Gut Bacteria Products Prevent AKI Induced by Ischemia-Reperfusion.

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification.

Delivery of daunorubicin to cancer cells with decreased toxicity by association with a lipidic nanoemulsion that binds to LDL receptors.

A lipidic nanoemulsion termed LDE concentrates in neoplastic cells after injection into the bloodstream and thus can be used as a drug carrier to tumour sites. The chemotherapeutic agent daunorubicin associates poorly with LDE; the aim of this study was to clarify whether the derivatization of daunorubicin by the attachment of an oleyl group increases the association with LDE, and to test the cytotoxicity and animal toxicity of the new preparation. The association of oleyl-daunorubicin (oDNR) to LDE showed high yield (93 +/- 2% and 84 +/- 4% at 1:10 and 1:5 drug:lipid mass, respectively) and was stable for at least 20 days. Association with oDNR increased the LDE particle diameter from 42 +/- 4 nm to 75 +/- 6 nm. Cytotoxicity of LDE-oDNR was reduced two-fold in HL-60 and K-562 cell lines, fourteen-fold in B16 cells and nine-fold in L1210 cells when compared with commercial daunorubicin. When tested in mice, LDE-oDNR showed remarkable reduced toxicity (maximum tolerated dose > 253 micromol kg(-1), compared with <3 micromol kg(-1) for commercial daunorubicin). At high doses, the cardiac tissue of LDE-oDNR-treated animals had much smaller structural lesions than with commercial daunorubicin. LDE-oDNR is therefore a promising new preparation that may offer superior tolerability compared with commercial daunorubicin.

Prophylactic Supplementation of Bifidobacterium longum 51A Protects Mice from Ovariectomy-Induced Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness.

Asthma is a chronic inflammatory disease that affects more females than males after puberty, and its symptoms and severity in women change during menstruation and menopause. Recently, evidence has demonstrated that interactions among the microbiota, female sex hormones, and immunity are associated with the development of autoimmune diseases. However, no studies have investigated if therapeutic gut microbiota modulation strategies could affect asthma exacerbation during menstruation and menopause. Here we aimed to examine the preventive effects of a probiotic, Bifidobacterium longum 51A, on airway inflammation exacerbation in allergic ovariectomized mice. We first evaluated the gut microbiota composition and diversity in mice 10 days after ovariectomy. Next, we examined whether re-exposure of ovariectomized allergic mice to antigen (ovalbumin) would lead to exacerbation of lung inflammation. Finally, we evaluated the preventive and treatment effect of B. longum 51A on lung inflammation and airway hyperresponsiveness. Our results showed that whereas ovariectomy caused no alterations in the gut microbiota composition and diversity in this animal model, 10 days after ovariectomy, preventive use administration of B. longum 51A, rather than its use after surgery was capable of attenuate the exacerbated lung inflammation and hyperresponsiveness in ovariectomized allergic mice. This prophylactic effect of B. longum 51A involves acetate production, which led to increased fecal acetate levels and, consequently, increased Treg cells in ovariectomized allergic mice.

Plasma kinetics of a cholesterol-rich microemulsion (LDE) in patients with Hodgkin's and non-Hodgkin's lymphoma and a preliminary study on the toxicity of etoposide associated with LDE.

BACKGROUND: Neoplastic diseases are often associated with low plasma low-density lipoprotein (LDL) cholesterol and diminished LDL clearance due to upregulation in cancer cells of the receptors that internalize the lipoprotein. Thus, it is possible to use LDL or cholesterol-rich microemulsions (LDE) that bind to LDL receptors as carriers of antineoplastic agents to concentrate those drugs into cancer tissues. Our aim was to determine whether LDL cholesterol concentration plus LDE increased clearance occur in lymphomas. PATIENTS AND METHODS: The LDE labeled with [(3) H]-cholesteryl oleate was injected into four Hodgkin's and 12 non-Hodgkin's lymphoma patients and into 16 healthy control subjects and the LDE plasma residence time (RT) was determined from sequential plasma samples. Two volunteers with relapsed/refractory lymphoma were treated with 300 mg/m(2) body surface etoposide associated with LDE in six cycles at 3-week intervals. RESULTS: The LDL cholesterol was lower in lymphoma patients than in controls (94+/-52 and 115+/-16 mg/dL, p=0.0362, respectively). The LDE RT was 49% smaller in lymphoma patients than in controls (RT=21.9 and 45.7 h; p=0.0134), with positive correlation between RT and LDL cholesterol. LDE-etoposide showed no considerable toxicity in all cycles in the two treated patients and the disease remained stable during the treatment. CONCLUSIONS: Our results suggest that lymphomas overexpress LDL receptors that make room for using LDE as drug-targeting vehicle and that the LDE-etoposide preparation is suitable for patient use.

Evaluation in melanoma-bearing mice of an etoposide derivative associated to a cholesterol-rich nano-emulsion.

A cholesterol-rich nano-emulsion (LDE) may be used as a vehicle to target antineoplastic drugs against cancer cells. The association of an etoposide derivative to LDE is stable and retains the cytotoxic activity of etoposide. We have evaluated the toxicity and antitumoral action of this new preparation in-vivo. Melanoma-bearing mice and control mice were administered LDE-etoposide oleate or commercial etoposide, either with or without radioactive labelling. The maximum tolerated dose (MTD), tissue distribution, plasma decay curves, pharmacokinetic parameters and antitumoral activity were determined. Association to LDE drastically reduced the drug toxicity, since MTD was approximately five-fold greater than in commercial etoposide. LDE-etoposide oleate was concentrated four-fold in the tumour compared with the normal adjacent tissues, was removed faster from plasma in tumour-bearing mice than in controls, and remained in the bloodstream longer than commercial etoposide. The tumour growth inhibition rate and survival were greater in animals treated with LDE-etoposide oleate compared with commercial etoposide. However, increasing the dose from 17 to 85 microM kg(-1) did not result in further improvement of the antitumour action. The incorporation of etoposide oleate to LDE resulted in markedly reduced toxicity and superior antitumoral activity. LDE-etoposide oleate is a promising new weapon for cancer treatment.

Improvement of paclitaxel therapeutic index by derivatization and association to a cholesterol-rich microemulsion: in vitro and in vivo studies.

A cholesterol-rich microemulsion or nanoparticle termed LDE concentrates in cancer tissues after injection into the bloodstream. Here the cytotoxicity, pharmacokinetics, toxicity to animals and therapeutic action of a paclitaxel lipophilic derivative associated to LDE is compared with those of the commercial paclitaxel. Results show that LDE-paclitaxel oleate is stable. The cytostatic activity of the drug in the complex is diminished compared with the commercial paclitaxel due to the cytotoxicity of the vehicle Cremophor EL used in the commercial formulation. Competition experiments in neoplastic cultured cells show that paclitaxel oleate and LDE are internalized together by the LDL receptor pathway. LDE-paclitaxel oleate arrests the G(2)/M phase of cell cycle, similarly to commercial paclitaxel. Tolerability to mice is remarkable, such that the lethal dose (LD(50)) was ninefold greater than that of the commercial formulation (LD(50) = 326 microM and 37 microM, respectively). LDE concentrates paclitaxel oleate in the tumor roughly fourfold relative to the normal adjacent tissues. At equimolar doses, the association of paclitaxel oleate with LDE results in remarkable changes in the drug pharmacokinetic parameters when compared to commercial paclitaxel (t(1/2)=218 min and 184 min, AUC=1,334 microg h/ml and 707 microg h/ml and CL=0.125 ml/min and 0.236 ml/min, respectively). Finally, the therapeutic efficacy of the complex is pronouncedly greater than that of the commercial paclitaxel, as indicated by the reduction in tumor growth, increase in survival rates and % cure of treated mice. In conclusion, LDE-paclitaxel oleate is a stable complex and compared with paclitaxel toxicity is considerably reduced and activity is enhanced, which may lead to improved therapeutic index in clinical use.

Use of a cholesterol-rich microemulsion that binds to low-density lipoprotein receptors as vehicle for etoposide.

A cholesterol-rich microemulsion (LDE) that binds to low-density lipoprotein (LDL) receptors is selectively taken up by malignant cells that overexpress those receptors and may be used as vehicle for antineoplastic agents. This study aimed to develop the association of etoposide with LDE. It was firstly observed that etoposide poorly associates with the microemulsion, therefore the experiments were performed with a lipophilic fatty acid derivative of the drug. The association of etoposide oleate with LDE was almost 100% and was tested for physical and chemical stability, as well as for cellular uptake, toxicity in mice and cytotoxic activity against a neoplastic cell line (NCI-H292). Uptake and cytotoxic activity of LDE-etoposide oleate by NCI-H292 cells was mediated by LDL receptors. The anti-proliferative activity of LDE-etoposide oleate against the neoplastic cells was smaller than that of etoposide oleate (IC50 (drug concentration required to inhibit 50% of the cell growth) = 0.48 and 0.19 mM, respectively). This difference, however, can be ascribed to the activity of the commercially used vehicle and not the drug itself because when this vehicle was added to the cultures with LDE-etoposide oleate, the IC50 decreased. On the other hand, the tolerability of LDE-etoposide oleate to mice was remarkable, such that its lethal dose (LD50) was about five-fold that of the commercial formulation (LD50 = 315 and 58 mg kg(-1), respectively). In conclusion, LDE-etoposide oleate association is stable and the cytostatic activity of the drug is preserved while its toxicity to animals is small. By diminishing the side effects and directing etoposide to neoplastic tissues, LDE may be regarded as an advance in chemotherapy with this drug.

Novel formulation of a methotrexate derivative with a lipid nanoemulsion.

BACKGROUND: Lipid nanoemulsions that bind to low-density lipoprotein receptors can concentrate chemotherapeutic agents in tissues with low-density lipoprotein receptor overexpression and decrease the toxicity of the treatment. The aim of this study was to develop a new formulation using a lipophilic derivative of methotrexate, ie, didodecyl methotrexate (ddMTX), associated with a lipid nanoemulsion (ddMTX-LDE). METHODS: ddMTX was synthesized by an esterification reaction between methotrexate and dodecyl bromide. The lipid nanoemulsion was prepared by four hours of ultrasonication of a mixture of phosphatidylcholine, triolein, and cholesteryloleate. Association of ddMTX with the lipid nanoemulsion was performed by additional cosonication of ddMTX with the previously prepared lipid nanoemulsion. Formulation stability was evaluated, and cell uptake, cytotoxicity, and acute animal toxicity studies were performed. RESULTS: The yield of ddMTX incorporation was 98% and the particle size of LDE-ddMTX was 60 nm. After 48 hours of incubation with plasma, approximately 28% ddMTX was released from the lipid nanoemulsion. The formulation remained stable for at least 45 days at 4°C. Cytotoxicity of LDE-ddMTX against K562 and HL60 neoplastic cells was higher than for methotrexate (50% inhibitory concentration [IC(50)] 1.6 versus 18.2 mM and 0.2 versus 26 mM, respectively), and cellular uptake of LDE-ddMTX was 90-fold higher than that of methotrexate in K562 cells and 75-fold in HL60 cells. Toxicity of LDE-ddMTX, administered at escalating doses, was higher than for methotrexate (LD(50) 115 mg/kg versus 470 mg/kg; maximum tolerated dose 47 mg/kg versus 94 mg/kg) in mice. However, the hematological toxicity of LDE-ddMTX was lower than for methotrexate. CONCLUSION: LDE-ddMTX was stable, and uptake of the formulation by neoplastic cells was remarkably greater than of methotrexate, which resulted in markedly greater cytotoxicity. LDE-ddMTX is thus a promising formulation to be tested in future animal models of cancer or rheumatic disease, wherein methotrexate is widely used.

Use of cholesterol-rich nanoparticles that bind to lipoprotein receptors as a vehicle to paclitaxel in the treatment of breast cancer: pharmacokinetics, tumor uptake and a pilot clinical study.

PURPOSE: In animal experiments paclitaxel oleate associated with a cholesterol-rich nanoemulsion concentrated in the neoplastic tissues and showed reduced toxicity and increased antitumor activity compared with paclitaxel-Cremophor EL. Here, a clinical study was performed in breast cancer patients to evaluate the tumoral uptake, pharmacokinetics and toxicity of paclitaxel associated to nanoemulsions. METHODS: Twenty-four hours before mastectomy [(3)H]-paclitaxel oleate associated with [(14)C]-cholesteryl oleate-nanoemulsion or [(3)H]-paclitaxel in Cremophor EL were injected into five patients for collection of blood samples and fragments of tumor and normal breast tissue. A pilot clinical study of paclitaxel-nanoemulsion administered at 3-week intervals was performed in four breast cancer patients with refractory advanced disease at 175 and 220 mg/m(2) dose levels. RESULTS: T (1/2) of paclitaxel oleate associated to the nanoemulsion was greater than that of paclitaxel (t (1/2) = 15.4 +/- 4.7 and 3.5 +/- 0.80 h). Uptake of the [(14)C]-cholesteryl ester nanoemulsion and [(3)H]-paclitaxel oleate by breast malignant tissue was threefold greater than the normal breast tissue and toxicity was minimal at the two dose levels. CONCLUSIONS: Our results suggest that the paclitaxel-nanoemulsion preparation can be advantageous for use in the treatment of breast cancer because the pharmacokinetic parameters are improved, the drug is concentrated in the neoplastic tissue and the toxicity of paclitaxel is reduced.

Paclitaxel associated with cholesterol-rich nanoemulsions promotes atherosclerosis regression in the rabbit.

A cholesterol-rich nanoemulsion (LDE) that resembles LDL binds to the LDL receptors and after injection into the blood stream may concentrate in cells with LDL receptor overexpression, as occurs in neoplasias and other proliferative processes. Thus, LDE can be used as vehicle to target drugs against those cells. The current study was designed to verify in rabbits whether LDE concentrates in the lesioned rabbit artery and whether a paclitaxel derivative, paclitaxel oleate, associated to LDE could reduce the atherosclerotic lesions. Sixteen male New Zealand rabbits were fed a 1% cholesterol diet for 60 days. Starting from day 30 under cholesterol feeding, eight animals were treated with four weekly intravenous injections of LDE-paclitaxel (4 mg/kg) and eight with four weekly intravenous saline solution injections for additional 30 days. On day 60, the animals were sacrificed for analysis. The uptake of LDE labeled with [(14)C]-cholesteryl oleate by the aortic arch of cholesterol-fed rabbits was twice as much that observed in animals fed only regular chow. LDE-paclitaxel reduced the lesion areas of cholesterol-fed animals by 60% and intima-media ratio fourfold and inhibited the macrophage migration and the smooth muscle cell proliferation and invasion of the intima. LDE-paclitaxel treatment had no toxicity. In conclusion, LDE-paclitaxel produced pronounced atherosclerosis regression without toxicity and has shown remarkable potential in cardiovascular therapeutics.

Plasma kinetics and uptake by the tumor of a cholesterol-rich microemulsion (LDE) associated to etoposide oleate in patients with ovarian carcinoma.

OBJECTIVES: Previously, we reported that etoposide oleate associated to a cholesterol-rich microemulsion (LDE) is taken up by malignant cells overexpressing low-density lipoprotein (LDL) receptors. The association is stable, preserves antiproliferative activity of the drug, and reduces toxicity to animals. Here, we determined in patients the plasma kinetics of LDE-etoposide oleate and verified whether the complex concentrates in ovarian carcinomas. METHODS: [(3)H]-etoposide oleate associated to LDE labeled with [(14)C]-cholesteryl oleate was intravenously injected into four ovarian carcinoma patients (50 +/- 8.7 years) 24 h before surgery. Blood samples were collected over a 24-h period to determine the radioactivity plasma decay curves, and the plasma fractional clearance rate (FCR) was calculated by compartmental analysis. Specimens of tumors and normal ovaries excised during the surgery were collected for lipid extraction and radioactive counting. RESULTS: FCRs of LDE label and of the drug were similar (0.0985 and 0.1722, respectively, P = 0.2422). [(14)C]-LDE uptake was 4.9 times and [(3)H]-etoposide oleate uptake was 4.1 times greater in the ovarian tumors than in the contralateral normal ovaries (LDE uptake, in cpm/g = 560 +/- 171 and 146 +/- 59; etoposide oleate uptake = 346 +/- 75 and 103 +/- 56, respectively). CONCLUSIONS: Most of the drug is retained in the microemulsion particles until its removal from the circulation and internalization by the cells. In addition, LDE-etoposide oleate has the ability to concentrate in malignant ovarian tissues. Therefore, the complex may be used to direct and concentrate etoposide oleate in ovarian carcinomas.