Use of combined chemotherapy with etoposide and methotrexate, both associated to lipid nanoemulsions for atherosclerosis treatment in cholesterol-fed rabbits.

PURPOSE: Treatment of atherosclerotic rabbits with intravenous methotrexate or etoposide carried in lipid nanoemulsions (LDE) markedly reduced the lesions in the aorta. Here, the combined treatment with LDE-methotrexate and LDE-etoposide was investigated aiming to increase the anti-atherosclerosis effect. METHODS: Thirty-six male rabbits received a diet with 1 % cholesterol for 2 months. After the first month, the animals received 4 weekly intravenous injections of LDE-methotrexate (4 mg/kg dose), LDE-etoposide (6 mg/kg), or a combination of those two drugs, while the control animals were injected with LDE (n = 9 for each group). RESULTS: LDE-methotrexate+LDE-etoposide reduced aortic lesion areas by 95 % compared with controls and the intima-media ratio was reduced five-fold, whereas LDE-methotrexate reduced the lesions by 81 % and LDE-etoposide by 83 %. Compared to controls, the positive area of macrophages and MMP-9 in the arterial intima was significantly reduced in all treated groups (p < 0.001), but the MMP9 reduction was greater with the combined chemotherapy than the reduction achieved by the isolated treatments. Presence of CD3 positive cells was equal in controls and LDE-methotrexate+LDE-etoposide treated animals. However, FOXP3 positive T lymphocytes in the intima were increased in the LDE-methotrexate+LDE-etoposide rabbits. Weight, food intake evolution and the hematologic parameters suggested that the treatment had very low toxicity. CONCLUSIONS: Compared to the single treatments with LDE-methotrexate and LDE-etoposide, the combined treatment was more effective in reducing the atherosclerotic lesions. Because the toxicity of the novel drug-target combined scheme was low, those results favor the possibility of future clinical studies in patients with cardiovascular disease.

Development of anti-atherosclerosis therapy based on the inflammatory and proliferative aspects of the disease.

Atherosclerotic cardiovascular diseases are a worldwide major public health concern. Atherosclerosis is driven by a chronic inflammatory process which is present since the early stages of the disease, as a response to endothelium aggression by a variety of offending agents, to subsequent formation of foam cells, atheromatous plaque development and the clinical complications of the disease, due to plaque rupture and thromboembolic acute episodes. However, drug therapies directed to inflammation are lacking in the clinical practice, despite an increasing effort of research and identification of several potential molecular targets. Effective medical treatments available for primary and secondary prevention are restricted to cholesterol lowering statins and anti-platelet drugs such as aspirin. Here, steps of atherogenesis, cells involved in the process, secreted pro- and anti-inflammatory factors, the concept of unstable and stable atheroma plaques, the intertwining among inflammation, lipid arterial deposit, thrombus formation, therapeutically targetable mechanisms, monoclonal antibodies, enzymatic inhibitors, phytotherapeutic compounds and anti-proliferative agents used in cancer chemotherapy, drugs tested in experimental animals and at the clinical stage are shortly reviewed. Because statins and anti-platelet drugs use do not prevent more than 30-40% of the major cardiovascular events, the development of novel therapeutic tools is desirable. Nonetheless, atherosclerosis is a chronic process presumably demanding long-standing treatments, so that the safety, opportunity, cost-effectiveness and development of drug resistance are major issues that challenge the introduction of novel, inflammation-oriented therapies.

Anti-atherogenic effects of methotrexate carried by a lipid nanoemulsion that binds to LDL receptors in cholesterol-fed rabbits.

PURPOSE: Nanoemulsions (LDE) with a lipid composition resembling that of LDL can concentrate in aortic lesions and when associated with anti-blastic agents, such as paclitaxel or etoposide, decrease atherosclerotic lesions induced in rabbits. Our aim was to test the association of a lipophilic derivative of methotrexate, didodecyl-methotrexate (ddMTX) to LDE on the lesions and on the expression of pro-inflammatory and anti-inflammatory genes. METHODS: Twenty male New Zealand rabbits were fed 1 % cholesterol diet for 60 days. Starting from day 30, 10 animals were treated with 4 weekly LDE-ddMTX injections (4 mg/kg, I.V.) and 10 with LDE injections (20 mg LDE total lipid mass/kg). RESULTS: LDE-ddMTX reduced the size of the lesion areas by 65 % and the intima-media ratio by 2-fold. Reduction of intimal macrophage was 67 % and of apoptotic cells was 88 %. Smooth muscle cells migration into the intima was unaffected. LDE-ddMTX treatment diminished metalloproteinase-9 in the intima. In aortas of atherosclerotic rabbits, downregulation of 6 pro-inflammatory genes, TNF-α, MCP-1, IL-1ß, IL-18, MMP-9, MMP-12 and upregulation of the anti-inflammatory IL-10 gene were observed. Incubation of LDE-ddMTX with HUVEC cells led to downregulation of TNF-α IL1-ß VAP-1, TLR2 and CXCL2. CONCLUSIONS: LDE-ddMTX is potentially useful to threat atherosclerosis by acting on inflammatory processes which are instrumental in the development of the disease.