Hila Epstein

Delivery of serotonin to the brain by monocytes following phagocytosis of liposomes

Many drugs are not able to enter the brain due to the presence of the blood-brain barrier (BBB) and therefore cannot be used in the treatment of diseases of the brain. Since it is now known that the brain is under immunological surveillance, we hypothesized that phagocytic cells of the innate immune system, mainly neutrophils and monocytes, can be exploited as transporters of drugs to the brain. To target circulating mononuclear phagocytic cells, negatively-charged nano-sized liposomes were formulated encapsulating serotonin, a BBB impermeable neurological drug. Brain uptake, biodistribution, and the mechanism of brain transport were examined in vitro and in rats and rabbits by utilizing double-radiolabeled (3)H (in the membrane) and (14)C-serotonin (in the core), and liposomes with fluorescent markers (membrane and core). The brain uptake of liposomal serotonin was significantly higher (0.138%+/-0.034 and 0.097%+/-0.011, vs. 0.068%+/-0.02 and 0.057%+/-0.01, 4 h and 24 h after IV administration in rats, serotonin liposomes and in solution, respectively). The same brain uptake of both empty and serotonin liposomes, the co-localization in the brain of both markers, and the unchanged ratio of (3)H:(14)C suggest that intact liposomes entered the brain. Since treatment of animals by liposomal alendronate resulted with inhibition of monocytes but not of neutrophils, and with no brain delivery, it is suggested that monocytes are the main transporters of liposomes to the brain.

Liposomal Alendronate Inhibits Systemic Innate Immunity and Reduces In-Stent Neointimal Hyperplasia in Rabbits

Background—Innate immunity is of major importance in vascular repair. The present study evaluated whether systemic and transient depletion of monocytes and macrophages with liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation. Methods and Results—Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment. Liposomal alendronate (3 or 6 mg/kg) was given concurrently with stenting. Monocyte counts were reduced by >90% 24 to 48 hours after a single injection of liposomal alendronate, returning to basal levels at 6 days. This treatment significantly reduced intimal area at 28 days, from 3.88±0.93 to 2.08±0.58 and 2.16±0.62 mm2. Lumen area was increased from 2.87±0.44 to 3.57±0.65 and 3.45±0.58 mm2, and arterial stenosis was reduced from 58±11% to 37±8% and 38±7% in controls, rabbits treated with 3 mg/kg, and rabbits treated with 6 mg/kg, respectively (mean±SD, n=8 rabbits/group, P <0.01 for all 3 parameters). No drug-related adverse effects were observed. Reduction in neointimal formation was associated with reduced arterial macrophage infiltration and proliferation at 6 days and with an equal reduction in intimal macrophage and smooth muscle cell content at 28 days after injury. Conversely, drug regimens ineffective in reducing monocyte levels did not inhibit neointimal formation. Conclusions—Systemic transient depletion of monocytes and macrophages, by a single liposomal bisphosphonates injection concurrent with injury, reduces in-stent neointimal formation and arterial stenosis in hypercholesterolemic rabbits.

Systemic depletion of macrophages by liposomal bisphosphonates reduces neointimal formation following balloon-injury in the rat carotid artery.

Objectives Macrophage depletion by liposomal clodronate inhibits neointimal formation after balloon-injury. The present study examined bisphosphonates (BPs) potency-effect relationship and the role of systemic versus local monocytes in vascular repair. Methods and Results Liposomal preparations of clodronate, pamidronate, alendronate, and ISA-13-1 inhibited RAW-264 macrophages growth in a dose-response manner. Administration to balloon-injured rats suppressed neointimal growth. Neointima to media ratio (N/M) at 14 days was reduced from 1.35 ± 0.22 (control) to 0.4 ± 0.1 and 0.9 ± 0.17 by liposomal alendronate (1.5 mg/kg, i.v.) and liposomal ISA-13-1 (15 mg/kg), respectively (n = 8–10, P < 0.05). Suppression of neointimal formation was preserved at 30 days. Subcutaneous administration of liposomal BP (LBP) was also effective in suppressing neointimal formation, while short local intraluminal application had no effect. Immunostaining for ED-1 and ED-2 revealed no resident macrophages in the arterial wall, and reduced macrophage infiltration in LBP-treated animals. Arterial PDGF-B chain and PDGF-&bgr; receptor activation were reduced in LBP-treated animals and up-regulation of the PDGF receptor was noted. Conclusions Systemic transient inactivation of monocytes and macrophages by LBPs reduced macrophage infiltration and neointimal formation in the rat carotid injury model. The findings demonstrate a BP potency-effect relationship, and highlight the role of circulating monocytes in vascular injury and repair.

Route of administration-dependent anti-inflammatory effect of liposomal alendronate

Innate immunity and inflammation are of major importance in various pathological conditions. Intravenous (IV) and intraperitoneal (IP) liposomal alendronate (LA) treatments have been shown to deplete circulating monocytes and peritoneal macrophages resulting in the inhibition of restenosis and endometriosis (EM), respectively. Nevertheless, the correlation between the extent of circulating monocyte depletion and liposome biodistribution is unknown, and the route of administration-dependent bioactivity in restenosis and EM has not been determined. We found that, LA treatment resulted in a dose-response modified biodistribution following both IV and IP administrations. The biodistribution of high-dose LA (10mg/kg), but not that of the low-dose (1mg/kg), was similar in healthy and diseased animals. It is concluded that LA impedes its own elimination from the circulation by depleting circulating monocytes and/or inhibiting their endocytic activity, in a dose-dependent manner. Both IV and IP administration of LA mediated by the partial and transient depletion of circulating monocytes effected inhibition of restenosis. Inhibition of EM was effected only by IP administration, which depleted both intraperitoneal and circulating monocytes. Thus, EM should be considered as a local inflammatory condition with systemic manifestations as opposed to restenosis, a systemic inflammatory disease.

31P-NMR and Differential Scanning Calorimetry Studies for Determining VesicleâÂÂs Drug Physical State and Fraction in Alendronate Liposomes

Background: A liposomal delivery system requires a complete understanding of the physicochemical characteristics of the drug– liposome system in order to predict their behavior and stability in-vitro and in-vivo . Objectives: Develop a rapid and simple experimental method to determine the fractions of the drug, alendronate (ALN), encapsulated and as a free form distributed in the liposomal suspension, and the physical state of the encapsulated drug. Methods: 31 P-NMR measurements utilizing Ga +3 as a shifting reagent in comparison to HPLC determinations, theoretical calculations and differential scanning calorimetry (DSC) studies of various liposomal ALN formulations. Results: The 31 P-NMR demonstrated that titrating liposomal ALN with increasing amounts of Ga +3 induced a signi fi cant shift in the exterior fraction without changing the interior fraction. Quantitative determination of the encapsulated and non-encapsulated f ractions of ALN has been achieved at Ga +3 concentrations of 3.2-25mM. The DSC study revealed that none of the formulation ingredients is in a solid phase. Conclusions: 31 P-NMR was found to be sensitive enough to allow accurate differentiation of the distributed fractions of ALN, encapsulated and the non-encapsulated free form. Based on theoretical calculations and DSC analysis it can be concluded that AL N is dissolved in the aqueous core of the liposome.

Biodistribution and imaging studies of 67Ga-labeled liposomes in rabbits with a vascular injury

Innate immunity and inflammation are of major importance in vascular repair. Monocytes and neutrophils adhesion and infiltration occur immediately after vascular injury. We hypothesized that the systemic inflammatory state following vascular injury would result in altered biodistribution of the administered liposomes. The purpose of this study was to assess the biodistribution of liposomes in intact and balloon-injured rabbit carotid arteries. Real-time imaging, single photon emission computed tomography (SPECT) and dynamic tomography were used to evaluate the dynamic biodistribution. In addition, γ-counter scintigraphy of organ explants was used. Increased accumulation of liposomes was observed in the liver and spleen. Furthermore, a significantly higher concentration of liposomes was observed in the injured carotid in comparison to the intact artery, 0.091 ± 0.040% and 0.033 ± 0.016%, 24 h post-injection, respectively. Our findings emphasize the importance of assessing the distribution of drug and liposomal formulations under pathological conditions which are associated with an inflammatory process.

Predicting in vivo efficacy of potential restenosis therapies by cell culture studies: species-dependent susceptibility of vascular smooth muscle cells.

Although drug-eluting stents (DES) are successfully utilized for restenosis therapy, the development of local and systemic therapeutic means including nanoparticles (NP) continues. Lack of correlation between in vitro and in vivo studies is one of the major drawbacks in developing new drug delivery systems. The present study was designed to examine the applicability of the arterial explant outgrowth model, and of smooth muscle cells (SMC) cultures for prescreening of possible drugs. Elucidation of different species sensitivity (rat, rabbit, porcine and human) to diverse drugs (tyrphostins, heparin and bisphsophonates) and a delivery system (nanoparticles) could provide a valuable screening tool for further in vivo studies. The anticipated sensitivity ranking from the explant outgrowth model and SMC mitotic rates (porcine>rat>>rabbit>human) do not correlate with the observed relative sensitivity of those animals to antiproliferative therapy in restenosis models (rat≥rabbit>porcine>human). Similarly, the inhibitory profile of the various antirestenotic drugs in SMC cultures (rabbit>porcine>rat>>human) do not correlate with animal studies, the rabbit- and porcine-derived SMC being highly sensitive. The validity of in vitro culture studies for the screening of controlled release delivery systems such as nanoparticles is limited. It is suggested that prescreening studies of possible drug candidates for restenosis therapy should include both SMC cell cultures of rat and human, appropriately designed with a suitable serum.