratio (y) against the analyte concentration (x) in nanograms per milliliter. The linearity of the relationship between peak area ratio and concentration was demonstrated by the correlation coefficient (r) obtained for the linear regression. The relative SD was calculated for all the standards. The intra- and interday assays of the method were evaluated by quintuplicate analyses of 3 quality control samples. The calibration standards were analyzed on 3 different days in order to determine intra- and interday precision and accuracy. The accepted criteria were that the relative SD and accuracy should not exceed 15%. Pharmacokinetic Data Analysis Pharmacokinetic parameters related to the absorption, distribution, and elimination of the BPT derivative were calculated using Kinetica 4.4.1 software (Thermo Fisher Scientific, Waltham, Mass., USA). Akaike’s information criterion was applied to select the compartment model to be used for data analysis [19]. Based on linear pharmacokinetics, the plasma disposition of the BPT derivative was best described by a one- or two-compartment open body model after oral and i.p. administration, respectively. The maximum plasma concentration (Cmax) and the time to reach the maximum concentration (Tmax) were directly determined from the plasma concentration-time curves. The other parameters, including the distribution volume of the central compartment (Vd/F), the elimination rate constant, and the microconstants, were estimated. Drug clearance (Cl/F) was determined directly from the plasma drug concentration-time curve. The area under the concentration-time curves from time 0 to infinity (AUC0–∞) were calculated from the model-derived parameters, and the elimination half-lives were calculated from the slope of the terminal elimination phase. The mean residence time (MRT) was calculated as the area under the moment curve extrapolated to infinity (AUMC0–∞)/AUC0–∞. The relative bioavailability was estimated from the ratios of the AUC0–∞ values for oral and i.p. administration. All values are expressed as means.
Bourdon/Lecoeur/Lebègue/Gressier/ Luyckx/Odou/Dine/Goossens/Kambia
Downloaded by: Selçuk Universitesi 193.255.248.150 - 1/22/2015 12:36:49 PM
administration) for the pharmacokinetic studies. All animal care and experimental procedures were approved by the institutional Experimental Animal Ethics Committee (EAEC-75 Nord-Pas de Calais) of INSERM U1011 (Pasteur Institute, Lille, France).
ŽŶĐĞŶƚƌĂƟŽŶƐ (μg/ml)
1
0.1
0.01
0
50
100 Time (min)
150
200
0
50
100 Time (min)
150
200
a
ConcentraƟŽŶƐ (μg/ml)
1.0
0.01 b
Results
pharmacokinetic studies of the compound were characterized after both i.p. and oral administration in Wistar rats using 5 times less than the test dose, i.e. 10 mg/kg. The good sensitivity of our analytical method allows the detection of plasmatic concentrations.
Toxicity Test and Dose Selection Before carrying out the pharmacokinetic study of the BPT derivative, it was important to find the nontoxic dose. After a single i.p. or oral administration of the compound (50 mg/kg), the treatment was well tolerated without loss of body weight or decrease in the amount of food and tap water consumed during the study period (i.e. 7 days) in comparison with the untreated group. Moreover, there were no treatment-associated deaths. Thus, pilot
Method Validation The analytical methods described above were validated to simultaneously determine the parent drug and its metabolites with respect to linearity, accuracy, and precision. The HPLC-UV method has high selectivity,
Pharmacokinetic Evaluation of a Novel BPT Derivative
Pharmacology 2014;94:170–178 DOI: 10.1159/000368084
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Fig. 2. Mean plasma concentration-time profiles of the BPT derivative in rats after i.p. (a) and oral administration (b) of a dose of 10 mg/kg. The points represent the mean concentrations of 4 rats.
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sensitivity, and specificity, which enables the determination of the BPT derivative in rat plasma with a limit of quantification of 11.45 ng/ml based on a signal-tonoise ratio of 10. Under the described chromatographic conditions, the compound, the IS, and the metabolites were eluted without an interference peak from the blank rat plasma. The standard curve obtained from the detection of plasma containing known amounts of the BPT derivative was linear over the concentration ranges from 19.1 to 382 ng/ml. The calibration curve was found to be linear and could be described by the regression equation y = 0.00248x – 0.0255 (with r ≥0.998). This sensitivity has proven useful in the analysis of pharmacokinetic data on rats treated both intraperitoneally and orally. The intra- and interday precision and the accuracy based on peak area ratios were determined. The overall intraday precision was
