Pharmacokinetic Investigation of Quetiapine Transport across Blood

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PHARMACOKINETIC INVESTIGATION OF QUETIAPINE TRANSPORT ACROSS BLOOD-BRAIN BARRIER MEDIATED BY LIPID CORE NANOCAPSULES USING BRAIN MICRODIALYSIS IN RATS Fernando Carreño, Karina Paese, Carolina Miranda Silva, Silvia S. Guterres, and Teresa Dalla Costa Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00875 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016

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Molecular Pharmaceutics

PHARMACOKINETIC INVESTIGATION OF QUETIAPINE TRANSPORT ACROSS BLOOD-BRAIN BARRIER MEDIATED BY LIPID CORE NANOCAPSULES USING BRAIN MICRODIALYSIS IN RATS Fernando Carreño1, Karina Paese1, Carolina Miranda Silva1, Silvia S. Guterres1, Teresa Dalla Costa1* 1

Pharmaceutical Sciences Graduate Program, College of Pharmacy, Federal

University of Rio Grande do Sul, Av. Ipiranga, 2759, 90610-000 – Porto Alegre, Rio Grande do Sul, RS, Brazil. Tel.: +55 51 3308-5499.

Emails: Fernando Carreño: [email protected] Karina Paese: [email protected] Carolina Miranda Silva: [email protected] Sílvia S. Guterres: [email protected] Teresa Dalla Costa: [email protected]

* Corresponding author: Teresa Dalla Costa [email protected] Federal University of Rio Grande do Sul College of Pharmacy Pharmaceutical Sciences Graduate Program Av. Ipiranga, 2759, 90610-000 – Porto Alegre, Rio Grande do Sul, RS, Brazil. Phone: +55 51 3308-5418.

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GRAPHICAL ABSTRACT


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ABSTRACT Lipid-core nanocapsules (LCN) have been proposed as drug carriers to improve brain delivery by modulating drug pharmacokinetics (PK). However, it is not clear whether the LCN carry the drug through the BBB or increase free drug penetration due to changes in the barrier permeability. Quetiapine (QTP) penetration to the brain is mediated by influx transporters and therefore might be reduced by drug transporters inhibitiors as probenecid . The goal of this work was to investigate the role of type III LCN on brain penetration of quetiapine (QTP) using microdialysis in the presence

probenecid. Quetiapine loaded LCN (QLNC) was successfully

obtained with a small particle size (143 ± 6 nm), low polydispersity index (PI < 0.1) and high encapsulation efficiency (95.4 ± 1.82 %.). Total and free drug concentration in plasma and free drug concentration in brain were analyzed following i.v. bolus dosing of non-encapsulated drug (FQ) and QLNC formulations alone and in association with probenecid to male Wistar rats. QTP free plasma fraction right after administration of QLNC was smaller than the fraction observed after FQ dosing; however it increased over time until attaining similar free drug levels , suggesting that type III LNC produce an short in vivo sustained release of the drug. The inhibition of influx transporters by PB led to a reduction of free QTP brain penetration, as observed by the reduction of penetration factor from 1.55 ± 0.17 to a value closer to unit (0.94 ± 0.15). However, when the drug was nanoencapsulated the inhibition of influx transporters had no effect on the brain penetration factor (0.88 ± 0.21 to 0.92 ± 0.13) probably because QTP is loaded into LNC and not available to interact with transporters. Taking together these results suggested that LNC type III carried QTP in the blood stream and delivered the drug to the brain. Key-words: Lipid-core nanocapsules, quetiapine, BBB transport, microdialysis



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1. INTRODUCTION

The blood-brain-barrier (BBB) is a challenge for the development of effective drug-delivery therapies targeting the central nervous system (CNS)1–3 due to the endothelial cells tight junctions which restrict paracellular transport of substances and the expression of transporter proteins which modulate the influx and efflux of endogenous and xenobiotic compounds within the brain4. In an attempt to overcome these limitations, different types of nanocarriers have been proposed as drug delivery system for CNS therapeutics. Huang et al5 have found increased area under the curve per dose ratio (AUC/dose) and mean residence time (MRT) of temozolomide in brain and reticuloendothelial cells of rabbits after drug intravenous administration as a solid lipid nanoparticles suspension. Wilson et al.6 showed that brain concentrations of intravenously injected rivastigmine in Wistar rats were over 3.8 fold higher than pure drug when it was bounded to poly(n-butylcyanoacrylate) nanoparticles coated with polysorbate 80 (P-80). In 2009, Jäger et al.7,8 developed a novel polymeric nanocarriers system, named lipid-core nanocapsules (LNC), composed by a core dispersion of medium chain

triacylglycerol

and

sorbitan

monoestearate

enveloped

by

poly(ǫ-

caprolactone) as polymeric wall. Analysis of the obtained vesicular structures by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) revealed a dispersion of solid lipid in the oil core, which confers


these polymeric

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nanocapsules an sustainable release kinetics for drugs dispersed within the lipid core7–9. The use of LNC has been investigated for cerebral drug delivery of different drugs such as indomethacin, trans-resveratrol and olanzapine, resulting in increased cerebral distribution of these molecules, as assessed by tissue homogenates, with significant reduction in side effects and toxicity10–14. Although it has been shown an increase in brain drug concentrations after LNC administration it is not clear whether the nanoparticles carry the drug through the BBB or increase drug penetration due to changes in the barrier permeability. Despite these lipid-core nanocapsules improve targeting to CNS by modulating drug’s pharmacokinetics (PK)11, little is known about this process at BBB level and how changes in plasma PK due to encapsulation can lead to changes in drug’s brain distribution. There is a broad spectrum of in vivo approaches to study BBB targeted delivery via nanocarriers, with most of them requiring the determination of drug concentrations in both blood and brain tissue15,16. Currently, microdialysis (MD) is a valuable tool to study the rate of unbound extracellular drug distribution within the brain, providing data regarding the active substance release from the nanoparticle and the brain drug delivery process1. Moreover, since several influx and efflux transporter superfamilies are expressed at BBB and mediate access of therapeutic drugs within brain parenchyma, the addition of drug transporter inhibitors as verapamil and probenecid in those studies enables to investigate the potential ability of LNC to bypass these membrane transporters and increase the entry and persistence of drug molecules in the brain. 5 ACS Paragon Plus Environment


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Therefore the present work aims to evaluate if LNC type III carry the drug through the BBB. For that purpose QTP was chosen as test compound due to its physicochemical properties (logD7,4 and logP), which lead to the formation of a LCN with higher percentage of the drug distributed into the core of the particle17. Furthermore, QTP is described as a substrate for efflux and influx transporters at BBB18,19, what might provide information regarding ability of LNC to bypass membrane transporters.

2. EXPERIMENTAL SECTION

2.1 Chemicals

QTP

hemifumarate

was

generously

donated

by

Prati

Donaduzzi

Pharmaceutical Industry (Brazil). Caprylic/capric triglyceride and polysorbate 80 were purchased from Delaware (Brazil). Poly(ε-caprolactone) (Mw 60.000 g.mol-1), sorbitanmonostearate (Span 60®) and probenecid were acquired from SigmaAldrich (Brazil). Methanol and acetonitrile with HPLC grade were obtained from Tedia (Brazil). All other chemicals and reagents were of analytical grade.

2.2 Free base QTP conversion and QLNC preparation

QTP hemifumarate salt was converted into the quetiapine free base through a liquid-liquid extraction procedure with ethyl acetate/ammoniun hydroxide 6 ACS Paragon Plus Environment

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saturated solution (50%,v/v)17. The ethyl acetate phase was concentrated under reduced pressure to remove the solvents and the faint yellow oily material was placed into a desiccator cabinet protected from light. Samples were analyzed 72 hours after the extraction procedure by infrared spectroscopy to confirm fumaric acid removal (data not shown). This procedure was necessary in order to increase QTP lipophilicity and improve its encapsulation efficiency. Before each animal experiment, QLNC – 1 mg.mL-1, were prepared by interfacial

deposition

of

pre-formed

polymer

using

a

self-assembling

mechanism8,20. At 40 ºC, the poly(ǫ-caprolactone) (PCL) (0.1g), medium chain triglycerides (160 µL), sorbitan monoestearate (0.019 g) and QTP (0.01 g) were dissolved in acetone (20 mL) and ethanol (3 mL). After complete dissolution, the obtained organic phase was injected into 53 mL of ultrapurifed water containing polysorbate 80 (P-80) (0.078g), at room temperature. The suspension was homogenized for 10 min under magnetic stirring and then concentrated to 10 mL, under reduced pressure, to completely remove ethanol and acetone. Blank lipid core nanocapsules (n = 3 batches) were also prepared in the same manner, but QTP was not added to the organic phase. Non-encapsulated drug formulation, FQ – 2.5 mg.mL-1 was also prepared before each animal experiment. QTP was dissolved in a glucose 5% solution containing P-80 in the same concentration as in QLNC suspension. FQ formulation was placed in the ultrasonic bath for 15 min before use.

2.3 QLNC Physicochemical Characterization



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In order to verify the absence of microparticles and to ensure the particle size were submicrometric, the QLNC and blank nanocapsules suspension were analyzed by laser diffraction technique using a Mastersizer® 2000 instrument (Malvern Instruments, UK). Experiments were carried out with three different batches for each formulation. Diameters were expressed by volume of correspondent sphere D4.3 and the size distribution was expressed by the SPAN value, calculated as describe previously by Venturini et al.8 Mean diameters (z-average) and polydispersity index were determined by dynamic light scattering using Zeta Sizer ZS (Malver Instruments, UK). Samples (n = 3 batches/formulation) were diluted previously (500 x) in ultrapure water and the dilution media were filtered (0.45 µm) before analysis. All measurements were taken at 25 ºC and the results described as mean diameters. The zeta potential was determined on a ZetaSizer Nano ZS (Malvern Instrument, UK) by electrophoretic mobility in triplicate batches of QLNC and blank formulation. Samples were diluted (500x) just before the analysis in NaCl aqueous solution (10 mmol.L-1) and the determinations were carried at 25 ºC. The pH values of formulations were measured in triplicate in three different batches of QLNC and blank nanocapsules, immediately (Day 0) and seven days after (Day 7) the preparation of the formulations using a calibrated potentiometer (Digimed, Brazil) at 25 ºC. Morphological analysis of QLNC were performed using a transmission electron microscope (Jeol JEM 1200, Japan) located at the Center of Eletronic Microscopy of the University. Samples were diluted (1:10) with ultrapurifed water, deposited on a grid (Formvar-Carbon support film, Electron Microscopy Sciences), 8 ACS Paragon Plus Environment

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negatively stained with uranyl acetate solution (2% w/v) and placed at the dessicator for at least 24 h. After that period the analyses were performed at 80kV with 50K, 100K and 300K times magnification. Total drug content and encapsulation efficiency of QTP in QLNC formulation was accessed using a validated LC/UV method17. For total drug content measurement QTP was extracted from QLNC through dilution in methanol (1:100) followed by ultrasonic bath for 20 min. 1000 µL aliquots of the obtained samples were centrifuged at 12.000 rpm for 10 min, the supernatant was filtered (0.45 µm) and 35 µL were injected into the LC system. In order to obtain the encapsulation efficiency free QTP concentrations were determined after formulation centrifugation and ultrafiltration using an Ultracel YM-100 (Amicon®Milipore). The amount of nanoencapsulated QTP was calculated by the difference between total and free QTP concentrations measured in the QLNC and ultrafitrated samples, respectively.

2.4 Pharmacokinetic evaluation

2.4.1 Animals

All in vivo studies were approved by the University’s Ethics Committee in Animal Use (UFRGS/CEUA protocol #25737) and were in accordance with the Council for Control of Animal Experiments (CONCEA) and the National Institutes of Health (NIH) principles of laboratory animal care 21 Male Wistar rats (200 – 250g) were obtained from the University’s animal facilities (CREAL/UFRGS). The animals were acclimatized (5 rats/cage) for at least 9 ACS Paragon Plus Environment


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72 h before the experiments in controlled conditions (22 ± 2 ºC, 65% humidity in a 12 h light/dark cycle) with unrestricted access to food and water.

2.4.2 QLNC plasma pharmacokinetics and brain penetration

QTP total plasma pharmacokinetics and unbound brain concentrations, determined by microdialysis, were evaluated in awake animals after a single intravenous (i.v) bolus dose (5 mg.kg-1) of non-encapsulated QTP(FQ-5 group; n = 7 animals) or QLNC (QLNC-5 group; n = 8 animals) via lateral tail vein. Linearity in QTP plasma pharmacokinetics was investigated by comparing the pharmacokinetic parameters obtained for the FQ-5 group with those obtained after a 10 mg.kg-1 i.v bolus administration to awake rats (FQ-10 group; n = 7 animals). To evaluate the influence of LNC on QTP brain penetration, probenecid (PB), a BBB drug-transporter inhibitor, was used in two groups of animals. PB solution (15 mg.mL-1) was prepared by dissolving the proper amount of drug in glucose 5% solution basified with NaOH (0.5 M, pH 9.0), followed by 15 min in the ultrassonic bath, The final pH of the formulation was set to 7.0 Through the addittion of HCl (0.5 M). Intravenous bolus dose (30 mg.kg-1 ) of PB was administered via right lateral tail vein to animals 30 minutes before i.v. bolus dosing non-encapsulated QTP (10 mg.kg-1 ; FQPB-10 group; n = 6 animals) or QLNC (5 mg.kg-1 - QLNCPB5; n = 7 animals) via the left lateral tail vein. Blood and brain microdialysis samples were harvested up to 8 h after dosing.Blood and microdialysis sampling were performed through ugular vein cannulation and microdialysis probe insertion, 10 ACS Paragon Plus Environment

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respectively.

For

the

surgery,

Wistar

rats

were

anesthetized

with

a

ketamine/xilazine mixture (100 mg.kg-1/10 mg.kg-1,respectively). For blood sampling, a Silastic® medical-grade tubing (Dow Corning, USA) was placed in the right jugular vein and subcutaneously passed to the posterior surface of the neck, out of the reach of the animal. To avoid clotting, the catheter was filled with heparin solution (100 IU/mL in phosphate buffer pH: 7.4 ± 0.1). Once terminated the jugular vein cannulation surgery, the animal was positioned in a stereotaxic apparatus (ASI instruments, USA). A CMA 12 guide cannula was inserted into the rat hippocampus (A:-5.20mm; L: +4.80 mm; V: -4.50 mm)22 and fixed with two screws and dental cement (Vip Flash, Brazil). The rats recovered from the surgery in individual polypropylene boxes for 48 h. One hour before the experiment the animals were placed in a CMA-120 system for freely moving animals (CMA Microdialysis, Sweden) and the guide cannula was carefully replaced with a previously calibrated CMA 12 probe (3 mm PAES membrane and 20 kDa cutoff; CMA Microdialysis, Sweden) for brain microdialysis. In order to stabilize the system, assembled probes were perfused with artificial cerebrospinal fluid (ACF - NaCl 145 mM; KCl 2.7 mM; CaCl2 1.2 mM; MgCl2 1mM, pH 7,4) in a flow rate of 1.5 µL.min-1 for at least 1 h before the start of the drug administration,. Brain microdialysis samples were collected every 30 min up to 8 h after drug dosing; dialysate was analyzed just after the experiment using a previously validated LC-UV method

19

. CMA-12 microdialysis probe relative

recovery in brain (22.9 ± 4.9 %)19 was used to determine the real QTP free brain concentrations.



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Blood samples were collected through the jugular vein at scheduled time points (0 – pre-dose; 0.083; 0.25; 0.5; 1; 2; 4; 6; 8 h after dosing) into heparinized Eppendorf tubes with the replacement of the removed blood volume by an equivalent volume of saline (0.9%) added with heparin (2%). Immediately after the collection, blood samples were centrifuged (5000 rpm for 10 min at 4 ± 2 ºC), 100 µL of plasma was separated and stored at -80 ºC ± 1 ºC until QTP extraction and quantification by a LC/UV method previously validated23. Pharmacokinetic evaluation plasma and brain profiles was performed using the Phoenix (Pharsight) software. The pharmacokinetic parameters: elimination rate constant (λ), area under the curve (AUC0-∞), clearance (CL), half-life (t1/2), mean residence time (MRT) and volume of distribution (Vd) were calculated through Non-compartmental analysis Statistical analysis was performed using the software Prism v. 5.0 (GraphPad Prism, USA). Pharmacokinetic parameters were compared between groups using non-parametric Mann-Whitney test or Kruskal-Wallis analysis of variance, with the significance level set at 0.05.

2.4.3 Unbound QTP plasma concentration-time profile determined by microdialysis in vivo Plasma microdialysis was performed in vivo in order to evaluate the influence of the nanoparticles formulation on QTP plasma protein binding. Two groups of animals (n = 6/group) were used in this experiment. Each group received FQ (10 mg.kg-1) or QLNC (5 mg. kg-1) i.v. bolus administration via lateral tail vein.


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The in vivo free plasma concentrations were determined by microdialysis through the insertion of a CMA 20 probe into the jugular vein. A CMA-20 probe (4 mm membrane length, 20 kDa cutoff; CMA Microdialysis, Sweden) was inserted on the right jugular vein following anesthesia with ketamine/xilazine mixture (100 mg.kg-1/10 mg.kg-1). Before and after the insertion, the probes were perfused for 10 min with a heparinized Ringer’s solution (147 mM NaCl, 1.3 mM CaCl2 and 4 mM KCl, 100 IU / mL heparin) in a flow rate of 3 µL.min-1 to prevent blood clotting in the dialysis membrane. The rats recovered from the surgery in individual polypropylene boxes. Forty eight hours after surgery the CMA-20 probe were perfused with a heparinized Ringer’s solution with a flow rate of 2.0 µL.min-1 for 1 h before the beginning of drug administration, in order to stabilize the system. Rats were dosed by intravenous injection of FQ (FFQ-10; 10 mg.kg-1) or QLNC (FQLNC-5;5 mg.kg1

). Plasma microdialysate samples were collected every 30 min up to 5 h after

dosing and analyzed at the end of the experiment using a validated LC-UV method. The previously described bioanalytical method23 used for the quantification of QTP in brain microdialisate samples was co-validated, according to FDA guidelines24, in order to guarantee that the analytical procedure is suitable for the quantification of the drug in heparinezed Ringer solution. Standard curves (n = 3) ranging from 25 to 1000 ng.mL-1 and QCs samples (n = 6/each) (QCL: 75 ng. mL-1; QCM: 450 ng.mL-1; QCH: 800 ng.mL-1) were used to acess linearity, intra-day precision and accuracy of the method. The method was linear (r2> 0.98), with intraday precision ranging from 2.7 – 6.3% and accuracy ranging from 88.0 – 112%. 13 ACS Paragon Plus Environment


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CMA-20 probes were previously calibrated in vitro by dialysis and retrodialysis, in order to determine the influence of flow rate and QTP concentration and optimize the best conditions for the in vivo experiments (n = 4 probes) as previously described for QTP brain microdialysis

19

. The same probes were also

calibrated in vivo by retrodialysis, to established the relative recovery which was used to correct the free plasma concentrations obtained (n = 4 probes). Briefly, after the surgery recovery period the rats were placed in a CMA/120 system for freely moving animals and microdialysis was perfused with heparinized Ringer (HR) solution at a flow rate of 2.0 µL.min-1 for 40 min. The perfusion fluid was then replaced by QTP 350 ng.mL-1 solution in HR perfused at same flow rate. Dialysate samples were collected each 30 min up to 120 min and injected directly into the LC system for quantification. In vivo RRRD was calculated as described previously by Araujo et al. 25 In order to obtain in vivo plasma protein binding at each free plasma sempling time a compartmental analyses was performed on the total QTP plasma concentration-time

profile,

described

in

section

4.2,

using

the

software

Phoenix®(Pharsight®). The model that best fitted the experimental data was chosen based on the the random distribution of residuals. The model was used to simulated total plasma concentrations for each plasma microdialsys sampling time. The ratio btween the mean QTP free concentrations obtained by plasma microdialysis by the total simulated plasma concentration for each time point was used to determine QTP free fraction in plasma over time.


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2.4.4 FQ and QLCN erythrocytes partition coefficient

To compare the erythrocytes distribution of both formulations (FQ and QLNC) erythrocytes partition coefficient determination was conducted adapting a method from a previously published work26. Red blood cells (RBC) were obtained, via cardiac puncture, from anesthetized male Wistar rats. Total blood was transferred to Falcon tubes containing EDTA solution 10% (w/v) (50 µL EDTA:5 mL of blood). Blood was centrifuged for 15 min at 1500 rpm, the plasma was removed and an isotonic glucose solution (5% w/v), equilvalent to three times the RBC volume, was added to the tubes, the erythrocytes wwere gently ressuspended and centrifuged for 15 min at 2000 rpm. The supernatant was discarded and the washing procedure was repeated four times to remove all plasma proteins. RBC suspension were spiked with QLNC or FQ formulations to yield total drug concentration of 2500 ng.mL-1 and an haematocrit of 0.45. The spiked suspensions were incubated for 15 min before the insertion of a CMA-20 microdialysis probe, previously calibrated by retrodialysis in vitro (RR = 20.5 ± 2.3%), into the RBC medium at 37 ± 1 ºC. Samples were colected every 30 min up to 2 h (Cpw). A concentration reference solution, without erythrocytes, with the same amount of drug an the same volume was prepared in order to obtained the total QTP concentration (Cref). Samples were analyzed using LC/UV validated procedure and the RBC partition coefficiente was calculated, using the folowing equation:



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where Crefis the concentration in the reference solution, Cpw is the free QTP concentration in the plasma water obtained by microdialysis and H is the haematocrit.

3. RESULTS AND DISCUSSION

3.1 Physicochemical characterization of QLNC and blank formulation

The macroscopical analysis of QLNC and blank formulation showed homogeneous suspensions with bluish opalescent aspect in which it was possible to notice the characteristic light scattering of a colloid suspension known as Tyndall effect. The laser diffraction analysis confirmed the presence of particles only on the nanometric scale for all formulations investigated with a mean particle size (D4.3) of 143 ± 6 nm with a span value of 1.54 ± 0.09 for QLNC formulations and 167 ± 8 nm with a span value of 1.75 ± 0.05 for the blank formulations. Span values below 2.0 indicate the narrow particle size distribution for these formulations. The z-average size, mean hydrodynamic diameter, for QLNC formulation was 176 ± 2 nm with monomodal size distribution indicated by a polydispersity index (PI) of 0.068 ± 0.018. Blank nanocapsules presented similar results with a zaverage size of 194 ± 2 nm and a PI of 0.075 ± 0.017.


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QLNC formulation presented a zeta potential, measured based on electrophoretic mobility of the nanoparticles, of –7.65 ± 0.815 mV while blank nanocapsules showed zeta potential of –8.16 ± 0.83 mV. The negative zeta potential is due to the presence of ester groups in the PCL polymeric shell and its close to zero since P-80, which is coating the nanoparticles, causes an steric effect and contributes to the stability of the colloidal suspension7. Morphological analysis of QLNC by transmission electron micrograph (TEM) images showed isolated spherically shaped nanoparticles in all magnifications investigated (Fig. 1).

(A)

(B)

(C)

Figure 1. Photomicrographs obtained by TEM of quetiapine lipid-core nanocapsules: (A) QLNC – 100K magnification (bar: 0.2 µm); (B) QLNC – 300K magnification (bar: 100 nm); (C) QLNC - 500K magnification (bar: 50 nm).

QLNC formulations showed a QTP total concentration of 0.975 ± 0.023 mg.mL-1 with an encapsulation efficiency of 95.4 ± 1.82 %. Taking together, the results of QLNC physico-chemical characterization are in accordance with previous reported results for the same formulation17 and similar LNC

formulations

containing

other

drugs

such


as

indomethacin

and


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olanzapine10,12,14. This type of nanocarrier is composed of biodegradable and biocompatible materials27, and the physico-chemical characterization showed that the formulation has appropriated characteristics (small and homogeneous particle size, higher encapsulation efficiency, type III mechanism of distribution drug distribution in the particle pseudo-phases) for in vivo studies.

3.2 Pharmacokinetic Studies

Mean QTP plasma concentration/dose-time profiles after FQ and QLNC administration to Wistar rats, in the presence and absence of PB, are represented in Fig. 2. Because two different doses of the FQ formulation (5 mg.kg-1 and 10 mg.kg-1) were used during this work due to a QTP reduction in brain exposure in the presence of PB, the concentration-time profiles and area under concentration curve were expressed as a ratio concentration/dose and AUC/dose allowing direct comparisons of the results.


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1000 Concentration (ng.mL-1) / Dose

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100

10 0

2

4

6

8

Time (h)

Figure 2. Average QTP plasma concentration/dose–time profiles obtained after i.v. dosing of FQ-10 (O) (n = 7); QLNC-5 (□) (n = 8) alone or 30 min after PB 30 mg.kg-1 i.v. dosing: PBFQ-10 (●) (n = 6) and PBQLNC-5 (■) (n = 7). Data show mean ± S.D.

Plasma

concentration/dose–time

profiles

show

differences

in

QTP

distribution pattern and total expouse to the drug. FQ-10 group has a faster distribution phase compared to the others groups. Co-administration of nonencapsulated QTP together with probenecid increases the plasma exposure and decreases the velocity of QTP distribution phase due to a reduction in tissue uptake caused by PB. The mean QTP total plasma pharmacokinetic parameters estimated by noncompartmental approach after FQ and QLNC formulation administration alone or 30 min following PB i.v. dosing (30 mg.kg-1) are presented in Table 1.



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Table 1. QTP pharmacokinetic parameters estimated by non-compartmental analysis after i.v bolusadministration of FQ and QLNC formulations in the absence or following i.v. bolus dosing of PB 30 mg.kg-1 Plasma PK

FQ-5

FQ-10

QLNC-5

PBFQ-10

PBQLNC-5

Parameters*

(n = 7/group)

(n = 7/group)

(n = 8/group)

(n = 6/group)

(n = 7/group)

λ (h-1)

0.27 ± 0.10

0.28 ± 0.04

0.17 ± 0.04

0.20 ± 0.05

0.14 ± 0.05

t½ (h)

3.0 ± 1.5

2.5 ± 1.4

4.2 ± 1.1

3.6 ± 1.0

5.6 ± 2.3

600 ± 101 a

583 ± 143a,b

1443 ± 201

833 ± 122a

1290 ± 187

2.79 ± 1.36

2.07 ± 1.21

4.81 ± 1.35

3.44 ± 1.58

6.18 ± 3.15

1.72 ± 0.29a,b

1.71 ± 0.12 a,b

0.70 ± 0.19

1.14 ± 0.27 a,b

0.87 ± 0.23b

4.5 ± 1.5

3.5 ± 0.5

3.2 ± 0.5

3.6 ± 1.3

4.6 ± 1.3

AUC0-∞/Dose -1

-1

(ng.h.kg.mL .mg ) MRT (h) CL -1

-1

(L.h .kg ) Vdss (L.kg-1)

Values represent mean ± SD. λ: elimination rate constant; t1/2, half-life; AUC: area under the concentration–time curve; MRT: mean residence time; -1 CL: clearance; Vdss: volume of distribution. FQ-5 group: free QTP dosing of 5 mg.kg 1; FQ-10 group: free QTP dosing of 10 mg.kg ; QLNC-5 -1 -1 -1 1 group: QLNC dosing of 5 mg.kg PBFQ-10 group: free QTP dosing of 10 mg.kg 30 min after PB (30 mg.kg administration; PBQLNC-5 group: -1 -1 QLNC dosing of 5 mg.kg 30 min after PB (30 mg.kg administration. Kruskal-Wallis analysis of variance (α: 0.05) followed by Dunn’s multiple a b comparison test. Statistically different from QLNC-5 (p < 0.05); Statistically different from PBFQ-10 (p < 0.05).


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No differences were founded when the pharmacokinetic parameters half-life, AUC0-∞/Dose ratio and clearance obtained for the group FQ-5 and FQ-10 were compared (p > 0.05). This indicates that, for QTP, blood levels in Wistar rats change proportionally to the dose administered on the 5-10 mg.kg-1 dose range. Nemeroff et al.

28

have already shown linear QTP pharmacokinetics in a clinical

trial with healthy volunteers. The AUC0-∞/Dose was significantly higher in the QLNC-5 group (1443 ± 201 ng.h.mL-1.kg-1) than in the FQ-5 group (600 ± 101 ng.h.mL-1.kg-1) (p < 0.05) due to a significant decrease in total clearance (QLNC-5: 0.70 ± 0.15 L.h-1.kg-1 and FQ-5: 1. 72 ± 0.29L.h-1.kg-1) (p

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