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Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 405–412
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Preclinical pharmacokinetic evaluation of praziquantel loaded in poly (methyl methacrylate) nanoparticle using a HPLC–MS/MS Mayara Malhado a , Douglas P. Pinto b , Aline C.A. Silva b , Gabriel P.E. Silveira b , Heliana M. Pereira b , Jorge G.F. Santos Jr. c , Carla V.V. Guilarducci-Ferraz a , Alessandra L. Vic¸osa d , Márcio Nele e , Laís B. Fonseca b , José Carlos C.S. Pinto f , Sabrina Calil-Elias a,∗ a Laboratório de Farmacologia, Departamento de Farmácia e Administrac¸ão Farmacêutica, Programa de Pós-graduac¸ão em Ciências Aplicadas a Produtos para Saúde, Faculdade de Farmácia, Universidade Federal Fluminense, Niterói, RJ, Brazil b Laboratório de Farmacocinética, Vice-Presidência de Produc¸ão e Inovac¸ão em Saúde, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, Brazil c Laboratório de Engenharia de Polimerizac¸ão, COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil d Instituto de Tecnologia em Fármaco, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, Brazil e Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil f Engenharia Química, COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 8 May 2015 Received in revised form 17 September 2015 Accepted 18 September 2015 Available online 25 September 2015 Keywords: Praziquantel Poly (methyl methacrylate) nanoparticle Pharmacokinetic HPLC–MS/MS
a b s t r a c t Praziquantel (PZQ) is the drug recommended by the World Health Organization for treatment of schistosomiasis. However, the treatment of children with PZQ tablets is complicated due to difficulties to adapt the dose and the extremely bitter taste of PZQ. For this reason, poly (methyl methacrylate) nanoparticles loaded with Praziquantel (PZQ-NP) were developed for preparation of a new formulation to be used in the suspension form. For this reason, the main aim of the present study was to evaluate the pharmacokinetic (PK) profile of PZQ-NP, through HPLC–MS/MS assays. Analyses were performed with an Omnisphere C18 column (5.0 m × 4.6 mm × 150.0 mm), using a mixture of an aqueous solution containing 0.1 wt% of formic acid and methanol (15:85—v/v) as the mobile phase at a flow rate of 0.800 mL/min. Detection was performed with a hybrid linear ion-trap triple quadrupole mass spectrometer with multiple reactions monitoring in positive ion mode via electrospray ionization. The monitored transitions were m/z 313.18 > 203.10 for PZQ and m/z 285.31 > 193.00 for the Internal Standard. The method was validated with the quantification limit of 1.00 ng/mL, requiring samples of 25 L for analyses. Analytic responses were calibrated with known concentration data, leading to correlation coefficients (r) higher than 0.99. Validation performed with rat plasma showed that PZQ was stable for at least 10 months when stored below −70 ◦ C (long-term stability), for at least 17 h when stored at room temperature (RT, 22 ◦ C) (short-term stability), for at least 47 h when stored at room temperature in auto-sampler vials (post-preparative stability) and for at least 8 successive freeze/thaw cycles at −70 ◦ C. For PK assays, Wistar rats, weighing between 200 and 300 g were used. Blood samples were collected from 0 to 24 h after oral administration of single doses of 60 mg/kg of PZQ-NP or raw PZQ (for the control group). PZQ was extracted from plasma by liquid–liquid extraction with terc-butyl methyl ether. The values obtained for maximum concentration (Cmax ) and area under curve (AUC) for the PZQ-NP group were about 3 times smaller than the respective values obtained for the control group. However, the time for achieving maximum concentration (Tmax ), the elimination constant (Ke) and the half-life time of elimination (T½ ) were not statistically different. These results suggest that PZQ absorption is probably the rate-limiting step for obtainment of better PK parameters for PZQ-NP. Thus, further studies are needed to understand both the PZQ-NP absorption mechanisms and the drug diffusion process through the polymer matrix in vivo, in order to improve the PZQ-NP release profile. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Rua Mario Viana, 523–Santa Rosa–Niterói – RJ, CEP: 24241-000, Brazil. E-mail address: [email protected] (S. Calil-Elias). http://dx.doi.org/10.1016/j.jpba.2015.09.023 0731-7085/© 2015 Elsevier B.V. All rights reserved.
Schistosomiasis is a parasite disease caused by worms of the genus Schistosoma. According to World Health Organization
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2. Materials and methods 2.1. Chemicals and reagents
Fig. 1. Chemical structures of Praziquantel.
(WHO), schistosomiasis is one of the major public health problems worldwide, associated with severe mortality and morbidity and affecting approximately 240 million people in the world. More than 700 million people live in schistosomiasis endemic areas, with higher infection rates in children than in adults. In tropical and subtropical areas, schistosomiasis is the second most important disease in terms of socio-economic aspects, exceeded only by malaria [1]. The strategy to control schistosomiasis aims to prevent morbidity through regular treatment with praziquantel (PZQ) (Fig. 1a), which is currently the only drug recommended for schistosomiasis treatment [1]. However, the treatment of children with PZQ tablets is especially complicated because of both the difficulty to adjust the doses (the recommended dose is 40 mg/kg) and the nonadherence to therapy because of the extremely bitter taste of the active pharmaceutical ingredient (API) praziquantel [2]. The development of pharmaceutical forms containing PZQ as active ingredient is difficult due to the hydrophobicity of this drug. However, the preparation in polymer nanoparticles has emerged as a promising alternative for development of drugs with poor water solubility [3]. In addition, nanoparticles can increase the bioavailability, improve the doses proportionality, reduce the variability in fed or fasted individuals, reduce inter-patient variability, improve the absorption rate and mask the taste [4,5]. Based on the previous remarks, poly (methyl methacrylate) (PMMA) nanoparticles loaded with PZQ (PZQ-NP) were synthesized [4] for posterior preparation of PZQ suspensions. The PMMA nanoparticles loaded with PZQ were prepared by “in situ” freeradical miniemulsion polymerization. The preparation comprises two phases, organic and aqueous. First PZQ was dissolved in the mixture with MMA, mineral oil (used as a co-stabilizer) and Ethylene Glycol Dimethacrylate (EDGMA) (used as crosslinking agent) using a magnetic stirrer. The aqueous phase was prepared with water, sodium dodecyl sulfate and bicarbonate. The phases were emulsified with the help of the high-pressure homogenizer with pressure drop of 800 bar for 10 min at 85 ◦ C. After this process, the emulsion was transferred to the reactor and the initiator (potassium persulfate) was added to the reaction medium under agitation of 1000 rpm for 120 min. The system was kept to 90 ◦ C. The final emulsion was dried by lyophilization at −50 ◦ C and 0.4 mbar. Before preparing the new formulation, though, it is necessary to understand the pharmacokinetics (PK) of the new product. It is important to mention that the study of drug pharmacokinetics in animal models is included as a preclinical stage during the development of new drugs [6–8]. For this reason, the main objective of the present study was to evaluate the pharmacokinetic profile of PZQ-NP and to compare obtained results with the pharmacokinetic profile of PZQ in the free form, after oral administration in rats. In order to do that, a sensitive and specific analytical method for analysis of PZQ in rat plasma was developed, using coupled high performance liquid chromatography and efficiency tandem mass spectrometry (HPLC–MS/MS).
PMMA Nanoparticles loaded with PZQ were provided by Laboratório de Engenharia de Polímeros (EngePol/COPPE), Universidade Federal do Rio de Janeiro (Rio de Janeiro, RJ, BRA). PZQ was provided as a pharmaceutical grade by Farmanguinhos (Maker Yixing City Xing Yu Medicine Chem., Yixing, JS, CHN). MMA (purity of 99.9%) was purchased from Vetec (Duque de Caxias, RJ, BRA), EGDMA (purity of 98%) provided by Sigma–Aldrich (St. Louis, MO, USA), sodium dodecylsulfate, sodium bicarbonate and potassium persulfate were provided as commercial grades by Vetec (Duque de Caxias, RJ, BRA). The mineral oil was provided as a pharmaceutical grade by Isofar (Duque de Caxias, RJ, BRA) and water was distilled and deionized prior to use. The PZQ reference standard were provided by United States Pharmacopoeia (Rockville, MD, USA) and the internal standard (I.S.), Diazepam (DZP), was provided by INCQS—Instituto Nacional de Controle de Qualidade em Saúde (Rio de Janeiro, RJ, BRA) (both with minimum purity of 99.9%). Acetonitrile and methanol were purchased from Tedia (Fairfield, OH, USA). Terc-butyl methyl ether (TBME) was purchased from J.T Baker (Phillipsburg, MT, USA). All solvents used in the present study presented HPLC grade. Ultrapure water was provided by a MilliQ system from Millipore (Molshein, AL, FRA). Formic Acid 96% was purchased from Spectrum (New Brunswick, NJ, USA). 2.2. Instrumentation and HPLC–MS/MS conditions 2.2.1. Equipment Analyses were performed with a EkspertTM ultra LC 110 system (Eksigent/AB Sciex, Redwood City, CA, USA) coupled to a QTrap 5500 linear ion trap quadrupole mass spectrometer (AB Sciex, Franingham, MA, USA). Obtained data were processed with the proprietary Analyst softwareTM (version 1.6.1). 2.2.2. Analytical conditions Chromatographic separation was carried out with a Omnisphere C18 column (5 m × 4.6 mm × 150 mm) (Agilent Technologies, Santa Clara, CA, USA), using the isocratic elution technique. The mobile phase consisted of a mixture of methanol and an aqueous solution containing 0.1 wt% of formic acid (85:15, v/v). The flow rate was equal to 0.800 mL/min (split ratio 1/1). The autosampler and column oven temperatures were equal to 15 ◦ C and 40 ◦ C, respectively. Sample injection volumes of 10 L were used. The mass spectrometer was operated with electrospray ionization (ESI) in the positive mode. Nitrogen was used as the nebulizer and auxiliary gas. Important operation parameters were: (i) source temperature of 500 ◦ C, (ii) curtain gas at 25 psi, (iii) nebulizer gas (GS1) at 60 psi and (iv) heater gas (GS2) at 60 psi. Collision-activated dissociation (CAD) gas was medium. The ion spray voltage was equal to 5500 V, while the entrance potential (EP) was equal to 10 V. The declustering potential (DP), collision energy (CE) and collision exit potential (CXP) were equal to 130, 15 and 20 V for PZQ and 91, 29 and 12 V for DZP, respectively. The detector was operated at 1900 V. Quantification was carried out using multiple reaction monitoring (MRM). The MRM transitions selected were m/z 313.18 > 203.10 for PZQ and m/z 285.31 > 193.00 for DZP. 2.3. Sample preparation 2.3.1. Preparation of calibration standards and quality control samples The stock of PZQ solution was prepared in methanol/water (50:50—v/v) at a concentration of 1.0 mg/mL. Working solutions of PZQ were obtained by diluting the stock solution
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with the same diluent (methanol/wate—50:50–v/v) until the desired final concentrations in the range of 1.0–1000 ng/mL. The I.S. solution was prepared in methanol/water (50:50—v/v) at a concentration of 500 ng/mL. The standard calibration samples were prepared by spiking 25 L of blank rat plasma with 25 L of corresponding working solution to yield eight standards with concentration of 1.0 ng/mL, 2.5 ng/mL, 5.0 ng/mL, 10.0 ng/mL, 100 ng/mL, 400 ng/mL, 800 ng/mL and 1000 ng/mL. For validation, quality control (QC) samples were prepared with similar procedures at five concentrations: lower limit quantification quality control (LLQQC) = 1.0 ng/mL, low quality control (LQC) = 2.5 ng/mL, medium quality control (MQC) = 400 ng/mL, high quality control (HQC) = 800 ng/mL and dilution quality control (DQC) = 1000 ng/mL, diluted to 800 ng/mL. All standard calibration samples and quality control samples were spiked with 500 ng/mL of I.S.
2.3.2. Plasma preparation Aliquots of 25 L of I.S solution were added to 25 L of plasma samples and mixed for 1 min. Then, 1000 L of the extraction solution, TBME (100%), were added. After mixing in automatic shakers for 10 min, samples were submitted to centrifugation at 14,000 rpm at −10 ◦ C for 5 min. Then, 900 L of the organic phase were transferred to a clean tube and evaporated under nitrogen flow. The residue was reconstituted in 400 L of the mobile phase (methanol/0.1% formic acid in water—60:40—v/v) and 10 L were injected into the HPLC–MS/MS system for analysis.
2.4. Validation method In all validation tests, precision was expressed as the CV (%), and accuracy was expressed as the Bias (%), as deviation of the obtained mean from nominal concentration values. The precision (CV%) must not exceed 15% for all levels (20% for the LLQQC as exception), and the accuracy (Bias%) must be within ±15% of the nominal value for all levels (±20% of the nominal value for the LLQQC as exception). The method was validated based on FDA Guidance for industry [9].
2.4.1. Linearity The calibration curve consisted of a double blank sample (rat plasma sample processed without I.S), a blank sample (rat plasma processed with I.S), and eight calibration samples at concentrations of 1.0 ng/mL, 2.5 ng/mL, 5.0 ng/mL, 10.0 ng/mL, 100 ng/mL, 400 ng/mL, 800 ng/mL and 1000 ng/mL.. For the calibration and the run to be valid, the coefficient of correlation (r) should be greater than 0.99, and the accuracy of at least 75% of calibration samples had to remain within ±15%. 2.4.2. Accuracy and precision Eight replicates of QC samples at five concentration levels (LLQQC, LQC, MQC, HQC and DQC) were processed as described in Section 2.3.2 on three different days to determine intra-day and inter-day accuracies and precision. The dilution quality control was used in order to assess the reliability of the method at concentration levels outside of the upper limit of quantification (1000 ng/mL).
2.4.3. Freeze and thaw stability Eight replicates at QC sample concentrations of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were submitted to eight freeze/thaw cycles (freezing at −70 ◦ C for at least 12 h and thaw at 22 ± 2 ◦ C). These samples were then processed and quantified with a set of calibration samples that were not submitted to the freeze/thaw cycles.
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2.4.4. Stability on bench-top at room temperature (short-term stability) Eight replicates at QC sample concentrations of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were prepared and kept at RT for 17 h: 54 min before processing, as described in Section 2.3.2, and quantifying with a set of calibration samples that were processed immediately. The I.S. was added into QC samples only when it was also added into the calibration samples, in order to ensure that samples were analyzed under similar conditions. 2.4.5. Post-preparative stability Eight replicates of QC samples at concentration of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were processed and quantified. After 47 h: 55 min of storage in the auto-sampler (RT and protected from light), the run was re-injected and re-analyzed with freshly prepared calibration samples. 2.4.6. Long-term stability Eight replicates of QC samples at concentrations of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were prepared and kept at −70 ◦ C for 10 months. Afterwards, the samples were processed as described in Section 2.3.2 and quantified with a set of calibration samples that were processed immediately. The I.S. was added into the QC samples only when it was also added into the calibration samples, in order to ensure that samples were analyzed under similar conditions. 2.5. Pharmacokinetic study 2.5.1. Animals Twenty female Wistar rats (weighing between 200 and 300 g) from Centro de Animais de Laboratório of Universidade Federal Fluminense (Rio de Janeiro, RJ, BRA) were used. The rats were maintained on a 12 h/12 h light/dark cycle, housed in plastic cages and received a standard chow and water ad libitum. Before and after PZQ oral administration, rats were fasted for food (2 h) and water (1 h). All animal experiments were performed according to the policies and guidelines of the ethics committee of animal use of Universidade Federal Fluminense (Niterói, RJ, BRA) and the study was approved by its ethics committee (ethical permission number 378/2013). 2.5.2. Design of pharmacokinetic experiments Animals were divided into three groups. The control group (n = 5) received 1 mL of vehicle (aqueous solution containing 2% of Cremophor EL); the group PZQ-NP (n = 6) received 60 mg/kg of PZQNP dissolved in 1 mL of vehicle; and group PZQ-L (n = 9) received 60 mg/kg of PZQ in free form dissolved in 1 mL of vehicle. Approximately 150 L of blood was collected into heparinized syringes from tail vein at 0 (pre-dose), 5 min, 10 min, 20 min, 30 min, 1 h, 1 h: 30 min, 2 h, 4 h, 8 h, 10 h, 12 h and 24 h after oral administration of a single dose. Blood samples were centrifuged at 4000 rpm for 10 min. Then, the plasma was stored at −20 ◦ C until analysis. 2.5.3. Sample analysis Plasma samples were processed using the liquid–liquid extraction procedure described in Section 2.3.2 and analyzed using HPLC–MS/MS method described in Section 2.2. A calibration curve was built using weighted linear regression, as described in Section 2.4.1, and plasma sample concentrations were calculated. 2.5.4. Pharmacokinetic data analysis Non-compartmental analyses were performed with the Phoenix WinNonlin software (version 6.3, Pharsight, St. Louis, MO, USA). The pharmacokinetic (PK) parameters determined were the maximum concentration (Cmax ), time to achieve maximum concentration
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(Tmax ), area under the curve (AUC0-t and AUC 0−∞ ), the terminal elimination rate constant (Ke) and the terminal elimination halflife (T½  ). Parameters Cmax and Tmax were extracted directly from the plasma concentration (Cp) versus time curve, AUC 0−t was calculated with a linear trapezoidal method, AUC0−∞ was calculated by combining AUC0−tlast with Ctlast /Ke, Ke was obtained by calculating the slope of the decay phase from the graph of ln(Cp) versus time and T½  was calculated using the equation: T½  = 0.693/ Ke. Outliers were detected with the Dixon and Chauvente tests. 2.6. Statistical analysis The graphics were built using GraphPad Prism® software (version 5.0, San Diego, CA, USA). Mann–Whitney Test was used for comparison between two groups of data. The results were expressed as the mean ± standard error. Significance was evaluated at P < 0.05. 3. Results and discussion 3.1. HPLC–MS/MS analysis The MS spectra of PZQ and I.S. (DZP) showed a retention times equal to 2.78 min for PZQ and 2.98 min for I.S and the run time was equal to 4 min. The range of linear response of 1.0–1000 ng/mL was established by determining the accuracy and precision of calibration curves. For all runs, calibration curves and QC samples met the required acceptance criteria. Typical MRM (Multiple Reaction Monitoring) chromatograms of blank rat plasma spiked with PZQ and I.S. are shown in Fig. 2. Bioanalytical method was developed and validated using high performance liquid chromatography tanden mass spectrometry (HPLC–MS). Analyses were shown to be rapid, sensitive, accurate, reproducible and simple (sample preparation process held in a single step) for analyses of PZQ in rat plasma. In tests conducted by Bonato et al. [10] and Hanpitakpong et al. [11] for plasma analyses in rats using HPLC, the limit of quantitation was 5.0 ng/ml and sample volumes of 1 mL were employed. However, in the present study, we developed an analytical method with range of quantification of 1.0–1000 ng/mL, using 25 L of plasma samples. This is a great advantage for experiments where multiple blood samples must be characterized in short times, especially because of the wide concentration interval, as in most pharmacokinetics and toxicology studies. The application of mass spectrometry ensures the selectivity and specificity of the method, since this technique monitors fragments of highly specific mass/charge (m/z) ratios. 3.2. Validation 3.2.1. Linearity test The response versus concentration data, in the range 1.0–1000 ng/mL of PZQ, was assessed by analyzing the calibration curves using the peak area ratios of analyte/I.S versus the nominal concentrations of the analyte/I.S. calibration standard with a weighting factor (1/x2 ). Obtained correlation coefficients (r) were always higher than 0.99, indicating that all calibration curves met the acceptance criteria. 3.2.2. Accuracy and precision The intra-day and inter-day precision CV% did not exceed 14.01% at any QC concentration levels. The intra-day and inter-day accuracy (Bias%) was between −10.43% and +8.88%, and between −10.42% and +10.52% respectively (Table 1), indicating the occurrence of random fluctuations and absence of consistent bias. Since
both intra-day and inter-day precision were below 15% and accuracy was within ±15%, the proposed bioanalytical method could be regarded as precise and accurate. The DQC results showed that the dilution process could be realized for samples with concentrations above upper limit of quantification. 3.2.3. Freeze–thaw stability After eight freeze–thaw cycles for two concentration samples (2.5 and 800 ng/mL), the precision (CV%) did not exceed 5.81%. The accuracy (Bias%) was between −0.30% and −6.72% (Table 2), indicating the possible occurrence of negative bias. However, based on the proposed performance indices, PZQ in rat plasma could be regarded as stable after eight freeze/thaw cycles at −70 ◦ C. 3.2.4. Bench-top stability at room temperature After storage for 17 h: 54 min at room temperature, the precision (CV%) of the QC samples did not exceed 4.68% and the accuracy (Bias%) was between −6.50 % and 1.59 % (Table 2), indicating the occurrence of random fluctuations and absence of consistent bias. This indicated that PZQ in rat plasma was stable after storing at room temperature prior to processing. 3.2.5. Post-preparative stability Before and after 47 h: 55 min of storage (RT, protected from light) of the QC samples, the precision (CV%) did not exceed 9.93% and the accuracy (Bias%) was between −3.50% and −8.88% (Table 2), indicating the possible occurrence of negative bias. However, based on the proposed performance indices, PZQ in rat plasma could be regarded as stable after post-preparative process. 3.2.6. Long-term stability After storage for 10 months at −70 ◦ C, the precision (CV%) of the QC samples did not exceed 2.75% and the accuracy (Bias%) was between −3.13% and 11.43% (Table 2), indicating the occurrence of random fluctuations and absence of consistent bias. This indicated that, when stored at −70 ◦ C, PZQ in rat plasma could be regarded as stable for a minimum of 10 months without significant decomposition of the drug. 3.3. Pharmacokinetic data analysis Pharmacokinetic studies were performed to determine the absorption, bioavailability and elimination profile of praziquantel administered in PZQ-NP after single oral doses and to compare the obtained PK profiles with the pharmacokinetic profile obtained for PZQ administered in its free form. The values obtained for Cmax and AUC for PZQ-NP group were about 3 times lower than obtained for the PZQ-L group. Tmax, Ke and T 1/2 were not statistically different. The present study demonstrated that PZQ is released in vivo from PZQ-NP and is partially absorbed in the gastrointestinal tract after oral administration in rats. After outlier detection, two animals from the PZQ-L group and one animal from the PZQ-NP group were excluded, according to criteria established by the Dixon and Chauvente’s tests. Then, the pharmacokinetic parameters was calculated with five animals in control group (n = 5), seven animals in PZQ-L group (n = 7) and five animals in PZQ-NP group (n = 5). PZQ in rat plasma was observed from 5 mins until 24 h after oral administration in PZQ-L and PZQ-NP groups. In the control group, PZQ was not detected in any of the analyzed time, demonstrating that the vehicle used (water with 2% Cremophor EL) was free of the drug, Cremophor EL does not interfere with the analytical setup and there was no cross-contamination during sample preparation and analysis. The absorption was approximately three times lower in the PZQNP group when compared to PZQ-L. PZQ is highly lipophilic and crosses the gastrointestinal tract membranes by passive diffusion,
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Fig. 2. The HPLC–MS/MS chromatograms of (A) blank of plasma spiked with PZQ and DZP (10 ng/mL and 500 ng/mL, respectively), (B) plasma sample from a rat obtained at 5 min after oral administration of 60 mg/kg of PZQ loaded in PZQ-NP with I.S. (11.52 ng/mL and 500 ng/mL, respectively).
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Table 1 Summary of precision and accuracy assay (intra-day and inter-day) for PZQ in rat plasma. Nominal concentration (n = 8) Precision and accuracy of control Intra-day
Inter-day
Day 1
1.0 ng/mL 2.5 ng/mL 400 ng/mL 800 ng/mL 800 ng/mL
Day 2
Mean ± S.D
CV(%) Bias(%) Mean ± S.D
1.09 ± 0.10 2.39 ± 0.11 361.26 ± 34.04 725.05 ± 19.35 716.53 ± 30.52
9.51 4.42 9.42 2.67 4.26
8.88 −4.30 −9.69 −9.37 −10.43
1.20 ± 0.17 2.28 ± 0.19 388.80 ± 18.43 798.68 ± 23.69 817.99 ± 35.84
Day 3 CV(%)
Bias(%) Mean ± S.D
14.01 8.45 4.74 2.97 4.38
19.75 −8.85 −2.80 −0.16 2.25
0.97 ± 0.10 2.21 ± 0.11 362.37 ± 10.12 713.71 ± 16.50 721.10 ± 15.77
Overall CV(%) Bias(%) Mean ± S.D 10.35 4.93 2.79 2.31 2.19
−3.38 −11.70 −9.41 −10.79 −9.86
1.08 ± 0.12 2.24 ± 0.04 374.98 ± 13.25 715.82 ± 8.45 751.87 ± 57.31
CV(%)
Bias(%)
10.67 1.61 3.53 1.18 7.62
8.42 −10.42 −6.26 10.52 −6.02
S.D = standard deviation; CV(%) = coefficient of variant; Bias(%) = deviation of mean from nominal value.
Table 2 Stabilities during storage in different conditions (n = 8). Nominal concentration (n = 8)
2.5 (ng/mL) 800 (ng/mL)
Post-preparative stability
Freeze–thaw stability
Mean ± S.D
CV(%)
Bias(%)
Mean ± S.D
CV(%)
Bias(%)
Bench-top stability at RT Mean±S.D
CV(%)
Bias(%)
Long-term stability Mean ± S.D
CV(%)
Bias(%)
2.41 ± 0.24 728.96 ± 44.04
9.93 6.04
−3.5 −8.8
2.49 ± 0.14 746.26 ± 19.26
5.81 2.58
−0.30 −6.72
2.66 ± 0.12 812.75 ± 38.07
4.58 4.68
6.50 1.59
2.79 ± 0.08 774.98 ± 9.43
2.75 1.22
11.43 −3.13
RT = room temperature; S.D = standard deviation; CV(%) = coefficient of variant; Bias(%) = deviation of mean from nominal value.
reaching the bloodstream very fast [12]. The difference in concentrations may be explained by the fact that the absorption of the PZQ trapped in nanoparticles is dependent on the diffusion of the drug through the polymer matrix. Although bioavailability was significantly lower in the PZQ -NP group, more studies are needed to evaluate whether plasma concentrations of PZQ-NP achieves therapeutic margin in the desired dose, and to evaluate their effectiveness. The plasma concentration versus time curves of PZQ obtained after oral administration of a single dose (60 mg/kg) of PZQ in PZQNP are shown in Fig. 3. The oral pharmacokinetic parameters are listed in Table 3. Cmax values in the PZQ-L group were approximately 3 times higher than Cmax values in the PZQ-NP group, with statistically significant difference. Similar behavior was observed for area under curve (AUCo−t and AUCo−∞ ). In order to evaluate this significant AUC difference between the groups, the relative bioavailability (FR) of PZQ in the PZQ-NP, in relation to the bioavailability of PZQ in free form, was calculated in accordance with the following equation: FR =
FR =
ASC
NP ASC0−∞ L 0−∞
QL ×
Q NP
19086.28 15.03 63344.48
×
15.72
FR = 0.2881(28.81%) Note: ASC0−∞ NP: bioavailability from time zero to infinity calculated for the PZQ NP group; ASC0−∞ L: bioavailability from time zero to infinity calculated for the PZQ L group; Q L: average amount (mg) administered of PZQ to PZQ L group; Q NP: average amount (mg) administered of PZQ to PZQ NP group. This means that only 28.81% of the PZQ-NP became bioavailable in respect to the free PZQ. Interestingly both Cmax and AUC from PZQ-NP group were approximately 30% of the values obtained for the PZQ-L group. These results suggest an apparent proportionality between the tested suspensions regarding the pharmacokinetics of praziquantel, since reductions observed in Cmax and AUC from PZQ-NP group are related to reduce in the same proportion of the bioavailable drug in this group. The bioavailability of PZQ in PZQ-NP group was significantly lower. These results differ from those found by Xie et al. [13] and
Yang et al. [14] where solid lipid nanoparticles increased the AUC of PZQ when compared to its free form and tablet, respectively. However, it is known that the absorption process (and consequent bioavailability) depends on the particles physicochemical properties [15–17] and the polymer used in this study and those used by these authors are different. According to Boegh et al. [18] nanoparticles synthesized with bioadhesive polymers may result in greater drug absorption by increasing the carrier system interaction with the intestinal cells. However, the polymer used as the matrix for the development of nanoparticle in study, PMMA, is not bioadhesive [19] and this may be the reason why it does not promote the increase of the bioavailability of PZQ. Thus, studies to determine the diffusion rate, viscoelasticity, speed, direction and transport of PMMA nanoparticles in the mucus need to be performed. Although PMMA is not bioadhesive, it has been widely used in pharmaceutical and biomedical field due to its known biocompatibility, dimensional stability and resistance [20]. Additionally, the use of Cremophor EL was necessary to increase the aqueous solubility of nanoparticles, since these suspension only with water obstructed the gavage cannula. Cremophor EL is a solubilizing and emulsifier agent used by pharmaceutical industries for aqueous preparations of hydrophobic active ingredients. Different studies have used free praziquantel or its reference tablet solubilized in 2% Cremophor EL [21–23] and any interference on the drug pharmacokinetics/pharmacodynamics was reported. However, no studies were found about interactions between PMMA and Cremophor EL. Thus, one hypothesis is the addition of this solubilizing agent may have caused changes in the surface of the nanoparticles which avoided the complete release of drug from the polymer matrix. Another possible hypothesis is that PMMA in biological fluids does not dissolve completely and this may hinder the release of PZQ. PZQ is a chiral compound marketed as the racemate mixture, although its (+)–(S) enantiomer has no proven pharmacological activity [24]. It is biotransforrmed in the liver and its active metabolite (−)-(R)-trans-4-hydroxypraziquantel (4-OHPZQ) seems to possess antischistosomal activity [25]. Lima et al. developed and validated a method for enantioselective analysis of praziquantel and trans-4-hydroxypraziquantel in human plasma by chiral LC–MS/MS [26]. However, in this study it was not possible to evaluate the influence of nanoparticles on biotransformation of PZQ by
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Fig. 3. Concentration-time curve after a single oral dose, in rats, of PZQ-L (n = 7) and PZQ-NP (n = 5). The control group received 1 mL of vehicle. Results are expressed as mean ± SE. Featured: Plasma concentration-time of PZQ until 1 h on groups. Table 3 The non-compartimental pharmacokinetic parameter of PZQ in rat plasma after oral administration of PZQ-NP (n = 5) and PZQ-L (n = 7). GROUP
Pharmacokinetic parameters Cmax {g/L}
Tmax
PZQ-L PZQ-NP
554.23 ± 94.19 172.09 ± 43.84*
0.33 ± 0.04 0.38 ± 0.10
{h}
ASC0−t {g h/L}
ASC0−∞ {g h/L}
Ke {1/h}
T1/2 {h}
1111.91 ± 195.51 326.79 ± 35.54*
1125.00 ± 196.33 336.09 ± 34.63*
0.27029 ± 0.03886 0.27032 ± 0.04276
2.91 ± 0.45 2.85 ± 0.47
Data are mean ± SE. P < 0.05 versus PZQ-L.
*
difficulty in the acquisition of cis and trans-4-hidroxypraziquantel standards, the major PZQ biotransformation’s products reported in the literature [12]. Since nanoparticles have demonstrated to protect the molecules of the first-pass effect in the liver [27,28], research is needed to determine qualitatively and quantitatively the biotransformation products from PZQ loaded in PMMA nanoparticle and to evaluate if they have anthelmintic activity. Both PZQ-NP and PZQ-L reached the maximum concentration (Tmax ) at approximately 20 mins. The Tmax results showed the PZQ-NP do not have a delayed release effect. Similar result was found by Xie et al. [13] for solid lipid nanoparticles containing PZQ. On the other hand, Zhang et al. [29] demonstrated that lipid nanosuspensions significantly increased the Tmax . Since polymer’s nanosystem used in this study is different from those used by the authors cited above, again it is emphasized the importance of chemical and physical characteristics of the material used as matrix for the nanoparticle drug absorption. T½  in PZQ-NP and PZQ-L group was statistically similar, about 3 h. The T1/2 of the free PZQ was approximately 10 times lower than found by Xie et al. [13]. These authors, however, conducted the study in dogs and PZQ were administrated subcutaneously at a dose of 5 mg/kg. This demonstrates that the kinetics of PZQ may vary considerably depending on the species used. Both in PZQ-L and PZQ-NP Ke demonstrated that approximately 27% of the drug had been eliminated from the body per hour. This result Ke suggest that there is no saturation of the elimination process, because although ASC0−∞ of PZQ-NP group was about three times larger, Ke was similar between the groups.
4. Conclusion The analytical method proposed and developed here for determination of praziquantel in rat plasma meets the criteria for application in pharmacokinetic studies. The advantage over previously reported methods are its rapidity, with total run time of four minutes, and its high sensitivity (LOQ, 1.0 ng/mL). Besides, rat plasma sample can be diluted without affecting the precision and accuracy. The pharmacokinetic results suggest the low plasma concentration of PZQ in the group treated with PZQ-NP can be due to in vivo PZQ incomplete release from polymer matrix, which limits the amount of PZQ available to be absorbed via molecular passive diffusion. Further, since PMMA is not a bioadhesive polymer, it is also possible that the residence time of the nanoparticle in the gastrointestinal tract was not large enough to increase the interaction of the carrier system with the intestinal cells, which could be resulted in a lower rate of nanoparticle internalization. In both cases, more studies are needed to understand these nanoparticles behavior in the body in order to improve this nanosystem. In addition, tests must be conducted in order to assess whether PZQ-NP reaches the therapeutic range and if it is effective in the schistosomiasis treatment.
Conflict of interest The authors state no conflict of interest.
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Acknowledgments This research was supported by grants from FAPERJ (Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FINEP (Financiadora de Estudos e Projetos) and BNDES (Banco Nacional de Desenvolvimento Econômico e Social). The authors also thank Farmanguinhos for technical support. References [1] WHO, Schistosomiais (2013) http://www.who.int/schistosomiasis/en. [2] T. Meyer, H. Sekljic, S. Fuchs, H. Bothe, D. Schollmeyer, C. Miculka, Taste, a new incentive to switch to (R)-praziquantel in schistosomiasis treatment, PLoS Negl. Trop. Dis. 3 (2009) e357, http://dx.doi.org/10.1371/journal.pntd. 0000357). [3] K. Landfester, A. Musyanovych, V. Mailander, From polymeric particles to multifunctional nanocapsules for biomedical applications using the miniemulsion process, J. Polym. Sci., Part A 48 (2010) 493–515. [4] L.B. Fonseca, A.L. Vic¸osa, A.C.A. Matos, P.M.Z. Coelho, N. Araújo, H.P.S. Zamith, N.M. Volpato, M. Nele, J.C.C.S. Pinto, Development of a brazilian nanoencapsulated drug for schistosomiasis treatment, Vig. Sanit. Debate 1 (4) (2013) 85–91. [5] R.H. Muller, C. Jacobs, O. Kayser, Nanosuspensions as particulate drug formulations in therapy. Rationale for development and what we can expect for the future, Adv. Drug Deliv. Rev. 47 (2001) 3–19. [6] W. Tang, A.Y.H. Lu, Drug metabolism and pharmacokinetics in support of drug design, Curr. Pharm. Des. 15 (19) (2009) 2170–2183. [7] C.M. Masimirembwa, U. Bredberg, T.B. Andersson, Metabolic stability for drug discovery and development. Pharmacokinetic and biochemical challenges, Clin. Pharmacokinet. 42 (6) (2003) 515–528. [8] R. Panghagnula, N.S. Thomas, Biopharmaceutics and pharmacokinetics in drug research, Int. J. Pharm. 201 (2) (2000) 131–150. [9] FDA, Guidance Ind. (Draft Guidance) (2013) htttp://www.fda.gov/downloads/drug/guidancecomplianceregulatoryinformation/guidances/ucm368107.pdf. [10] P.S. Bonato, A.R.M. Oliveira, F.J.M. Santana, B.H.D. Fernandes, V.L. Lanchote, A.E. Gonzalez, H.H. Garcia, O.M. Takayanagui, Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry, J. Pharm. Biomed. 44 (2007) 558–563. [11] W. Hanpitakpong, V. Banmairuroi, B. Kamanikom, A. Choemung, K. Na-Bangchang, A high-performance liquid chormatografic method for determination of praziquantel in plasma, J. Pharm. Biomed. 36 (2014) 871–876. [12] B.H. Ali, A short review of some pharmacological, therapeutic and toxicological properties of praziquantel in man and animals, Pak. J. Pharm. Sci. 19 (2) (2006) 170–175. [13] S. Xie, B. Pan, B. Shi, Z. Zhang, X. Zhang, M. Wang, W. Zhou, Solid lipid nanoparticle suspension enhanced the therapeutic efficacy of praziquantel against tapeworm, Int. J. Nanomed. 6 (2011) 2367–2374.
[14] L. Yang, Y. Geng, H. Li, J. You, Y. Chang, Enhancement the oral bioavailability of Praziquantel by incorporation into solid lipid nanoparticles, Pharmazie 4 (2009) 86–89. [15] H.M. Ding, Y.Q. Ma, Theoretical and computational investigations of nanoparticle-biomembrane interactions in cellular delivery, Small (2014). [16] L. Shang, K. Nienhaus, X. Jiang, L. Yang, K. Landfester, V. Mailander, T. Simmet, U. Nienhaus, Nanoparticle interaction with live cell: quantitative fluorescence microscopy of nanoparticle size effects, Beilstein J. Nanotechnol. 5 (2014) 2388–2397. [17] H. Kettiger, A. Schipanski, P. Wick, J. Huwyler, Engineered nanomaterial uptake and tissue distribuition: from cell to organism, Int. J. Nanomed. 8 (2013) 3255–3269. [18] M. Boegh, C. Foged, A. Mullertz, H.M. Nielsen, Mucosal drug delivery: barriers, in vitro models and formulation strategies, J. Drug. Deliv. Sci. Technol. 23 (4) (2013) 383–391. [19] J. Reineke, D.Y. Cho, Y.L. Dingle, P. Cheifetz, B. Laulicht, D. Lavin, S. Furtado, E. Mathiowitz, Can bioadhesive nanoparticle allow for more effective particle uptake from the small intestine? J. Controlled Release 170 (2013) 477–484. [20] F. Chen, D. Lou, J. Yang, M. Zhong, Mechanical and thermal properties of attapulgite clay reinforced polymethylmethacrylate nanocomposites, Polym. Adv. Technol. 22 (2010) 1912–1918. [21] N.M. El-Lakkany, S.H. Seif El-Din, A.A. Sabra, O.A. Hammam, Pharmacodynamics of mefloquine and praziquantel combination therapy in mice harbouring juvenile and adult Schistosoma mansoni, Mem Inst Oswaldo Cruz 106 (7) (2011) 814–822. [22] L.A. Barros, M. Costa-Silva, C.L. Biolchini, R.H. Neves, J.R. Machado-Silva, Effect of praziquantel administration on hepatic stereology of mice infected with Schistosoma mansoni and fed a low-protein diet, Braz. J. Med. Biol. Res. 42 (2009) 812–815. [23] S.S. Botros, O.A. Hammam, N.M. El-Lakkany, S.H. Seif El-Din, F.A. Ebeid, Schistosoma haematobium (egyptian strain): rate of development and effect of praziquantel treatment, J. Parasitol. 94 (2) (2008) 386–394. [24] H. Wang, Z. Fang, Y. Zheng, K. Zhou, C. Hu, K.W. Krausz, D. Sun, J.R. Idle, F.J. Gonzalez, Metabolic profiling of praziquantel enantiomers, Biochem. Pharmacol. 90 (2) (2014) 166–178. [25] J. Utzinger, E.K. N’Goran, C.R. Caffrey, J. Keiser, From innovation to application: social-ecological context, diagnostics, drugs and integrated control of schistosomiasis, Acta Trop. 120 (2011) 121–137. [26] R.M. Lima, M.A.D. Ferreira, T.M.J. Ponte, M.P. Marques, O.M. Takayanagui, H.H. Garcia, E.B. Coelho, P.S. Bonato, V.L. Lanchote, Enantioselective analysis of praziquantel and trans-4-hydroxypraziquantel in human plasma by chiral LC–MS/MS: application to pharmacokinetics, J. Chromatogr. B 877 (2009) 3083–3088. [27] R.S. Kadam, D.W. Bourne, U.B. Kompella, Nano-advantage in enhanced drug delivery with biodegradable nanoparticles: contribution of reduced clearance, Drug Metab. Dispos. 40 (7) (2012) 1308–1388. [28] J.T. Italia, D.K. Bhatt, V. Bhardwaj, K. Tikoo, M.N.V. Ravi Kumar, PLGA nanoparticles for oral delivery of cyclosporine: Nephrotoxicity and pharmacokinetic studies in comparison to Sandimmune Neoral® , J. Control Release 119 (2007) 197–206. [29] J. Zhang, M. Li, Z. Liu, L. Wang, Y. Liu, N. Zhang, Preclinical studies of N3-O-toluyl-fluorouracil-loaded lipid-based nanosuspensions in H22—bearing mice, Int. J. Nanomed. 9 (2014) 2741–2751.
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Preclinical pharmacokinetic evaluation of praziquantel loaded in poly (methyl methacrylate) nanoparticle using a HPLC–MS/MS Mayara Malhado a , Douglas P. Pinto b , Aline C.A. Silva b , Gabriel P.E. Silveira b , Heliana M. Pereira b , Jorge G.F. Santos Jr. c , Carla V.V. Guilarducci-Ferraz a , Alessandra L. Vic¸osa d , Márcio Nele e , Laís B. Fonseca b , José Carlos C.S. Pinto f , Sabrina Calil-Elias a,∗ a Laboratório de Farmacologia, Departamento de Farmácia e Administrac¸ão Farmacêutica, Programa de Pós-graduac¸ão em Ciências Aplicadas a Produtos para Saúde, Faculdade de Farmácia, Universidade Federal Fluminense, Niterói, RJ, Brazil b Laboratório de Farmacocinética, Vice-Presidência de Produc¸ão e Inovac¸ão em Saúde, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, Brazil c Laboratório de Engenharia de Polimerizac¸ão, COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil d Instituto de Tecnologia em Fármaco, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, Brazil e Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil f Engenharia Química, COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 8 May 2015 Received in revised form 17 September 2015 Accepted 18 September 2015 Available online 25 September 2015 Keywords: Praziquantel Poly (methyl methacrylate) nanoparticle Pharmacokinetic HPLC–MS/MS
a b s t r a c t Praziquantel (PZQ) is the drug recommended by the World Health Organization for treatment of schistosomiasis. However, the treatment of children with PZQ tablets is complicated due to difficulties to adapt the dose and the extremely bitter taste of PZQ. For this reason, poly (methyl methacrylate) nanoparticles loaded with Praziquantel (PZQ-NP) were developed for preparation of a new formulation to be used in the suspension form. For this reason, the main aim of the present study was to evaluate the pharmacokinetic (PK) profile of PZQ-NP, through HPLC–MS/MS assays. Analyses were performed with an Omnisphere C18 column (5.0 m × 4.6 mm × 150.0 mm), using a mixture of an aqueous solution containing 0.1 wt% of formic acid and methanol (15:85—v/v) as the mobile phase at a flow rate of 0.800 mL/min. Detection was performed with a hybrid linear ion-trap triple quadrupole mass spectrometer with multiple reactions monitoring in positive ion mode via electrospray ionization. The monitored transitions were m/z 313.18 > 203.10 for PZQ and m/z 285.31 > 193.00 for the Internal Standard. The method was validated with the quantification limit of 1.00 ng/mL, requiring samples of 25 L for analyses. Analytic responses were calibrated with known concentration data, leading to correlation coefficients (r) higher than 0.99. Validation performed with rat plasma showed that PZQ was stable for at least 10 months when stored below −70 ◦ C (long-term stability), for at least 17 h when stored at room temperature (RT, 22 ◦ C) (short-term stability), for at least 47 h when stored at room temperature in auto-sampler vials (post-preparative stability) and for at least 8 successive freeze/thaw cycles at −70 ◦ C. For PK assays, Wistar rats, weighing between 200 and 300 g were used. Blood samples were collected from 0 to 24 h after oral administration of single doses of 60 mg/kg of PZQ-NP or raw PZQ (for the control group). PZQ was extracted from plasma by liquid–liquid extraction with terc-butyl methyl ether. The values obtained for maximum concentration (Cmax ) and area under curve (AUC) for the PZQ-NP group were about 3 times smaller than the respective values obtained for the control group. However, the time for achieving maximum concentration (Tmax ), the elimination constant (Ke) and the half-life time of elimination (T½ ) were not statistically different. These results suggest that PZQ absorption is probably the rate-limiting step for obtainment of better PK parameters for PZQ-NP. Thus, further studies are needed to understand both the PZQ-NP absorption mechanisms and the drug diffusion process through the polymer matrix in vivo, in order to improve the PZQ-NP release profile. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Rua Mario Viana, 523–Santa Rosa–Niterói – RJ, CEP: 24241-000, Brazil. E-mail address: [email protected] (S. Calil-Elias). http://dx.doi.org/10.1016/j.jpba.2015.09.023 0731-7085/© 2015 Elsevier B.V. All rights reserved.
Schistosomiasis is a parasite disease caused by worms of the genus Schistosoma. According to World Health Organization
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2. Materials and methods 2.1. Chemicals and reagents
Fig. 1. Chemical structures of Praziquantel.
(WHO), schistosomiasis is one of the major public health problems worldwide, associated with severe mortality and morbidity and affecting approximately 240 million people in the world. More than 700 million people live in schistosomiasis endemic areas, with higher infection rates in children than in adults. In tropical and subtropical areas, schistosomiasis is the second most important disease in terms of socio-economic aspects, exceeded only by malaria [1]. The strategy to control schistosomiasis aims to prevent morbidity through regular treatment with praziquantel (PZQ) (Fig. 1a), which is currently the only drug recommended for schistosomiasis treatment [1]. However, the treatment of children with PZQ tablets is especially complicated because of both the difficulty to adjust the doses (the recommended dose is 40 mg/kg) and the nonadherence to therapy because of the extremely bitter taste of the active pharmaceutical ingredient (API) praziquantel [2]. The development of pharmaceutical forms containing PZQ as active ingredient is difficult due to the hydrophobicity of this drug. However, the preparation in polymer nanoparticles has emerged as a promising alternative for development of drugs with poor water solubility [3]. In addition, nanoparticles can increase the bioavailability, improve the doses proportionality, reduce the variability in fed or fasted individuals, reduce inter-patient variability, improve the absorption rate and mask the taste [4,5]. Based on the previous remarks, poly (methyl methacrylate) (PMMA) nanoparticles loaded with PZQ (PZQ-NP) were synthesized [4] for posterior preparation of PZQ suspensions. The PMMA nanoparticles loaded with PZQ were prepared by “in situ” freeradical miniemulsion polymerization. The preparation comprises two phases, organic and aqueous. First PZQ was dissolved in the mixture with MMA, mineral oil (used as a co-stabilizer) and Ethylene Glycol Dimethacrylate (EDGMA) (used as crosslinking agent) using a magnetic stirrer. The aqueous phase was prepared with water, sodium dodecyl sulfate and bicarbonate. The phases were emulsified with the help of the high-pressure homogenizer with pressure drop of 800 bar for 10 min at 85 ◦ C. After this process, the emulsion was transferred to the reactor and the initiator (potassium persulfate) was added to the reaction medium under agitation of 1000 rpm for 120 min. The system was kept to 90 ◦ C. The final emulsion was dried by lyophilization at −50 ◦ C and 0.4 mbar. Before preparing the new formulation, though, it is necessary to understand the pharmacokinetics (PK) of the new product. It is important to mention that the study of drug pharmacokinetics in animal models is included as a preclinical stage during the development of new drugs [6–8]. For this reason, the main objective of the present study was to evaluate the pharmacokinetic profile of PZQ-NP and to compare obtained results with the pharmacokinetic profile of PZQ in the free form, after oral administration in rats. In order to do that, a sensitive and specific analytical method for analysis of PZQ in rat plasma was developed, using coupled high performance liquid chromatography and efficiency tandem mass spectrometry (HPLC–MS/MS).
PMMA Nanoparticles loaded with PZQ were provided by Laboratório de Engenharia de Polímeros (EngePol/COPPE), Universidade Federal do Rio de Janeiro (Rio de Janeiro, RJ, BRA). PZQ was provided as a pharmaceutical grade by Farmanguinhos (Maker Yixing City Xing Yu Medicine Chem., Yixing, JS, CHN). MMA (purity of 99.9%) was purchased from Vetec (Duque de Caxias, RJ, BRA), EGDMA (purity of 98%) provided by Sigma–Aldrich (St. Louis, MO, USA), sodium dodecylsulfate, sodium bicarbonate and potassium persulfate were provided as commercial grades by Vetec (Duque de Caxias, RJ, BRA). The mineral oil was provided as a pharmaceutical grade by Isofar (Duque de Caxias, RJ, BRA) and water was distilled and deionized prior to use. The PZQ reference standard were provided by United States Pharmacopoeia (Rockville, MD, USA) and the internal standard (I.S.), Diazepam (DZP), was provided by INCQS—Instituto Nacional de Controle de Qualidade em Saúde (Rio de Janeiro, RJ, BRA) (both with minimum purity of 99.9%). Acetonitrile and methanol were purchased from Tedia (Fairfield, OH, USA). Terc-butyl methyl ether (TBME) was purchased from J.T Baker (Phillipsburg, MT, USA). All solvents used in the present study presented HPLC grade. Ultrapure water was provided by a MilliQ system from Millipore (Molshein, AL, FRA). Formic Acid 96% was purchased from Spectrum (New Brunswick, NJ, USA). 2.2. Instrumentation and HPLC–MS/MS conditions 2.2.1. Equipment Analyses were performed with a EkspertTM ultra LC 110 system (Eksigent/AB Sciex, Redwood City, CA, USA) coupled to a QTrap 5500 linear ion trap quadrupole mass spectrometer (AB Sciex, Franingham, MA, USA). Obtained data were processed with the proprietary Analyst softwareTM (version 1.6.1). 2.2.2. Analytical conditions Chromatographic separation was carried out with a Omnisphere C18 column (5 m × 4.6 mm × 150 mm) (Agilent Technologies, Santa Clara, CA, USA), using the isocratic elution technique. The mobile phase consisted of a mixture of methanol and an aqueous solution containing 0.1 wt% of formic acid (85:15, v/v). The flow rate was equal to 0.800 mL/min (split ratio 1/1). The autosampler and column oven temperatures were equal to 15 ◦ C and 40 ◦ C, respectively. Sample injection volumes of 10 L were used. The mass spectrometer was operated with electrospray ionization (ESI) in the positive mode. Nitrogen was used as the nebulizer and auxiliary gas. Important operation parameters were: (i) source temperature of 500 ◦ C, (ii) curtain gas at 25 psi, (iii) nebulizer gas (GS1) at 60 psi and (iv) heater gas (GS2) at 60 psi. Collision-activated dissociation (CAD) gas was medium. The ion spray voltage was equal to 5500 V, while the entrance potential (EP) was equal to 10 V. The declustering potential (DP), collision energy (CE) and collision exit potential (CXP) were equal to 130, 15 and 20 V for PZQ and 91, 29 and 12 V for DZP, respectively. The detector was operated at 1900 V. Quantification was carried out using multiple reaction monitoring (MRM). The MRM transitions selected were m/z 313.18 > 203.10 for PZQ and m/z 285.31 > 193.00 for DZP. 2.3. Sample preparation 2.3.1. Preparation of calibration standards and quality control samples The stock of PZQ solution was prepared in methanol/water (50:50—v/v) at a concentration of 1.0 mg/mL. Working solutions of PZQ were obtained by diluting the stock solution
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with the same diluent (methanol/wate—50:50–v/v) until the desired final concentrations in the range of 1.0–1000 ng/mL. The I.S. solution was prepared in methanol/water (50:50—v/v) at a concentration of 500 ng/mL. The standard calibration samples were prepared by spiking 25 L of blank rat plasma with 25 L of corresponding working solution to yield eight standards with concentration of 1.0 ng/mL, 2.5 ng/mL, 5.0 ng/mL, 10.0 ng/mL, 100 ng/mL, 400 ng/mL, 800 ng/mL and 1000 ng/mL. For validation, quality control (QC) samples were prepared with similar procedures at five concentrations: lower limit quantification quality control (LLQQC) = 1.0 ng/mL, low quality control (LQC) = 2.5 ng/mL, medium quality control (MQC) = 400 ng/mL, high quality control (HQC) = 800 ng/mL and dilution quality control (DQC) = 1000 ng/mL, diluted to 800 ng/mL. All standard calibration samples and quality control samples were spiked with 500 ng/mL of I.S.
2.3.2. Plasma preparation Aliquots of 25 L of I.S solution were added to 25 L of plasma samples and mixed for 1 min. Then, 1000 L of the extraction solution, TBME (100%), were added. After mixing in automatic shakers for 10 min, samples were submitted to centrifugation at 14,000 rpm at −10 ◦ C for 5 min. Then, 900 L of the organic phase were transferred to a clean tube and evaporated under nitrogen flow. The residue was reconstituted in 400 L of the mobile phase (methanol/0.1% formic acid in water—60:40—v/v) and 10 L were injected into the HPLC–MS/MS system for analysis.
2.4. Validation method In all validation tests, precision was expressed as the CV (%), and accuracy was expressed as the Bias (%), as deviation of the obtained mean from nominal concentration values. The precision (CV%) must not exceed 15% for all levels (20% for the LLQQC as exception), and the accuracy (Bias%) must be within ±15% of the nominal value for all levels (±20% of the nominal value for the LLQQC as exception). The method was validated based on FDA Guidance for industry [9].
2.4.1. Linearity The calibration curve consisted of a double blank sample (rat plasma sample processed without I.S), a blank sample (rat plasma processed with I.S), and eight calibration samples at concentrations of 1.0 ng/mL, 2.5 ng/mL, 5.0 ng/mL, 10.0 ng/mL, 100 ng/mL, 400 ng/mL, 800 ng/mL and 1000 ng/mL.. For the calibration and the run to be valid, the coefficient of correlation (r) should be greater than 0.99, and the accuracy of at least 75% of calibration samples had to remain within ±15%. 2.4.2. Accuracy and precision Eight replicates of QC samples at five concentration levels (LLQQC, LQC, MQC, HQC and DQC) were processed as described in Section 2.3.2 on three different days to determine intra-day and inter-day accuracies and precision. The dilution quality control was used in order to assess the reliability of the method at concentration levels outside of the upper limit of quantification (1000 ng/mL).
2.4.3. Freeze and thaw stability Eight replicates at QC sample concentrations of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were submitted to eight freeze/thaw cycles (freezing at −70 ◦ C for at least 12 h and thaw at 22 ± 2 ◦ C). These samples were then processed and quantified with a set of calibration samples that were not submitted to the freeze/thaw cycles.
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2.4.4. Stability on bench-top at room temperature (short-term stability) Eight replicates at QC sample concentrations of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were prepared and kept at RT for 17 h: 54 min before processing, as described in Section 2.3.2, and quantifying with a set of calibration samples that were processed immediately. The I.S. was added into QC samples only when it was also added into the calibration samples, in order to ensure that samples were analyzed under similar conditions. 2.4.5. Post-preparative stability Eight replicates of QC samples at concentration of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were processed and quantified. After 47 h: 55 min of storage in the auto-sampler (RT and protected from light), the run was re-injected and re-analyzed with freshly prepared calibration samples. 2.4.6. Long-term stability Eight replicates of QC samples at concentrations of 2.5 ng/mL (LQC) and 800 ng/mL (HQC) were prepared and kept at −70 ◦ C for 10 months. Afterwards, the samples were processed as described in Section 2.3.2 and quantified with a set of calibration samples that were processed immediately. The I.S. was added into the QC samples only when it was also added into the calibration samples, in order to ensure that samples were analyzed under similar conditions. 2.5. Pharmacokinetic study 2.5.1. Animals Twenty female Wistar rats (weighing between 200 and 300 g) from Centro de Animais de Laboratório of Universidade Federal Fluminense (Rio de Janeiro, RJ, BRA) were used. The rats were maintained on a 12 h/12 h light/dark cycle, housed in plastic cages and received a standard chow and water ad libitum. Before and after PZQ oral administration, rats were fasted for food (2 h) and water (1 h). All animal experiments were performed according to the policies and guidelines of the ethics committee of animal use of Universidade Federal Fluminense (Niterói, RJ, BRA) and the study was approved by its ethics committee (ethical permission number 378/2013). 2.5.2. Design of pharmacokinetic experiments Animals were divided into three groups. The control group (n = 5) received 1 mL of vehicle (aqueous solution containing 2% of Cremophor EL); the group PZQ-NP (n = 6) received 60 mg/kg of PZQNP dissolved in 1 mL of vehicle; and group PZQ-L (n = 9) received 60 mg/kg of PZQ in free form dissolved in 1 mL of vehicle. Approximately 150 L of blood was collected into heparinized syringes from tail vein at 0 (pre-dose), 5 min, 10 min, 20 min, 30 min, 1 h, 1 h: 30 min, 2 h, 4 h, 8 h, 10 h, 12 h and 24 h after oral administration of a single dose. Blood samples were centrifuged at 4000 rpm for 10 min. Then, the plasma was stored at −20 ◦ C until analysis. 2.5.3. Sample analysis Plasma samples were processed using the liquid–liquid extraction procedure described in Section 2.3.2 and analyzed using HPLC–MS/MS method described in Section 2.2. A calibration curve was built using weighted linear regression, as described in Section 2.4.1, and plasma sample concentrations were calculated. 2.5.4. Pharmacokinetic data analysis Non-compartmental analyses were performed with the Phoenix WinNonlin software (version 6.3, Pharsight, St. Louis, MO, USA). The pharmacokinetic (PK) parameters determined were the maximum concentration (Cmax ), time to achieve maximum concentration
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(Tmax ), area under the curve (AUC0-t and AUC 0−∞ ), the terminal elimination rate constant (Ke) and the terminal elimination halflife (T½  ). Parameters Cmax and Tmax were extracted directly from the plasma concentration (Cp) versus time curve, AUC 0−t was calculated with a linear trapezoidal method, AUC0−∞ was calculated by combining AUC0−tlast with Ctlast /Ke, Ke was obtained by calculating the slope of the decay phase from the graph of ln(Cp) versus time and T½  was calculated using the equation: T½  = 0.693/ Ke. Outliers were detected with the Dixon and Chauvente tests. 2.6. Statistical analysis The graphics were built using GraphPad Prism® software (version 5.0, San Diego, CA, USA). Mann–Whitney Test was used for comparison between two groups of data. The results were expressed as the mean ± standard error. Significance was evaluated at P < 0.05. 3. Results and discussion 3.1. HPLC–MS/MS analysis The MS spectra of PZQ and I.S. (DZP) showed a retention times equal to 2.78 min for PZQ and 2.98 min for I.S and the run time was equal to 4 min. The range of linear response of 1.0–1000 ng/mL was established by determining the accuracy and precision of calibration curves. For all runs, calibration curves and QC samples met the required acceptance criteria. Typical MRM (Multiple Reaction Monitoring) chromatograms of blank rat plasma spiked with PZQ and I.S. are shown in Fig. 2. Bioanalytical method was developed and validated using high performance liquid chromatography tanden mass spectrometry (HPLC–MS). Analyses were shown to be rapid, sensitive, accurate, reproducible and simple (sample preparation process held in a single step) for analyses of PZQ in rat plasma. In tests conducted by Bonato et al. [10] and Hanpitakpong et al. [11] for plasma analyses in rats using HPLC, the limit of quantitation was 5.0 ng/ml and sample volumes of 1 mL were employed. However, in the present study, we developed an analytical method with range of quantification of 1.0–1000 ng/mL, using 25 L of plasma samples. This is a great advantage for experiments where multiple blood samples must be characterized in short times, especially because of the wide concentration interval, as in most pharmacokinetics and toxicology studies. The application of mass spectrometry ensures the selectivity and specificity of the method, since this technique monitors fragments of highly specific mass/charge (m/z) ratios. 3.2. Validation 3.2.1. Linearity test The response versus concentration data, in the range 1.0–1000 ng/mL of PZQ, was assessed by analyzing the calibration curves using the peak area ratios of analyte/I.S versus the nominal concentrations of the analyte/I.S. calibration standard with a weighting factor (1/x2 ). Obtained correlation coefficients (r) were always higher than 0.99, indicating that all calibration curves met the acceptance criteria. 3.2.2. Accuracy and precision The intra-day and inter-day precision CV% did not exceed 14.01% at any QC concentration levels. The intra-day and inter-day accuracy (Bias%) was between −10.43% and +8.88%, and between −10.42% and +10.52% respectively (Table 1), indicating the occurrence of random fluctuations and absence of consistent bias. Since
both intra-day and inter-day precision were below 15% and accuracy was within ±15%, the proposed bioanalytical method could be regarded as precise and accurate. The DQC results showed that the dilution process could be realized for samples with concentrations above upper limit of quantification. 3.2.3. Freeze–thaw stability After eight freeze–thaw cycles for two concentration samples (2.5 and 800 ng/mL), the precision (CV%) did not exceed 5.81%. The accuracy (Bias%) was between −0.30% and −6.72% (Table 2), indicating the possible occurrence of negative bias. However, based on the proposed performance indices, PZQ in rat plasma could be regarded as stable after eight freeze/thaw cycles at −70 ◦ C. 3.2.4. Bench-top stability at room temperature After storage for 17 h: 54 min at room temperature, the precision (CV%) of the QC samples did not exceed 4.68% and the accuracy (Bias%) was between −6.50 % and 1.59 % (Table 2), indicating the occurrence of random fluctuations and absence of consistent bias. This indicated that PZQ in rat plasma was stable after storing at room temperature prior to processing. 3.2.5. Post-preparative stability Before and after 47 h: 55 min of storage (RT, protected from light) of the QC samples, the precision (CV%) did not exceed 9.93% and the accuracy (Bias%) was between −3.50% and −8.88% (Table 2), indicating the possible occurrence of negative bias. However, based on the proposed performance indices, PZQ in rat plasma could be regarded as stable after post-preparative process. 3.2.6. Long-term stability After storage for 10 months at −70 ◦ C, the precision (CV%) of the QC samples did not exceed 2.75% and the accuracy (Bias%) was between −3.13% and 11.43% (Table 2), indicating the occurrence of random fluctuations and absence of consistent bias. This indicated that, when stored at −70 ◦ C, PZQ in rat plasma could be regarded as stable for a minimum of 10 months without significant decomposition of the drug. 3.3. Pharmacokinetic data analysis Pharmacokinetic studies were performed to determine the absorption, bioavailability and elimination profile of praziquantel administered in PZQ-NP after single oral doses and to compare the obtained PK profiles with the pharmacokinetic profile obtained for PZQ administered in its free form. The values obtained for Cmax and AUC for PZQ-NP group were about 3 times lower than obtained for the PZQ-L group. Tmax, Ke and T 1/2 were not statistically different. The present study demonstrated that PZQ is released in vivo from PZQ-NP and is partially absorbed in the gastrointestinal tract after oral administration in rats. After outlier detection, two animals from the PZQ-L group and one animal from the PZQ-NP group were excluded, according to criteria established by the Dixon and Chauvente’s tests. Then, the pharmacokinetic parameters was calculated with five animals in control group (n = 5), seven animals in PZQ-L group (n = 7) and five animals in PZQ-NP group (n = 5). PZQ in rat plasma was observed from 5 mins until 24 h after oral administration in PZQ-L and PZQ-NP groups. In the control group, PZQ was not detected in any of the analyzed time, demonstrating that the vehicle used (water with 2% Cremophor EL) was free of the drug, Cremophor EL does not interfere with the analytical setup and there was no cross-contamination during sample preparation and analysis. The absorption was approximately three times lower in the PZQNP group when compared to PZQ-L. PZQ is highly lipophilic and crosses the gastrointestinal tract membranes by passive diffusion,
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Fig. 2. The HPLC–MS/MS chromatograms of (A) blank of plasma spiked with PZQ and DZP (10 ng/mL and 500 ng/mL, respectively), (B) plasma sample from a rat obtained at 5 min after oral administration of 60 mg/kg of PZQ loaded in PZQ-NP with I.S. (11.52 ng/mL and 500 ng/mL, respectively).
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Table 1 Summary of precision and accuracy assay (intra-day and inter-day) for PZQ in rat plasma. Nominal concentration (n = 8) Precision and accuracy of control Intra-day
Inter-day
Day 1
1.0 ng/mL 2.5 ng/mL 400 ng/mL 800 ng/mL 800 ng/mL
Day 2
Mean ± S.D
CV(%) Bias(%) Mean ± S.D
1.09 ± 0.10 2.39 ± 0.11 361.26 ± 34.04 725.05 ± 19.35 716.53 ± 30.52
9.51 4.42 9.42 2.67 4.26
8.88 −4.30 −9.69 −9.37 −10.43
1.20 ± 0.17 2.28 ± 0.19 388.80 ± 18.43 798.68 ± 23.69 817.99 ± 35.84
Day 3 CV(%)
Bias(%) Mean ± S.D
14.01 8.45 4.74 2.97 4.38
19.75 −8.85 −2.80 −0.16 2.25
0.97 ± 0.10 2.21 ± 0.11 362.37 ± 10.12 713.71 ± 16.50 721.10 ± 15.77
Overall CV(%) Bias(%) Mean ± S.D 10.35 4.93 2.79 2.31 2.19
−3.38 −11.70 −9.41 −10.79 −9.86
1.08 ± 0.12 2.24 ± 0.04 374.98 ± 13.25 715.82 ± 8.45 751.87 ± 57.31
CV(%)
Bias(%)
10.67 1.61 3.53 1.18 7.62
8.42 −10.42 −6.26 10.52 −6.02
S.D = standard deviation; CV(%) = coefficient of variant; Bias(%) = deviation of mean from nominal value.
Table 2 Stabilities during storage in different conditions (n = 8). Nominal concentration (n = 8)
2.5 (ng/mL) 800 (ng/mL)
Post-preparative stability
Freeze–thaw stability
Mean ± S.D
CV(%)
Bias(%)
Mean ± S.D
CV(%)
Bias(%)
Bench-top stability at RT Mean±S.D
CV(%)
Bias(%)
Long-term stability Mean ± S.D
CV(%)
Bias(%)
2.41 ± 0.24 728.96 ± 44.04
9.93 6.04
−3.5 −8.8
2.49 ± 0.14 746.26 ± 19.26
5.81 2.58
−0.30 −6.72
2.66 ± 0.12 812.75 ± 38.07
4.58 4.68
6.50 1.59
2.79 ± 0.08 774.98 ± 9.43
2.75 1.22
11.43 −3.13
RT = room temperature; S.D = standard deviation; CV(%) = coefficient of variant; Bias(%) = deviation of mean from nominal value.
reaching the bloodstream very fast [12]. The difference in concentrations may be explained by the fact that the absorption of the PZQ trapped in nanoparticles is dependent on the diffusion of the drug through the polymer matrix. Although bioavailability was significantly lower in the PZQ -NP group, more studies are needed to evaluate whether plasma concentrations of PZQ-NP achieves therapeutic margin in the desired dose, and to evaluate their effectiveness. The plasma concentration versus time curves of PZQ obtained after oral administration of a single dose (60 mg/kg) of PZQ in PZQNP are shown in Fig. 3. The oral pharmacokinetic parameters are listed in Table 3. Cmax values in the PZQ-L group were approximately 3 times higher than Cmax values in the PZQ-NP group, with statistically significant difference. Similar behavior was observed for area under curve (AUCo−t and AUCo−∞ ). In order to evaluate this significant AUC difference between the groups, the relative bioavailability (FR) of PZQ in the PZQ-NP, in relation to the bioavailability of PZQ in free form, was calculated in accordance with the following equation: FR =
FR =
ASC
NP ASC0−∞ L 0−∞
QL ×
Q NP
19086.28 15.03 63344.48
×
15.72
FR = 0.2881(28.81%) Note: ASC0−∞ NP: bioavailability from time zero to infinity calculated for the PZQ NP group; ASC0−∞ L: bioavailability from time zero to infinity calculated for the PZQ L group; Q L: average amount (mg) administered of PZQ to PZQ L group; Q NP: average amount (mg) administered of PZQ to PZQ NP group. This means that only 28.81% of the PZQ-NP became bioavailable in respect to the free PZQ. Interestingly both Cmax and AUC from PZQ-NP group were approximately 30% of the values obtained for the PZQ-L group. These results suggest an apparent proportionality between the tested suspensions regarding the pharmacokinetics of praziquantel, since reductions observed in Cmax and AUC from PZQ-NP group are related to reduce in the same proportion of the bioavailable drug in this group. The bioavailability of PZQ in PZQ-NP group was significantly lower. These results differ from those found by Xie et al. [13] and
Yang et al. [14] where solid lipid nanoparticles increased the AUC of PZQ when compared to its free form and tablet, respectively. However, it is known that the absorption process (and consequent bioavailability) depends on the particles physicochemical properties [15–17] and the polymer used in this study and those used by these authors are different. According to Boegh et al. [18] nanoparticles synthesized with bioadhesive polymers may result in greater drug absorption by increasing the carrier system interaction with the intestinal cells. However, the polymer used as the matrix for the development of nanoparticle in study, PMMA, is not bioadhesive [19] and this may be the reason why it does not promote the increase of the bioavailability of PZQ. Thus, studies to determine the diffusion rate, viscoelasticity, speed, direction and transport of PMMA nanoparticles in the mucus need to be performed. Although PMMA is not bioadhesive, it has been widely used in pharmaceutical and biomedical field due to its known biocompatibility, dimensional stability and resistance [20]. Additionally, the use of Cremophor EL was necessary to increase the aqueous solubility of nanoparticles, since these suspension only with water obstructed the gavage cannula. Cremophor EL is a solubilizing and emulsifier agent used by pharmaceutical industries for aqueous preparations of hydrophobic active ingredients. Different studies have used free praziquantel or its reference tablet solubilized in 2% Cremophor EL [21–23] and any interference on the drug pharmacokinetics/pharmacodynamics was reported. However, no studies were found about interactions between PMMA and Cremophor EL. Thus, one hypothesis is the addition of this solubilizing agent may have caused changes in the surface of the nanoparticles which avoided the complete release of drug from the polymer matrix. Another possible hypothesis is that PMMA in biological fluids does not dissolve completely and this may hinder the release of PZQ. PZQ is a chiral compound marketed as the racemate mixture, although its (+)–(S) enantiomer has no proven pharmacological activity [24]. It is biotransforrmed in the liver and its active metabolite (−)-(R)-trans-4-hydroxypraziquantel (4-OHPZQ) seems to possess antischistosomal activity [25]. Lima et al. developed and validated a method for enantioselective analysis of praziquantel and trans-4-hydroxypraziquantel in human plasma by chiral LC–MS/MS [26]. However, in this study it was not possible to evaluate the influence of nanoparticles on biotransformation of PZQ by
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Fig. 3. Concentration-time curve after a single oral dose, in rats, of PZQ-L (n = 7) and PZQ-NP (n = 5). The control group received 1 mL of vehicle. Results are expressed as mean ± SE. Featured: Plasma concentration-time of PZQ until 1 h on groups. Table 3 The non-compartimental pharmacokinetic parameter of PZQ in rat plasma after oral administration of PZQ-NP (n = 5) and PZQ-L (n = 7). GROUP
Pharmacokinetic parameters Cmax {g/L}
Tmax
PZQ-L PZQ-NP
554.23 ± 94.19 172.09 ± 43.84*
0.33 ± 0.04 0.38 ± 0.10
{h}
ASC0−t {g h/L}
ASC0−∞ {g h/L}
Ke {1/h}
T1/2 {h}
1111.91 ± 195.51 326.79 ± 35.54*
1125.00 ± 196.33 336.09 ± 34.63*
0.27029 ± 0.03886 0.27032 ± 0.04276
2.91 ± 0.45 2.85 ± 0.47
Data are mean ± SE. P < 0.05 versus PZQ-L.
*
difficulty in the acquisition of cis and trans-4-hidroxypraziquantel standards, the major PZQ biotransformation’s products reported in the literature [12]. Since nanoparticles have demonstrated to protect the molecules of the first-pass effect in the liver [27,28], research is needed to determine qualitatively and quantitatively the biotransformation products from PZQ loaded in PMMA nanoparticle and to evaluate if they have anthelmintic activity. Both PZQ-NP and PZQ-L reached the maximum concentration (Tmax ) at approximately 20 mins. The Tmax results showed the PZQ-NP do not have a delayed release effect. Similar result was found by Xie et al. [13] for solid lipid nanoparticles containing PZQ. On the other hand, Zhang et al. [29] demonstrated that lipid nanosuspensions significantly increased the Tmax . Since polymer’s nanosystem used in this study is different from those used by the authors cited above, again it is emphasized the importance of chemical and physical characteristics of the material used as matrix for the nanoparticle drug absorption. T½  in PZQ-NP and PZQ-L group was statistically similar, about 3 h. The T1/2 of the free PZQ was approximately 10 times lower than found by Xie et al. [13]. These authors, however, conducted the study in dogs and PZQ were administrated subcutaneously at a dose of 5 mg/kg. This demonstrates that the kinetics of PZQ may vary considerably depending on the species used. Both in PZQ-L and PZQ-NP Ke demonstrated that approximately 27% of the drug had been eliminated from the body per hour. This result Ke suggest that there is no saturation of the elimination process, because although ASC0−∞ of PZQ-NP group was about three times larger, Ke was similar between the groups.
4. Conclusion The analytical method proposed and developed here for determination of praziquantel in rat plasma meets the criteria for application in pharmacokinetic studies. The advantage over previously reported methods are its rapidity, with total run time of four minutes, and its high sensitivity (LOQ, 1.0 ng/mL). Besides, rat plasma sample can be diluted without affecting the precision and accuracy. The pharmacokinetic results suggest the low plasma concentration of PZQ in the group treated with PZQ-NP can be due to in vivo PZQ incomplete release from polymer matrix, which limits the amount of PZQ available to be absorbed via molecular passive diffusion. Further, since PMMA is not a bioadhesive polymer, it is also possible that the residence time of the nanoparticle in the gastrointestinal tract was not large enough to increase the interaction of the carrier system with the intestinal cells, which could be resulted in a lower rate of nanoparticle internalization. In both cases, more studies are needed to understand these nanoparticles behavior in the body in order to improve this nanosystem. In addition, tests must be conducted in order to assess whether PZQ-NP reaches the therapeutic range and if it is effective in the schistosomiasis treatment.
Conflict of interest The authors state no conflict of interest.
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Acknowledgments This research was supported by grants from FAPERJ (Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FINEP (Financiadora de Estudos e Projetos) and BNDES (Banco Nacional de Desenvolvimento Econômico e Social). The authors also thank Farmanguinhos for technical support. References [1] WHO, Schistosomiais (2013) http://www.who.int/schistosomiasis/en. [2] T. Meyer, H. Sekljic, S. Fuchs, H. Bothe, D. Schollmeyer, C. Miculka, Taste, a new incentive to switch to (R)-praziquantel in schistosomiasis treatment, PLoS Negl. Trop. Dis. 3 (2009) e357, http://dx.doi.org/10.1371/journal.pntd. 0000357). [3] K. Landfester, A. Musyanovych, V. Mailander, From polymeric particles to multifunctional nanocapsules for biomedical applications using the miniemulsion process, J. Polym. Sci., Part A 48 (2010) 493–515. [4] L.B. Fonseca, A.L. Vic¸osa, A.C.A. Matos, P.M.Z. Coelho, N. Araújo, H.P.S. Zamith, N.M. Volpato, M. Nele, J.C.C.S. Pinto, Development of a brazilian nanoencapsulated drug for schistosomiasis treatment, Vig. Sanit. Debate 1 (4) (2013) 85–91. [5] R.H. Muller, C. Jacobs, O. Kayser, Nanosuspensions as particulate drug formulations in therapy. Rationale for development and what we can expect for the future, Adv. Drug Deliv. Rev. 47 (2001) 3–19. [6] W. Tang, A.Y.H. Lu, Drug metabolism and pharmacokinetics in support of drug design, Curr. Pharm. Des. 15 (19) (2009) 2170–2183. [7] C.M. Masimirembwa, U. Bredberg, T.B. Andersson, Metabolic stability for drug discovery and development. Pharmacokinetic and biochemical challenges, Clin. Pharmacokinet. 42 (6) (2003) 515–528. [8] R. Panghagnula, N.S. Thomas, Biopharmaceutics and pharmacokinetics in drug research, Int. J. Pharm. 201 (2) (2000) 131–150. [9] FDA, Guidance Ind. (Draft Guidance) (2013) htttp://www.fda.gov/downloads/drug/guidancecomplianceregulatoryinformation/guidances/ucm368107.pdf. [10] P.S. Bonato, A.R.M. Oliveira, F.J.M. Santana, B.H.D. Fernandes, V.L. Lanchote, A.E. Gonzalez, H.H. Garcia, O.M. Takayanagui, Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry, J. Pharm. Biomed. 44 (2007) 558–563. [11] W. Hanpitakpong, V. Banmairuroi, B. Kamanikom, A. Choemung, K. Na-Bangchang, A high-performance liquid chormatografic method for determination of praziquantel in plasma, J. Pharm. Biomed. 36 (2014) 871–876. [12] B.H. Ali, A short review of some pharmacological, therapeutic and toxicological properties of praziquantel in man and animals, Pak. J. Pharm. Sci. 19 (2) (2006) 170–175. [13] S. Xie, B. Pan, B. Shi, Z. Zhang, X. Zhang, M. Wang, W. Zhou, Solid lipid nanoparticle suspension enhanced the therapeutic efficacy of praziquantel against tapeworm, Int. J. Nanomed. 6 (2011) 2367–2374.
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