Materials
Poly(D,L-lactic-co-glycolic acid) (PLGA; Resomer® RG504 molecular weight 42.000 Da Boheringer Ingelheim; Ingelheim; Germany), ATV calcium (Chemos GmbH; Regenstauf;
Germany), atorvastastin-d5 sodium salt, (TRC, Canada), PVAL or polyvinyl alcohol (Mowiol® 4 88; Kuraray Europe; Hattersheim am Main; Germany), chloroform (Chromasolv® plus for HPLC,
≥ 99.9 %, 0.5 - 1.0 % ethanol as stabilizer, Sigma- Aldrich Chemie GmbH, Steinheim, Germany).
Cross linked hyaluronic acid hydrogel (Fortelis Extra®) was a gift from Aptissen, Switzerland. All other chemicals were of analytical grade. MF-Millipore GSWP02500 filters (0.22 µm) were purchased from Millipore Merck, Darmstadt, Germany.
Methods
Microparticle preparation, characterization and sterilization
Microparticles were prepared by an oil-in-water (o/w) solvent emulsion-evaporation process as previously described [4]. Briefly, 375 mg of PLGA and 37.5 mg of ATV were dissolved in 7 g of chloroform before emulsification in a PVAL 2 % aqueous solution at 1500 rpm for 20 minutes using a paddle stirrer (Eurostar digital, IKA-Werke, Staufen, Germany). The emulsion was added to 50 mL of water, and chloroform was evaporated at room temperature overnight at a stirring rate of 500 rpm.
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
95 Unloaded microparticles were prepared similarly without ATV. The particles were washed and concentrated by successive steps of centrifugation/re-suspension before freeze-drying (Alpha 2-4 LSC, Christ, Kuhner, Switzerland).
For the quantification of ATV loading, microparticles (4 mg) were dissolved in 3 mL of acetonitrile/ethanol (50/50). The samples were analyzed by reversed phase HPLC (LC module I plus, Waters corporation, Milford, USA), and ATV was measured with a UV spectrometer set at λ = 245 nm, retention time 7.8 min. The column was a Nucleosil CC 125 / 4 120-5 C18 (Macherey-Nagel GmbH & Co. KG, Oensingen, Switzerland), maintained at 25°C.The mobile phase was 55 % acetate buffer (10 mM, pH 3) and 45 % acetonitrile, at a flow rate 1mL/min. The calibration curve was constructed by consecutive dilutions of ATV in acetonitrile/ethanol containing PLGA (0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 µg/mL). The method has been fully validated, and a limit of quantification of 500 ng/mL and limit of detection of 50 ng/mL were obtained. A trueness of 98 to 102 % was determined, and the intermediate precision was 2 %; moreover, the three replicates injected on three different days demonstrated the repeatability of the method. The quantification was conducted in triplicate. The results are presented as actual drug loading, corresponding to the percent mass ratio of the encapsulated drug to the mass of the microparticles recovered.
For sterilization, 380 mg of microparticles (containing 5 mg of ATV) and 5 mg of ATV in powder form were weighed in 10 mL vials. The vials wereput under an inert atmosphere of argon, sealed and sent for sterilization in dry ice. Two types of sterilization methods were tested: γ-ray treatment of 16kGy at 1.2 MeV (Ionisos, Pouzauges, France) and e-beam at 6.2 mA, at a dose of 2.06 mA/m/min, for 10 MeV (Leoni Studer AG, Daniken, Switzerland). A sterility test was performed according to Eur Pharmacopoeia 7.7 (paragraph 2.6.1).
Formulations preparation for characterization and in vivo testing
Lyophilized and sterilized 350 mg of microparticles were preserved in hermetically sealed vials under inert gas atmosphere. In the operating room, the content of a 1mL syringe of hyaluronic acid hydrogel was added in the vial and thoroughly mixed, under sterile conditions. The composition of the final formulations is presented in Table 1.
In addition, for the in vitro drug release assays and rheological characterization, we prepared GMatv, which consists in ATV-loaded microparticles dispersed into the (drug-free) gel phase. This allowed to study the sole contribution of the microparticles on drug release.
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
96 Table 1.: Amount of ATV loaded in the gel and/or the microparticle formulations applied per pig.
GM: gel and microparticles, GatvMatv: gel containing free ATV and microparticles encapsulating ATV.
Rheological measurements
The rheology of the preparations is described in Table 1; Fortelis Extra as received or after lyophilization (G) and Poloxamer 407 at 20 % w/w were characterized at 37 °C with a Haake Rheostress 1, Thermo Scientific fitted with a 35 mm / 2° cone-plate geometry. The instrument was set to perform the measurements with three repetitions and deliver the results as the mean. Rotation was performed over a shear rate ranging from 0.01 to 500 s-1. Oscillation was performed with a frequency ranging from 0.628 to 31.4 rad/sec.
In vitro ATV release
ATV release assays from the gel/particle formulation was performed using Vertical Franz's diffusion cells as described previously [4]. In this system, the gel is kept separated from the release medium by a 0.22-μm filter. The filter allows the free diffusion of ATV. Briefly, 500 mg of each formulation were placed in the donor compartment and 10 mL of PBS (0.1 M, pH 7.4)/SDS 0.1 % in the receptor compartment (ensuring sink conditions, solubility of ATV 600 μg/mL). The receptor compartment was under constant magnetic stirring. Franz's cells were placed in an incubator at 37 °C (EG 110 IR, Jouan, Saint-Herblain, France). At predetermined time intervals, 0.5 mL were withdrawn from the receptor compartment and replaced with fresh medium. The samples were analyzed by reversed phase HPLC as previously described. Each experiment was conducted in triplicate, and the values are expressed as the mean ± standard deviation.
In vivo model
Adult Yorkshire domestic swine were used (≈50 kg). All in vivo experiments were performed in compliance with the Swiss Federal Law on the Protection of the Animals, following a protocol accepted by the canton of Vaud Authority (Service cantonal de la consommation et des affaires vétérinaires), authorization n° VD2870. In total, three groups of eight animals each were operated.
The sham group received no treatment, the GM group received the formulation without ATV and the GatvMatv group received the ATV-loaded formulation.
The animals were anesthetized by intramuscular injection of a mixture of ketamine (10 mg/kg), xylazine (0.1 mg/kg) and atropine (2mg). Induction was done by propofol (3.5-5mg/Kg) and
Formulation ATV in Gel ATV in microparticles Total ATV
GM no ATV Unloaded microparticles -
GatvMatv 5 mg 5 mg 10 mg
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
97 portalgesic (1.5mg/Kg). General anesthesia was maintained using 1-2 % isoflurane introduced via tracheal intubation. The different steps of the intervention are illustrated on Fig. 1. After a longitudinal skin incision at the right lateral side of the neck, the subcutaneous and muscle were dissected to expose the common carotid artery as well as the internal jugular veins. Surgical loops were used to isolate both vessels. The internal jugular vein is harvested after ligation of the vein proximally and distally. The vein was flushed with saline solution and then opened longitudinally to get a venous patch. Intravenous heparin is then injected intravenously (50Ui/Kg) just before clamping the artery. A longitudinal arteriotomy (20 mm length) is performed. The venous patch is then sutured to the arterial wall using a 6.0 polypropylene stitch. In the group of pigs with gel, the application of the hydrogel is performed using a scoop. The hydrogel should be placed at the entire anastomotic surface covering the venous patch and the suture lines. Finally, the hemostasis is confirmed and the neck is closed using absorbable suture. Wound dressing using Opsite® spray is used at the end of the procedure. Post-operative analgesia was provided by intra-muscular injection of buprenorphine hydrochloride (0.01 mg/kg) and application of fentanyl patch (50 mg). Animals were returned to the farm and fed in a regular diet. Their temperature and weight were monitored daily. Antiobiotic (Augumentin, 2 g) treatment was administered over 5 days post-operatively and anti-platelet (Aspegic, 200 mg) therapy over 28 days. After 28 days, the arteries and tissues were collected and animals were sacrificed with a lethal dose of barbituric.
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
98 Figure 1. Schematic representation (upper panel) and photographs of the pig in vivo model (lower panel). A: Jugular vein and carotid artery are exposed. B: A longitudinal incision is performed on the artery. b: A piece of vein is longitudinally cut. C: The vein is sutured on the longitudinal incision of the artery. D: The gel is applied on the patch.
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
99 Histology
IH extent was assessed histomorphometricaly as the intima/media thickness ratio. Common carotid arteries receiving a venous patch or not, were explanted bilaterally from the pigs, 28 days after the surgery, fixed in 10 % buffered formalin and paraffin-embedded. Sections of jugular vein and fragments of the sternocleidomastoid muscle and the liver were also harvested. To observe the IH formation in the carotid arteries, three 5-µm thick transversal sections were performed at 5 mm intervals. Hematoxylin and eosin (HE) as well as Miller Elastin (MEL) staining were performed for histologic and morphometric analysis. A Panoramic MIDI slide scanner (3DHistech Kft., Hungary) was used to digitize the sections. Snapshots were extracted using Panoramic Viewer software. The animals presenting histological evidence of thrombi were excluded from the analysis. The intimal area was denoted as the area between the lumen and the internal elastic lamina, visible with the MEL staining. The intimal thickness for each animal was defined as the mean of 9 measurements of thickness of the intimal area (3 measurements on each of the 3 transversal sections of the same vascular segment). Medial thickness was defined accordingly. Inflammation was assessed from HE stained slides as presence of lymphocytes, eosinophils, plasma cells and foreign body reaction. A semi-quantitative histology scoring system was assessed and employed (0 no inflammation; 0.5 minimal; 1+ mild ; 2+ moderate ; 3+ severe).
For immunohistochemistry, a selection of the most representative carotid sections of 5 µm (one for each animal) were prepared from paraffin-embedded tissues. In brief, after deparaffinization and heat-mediated antigen retrieval, sections were incubated with a primary antibody against smooth muscle cell alpha actin (SMA) rabbit polyclonal, ab5694; Abcam 1:100), in blocking buffer (PBS, 2% BSA, 0.3% Triton X-100) at 4°C overnight. Samples were then incubated for 1 h at room temperature with either Alexa-Fluor-594- or -488-coupled secondary antibodies (Life Technologies), diluted 1:500 in PBS, covered with PBS containing 50% glycerol and 0.4μg/mL DAPI, and observed by fluorescence microscopy (Leica Leitz DMRB, Nidau, Switzerland). Two controls, in which we omitted the primary or secondary antibody, were included in each experiment.
For the analysis of vasa vasorum development, a manual microvessel counting was performed on a representative SMA-stained adventitial field (x 10).
Segments of liver and sternocleidomastoid muscle were also fixed in formalin and paraffin embedded. HE, Masson’s trichrome and Gomori’s reticulin staining were performed for the liver secitons and H&E for the muscle tissue. Snapshots were extracted using Panoramic Viewer software.
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
100
Results
Formulation characterization
Microparticles of 18 µm containing ATV at 1.2 % w/w were produced. The addition of microparticles in the gel (at a concentration of 10 %) affected its viscosity at low shear stress and its elastic modulus G’ increased (Fig. 2). As seen on the pictures, the formulation became soggy and lost its transparency. Despite the thicker texture, the gel was easily applied around the vessel (Fig.
1D).
Figure 2. Comparison of viscous and elastic moduli of the gel before and after microparticle addition. The viscosity and the elastic moduli increased.
The influence of the sterilization method (γ-ray or e-beam sterilization) on the release kinetic was assessed in vitro (Fig. 3). Microparticles sterilized by γ-rays sterilization presented a sustained release over 20 days, while e-beam treated microparticles maintained the release over 12 days. This corresponds to a 40 % (for γ-rays) and 65 % (for e-beam) reduction of the potential to sustain the release in time compared to non-sterilized microparticles. Interestingly no modification on the ATV peak on the HPLC chromatogram was observed after sterilization, suggesting than ATV was not molecularly affected by the process. For both techniques, sterilization was validated according to Eur. Pharmacopoeia 7.7 (paragraph 2.6.1).
Radiation with γ-rays is a well-established method to sterilize PLGA microparticles [15]. Radiation provokes polymeric chain cleavage and thus reduces the molecular weight of PLGA microparticles.
The microparticles molecular weight has been shown to be an important parameter affecting the drug release kinetics (see CHAPTER II of the thesis). Indeed, for both irradiation methods the shape of the curve was shown to shift from a triphasic release to a diffusion-like release curve. At the same time the duration of the release was significantly reduced, suggesting that the molecular weight went under the threshold of 20 kDa (see CHAPTER II of the thesis). The γ-ray sterilization was shown to be less destructive compared to the e-beam radiation despite the fact that the total radiation dose was lower for the e-beam. This might be due to the fact that the radiation energy of the e-beam is
8-CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
101 fold greater than for γ-rays. The shift of the release curve due to different irradiation energies is similar to the shift observed when different irradiation doses are applied [16]. This leads us to the conclusion that the irradiation energy is equally important to the dose with regards to the molecular weight degradation of PLGA microparticles.
0 1 0 2 0 3 0 4 0
0 2 0 4 0 6 0 8 0 1 0 0
T im e ( d a y s )
Atorvastatin (%)
not sterile Matv e - beam GMatv
- rays GMatv
Figure 3. Microparticles after γ-ray sterilization maintained their ability to release ATV over 20 days. Comparison of ATV releasing microparticles before and after γ-ray and e-beam treatment in vitro (PBS (0.1 M, pH 7.4) / SDS 0.1 %). Matv corresponds to ATV loaded microparticles, GMatv corresponds to ATV loaded microparticles incorporated in the gel.
Intimal hyperplasia development
The pig model of jugular venous patch placement on a carotid pig artery was evaluated in terms of IH development. The in vivo efficacy of the formulation containing ATV was consequently assessed.
Controls comprised sham operated and healthy pigs. Out of 24 animals operated, four exhibited thrombosis (two sham operated and two ATV receiving animals) and were excluded from the analysis. Representative histological samples of artery-patch sections as well as their histomorphometric evaluation are presented in Fig. 4.
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
102 Figure 4. Effect of ATV administration on intimal hyperplasia developed following the application of a venous patch on a carotid artery in a porcine model. Significant amounts of IH were generated from this model. No significant difference was observed in the intimal or medial thickness between ATV treated and sham operated pigs. Upper panel: Representative histological sections of carotid arteries receiving a venous patch (Miller elastin staining). A: arterial host, V: venous patch, m: media, i: intima. Middle and lower panels: Mean values of the intima/media ratio, intima and media thickness of the venous patch and the carotid host. GM: gel and microparticles, GatvMatv:
gel containing free ATV and microparticles encapsulating ATV. Data are presented as the mean values of 6 to 7 animals. Each bar is the mean ± SoEM. Ordinary one-way ANOVA with Tukey's multiple comparisons: P ≤ 0.01 (**), P < 0.001 (***), P ≤ 0.0001 (****) compared to the healthy vessels.
A pig model was developed to allow for IH development in hemodynamic conditions of unobstructed blood flow, as for human grafts. Significant occurrence of IH was observed on the venous patch area of the pig. This is an important accomplishment since it has been commented that pig models are more “thrombotic” and less “proliferative” than human restenosis [17]. Indeed, when injury was induced to the adventitial layer of the jugular vein, no IH development data is reported [10]. Instead,
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
103 in porcine models of vein to carotid interposition [12] or polytetrafluoroethylene graft placement [11, 14], the intimal hyperplasia development was sufficient to denote significant differences compared to treated animals. Moreover, the in vivo model we developed has the advantage of simultaneous evaluation of IH development both in an arterial and a venous substrate. In our set-up, the arterial side of the patch demonstrated minor IH development and no significant difference was observed when comparing sham to healthy animals. The short experimental time of 28 days is speculated to be the reason for this result. Another advantage of this model is the high success rates of surgery (100 % in our case). Indeed, when vein to artery interposition was employed, a 15 % rate of graft rupture was recorded [12].
The pigs receiving ATV did not show a significant difference in the development of IH compared to sham. This was not the case for the same formulation on a mouse model of carotid artery ligature [4]. ATV was previously shown to inhibit human VSMC proliferation [18]. Although the activity of statins on porcine VSMC in vitro has not been assessed, the mevalonate pathway is shared among mammals. Indeed statins are active as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors in the mevalonate pathway [19-21] and affect IH formation by reducing human VSMC proliferation, probably via inhibition of isoprenoid biosynthesis and subsequent protein prenylation [22]. Thus it is quite unlikely that the pig’s VSMC are deprived of the mevalonate pathway, through which VSMC proliferation is induced.
The results may be explained by the conservative dose approach. Indeed a dose of 0.2 mg/kg of ATV was administered in the porcine model for IH, while in the rodent model 50 mg/kg were given.
Statins have never been administered perivascularly in large animals in the past and thus the efficient dose range is unknown. Statin loaded intraluminal stents placed on pigs, successfully attenuated inflammation or stenosis at doses of 0.01 mg/kg for cerivastatin or pitavastatin [23, 24]. ATV administered per orally at doses as high as 2.5 and 5 mg/kg/day were necessary to inhibit IH in rabbit models of IH [25, 26]. However, those doses are close to the toxicity zone, as an oral administration of 10 mg/kg/day demonstrated extensive liver necrosis in rabbits [26]. All in all, a higher dose administration of ATV would be justified to control the efficacy of ATV perivascularly.
Angiogenesis
Carotid artery segments receiving a venous patch were evaluated in terms of vasa vasorum development (Fig. 5), by assessment of the microvessel presence on the adventition of SMA-stained sections. On the arterial side no angiogenesis was observed in all animal groups. Instead, on the venous patch side where the gel was mainly applied, the vasa vasorum development tripled in the ATV-receiving compared to the sham group. The presence of hyaluronic acid also induced the angiogenesis, as shown by 25 % in micro-vessel increased and as reported by other authors [27, 28].
CHAPTER IV - Local atorvastatin delivery from hydrogel and microparticles to prevent intimal hyperplasia in a pig model of arterial bypass grafting using a venous patch.
104 It is also of note that the vasa vasorum is mainly encountered on veins where the blood pressure is lower and the need for nutriments to reach the vessel wall from the outside of the vessel is more important.
The angiogenesis is stimulated by vascular injury (hypoxia and mechanical stress) and is regulated by endothelial cells and circulating endothelial progenitor cells through mechanism that remain to be deciphered [29, 30]. Statins are at least as potent as vascular endothelial growth factor (VEGF) in increasing proliferation and differentiation of endothelial progenitor cells [31, 32]. Importantly, it has been shown that the effect of orally administered statin on angiogenesis could be dose dependent [33-35]. Indeed the endothelial proliferation, migration and differentiation assessed in vitro and in vivo was promoted at low oral statin dose of 2.5 mg/kg/day and inhibited at high dosage. Others showed angiogenesis even at higher doses (4-8 mg/kg/day) of statins [29]. In our case, low doses of locally administered ATV enhanced vascular formation. When cytotoxic drugs are used for the inhibition of IH, a reduction in neoangiogenesis is observed [9]. The formation of vasa vasorum on
The angiogenesis is stimulated by vascular injury (hypoxia and mechanical stress) and is regulated by endothelial cells and circulating endothelial progenitor cells through mechanism that remain to be deciphered [29, 30]. Statins are at least as potent as vascular endothelial growth factor (VEGF) in increasing proliferation and differentiation of endothelial progenitor cells [31, 32]. Importantly, it has been shown that the effect of orally administered statin on angiogenesis could be dose dependent [33-35]. Indeed the endothelial proliferation, migration and differentiation assessed in vitro and in vivo was promoted at low oral statin dose of 2.5 mg/kg/day and inhibited at high dosage. Others showed angiogenesis even at higher doses (4-8 mg/kg/day) of statins [29]. In our case, low doses of locally administered ATV enhanced vascular formation. When cytotoxic drugs are used for the inhibition of IH, a reduction in neoangiogenesis is observed [9]. The formation of vasa vasorum on