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Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation
J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014
Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation F. Krier1*, R. Riva2, S. Defrère3, M. Mestdagt4, A. Van Langendonckt3, P. Drion5, J.-P. Dehoux6, J. Donnez3, J.-M. Foidart4, C. Jérome2, B. Evrard1 Laboratory of Pharmaceutical Technology, Department of Pharmacy, CIRM, University of Liège, B36, Tour 4, Level 2, Avenue de l’Hôpital, 1, 4000 Liège, Belgium 2 Center for Education and Research on Macromolecules (CERM), Department of Chemistry, University of Liège, Allée de la Chimie, 3, building B6a, level 3, 4000 Liège, Belgium 3 Pôle de gynécologie, Institut de Recherche expérimentale et clinique, Université Catholique de Louvain, Avenue Hippocrate 10, Level 9, 1200 Brussels, Belgium 4 Laboratory of Tumor and Development Biology, University of Liège, B 23, Tour 3, Level 4, avenue de l’Hôpital,1, 4000 Liège, Belgium 5 Animal facilities-GIGA, B 23 B, Avenue de l’Hôpital, 3, University of Liège, 4000 Liège, Belgium 6 Experimental Surgery Unit, ANIM, Université Catholique de Louvain, Avenue Hippocrate 55, bte B1.55.15, 1200 Brussels, Belgium Fabrice Krier and Raphaël Riva contributed equally to this work. *Correspondence: [email protected] 1
Local treatment using drug loaded implants allows decreasing seric concentrations of the active ingredient with the purpose of limiting side effects and reaching perfect observance. Nowadays, some diseases are already treated with implants, but generally, by subcutaneous or intra vaginal implantation. In this work, a new implant device dedicated to the intra-peritoneal cavity was developed. For this purpose, a core-membrane polymer implant was selected. We propose an original method to determine the most appropriate membrane to control the release based on the use of Franz cells. The ability of the implant to release a constant quantity of an active ingredient will be assessed by testing implants in vitro. Finally, intra peritoneal cavity and subcutaneous in vivo implantation has been achieved in order to confirm the controlled and local release of the active ingredient. Key words: Anastrozol – Implant – Diffusion – EVA – PDMS – Drug delivery system – Intra-peritoneal – Control release – Polymer – Franz cells.
for contraception (etonorgestrel) [11], but also for the conception of contraceptive delivery devices, such as the vaginal ring [13]. A recent study investigated the release of two drugs by an EVA system for the intraoral treatment of oral lesions, blisters, and fungal diseases [14]. The peritoneal cavity seems to be a convenient site for the implantation of a drug delivery system against endometriosis or gastric cancer because large parts of lesion are localized in this region [15, 16]. The in vivo biocompatibility of PDMS and EVA in the intraperitoneal cavity has already been confirmed in a previous publication [17]. The challenge of this research is the production of an implant able to release a constant quantity of active ingredient without any “burst effect” in the intraperitoneal cavity, but also with an accumulation of the active ingredient in this cavity. PDMS and EVA are thus largely applied in the biomedical field, but no example of implantation in the intra-peritoneal cavity of these polymers has been documented, at least to our knowledge.
Ideally, a drug delivery system must be able to deliver a drug to a specific site in a determined period of time with a specific release profile [1]. Controlled release formulations allow a decrease in the amount of the drug necessary to reach the therapeutic effect and maintain sustained drug release over a prolonged period of time, thereby eliminating fluctuations in the drug plasma concentration [2]. These formulations are known to limit side-effects and increase patient compliance, particularly during long-term treatment [3, 4]. Implantable drug delivery systems are one example of controlled drug release formulations already successfully applied for therapeutic use. In the present case, a sustained release of anastrozol between 10 to 25 µg/day over a period of one year (model molecule) has typically been targeted. For this goal, among the various categories of implants (i.e. biodegradable or non-biodegradable implants, implantable pump systems, and the newest atypical class of implants [5]), non-biodegradable implants appear to be the most relevant choice since they must be used over quite a long period while their physical properties remain constant. In this work, implants based on two non-biodegradable polymers were tested for the elaboration of intra-peritoneal drug delivery systems, namely polydimethylsiloxane (PDMS) and poly(ethylene-co-vinyl acetate) (EVA). The PDMS is a biocompatible polymer which has been used for a long time as a biomaterial in urology for the treatment of impotence [6] or incontinence [7], in ophthalmology for intraocular lens [8], and in plastic surgery for breast implants [9], whereas the EVA was initially described as a biomaterial in dental surgery [10]. Levonorgestrel loaded PDMS films have been largely applied as subcutaneously controlled drug delivery devices for contraception [11]. EVA has been used as a subcutaneously controlled drug delivery system for the treatment of alcoholism (nalmefene) [12], and
I. MATERIAL AND METHODS 1. Anastrozol assay for in vitro release studies
1.1. Chemicals and solvents Acetonitrile (LiChrosolv, purity: 99.9 %) was purchased from Merck (Darmstadt, Germany). Anastrozol was supplied by Apin Chemicals Limited (Abingdon, United Kingdom). The structure of anastrozol is presented in Figure 1. Water was purified by a Millipore system (18.2 MΩ/cm resistivity, Milli-Q) before being filtered through a 0.22 µm Millipore Millipak - 40 disposable filter units (Millipore Corporation, United States). The phosphate buffer solutions (pH 7.4; 50 mM and pH 3.0; 50 mM) were prepared with potassium dihydrogen phosphate (Merck), sodium hydroxide (VWR, Liège, Belgium) and sodium azide (Merck). 198
Device-based controlled local delivery of anastrozol into peritoneal cavity: J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014 in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
Table I - Transition, cone voltage and collision energy for anastrozol and dexchlorpheniramine. Analyte
Transition (m/z)
Cone voltage (V)
Collision energy (eV)
Anastrozol Dexchlorpheniramine
294 > 225 275 > 230
25 20
21 18
Table II - Gradient mode details of the UPLC system. Figure 1 - Chemical structure of anastrozol.
Time (min)
Mobile phase A (%)
Mobile phase B (%)
Flow rate (mL/ min)
0.0 5.0 5.1 8.1 8.2 10.1
95 50 5 5 95 95
5 50 95 95 5 5
0.6 0.6 0.6 0.6 0.6 0.6
1.2. Apparatus The chromatographic analyses were performed on an Agilent technologies HPLC 1100 series (Hewlett-Packard, PaloAlto, CA, United States) equipped with a solvent delivery binary pump G1312A, an on-line degasser G1379A, a thermostatised autosampler G1328A, a column oven G1316A and a diode-array detector G1315C. A HewlettPackard computer dc 5100 Mt with Chemstation was used to control the whole chromatographic system and to acquire, process and store all the data obtained. A Mettler Toledo (Schwerzenbach, Switzerland) AT261 scale was used to weigh all the compounds (precision: 10 µg). A Seven Easy Mettler Toledo pH meter was used to adjust the pH value.
formic acid and methanol were analytical grade and applicable for HPLC measurements and were supplied by Biosolve (Valkenswaard, The Netherland). The water was purified as already described in point I.1.1.
1.3. Chromatographic conditions The chromatographic analysis was performed on a Lichrospher 100 C18 end-capped column (250 × 4 mm i.d., 5 µm particle size) which was kept at 30 °C. The mobile phase was a mix of 45 % phosphate buffer (pH 3) and 55 % acetonitrile. The HPLC system was operated in isocratic mode at a flow rate of 1.0 mL/min and the volume injection was 100 µL. UV detection was performed at 210 nm. The method was successfully validated in the range of 50 to 2000 ng/mL by using e-noval version 2.0e (Arlenda s.a., Liège, Belgium). The trueness, precision and accuracy of the method were found to be acceptable. The limits of quantification were evaluated at 50.67 and 2027 ng/mL while the limit of detection was calculated to be 15.36 ng/mL. The accuracy profile obtained by considering linear regression through 0 fitted using the highest level only, is shown in Figure 2.
2.2. Apparatus The chromatographic analyses were performed on a Waters Acquity UPLC inlet system. The mass spectrometry detector was a Waters Quattro Premier XE equipped with an electrospray ionisation source (ESI) operating in positive ion mode. Mass chromatograms were acquired in the multiple reaction monitoring (MRM) mode. One specific transition was monitored for each compound of interest. The selection was made in order to reach the highest detectability. The capillary was set to 3.0 kV, the source temperature was set to 130 ± 5 °C, the desolve temperature was set to 345 ± 5 °C, the cone gas flow was set to 50 L/h. The parameters for transition, cone voltage and collision energy are summarized in Table I. Data acquisitions were achieved using MassLynx Version 4.1 software. An analytical balance was used to weight all compounds (Kern 870 - Kern DKD Labor, Germany).
2. Anastrozol assay for in vivo release studies
2.3. Chromatographic conditions The chromatographic analyses were performed on an Acquity Bech C18 column (100 × 2.1 mm i.d., 1.7 µm particle size) which was kept at 60 °C. The mobile phase was a mix of 10 mM ammonium formate with 0.1 % formic acid and methanol with 0.1 % formic acid. The UPLC system was operated in gradient mode (see Table II) at a flow rate of 0.6 mL/min and the volume injection was 6 µL.
2.1. Chemicals and solvents Chlorpheniramin maleate (internal standard) was purchased from Sigma-Aldrich (Diegem, Belgium). Acetonitrile, ammonium formate,
2.4. Standard solutions For this in vivo quantification method, two solutions of anastrozol in acetonitrile/water (50/50 v/v) were prepared at a concentration of 1 mg/mL, then aliquoted and stored at -20 °C. These solutions were used as standard solutions of anastrozol to prepare the calibration standard and QC samples respectively. The solution of internal standard (chlorpheniramin) was prepared in the same way in order to obtain a stock solution at a concentration of 1 mg/mL [18]. Two kinds of samples were prepared in an independent way: calibration standards and validation standards (or QC samples). One standard solution was used to prepare calibration standards. The calibration standards are plasma samples, containing known concentrations of the analyte of interest, and are only used for calibration. Calibration curves were generated daily over a concentration range between 0.2 and 100 ng/mL. Six concentration levels were selected as follows: 0.2, 1, 5, 25, 50 and 100 ng/mL. Two blank samples were also prepared, one containing only the internal standards.
Figure 2 - Accuracy profile obtained by considering linear regression through 0 fitted using the highest level only. Relative bias (—), ± 10 % acceptance limits (- - -), 95 % β-expectation tolerance limits (– – –), and relative back-calculated concentrations (l). 199
Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014
The validation standards were prepared from another standard solution. They are plasma samples containing known concentrations of the analytes of interest which are considered as true values by consensus. Three concentration levels (1, 25 and 100 ng/ml) representing the whole concentration range were selected for the QC samples. All these validation standards were analysed twice at each sequence of analysis.
formed during the PDMS cross-linking. The total extraction of these residues was assessed by gravimetry. 3.3.2. PDMS implants with PDMS membranes The anastrozol loaded PDMS rods were prepared as described above; they are embedded in a preformed PDMS membrane prepared with the same batch of PDMS/cross-linker/catalyser (Sterne, France). Typically, the PDMS membrane is swollen into heptane with the purpose of increasing the lumen of the tube, allowing the introduction of the anastrozol loaded rod inside the tube. Finally the heptane is removed under vacuum leading to the contraction of the PDMS membrane, which, at the end of the process, was strongly stuck to the rod. The total extraction of heptane was assessed by gravimetry.
2.5. Sample preparation The anastrozol and the internal standard were extracted from the plasma by a protein precipitation method. Fifty microlitres of the internal standard working solution (100 ng/mL) was added to 50 µL of the plasma sample in a micro tube and then the sample was put into a vortex. One hundred microlitres of methanol and 300 µL of acetonitrile were added successively. After 10 min of centrifugation, 150 µL of the supernatant was transferred to a HPLC vial where 50 µL of water was added before stirring with a vortex.
3.3.3. PDMS implants with EVA membranes The PDMS implants containing anastrozol (50 % w/w in the core of the implant) and bearing an EVA membrane were prepared by a technique already described in a previous paper [17]. In this technique, EVA tubes are used as a mould for the direct curing of PDMS by SiOPr4 in the presence of a catalytic amount of SnOct2. The EVA tubes were processed by extrusion of commercially available EVA pellets. Typically, PDMS (19.4 g) was transferred into a sterile container. Freshly phorphyrized anastrozol (19.5 g) was then added to the PDMS. The mixture was homogenized with an ultra-turrax T 25 basic (IKA, Staufen, Germany) with the purpose of obtaining a very efficient mix of the two compounds. After 5 min of mixing, a homogenous blend was obtained, which was kept at -20 °C for 1 h. 0.5 g of tetrapropyl orthosilicate and 0.1 g of SnOct2 were mixed together in a separate glass container. This mixture was then added into the cold PDMS mixture inside a laminar flow hood. The PDMS/anastrozol/cross-linker/ catalyst mixture was manually homogenized for 2 min before being placed under a vacuum for 5 min in order to remove the air bubbles trapped in the blend. Finally the PDMS mixture was transferred into a plastic syringe and kept at -20 °C, for at least 1 h. The PDMS-anastrozol mixture was then injected into the EVA tube (internal diameter of 3 mm, thickness of the wall 200 μm and 15 cm long) under a laminar flow hood. The ends of the tube were closed with a parafilm. Curing of the PDMS was performed at room temperature for 16 h in order to avoid deformation of the EVA tube during the process. Finally, the tubes were cut in order to obtain 2 cm long implants. The implant extremities were closed with MED-2000 adhesive silicone (Nusil technology, Carpinteria, CA, United States) and dried in a Vismara 65 vacuum oven at 950 mbar for 4 h at room temperature with the purpose of removing the propanol formed during the PDMS cross-linking. The total extraction of these residues was assessed by gravimetry. The schematic representation of implant is presented in Figure 3, whereas Figure 4 is a picture of the implant ready to be implanted.
3. Preparation of multi-biomaterial delivery systems
3.1. Membranes for diffusion studies The PDMS membranes were synthesized by the curing of linear PDMS in a mould. Typically, 0.125 g of tetrapropyl orthosilicate and 0.025 g of SnOct2 were mixed together in a glass container. This solution was then added to 4.854 g of PDMS, previously stored at -20 °C for at least 1 h. The PDMS/cross-linker/catalyst mixture was manually homogenized for 2 min before being placed under a vacuum for 5 min in order to remove the air bubbles trapped in the blend. Finally, the PDMS mixture was transferred into a plastic syringe and kept at -20 °C for at least 1 h. The PDMS mixture was then injected into a square-shaped moult (10 cm × 10 cm) covered by two Teflon films. The mould was then placed into a press (30 bars) at 80 °C for 15 min. The PDMS membrane was then collected and dried in a Vismara 65 vacuum oven at 950 mbar for 4 h at room temperature with the purpose of removing the propanol formed during the PDMS cross-linking. The total extraction of these residues was assessed by gravimetry. The EVA membranes were purchased from 3M Health Care (StPaul, MN, United States). 3.2. Materials for implant preparation Polydimethylsiloxane (PDMS, base, medical grade), tetrapropyl orthosilicate (cross-linker, medical grade), tin octoate (SnOct2, catalyst, medical grade), Anastrozol (APIN chemicals, batch 32993a), EVA (Elvax 3185, Dupont, batch 70106161) were used for the preparation of the implants. 3.3. Implant preparation 3.3.1. PDMS implants The PDMS implants containing anastrozol (5 % w/w) were synthesized by curing an anastrozol/PDMS mixture at 80 °C. Typically, PDMS (4.854 g) and anastrozol (0.192 g) were mixed together manually for 5 min. The blend was then kept at -20 °C for 1 h. 0.125 g of tetrapropyl orthosilicate and 0.025 g of SnOct2 were mixed together in a separate glass container. This mixture was then added into the cold PDMS mixture. The PDMS/anastrozol/cross-linker/catalyst mixture was manually homogenized for 2 min before being placed under a vacuum for 5 min in order to remove the air bubbles trapped in the blend. Finally the PDMS mixture was transferred into a plastic syringe and kept at -20 °C, for at least 1 h. The PDMS/anastrozol mixture was then injected into the cylindrical compartment (20 mm long, diameter = 3 mm) of an iron mould coated by Teflon. The mould was then placed into a press (30 bars) at 80 °C for 15 min. The cross-linked anastrozol loaded rods were then collected and dried in a Vismara 65 vacuum oven at 950 mbar for 4 h at room temperature with the purpose of removing the propanol
3.4. Sterilization of implants The implants were sterilized by the ethylene oxide procedure. The preconditioning step was performed for 8 h at 45 ± 5 °C with a relative humidity controlled between 50 and 80 %. The sterilization phase was pursued at 45 °C with a relative humidity level targeted at 60 ± 20 % for 3 h and at a gas concentration of 730 mg/L. Then the aeration process was carried out at 40 ± 5 °C with fresh air in bleed, for 72 h. The process was validated using a biological indicator (initial population 106 spores of Bacillus subtilis). The final ethylene oxide content in the implant after 2 days of airing was equal to 2.5 ppm.
4. In vitro drug diffusion and release kinetics
4.1. Anastrozol diffusion through EVA and PDMS membranes Diffusion studies were carried out using Franz type glass diffusion 200
Device-based controlled local delivery of anastrozol into peritoneal cavity: J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014 in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
4.2. Drug release kinetics from the device Release of anastrozol from the device was studied in triplicate under sink conditions. In order to determine the kinetics of the drug release, three implants of each formulation were placed in three different glass bottles containing 100 mL of phosphate buffer solution (pH 7.4) containing 0.025 % (w/w) of sodium azide. The bottles were placed in a shaking water bath (SW 22, Julabo, Seelback, Germany) at 37 °C. Samples of the buffer solution were collected at specific time intervals. After each collection, an equivalent amount of fresh buffer solution was transferred into each bottle to keep the volume of solution constant throughout the measurement. The amount of drug released was measured by HPLC. Implants without membranes and implants with a PDMS (600, 700, 800 µm) or an EVA membrane (10 weight % of VA) with three different thicknesses (100, 200 and 300 µm) were used in this experiment.
Figure 3 - Schematic representation of the EVA membrane coated implant.
5. In vivo kinetics
5.1. Intraperitoneal placing of anastrozol-loaded implants in rats All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the University of Louvain, Belgium (UCL/MD/2007/020). The “Guide for the Care and Use of Laboratory Animals”, prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, was followed carefully. Fifty-two 10-12 week old, female Wistar rats were used for the present study: 26 rats for subcutaneous (sc) and 26 for intraperitoneal (ip) implantation. The rats were anaesthetized with an injection of ketamin (ip, 30 mg/kg, Anesketin; Eurovet, Heusden-Zolder, Belgium) and medetomidin (ip, 50 µg/kg, Domitor; Pfizer, Cambridge, MA, United States). After surgery, the anaesthesia was reversed by the injection of atipamezol (sc, 0.25 mg/kg, Antisedan; Pfizer). For ip implantation, a median laparotomy of 1 cm was made. One implant per rat was put into the pelvic cavity, without any fixation. The abdominal wall and the skin were then sutured with absorbable suture. For subcutaneous (sc) implantation, a skin incision was made in the upper back of the animal and one implant was placed under the skin. The skin was then sutured with absorbable suture. At the end of the experiment, the rats were euthanatized by CO2 inhalation according to the standard euthanasia guidelines for rodents from the Institutional Animal Care and Use Committee (IACUC) (http://www.iacuc.ucsf.edu/Policies/awGlEuthR.asp).
Figure 4 - Picture of the implant tested in vivo.
cells (Hanson Research, CA, United States) consisting of a donor and a receiver chamber. Typically, the membrane was clamped between these two chambers. The receiver chamber was filled with 7.5 mL of phosphate buffer solution (pH 7.4; 50 mM), which was constantly stirred with a small magnetic bar and thermostated at 37 °C during all the experiments. The donor chamber was filled with a suspension of anastrozol (15 % w/w in the suspension) in an aqueous gel of Carbomer 980 (1 % in water). The test was performed in occlusive conditions. The surface of the membrane in contact with the gel was equal to 1.767 cm². After predetermined time intervals (24, 48, 72, 96 or 144 h), a sample of the receptor phase (2.0 mL) was collected with the purpose of measuring the anastrozol concentration [1]. After each collection, 2.0 mL of fresh phosphate buffer solution was added to the receiver chamber to keep the volume constant throughout the analysis. Several membranes of both polymers were tested: 4 different polymers were evaluated: PDMS, EVA 10 weight % of VA, EVA 28 weight % of VA and EVA of 40 weight % VA. The thicknesses of the different membranes are reported in Table III. This test was performed under “sink conditions” in the receptor compartment.
5.2. Pharmacokinetics studies Blood samples were collected from at least of 3 rats after 1, 4, 7 and 13 weeks for sc implantation and after 1, 5, 12 and 18 weeks for ip implantation for anastrozol measurements. Sera were stored at -80 °C until analysis. Six rats were euthanatized after 18 weeks for ip implantation and three rats after 13 weeks for sc implantation. Peritoneal fluids were then collected by peritoneal washing. Briefly, after ip injection of PBS (1 mL), the rats were gently shaken for 30 s. The peritoneal cavity was then opened, and the peritoneal fluid was collected and centrifuged at 400 g for 5 min. The supernatant was recovered and stored at -80 °C until the anastrozol assay.
Table III - Thickness of polymer membranes. Polymers
Thickness (µm)
PDMS PDMS EVA 10 weight% of VA EVA 10 weight% of VA EVA 10 weight% of VA EVA 28 weight% of VA EVA 28 weight% of VA EVA 40 weight% of VA EVA 40 weight% of VA
700 800 150 400 700 100 200 100 200
II. RESULTS AND DISCUSSIONS 1. In vitro drug diffusion and release kinetics
1.1. Drug release kinetics from PDMS implants without membranes Figure 5 shows the mean diffusion per 24 h of anastrozol in terms of time for PDMS implants without membranes. Clearly, the diffusion is not constant. The burst effect is very important. A diffusion of 431 µg is observed on the first day, while after 212 days of experiments, only 8 µg is released in 24 h. At the end of the experiment, the total drug 201
Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014
Figure 5 - Mean diffusion of anastrozol in the PDMS implant without membrane in terms of time.
Figure 6 - Mean diffusion of anastrozol in the PDMS implant with PDMS membrane in terms of time.
release represented 91.5 ± 5.1 % of the active substance present in the device. With the goal of regulating drug diffusion and achieving a constant release profile for more than 300 days, we decided to coat these implants with a drug-free PDMS membrane. 1.2. Drug release kinetics from PDMS implants with a PDMS membrane Figure 6 shows the mean diffusion per 24 h of anastrozol in terms of time for implants coated by a PDMS membrane (thickness: 600, 700 or 800 µm). The PDMS membrane allows an important decrease of the burst effect; after 24 h, the released drug is 5 times lower for the implant with the membrane. The amount of released anastrozol decreases more slowly than the implant without the membrane, after 212 days, only 84.21 ± 5.14 % of the anastrozol has diffused outside the implant. However, the diffusion is not really controlled and the observed release profile is not constant with such PDMS membranes. Thus, we decided to replace the PDMS membrane with a poly(ethylene-co-vinylacetate) (EVA) membrane whose polarity and crystallinity can be tuned by the vinyl acetate content, and compare them in terms of drug diffusion.
Figure 7 - Amount of anastrozol recovered in the Franz cell receptor phase in terms of time (µg/days) according to the membrane formulation (n = 1).
reduced as demonstrated by Figures 5 and 6. In addition, Figure 7 shows that the diffusion of anastrozol through EVA 28 % and EVA 40 % available membranes remains close to or higher than the tested PDMS membranes. That is why EVA membranes containing 10 weight % of VA have been selected to coat the PDMS implants. In order to release an acceptable quantity of anastrozol, this drug was increased to 50 % in weight in each core of the implant. The extremities of the implants were closed with MED 2000, an adhesive silicone as described in 2.3.3. Figure 8a and b show the mean diffusion per 24 h of anastrozol in terms of time from implants coated with a membrane of 100, 200 or 300 µm thickness. For the three formulations, a sustained release of the drug is obtained for more than 470 days. The burst effect is very limited in time (only 7 days) and in term of quantity (max 35 µg/24 h). Figure 9 shows a linear regression model of the diffusion result between days 50 and 450. The release of anastrozol from implants coated with a 100 µm membrane decreases in terms of time. For implants coated with thicker membranes, the release profile is constant during the 463 days of experiment. A significant influence of the thickness of the membrane can be observed since the mean diffusion per 24 h after 463 days are 22.10 ±1.17 and 16.67 ±3.87 µg for implants coated with 200 and 300 µm, respectively. Based on the targeted drug release, PDMS implants coated with a 200 µm EVA 10 membrane were chosen for in vivo studies.
1.3. Drug diffusion through EVA and PDMS membranes Figure 7 shows the amount of anastrozol detected in the receptor phase of the Franz cells in terms of time for various PDMS and EVA membranes. The diffusion of anastrozol through PDMS membranes of 700 and 800 µm (used in 3.1.2) appears weakly dependent on the membrane thickness and gives comparable results to EVA membranes containing 28 % of VA with a thickness between 100 and 200 µm. This clearly shows that EVA is a much more efficient barrier to drug diffusion than PDMS. It must be mentioned that the three curves for 10 % EVA membrane with three different thicknesses overlaid perfectly but it is impossible to distinguish this in Figure 7. In addition, when comparing EVA films of the same thickness, an increase of the VA content leads to an increase in the diffusion rate of the anastrozol. These observations are in line with a clear decrease in the drug diffusion rate, when the crystallinity of the membrane is increased. Indeed, PDMS is an amorphous rubber while the crystallinity of EVA decreases with the VA content. Interestingly, as particularly evidenced for EVA with 28 and 40 % of VA, the diffusion of anastrozol can be adjusted through the membrane thickness. The very low diffusion rate of the anastrozol for EVA 10 % prevents this observation on Figure 7. The more crystalline EVA thermoplastics appear, the better the material to tune the drug diffusion, decreasing the diffusion rate strongly as compared to PDMS membranes.
2. In vivo kinetics
The anastrozol concentrations in sera and in the peritoneal fluid were measured in rats receiving either intra peritoneal or subcutaneous implants. The in vivo release of anastrozol from sc implantation was constant (Figure 10). Sera concentrations of about 20-25 ng/mL per day were obtained over a 13 week period. Moreover, the burst
1.4. Drug release kinetics from PDMS implants with EVA membranes As a daily release of around 20 µg of anastrozol is targeted, the diffusion rate of this drug from the PDMS implants has to be efficiently 202
Device-based controlled local delivery of anastrozol into peritoneal cavity: J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014 in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
Figure 10 - Anastrozol concentration (mean and SD) in sera of rats receiving a subcutaneous implant (n = 4).
Figure 8 - (a) Mean diffusion of anastrozol in the PDMS implant with EVA 10 % membrane in terms of time. (b) Mean diffusion of anastrozol from the PDMS implant with EVA membrane in terms of time (only the first 60 days).
Figure 11 - Anastrozol concentration (mean and SD) in sera of rats receiving the implant in the peritoneal cavity (n = 4).
* A multi-component (core-membrane) drug loaded polymer implant, capable of delivering a constant quantity of the active ingredient each day for at least one year in the pelvic cavity, was successfully formulated. For this work, Franz Cells were used to choose the polymer membrane able to control the release kinetics of anastrozol. The developed technique for the implant preparation allowed the manufacturing of a large quantity of implants with a high reproducibility. After successful validation of the anastrozol assay in the phosphate buffer, an in vitro release kinetic demonstrated a controlled release of about 15-20 µg/day of anastrozol for at least 450 days with a very limited burst effect. A good correlation between the in vitro and the in vivo release kinetics was observed and confirmed the efficiency of the implant. Indeed, when the implant is introduced in the intra peritoneal cavity, a local accumulation of the active substance was measured, compared to subcutaneous implantation.
Figure 9 - Influence of the thickness of the EVA membrane to anastrozol diffusion.
effect was not observed for both ways of implantation. The variability of results obtained after intraperitoneal implantation is higher (Figure 11), indicating that the released quantity of anastrozol per day may not be constant. The implantation site does not influence the serum concentration of anastrozol. With regards to the drug concentration in peritoneal fluid washing, a concentration of anastrozol of 15.3 ± 5.6 and 7.5 ± 0.5 ng/mL can be achieved from ip or sc implantations respectively. This double concentration of ip fluid can be explained by an accumulation of anastrozol around the implant site. It must be mentioned that the measured intra peritoneal anastrozol concentration is underestimated. Indeed, the volume of the intra peritoneal fluid in rats is very low and impossible to collect quantitatively, even if the peritoneal cavity is washed with 1mL of PBS buffer.
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Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
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of burst release in matrix-controlled drug delivery systems.- J. Controlled Release, 73, 121-136, 2001. Dash A., Cudworth Ii G. - Therapeutic applications of implantable drug delivery systems.- J. Pharmacol. Toxicol. Methods, 40, 1-12, 1998. Beheri G. E. - Surgical treatment of impotence.- Plast. Reconstr. Surg., 38, 92-97, 1966. Brantley Scott F., Bradley W.E., Timm G.W. - Treatment of urinary incontinence by implantable prosthetic sphincter.- Urology, 1, 252-259, 1973. Amzallag T., Pynson J. - Biomatériaux dans la chirurgie du cristallin.- Journal Français d'Ophtalmologie, 30, 757-767, 2007. Garrido L., Young V.L. - Analysis of periprosthetic capsular tissue from women with silicone breast implants by magic-angle spinning NMR.- Magn. Reson. Med., 42, 436-441, 1999. Matsumura K., Hyon S.-H., Nakajima N., Peng C., Tsutsumi S. - Surface modification of poly(ethylene-co-vinyl alcohol) (EVA). Part I. Introduction of carboxyl groups and immobilization of collagen.- J. Biomed. Mater. Res., 50, 512-517, 2000. Shastri P.V. - Toxicology of polymers for implant contraceptives for women.- Contraception, 65, 9-13, 2002. Costantini L.C., Kleppner S.R., McDonough J., Azar M.R., Patel R. - Implantable technology for long-term delivery of nalmefene for treatment of alcoholism.- Int. J. Pharm., 283, 35-44, 2004. Roumen F.J.M.E., Dieben T.O.M. - Clinical acceptability of an ethylene-vinyl-acetate nonmedicated vaginal ring.- Contraception, 59, 59-62, 1999. Ramadevi A., Padmavathy T., Stigall G., Paquette D., Kalachandra S. - EVA copolymer matrix for intra-oral delivery of antimicrobial and antiviral agents.- J. Mater. Sci. Mater. Med., 19, 721-727, 2008.
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Massein A., Petit E., Darchen M. A., Loriau J., Oberlin O., Marty O., Sauvanet E., Afriat R., Girard F., Molinié V., Duchatelle V., Zins M. - Imaging of intestinal involvement in endometriosis.Diagnostic and Interventional Imaging. Sugarbaker P.H. - Adjuvant intraperitoneal chemotherapy for advanced primary gastric cancer.- Scand. J. Surg., 95, 270-273, 2006. Defrere S., Mestagdt M., Riva R., Krier F., Van Langendonckt A., Drion P., Jerome C., Evrard B., Dehoux J. P., Foidart J. M., Donnez J. - In vivo biocompatibility of three potential intraperitoneal implants.- Macromol Biosci., 11, 1336-1345, 2011. Mendes G.D., Hamamoto D., Ilha J., Pereira Ados S., De Nucci G. - Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies.- J. Chromatogr. B, 850, 553-559, 2007.
ACKNOWLEDGEMENTS Special thanks are due to ATC (Liège, Belgium) for the Pharmacokinetics studies. Thanks are due to the Walloon Region for the research grant (WALEO 2) and to Pierre Lebrun for the statistical studies.
MANUSCRIPT Received 11 July 2013, accepted for publication 14 February 2014.
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Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation F. Krier1*, R. Riva2, S. Defrère3, M. Mestdagt4, A. Van Langendonckt3, P. Drion5, J.-P. Dehoux6, J. Donnez3, J.-M. Foidart4, C. Jérome2, B. Evrard1 Laboratory of Pharmaceutical Technology, Department of Pharmacy, CIRM, University of Liège, B36, Tour 4, Level 2, Avenue de l’Hôpital, 1, 4000 Liège, Belgium 2 Center for Education and Research on Macromolecules (CERM), Department of Chemistry, University of Liège, Allée de la Chimie, 3, building B6a, level 3, 4000 Liège, Belgium 3 Pôle de gynécologie, Institut de Recherche expérimentale et clinique, Université Catholique de Louvain, Avenue Hippocrate 10, Level 9, 1200 Brussels, Belgium 4 Laboratory of Tumor and Development Biology, University of Liège, B 23, Tour 3, Level 4, avenue de l’Hôpital,1, 4000 Liège, Belgium 5 Animal facilities-GIGA, B 23 B, Avenue de l’Hôpital, 3, University of Liège, 4000 Liège, Belgium 6 Experimental Surgery Unit, ANIM, Université Catholique de Louvain, Avenue Hippocrate 55, bte B1.55.15, 1200 Brussels, Belgium Fabrice Krier and Raphaël Riva contributed equally to this work. *Correspondence: [email protected] 1
Local treatment using drug loaded implants allows decreasing seric concentrations of the active ingredient with the purpose of limiting side effects and reaching perfect observance. Nowadays, some diseases are already treated with implants, but generally, by subcutaneous or intra vaginal implantation. In this work, a new implant device dedicated to the intra-peritoneal cavity was developed. For this purpose, a core-membrane polymer implant was selected. We propose an original method to determine the most appropriate membrane to control the release based on the use of Franz cells. The ability of the implant to release a constant quantity of an active ingredient will be assessed by testing implants in vitro. Finally, intra peritoneal cavity and subcutaneous in vivo implantation has been achieved in order to confirm the controlled and local release of the active ingredient. Key words: Anastrozol – Implant – Diffusion – EVA – PDMS – Drug delivery system – Intra-peritoneal – Control release – Polymer – Franz cells.
for contraception (etonorgestrel) [11], but also for the conception of contraceptive delivery devices, such as the vaginal ring [13]. A recent study investigated the release of two drugs by an EVA system for the intraoral treatment of oral lesions, blisters, and fungal diseases [14]. The peritoneal cavity seems to be a convenient site for the implantation of a drug delivery system against endometriosis or gastric cancer because large parts of lesion are localized in this region [15, 16]. The in vivo biocompatibility of PDMS and EVA in the intraperitoneal cavity has already been confirmed in a previous publication [17]. The challenge of this research is the production of an implant able to release a constant quantity of active ingredient without any “burst effect” in the intraperitoneal cavity, but also with an accumulation of the active ingredient in this cavity. PDMS and EVA are thus largely applied in the biomedical field, but no example of implantation in the intra-peritoneal cavity of these polymers has been documented, at least to our knowledge.
Ideally, a drug delivery system must be able to deliver a drug to a specific site in a determined period of time with a specific release profile [1]. Controlled release formulations allow a decrease in the amount of the drug necessary to reach the therapeutic effect and maintain sustained drug release over a prolonged period of time, thereby eliminating fluctuations in the drug plasma concentration [2]. These formulations are known to limit side-effects and increase patient compliance, particularly during long-term treatment [3, 4]. Implantable drug delivery systems are one example of controlled drug release formulations already successfully applied for therapeutic use. In the present case, a sustained release of anastrozol between 10 to 25 µg/day over a period of one year (model molecule) has typically been targeted. For this goal, among the various categories of implants (i.e. biodegradable or non-biodegradable implants, implantable pump systems, and the newest atypical class of implants [5]), non-biodegradable implants appear to be the most relevant choice since they must be used over quite a long period while their physical properties remain constant. In this work, implants based on two non-biodegradable polymers were tested for the elaboration of intra-peritoneal drug delivery systems, namely polydimethylsiloxane (PDMS) and poly(ethylene-co-vinyl acetate) (EVA). The PDMS is a biocompatible polymer which has been used for a long time as a biomaterial in urology for the treatment of impotence [6] or incontinence [7], in ophthalmology for intraocular lens [8], and in plastic surgery for breast implants [9], whereas the EVA was initially described as a biomaterial in dental surgery [10]. Levonorgestrel loaded PDMS films have been largely applied as subcutaneously controlled drug delivery devices for contraception [11]. EVA has been used as a subcutaneously controlled drug delivery system for the treatment of alcoholism (nalmefene) [12], and
I. MATERIAL AND METHODS 1. Anastrozol assay for in vitro release studies
1.1. Chemicals and solvents Acetonitrile (LiChrosolv, purity: 99.9 %) was purchased from Merck (Darmstadt, Germany). Anastrozol was supplied by Apin Chemicals Limited (Abingdon, United Kingdom). The structure of anastrozol is presented in Figure 1. Water was purified by a Millipore system (18.2 MΩ/cm resistivity, Milli-Q) before being filtered through a 0.22 µm Millipore Millipak - 40 disposable filter units (Millipore Corporation, United States). The phosphate buffer solutions (pH 7.4; 50 mM and pH 3.0; 50 mM) were prepared with potassium dihydrogen phosphate (Merck), sodium hydroxide (VWR, Liège, Belgium) and sodium azide (Merck). 198
Device-based controlled local delivery of anastrozol into peritoneal cavity: J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014 in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
Table I - Transition, cone voltage and collision energy for anastrozol and dexchlorpheniramine. Analyte
Transition (m/z)
Cone voltage (V)
Collision energy (eV)
Anastrozol Dexchlorpheniramine
294 > 225 275 > 230
25 20
21 18
Table II - Gradient mode details of the UPLC system. Figure 1 - Chemical structure of anastrozol.
Time (min)
Mobile phase A (%)
Mobile phase B (%)
Flow rate (mL/ min)
0.0 5.0 5.1 8.1 8.2 10.1
95 50 5 5 95 95
5 50 95 95 5 5
0.6 0.6 0.6 0.6 0.6 0.6
1.2. Apparatus The chromatographic analyses were performed on an Agilent technologies HPLC 1100 series (Hewlett-Packard, PaloAlto, CA, United States) equipped with a solvent delivery binary pump G1312A, an on-line degasser G1379A, a thermostatised autosampler G1328A, a column oven G1316A and a diode-array detector G1315C. A HewlettPackard computer dc 5100 Mt with Chemstation was used to control the whole chromatographic system and to acquire, process and store all the data obtained. A Mettler Toledo (Schwerzenbach, Switzerland) AT261 scale was used to weigh all the compounds (precision: 10 µg). A Seven Easy Mettler Toledo pH meter was used to adjust the pH value.
formic acid and methanol were analytical grade and applicable for HPLC measurements and were supplied by Biosolve (Valkenswaard, The Netherland). The water was purified as already described in point I.1.1.
1.3. Chromatographic conditions The chromatographic analysis was performed on a Lichrospher 100 C18 end-capped column (250 × 4 mm i.d., 5 µm particle size) which was kept at 30 °C. The mobile phase was a mix of 45 % phosphate buffer (pH 3) and 55 % acetonitrile. The HPLC system was operated in isocratic mode at a flow rate of 1.0 mL/min and the volume injection was 100 µL. UV detection was performed at 210 nm. The method was successfully validated in the range of 50 to 2000 ng/mL by using e-noval version 2.0e (Arlenda s.a., Liège, Belgium). The trueness, precision and accuracy of the method were found to be acceptable. The limits of quantification were evaluated at 50.67 and 2027 ng/mL while the limit of detection was calculated to be 15.36 ng/mL. The accuracy profile obtained by considering linear regression through 0 fitted using the highest level only, is shown in Figure 2.
2.2. Apparatus The chromatographic analyses were performed on a Waters Acquity UPLC inlet system. The mass spectrometry detector was a Waters Quattro Premier XE equipped with an electrospray ionisation source (ESI) operating in positive ion mode. Mass chromatograms were acquired in the multiple reaction monitoring (MRM) mode. One specific transition was monitored for each compound of interest. The selection was made in order to reach the highest detectability. The capillary was set to 3.0 kV, the source temperature was set to 130 ± 5 °C, the desolve temperature was set to 345 ± 5 °C, the cone gas flow was set to 50 L/h. The parameters for transition, cone voltage and collision energy are summarized in Table I. Data acquisitions were achieved using MassLynx Version 4.1 software. An analytical balance was used to weight all compounds (Kern 870 - Kern DKD Labor, Germany).
2. Anastrozol assay for in vivo release studies
2.3. Chromatographic conditions The chromatographic analyses were performed on an Acquity Bech C18 column (100 × 2.1 mm i.d., 1.7 µm particle size) which was kept at 60 °C. The mobile phase was a mix of 10 mM ammonium formate with 0.1 % formic acid and methanol with 0.1 % formic acid. The UPLC system was operated in gradient mode (see Table II) at a flow rate of 0.6 mL/min and the volume injection was 6 µL.
2.1. Chemicals and solvents Chlorpheniramin maleate (internal standard) was purchased from Sigma-Aldrich (Diegem, Belgium). Acetonitrile, ammonium formate,
2.4. Standard solutions For this in vivo quantification method, two solutions of anastrozol in acetonitrile/water (50/50 v/v) were prepared at a concentration of 1 mg/mL, then aliquoted and stored at -20 °C. These solutions were used as standard solutions of anastrozol to prepare the calibration standard and QC samples respectively. The solution of internal standard (chlorpheniramin) was prepared in the same way in order to obtain a stock solution at a concentration of 1 mg/mL [18]. Two kinds of samples were prepared in an independent way: calibration standards and validation standards (or QC samples). One standard solution was used to prepare calibration standards. The calibration standards are plasma samples, containing known concentrations of the analyte of interest, and are only used for calibration. Calibration curves were generated daily over a concentration range between 0.2 and 100 ng/mL. Six concentration levels were selected as follows: 0.2, 1, 5, 25, 50 and 100 ng/mL. Two blank samples were also prepared, one containing only the internal standards.
Figure 2 - Accuracy profile obtained by considering linear regression through 0 fitted using the highest level only. Relative bias (—), ± 10 % acceptance limits (- - -), 95 % β-expectation tolerance limits (– – –), and relative back-calculated concentrations (l). 199
Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014
The validation standards were prepared from another standard solution. They are plasma samples containing known concentrations of the analytes of interest which are considered as true values by consensus. Three concentration levels (1, 25 and 100 ng/ml) representing the whole concentration range were selected for the QC samples. All these validation standards were analysed twice at each sequence of analysis.
formed during the PDMS cross-linking. The total extraction of these residues was assessed by gravimetry. 3.3.2. PDMS implants with PDMS membranes The anastrozol loaded PDMS rods were prepared as described above; they are embedded in a preformed PDMS membrane prepared with the same batch of PDMS/cross-linker/catalyser (Sterne, France). Typically, the PDMS membrane is swollen into heptane with the purpose of increasing the lumen of the tube, allowing the introduction of the anastrozol loaded rod inside the tube. Finally the heptane is removed under vacuum leading to the contraction of the PDMS membrane, which, at the end of the process, was strongly stuck to the rod. The total extraction of heptane was assessed by gravimetry.
2.5. Sample preparation The anastrozol and the internal standard were extracted from the plasma by a protein precipitation method. Fifty microlitres of the internal standard working solution (100 ng/mL) was added to 50 µL of the plasma sample in a micro tube and then the sample was put into a vortex. One hundred microlitres of methanol and 300 µL of acetonitrile were added successively. After 10 min of centrifugation, 150 µL of the supernatant was transferred to a HPLC vial where 50 µL of water was added before stirring with a vortex.
3.3.3. PDMS implants with EVA membranes The PDMS implants containing anastrozol (50 % w/w in the core of the implant) and bearing an EVA membrane were prepared by a technique already described in a previous paper [17]. In this technique, EVA tubes are used as a mould for the direct curing of PDMS by SiOPr4 in the presence of a catalytic amount of SnOct2. The EVA tubes were processed by extrusion of commercially available EVA pellets. Typically, PDMS (19.4 g) was transferred into a sterile container. Freshly phorphyrized anastrozol (19.5 g) was then added to the PDMS. The mixture was homogenized with an ultra-turrax T 25 basic (IKA, Staufen, Germany) with the purpose of obtaining a very efficient mix of the two compounds. After 5 min of mixing, a homogenous blend was obtained, which was kept at -20 °C for 1 h. 0.5 g of tetrapropyl orthosilicate and 0.1 g of SnOct2 were mixed together in a separate glass container. This mixture was then added into the cold PDMS mixture inside a laminar flow hood. The PDMS/anastrozol/cross-linker/ catalyst mixture was manually homogenized for 2 min before being placed under a vacuum for 5 min in order to remove the air bubbles trapped in the blend. Finally the PDMS mixture was transferred into a plastic syringe and kept at -20 °C, for at least 1 h. The PDMS-anastrozol mixture was then injected into the EVA tube (internal diameter of 3 mm, thickness of the wall 200 μm and 15 cm long) under a laminar flow hood. The ends of the tube were closed with a parafilm. Curing of the PDMS was performed at room temperature for 16 h in order to avoid deformation of the EVA tube during the process. Finally, the tubes were cut in order to obtain 2 cm long implants. The implant extremities were closed with MED-2000 adhesive silicone (Nusil technology, Carpinteria, CA, United States) and dried in a Vismara 65 vacuum oven at 950 mbar for 4 h at room temperature with the purpose of removing the propanol formed during the PDMS cross-linking. The total extraction of these residues was assessed by gravimetry. The schematic representation of implant is presented in Figure 3, whereas Figure 4 is a picture of the implant ready to be implanted.
3. Preparation of multi-biomaterial delivery systems
3.1. Membranes for diffusion studies The PDMS membranes were synthesized by the curing of linear PDMS in a mould. Typically, 0.125 g of tetrapropyl orthosilicate and 0.025 g of SnOct2 were mixed together in a glass container. This solution was then added to 4.854 g of PDMS, previously stored at -20 °C for at least 1 h. The PDMS/cross-linker/catalyst mixture was manually homogenized for 2 min before being placed under a vacuum for 5 min in order to remove the air bubbles trapped in the blend. Finally, the PDMS mixture was transferred into a plastic syringe and kept at -20 °C for at least 1 h. The PDMS mixture was then injected into a square-shaped moult (10 cm × 10 cm) covered by two Teflon films. The mould was then placed into a press (30 bars) at 80 °C for 15 min. The PDMS membrane was then collected and dried in a Vismara 65 vacuum oven at 950 mbar for 4 h at room temperature with the purpose of removing the propanol formed during the PDMS cross-linking. The total extraction of these residues was assessed by gravimetry. The EVA membranes were purchased from 3M Health Care (StPaul, MN, United States). 3.2. Materials for implant preparation Polydimethylsiloxane (PDMS, base, medical grade), tetrapropyl orthosilicate (cross-linker, medical grade), tin octoate (SnOct2, catalyst, medical grade), Anastrozol (APIN chemicals, batch 32993a), EVA (Elvax 3185, Dupont, batch 70106161) were used for the preparation of the implants. 3.3. Implant preparation 3.3.1. PDMS implants The PDMS implants containing anastrozol (5 % w/w) were synthesized by curing an anastrozol/PDMS mixture at 80 °C. Typically, PDMS (4.854 g) and anastrozol (0.192 g) were mixed together manually for 5 min. The blend was then kept at -20 °C for 1 h. 0.125 g of tetrapropyl orthosilicate and 0.025 g of SnOct2 were mixed together in a separate glass container. This mixture was then added into the cold PDMS mixture. The PDMS/anastrozol/cross-linker/catalyst mixture was manually homogenized for 2 min before being placed under a vacuum for 5 min in order to remove the air bubbles trapped in the blend. Finally the PDMS mixture was transferred into a plastic syringe and kept at -20 °C, for at least 1 h. The PDMS/anastrozol mixture was then injected into the cylindrical compartment (20 mm long, diameter = 3 mm) of an iron mould coated by Teflon. The mould was then placed into a press (30 bars) at 80 °C for 15 min. The cross-linked anastrozol loaded rods were then collected and dried in a Vismara 65 vacuum oven at 950 mbar for 4 h at room temperature with the purpose of removing the propanol
3.4. Sterilization of implants The implants were sterilized by the ethylene oxide procedure. The preconditioning step was performed for 8 h at 45 ± 5 °C with a relative humidity controlled between 50 and 80 %. The sterilization phase was pursued at 45 °C with a relative humidity level targeted at 60 ± 20 % for 3 h and at a gas concentration of 730 mg/L. Then the aeration process was carried out at 40 ± 5 °C with fresh air in bleed, for 72 h. The process was validated using a biological indicator (initial population 106 spores of Bacillus subtilis). The final ethylene oxide content in the implant after 2 days of airing was equal to 2.5 ppm.
4. In vitro drug diffusion and release kinetics
4.1. Anastrozol diffusion through EVA and PDMS membranes Diffusion studies were carried out using Franz type glass diffusion 200
Device-based controlled local delivery of anastrozol into peritoneal cavity: J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014 in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
4.2. Drug release kinetics from the device Release of anastrozol from the device was studied in triplicate under sink conditions. In order to determine the kinetics of the drug release, three implants of each formulation were placed in three different glass bottles containing 100 mL of phosphate buffer solution (pH 7.4) containing 0.025 % (w/w) of sodium azide. The bottles were placed in a shaking water bath (SW 22, Julabo, Seelback, Germany) at 37 °C. Samples of the buffer solution were collected at specific time intervals. After each collection, an equivalent amount of fresh buffer solution was transferred into each bottle to keep the volume of solution constant throughout the measurement. The amount of drug released was measured by HPLC. Implants without membranes and implants with a PDMS (600, 700, 800 µm) or an EVA membrane (10 weight % of VA) with three different thicknesses (100, 200 and 300 µm) were used in this experiment.
Figure 3 - Schematic representation of the EVA membrane coated implant.
5. In vivo kinetics
5.1. Intraperitoneal placing of anastrozol-loaded implants in rats All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the University of Louvain, Belgium (UCL/MD/2007/020). The “Guide for the Care and Use of Laboratory Animals”, prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, was followed carefully. Fifty-two 10-12 week old, female Wistar rats were used for the present study: 26 rats for subcutaneous (sc) and 26 for intraperitoneal (ip) implantation. The rats were anaesthetized with an injection of ketamin (ip, 30 mg/kg, Anesketin; Eurovet, Heusden-Zolder, Belgium) and medetomidin (ip, 50 µg/kg, Domitor; Pfizer, Cambridge, MA, United States). After surgery, the anaesthesia was reversed by the injection of atipamezol (sc, 0.25 mg/kg, Antisedan; Pfizer). For ip implantation, a median laparotomy of 1 cm was made. One implant per rat was put into the pelvic cavity, without any fixation. The abdominal wall and the skin were then sutured with absorbable suture. For subcutaneous (sc) implantation, a skin incision was made in the upper back of the animal and one implant was placed under the skin. The skin was then sutured with absorbable suture. At the end of the experiment, the rats were euthanatized by CO2 inhalation according to the standard euthanasia guidelines for rodents from the Institutional Animal Care and Use Committee (IACUC) (http://www.iacuc.ucsf.edu/Policies/awGlEuthR.asp).
Figure 4 - Picture of the implant tested in vivo.
cells (Hanson Research, CA, United States) consisting of a donor and a receiver chamber. Typically, the membrane was clamped between these two chambers. The receiver chamber was filled with 7.5 mL of phosphate buffer solution (pH 7.4; 50 mM), which was constantly stirred with a small magnetic bar and thermostated at 37 °C during all the experiments. The donor chamber was filled with a suspension of anastrozol (15 % w/w in the suspension) in an aqueous gel of Carbomer 980 (1 % in water). The test was performed in occlusive conditions. The surface of the membrane in contact with the gel was equal to 1.767 cm². After predetermined time intervals (24, 48, 72, 96 or 144 h), a sample of the receptor phase (2.0 mL) was collected with the purpose of measuring the anastrozol concentration [1]. After each collection, 2.0 mL of fresh phosphate buffer solution was added to the receiver chamber to keep the volume constant throughout the analysis. Several membranes of both polymers were tested: 4 different polymers were evaluated: PDMS, EVA 10 weight % of VA, EVA 28 weight % of VA and EVA of 40 weight % VA. The thicknesses of the different membranes are reported in Table III. This test was performed under “sink conditions” in the receptor compartment.
5.2. Pharmacokinetics studies Blood samples were collected from at least of 3 rats after 1, 4, 7 and 13 weeks for sc implantation and after 1, 5, 12 and 18 weeks for ip implantation for anastrozol measurements. Sera were stored at -80 °C until analysis. Six rats were euthanatized after 18 weeks for ip implantation and three rats after 13 weeks for sc implantation. Peritoneal fluids were then collected by peritoneal washing. Briefly, after ip injection of PBS (1 mL), the rats were gently shaken for 30 s. The peritoneal cavity was then opened, and the peritoneal fluid was collected and centrifuged at 400 g for 5 min. The supernatant was recovered and stored at -80 °C until the anastrozol assay.
Table III - Thickness of polymer membranes. Polymers
Thickness (µm)
PDMS PDMS EVA 10 weight% of VA EVA 10 weight% of VA EVA 10 weight% of VA EVA 28 weight% of VA EVA 28 weight% of VA EVA 40 weight% of VA EVA 40 weight% of VA
700 800 150 400 700 100 200 100 200
II. RESULTS AND DISCUSSIONS 1. In vitro drug diffusion and release kinetics
1.1. Drug release kinetics from PDMS implants without membranes Figure 5 shows the mean diffusion per 24 h of anastrozol in terms of time for PDMS implants without membranes. Clearly, the diffusion is not constant. The burst effect is very important. A diffusion of 431 µg is observed on the first day, while after 212 days of experiments, only 8 µg is released in 24 h. At the end of the experiment, the total drug 201
Device-based controlled local delivery of anastrozol into peritoneal cavity: in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014
Figure 5 - Mean diffusion of anastrozol in the PDMS implant without membrane in terms of time.
Figure 6 - Mean diffusion of anastrozol in the PDMS implant with PDMS membrane in terms of time.
release represented 91.5 ± 5.1 % of the active substance present in the device. With the goal of regulating drug diffusion and achieving a constant release profile for more than 300 days, we decided to coat these implants with a drug-free PDMS membrane. 1.2. Drug release kinetics from PDMS implants with a PDMS membrane Figure 6 shows the mean diffusion per 24 h of anastrozol in terms of time for implants coated by a PDMS membrane (thickness: 600, 700 or 800 µm). The PDMS membrane allows an important decrease of the burst effect; after 24 h, the released drug is 5 times lower for the implant with the membrane. The amount of released anastrozol decreases more slowly than the implant without the membrane, after 212 days, only 84.21 ± 5.14 % of the anastrozol has diffused outside the implant. However, the diffusion is not really controlled and the observed release profile is not constant with such PDMS membranes. Thus, we decided to replace the PDMS membrane with a poly(ethylene-co-vinylacetate) (EVA) membrane whose polarity and crystallinity can be tuned by the vinyl acetate content, and compare them in terms of drug diffusion.
Figure 7 - Amount of anastrozol recovered in the Franz cell receptor phase in terms of time (µg/days) according to the membrane formulation (n = 1).
reduced as demonstrated by Figures 5 and 6. In addition, Figure 7 shows that the diffusion of anastrozol through EVA 28 % and EVA 40 % available membranes remains close to or higher than the tested PDMS membranes. That is why EVA membranes containing 10 weight % of VA have been selected to coat the PDMS implants. In order to release an acceptable quantity of anastrozol, this drug was increased to 50 % in weight in each core of the implant. The extremities of the implants were closed with MED 2000, an adhesive silicone as described in 2.3.3. Figure 8a and b show the mean diffusion per 24 h of anastrozol in terms of time from implants coated with a membrane of 100, 200 or 300 µm thickness. For the three formulations, a sustained release of the drug is obtained for more than 470 days. The burst effect is very limited in time (only 7 days) and in term of quantity (max 35 µg/24 h). Figure 9 shows a linear regression model of the diffusion result between days 50 and 450. The release of anastrozol from implants coated with a 100 µm membrane decreases in terms of time. For implants coated with thicker membranes, the release profile is constant during the 463 days of experiment. A significant influence of the thickness of the membrane can be observed since the mean diffusion per 24 h after 463 days are 22.10 ±1.17 and 16.67 ±3.87 µg for implants coated with 200 and 300 µm, respectively. Based on the targeted drug release, PDMS implants coated with a 200 µm EVA 10 membrane were chosen for in vivo studies.
1.3. Drug diffusion through EVA and PDMS membranes Figure 7 shows the amount of anastrozol detected in the receptor phase of the Franz cells in terms of time for various PDMS and EVA membranes. The diffusion of anastrozol through PDMS membranes of 700 and 800 µm (used in 3.1.2) appears weakly dependent on the membrane thickness and gives comparable results to EVA membranes containing 28 % of VA with a thickness between 100 and 200 µm. This clearly shows that EVA is a much more efficient barrier to drug diffusion than PDMS. It must be mentioned that the three curves for 10 % EVA membrane with three different thicknesses overlaid perfectly but it is impossible to distinguish this in Figure 7. In addition, when comparing EVA films of the same thickness, an increase of the VA content leads to an increase in the diffusion rate of the anastrozol. These observations are in line with a clear decrease in the drug diffusion rate, when the crystallinity of the membrane is increased. Indeed, PDMS is an amorphous rubber while the crystallinity of EVA decreases with the VA content. Interestingly, as particularly evidenced for EVA with 28 and 40 % of VA, the diffusion of anastrozol can be adjusted through the membrane thickness. The very low diffusion rate of the anastrozol for EVA 10 % prevents this observation on Figure 7. The more crystalline EVA thermoplastics appear, the better the material to tune the drug diffusion, decreasing the diffusion rate strongly as compared to PDMS membranes.
2. In vivo kinetics
The anastrozol concentrations in sera and in the peritoneal fluid were measured in rats receiving either intra peritoneal or subcutaneous implants. The in vivo release of anastrozol from sc implantation was constant (Figure 10). Sera concentrations of about 20-25 ng/mL per day were obtained over a 13 week period. Moreover, the burst
1.4. Drug release kinetics from PDMS implants with EVA membranes As a daily release of around 20 µg of anastrozol is targeted, the diffusion rate of this drug from the PDMS implants has to be efficiently 202
Device-based controlled local delivery of anastrozol into peritoneal cavity: J. DRUG DEL. SCI. TECH., 24 (2) 198-204 2014 in vitro and in vivo evaluation F. Krier, R. Riva, S. Defrère, M. Mestdagt, A. Van Langendonckt, P. Drion, J.-P. Dehoux, J. Donnez, J.-M. Foidart, C. Jérome, B. Evrard
Figure 10 - Anastrozol concentration (mean and SD) in sera of rats receiving a subcutaneous implant (n = 4).
Figure 8 - (a) Mean diffusion of anastrozol in the PDMS implant with EVA 10 % membrane in terms of time. (b) Mean diffusion of anastrozol from the PDMS implant with EVA membrane in terms of time (only the first 60 days).
Figure 11 - Anastrozol concentration (mean and SD) in sera of rats receiving the implant in the peritoneal cavity (n = 4).
* A multi-component (core-membrane) drug loaded polymer implant, capable of delivering a constant quantity of the active ingredient each day for at least one year in the pelvic cavity, was successfully formulated. For this work, Franz Cells were used to choose the polymer membrane able to control the release kinetics of anastrozol. The developed technique for the implant preparation allowed the manufacturing of a large quantity of implants with a high reproducibility. After successful validation of the anastrozol assay in the phosphate buffer, an in vitro release kinetic demonstrated a controlled release of about 15-20 µg/day of anastrozol for at least 450 days with a very limited burst effect. A good correlation between the in vitro and the in vivo release kinetics was observed and confirmed the efficiency of the implant. Indeed, when the implant is introduced in the intra peritoneal cavity, a local accumulation of the active substance was measured, compared to subcutaneous implantation.
Figure 9 - Influence of the thickness of the EVA membrane to anastrozol diffusion.
effect was not observed for both ways of implantation. The variability of results obtained after intraperitoneal implantation is higher (Figure 11), indicating that the released quantity of anastrozol per day may not be constant. The implantation site does not influence the serum concentration of anastrozol. With regards to the drug concentration in peritoneal fluid washing, a concentration of anastrozol of 15.3 ± 5.6 and 7.5 ± 0.5 ng/mL can be achieved from ip or sc implantations respectively. This double concentration of ip fluid can be explained by an accumulation of anastrozol around the implant site. It must be mentioned that the measured intra peritoneal anastrozol concentration is underestimated. Indeed, the volume of the intra peritoneal fluid in rats is very low and impossible to collect quantitatively, even if the peritoneal cavity is washed with 1mL of PBS buffer.
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ACKNOWLEDGEMENTS Special thanks are due to ATC (Liège, Belgium) for the Pharmacokinetics studies. Thanks are due to the Walloon Region for the research grant (WALEO 2) and to Pierre Lebrun for the statistical studies.
MANUSCRIPT Received 11 July 2013, accepted for publication 14 February 2014.
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