Orexin A administration by OX26-mAb liposomes potentiates the nucleus accumbens shell dopamine responsiveness to food

INTRODUCTION The brain is one of the least accessible organs to circulating compounds because the blood-brain-barrier (BBB) restricts the most drug substances from the bloodstream into brain. For this reason, drug delivery across BBB is one of the major challenges in pharmaceutical research and development. BBB consists of a complex of structures that selectively regulate the passage of endogenous and exogenous substances from blood to CNS. Different strategies for the CNS drug administration have been developed: invasive administration (intracerebroventricular infusion, temporary disruption of the BBB, implants for drug delivery) and non-invasive administration (lipophylic prodrug, prodrug that utilize the physiological carrier mediated-CMT or receptor mediated-RMT active transport, PgP blockers). Among the different strategies evolved for permitting the administration of drugs in the CNS, the nanocarriers (liposomes, polymeric or lipidic nanoparticles, dendrimers, micells, etc) which utilize the Receptor-mediated transcytosis (RMT) have given encouraging results (Pardridge, 2005; Dawson et al ,2010). Orexin neurons originate in the hypothalamic region and project to different brain areas. They produce two different neuropeptides: orexin A (Orx A) and orexin B (Orx B) and two receptors for the orexin system have been characterized: OxR1 and OxR2. Orx A and Orx B neuropeptides are high hydrophilic compound and do not cross the BBB. Several studies reported that orexinergic neurons in the lateral hypothalamus (LH) are involved in motivated behavior for drugs of abuse as well as natural rewards. In particular, intraventricular administration of orexin A has been shown to stimulate food consumption, and orexin signaling in VTA is implicated in intake of high-fat food. It has been shown that LH orexin neurons project to the ventral tegmental area (VTA) indicating that the VTA is an important site of action for orexin's role in reward processing. Mesolimbic dopamine (DA) in the nucleus accumbens (NAc) shell is also involved in the rewarding mechanisms of food consumption. The aim of our study was the preparation and evaluation of long circulating liposomes for active Orx A targeting to SNC. To this purpose, anti-transferrin-monoclonal antibodies (OX26-mAb) was bounded to the liposome surface to transport the Orx A across the BBB by RMT. In vivo brain microdialysis was used to study the responsiveness of NAc shell DA transmission in food consumption after intravenous administration of Orx A loaded OX26-mAb liposomes (SLOX26) alone or in presence of an antagonist of the OxR1 (SB 334867). Not modified stealth liposomes were also prepared and tested as a reference. EXPERIMENTAL METHODS Surgery Male Sprague-Dawley rats (Harlan Italy, Udine, Italy) weighing 250-275 g were anesthetized with equitesin 5 ml/Kg i.p. A guide cannula (Plasticone, Roanoke, VA, USA) was stereotaxically implanted under the following coordinates: NAc shell (A:2.0; L: 1 from bregma, V: -3.6 from dura), according to the atlas of Paxinos & Watson (1998). During the same surgery session, rats were implanted with a catheter in the left femoral vein. After surgery, rats were housed in individual cages (45x21x24 cm) under the same conditions mentioned above. For 5 days after surgery, the catheters were flushed daily with 0.1 ml of gentamicin (40 mg/ml) as a preventive antibiotic therapy and with heparinized saline (heparin 250 U/ml in 0.9% sterile saline). To prevent the neophobia, five days before the microdialysis experiment, rats received once 10 sucrose pellets. Rats were kept with 15g of standard food and water ad libitum. Liposome preparation Liposomes were prepared using the thin-film hydration method. Distearoylphosphatidylcholine (DSPC, 5.2mmol), cholesterol (4.5mmol), DSPE-PEG (0.3mmol, Lipoid, Ludwigshafen, Germany,) and, for the preparation of immunoliposomes, DSPE-PEG–maleimide (0.18mmol, NOF Corporation ,Tokyo, Japan,) were dissolved in a chloroform/methanol (2:1 v/v) mixture. The solvent was evaporated under reduced pressure at room temperature. The obtained lipid film was hydrated under mechanical stirring with saline containing Orx A (292μg/ml, Tocris Bioscience,) at 65°C. Obtained liposomes were then subjected to sonication using a Soniprep 150 ultrasonic disintegrator (MSE Crowley, London, United Kingdom) until a clear opalescent dispersion was obtained. For the preparation of immunoliposomes (SLOX26), OX26-mAb (1 mg; 0.0058mmoles, AbD Serotec, Kidlington, UK,) was thiolated by reaction with iminothiolane (32mg; 0.23mmol) in 3ml of borate buffer solution adjusted at pH 8.1. EDTA solution (4mM) was added to chelate divalent metals eventually present in the solution. The mixture was stirred for 2 hours at room temperature. Thiolated OX26-mAb solutions were concentrated and the buffer replaced with phosphate buffer solution (pH 7.4) using a Centriprep-30 concentrator (molecular weight cut-off: 30,000). Finally, purified thiolated OX26-mAb was incubated with maleimide grafted liposomes overnight at room temperature. Immunoliposomes were separated from unentrapped orexin and free OX26-mAb by a gel filtration chromatography (Sepharose CL-4B) using phosphate saline as eluent. Microdialysis experiments The day before the microdialysis experiment rats were brought to the experimental room and placed in large hemispheric bowls with 15 g of standard food and water ad libitum. The next day, water and food were removed and a microdialysis probe was inserted through the guide cannula and perfused with a ringer solution at a constant flow of 1 μl/min. Dialysate samples were taken every 5 min and injected in the HPLC without purification. The microdialysis and behavioral experiments were performed in the same bowls. We studied: 1) the effect of the Orx A given by SLOX26 on NAc shell DA transmission; 2) the effect of the Orx A given by SLOX26 on NAc shell DA responsiveness during sucrose pellets consumption and on the number of sucrose pellets eaten; 3) the effect of the OxR1antagonist SB334867 (Tocris Bioscience) on the time-course of dialysate DA in the NAc shell during sucrose pellets consumption and number of sucrose pellets eaten (bars) after Orx A administration. Histology At the end of all experimental procedures, rats were anesthetized with Equitesin (5 ml/kg i.p.) and the brains were removed and postfixed for 5 days. The brains were cut in 100-μm-thick serial coronal slices on a Vibratome (Technical Products International, Saint Louis MO, USA) to establish the location of the dialysis probes. The location of the probes was reconstructed and referred to the atlas of Paxinos & Watson. RESULTS AND DISCUSSION All liposomes formulations showed a mean diameter around 120 nm, as determined by Dynamic Light Scattering and a polydispersity index smaller than 0.185, demonstrating a good monodisperse size distribution. Administration of OrX A by immunoliposomes SLOX26 increased DA basal levels in the NAc shell (Figure 1). Furthermore, OrX A potentiated the increase of NAc shell DA after sucrose feeding and also increased the number of eaten sucrose pellets (Figure 2). The injection of the OxR1 antagonist SB334867 completely blocked the increase of NAc shell DA observed during food consumption after OrX A administration and halved the number of eaten sucrose pellets. We can speculate that the strengthening of DA response during food consumption exerted by Orx A administration could increase the rewarding properties of food and could be one of the mechanisms that underlie food addiction Figure 1. Changes in dialysate DA in the NAc shell after Orexin A administration by immunoliposomes (SLOX 26) Figure 2. Time-course of dialysate DA in the NAc shell during sucrose pellets consumption and number of sucrose pellets eaten (bars) after Orexin A administration by immunoliposomes (SLOX 26) REFERENCES 1. Pardridge(2005) Mol biotech 30(1):57-69 2. Dawson LA et al (2010) Curr Pharm Des 16(3):344-57 3. Paxinos, G, Watson, C (1998) The rat brain in stereotaxic coordinates, 4th Ed. New York: Academic