Development of diethylcarbamazine-loaded poly(caprolactone) nanoparticles for anti-inflammatory purpose: Preparation and characterization

Abstract Diethylcarbamazine-loaded nanoparticles were previously evaluated for their anti-inflammatory activity. However, little is known regarding their physicochemical properties. Thus, the purpose of this study was to physiochemically characterize diethylcarbamazine-loaded poly(caprolactone) nanoparticles and evaluate their in vitro cytotoxicity. All formulations were prepared using the double-emulsion method. The average particle size was in the ranged between 298 and 364 nm and the polydispersity indexes were below 0.3. The zeta potential values were marginally negative, which may be related to drug loading, as higher loading led to an increase in the modulus of the zeta potential values. Fourier transform infrared spectroscopy (FT-IR) and X-ray powder diffraction (XRD) analysis did not reveal any chemical interactions between the chemicals used and the absence of drug in crystalline form on the nanoparticle surfaces. The in vitro drug release study revealed a concentration-dependent release from the nanoparticles into the medium. The in vitro cytotoxicity assay demonstrated the biocompatibility of the blank and loaded nanoparticles. Hence, all formulations presented good physicochemical and safety properties, corroborating the in vivo anti-inflammatory activity, previously reported by our group.

Metformin prevents p-tau and amyloid plaque deposition and memory impairment in diabetic mice.

Insulin deficiency or resistance can promote dementia and hallmarks of Alzheimer's disease (AD). The formation of neurofibrillary tangles of p-TAU protein, extracellular Aß plaques, and neuronal loss is related to the switching off insulin signaling in cognition brain areas. Metformin is a biguanide antihyperglycemic drug used worldwide for the treatment of type 2 diabetes. Some studies have demonstrated that metformin exerts neuroprotective, anti-inflammatory, anti-oxidant, and nootropic effects. This study aimed to evaluate metformin's effects on long-term memory and p-Tau and amyloid ß modulation, which are hallmarks of AD in diabetic mice. Swiss Webster mice were distributed in the following experimental groups: control; treated with streptozotocin (STZ) that is an agent toxic to the insulin-producing beta cells; STZ + metformin 200 mg/kg (M200). STZ mice showed significant augmentation of time spent to reach the target box in the Barnes maze, while M200 mice showed a significant time reduction. Moreover, the M200 group showed reduced GFAP immunoreactivity in hippocampal dentate gyrus and CA1 compared with the STZ group. STZ mice showed high p-Tau levels, reduced p-CREB, and accumulation of ß-amyloid (Aß) plaque in hippocampal areas and corpus callosum. In contrast, all these changes were reversed in the M200 group. Protein expressions of p-Tau, p-ERK, pGSK3, iNOS, nNOS, PARP, Cytochrome c, caspase 3, and GluN2A were increased in the parietal cortex of STZ mice and significantly counteracted in M200 mice. Moreover, M200 mice also showed significantly high levels of eNOS, AMPK, and p-AKT expression. In conclusion, metformin improved spatial memory in diabetic mice, which can be associated with reducing p-Tau and ß-amyloid (Aß) plaque load and inhibition of neuronal death.

Preventive role of metformin on peripheral neuropathy induced by diabetes.

Metformin is the first line drug in the treatment of type 2 diabetes, however, little is known about its therapeutic potential to prevent or delay damage to the peripheral nerve. Thus, the aim of this study was to investigate whether metformin is able to attenuate the neuroinflammatory response in sciatic nerve of insulin-dependent diabetic mice. Swiss Webster mice were divided into four groups: Control, Diabetic (STZ), Diabetic +100 mg/kg/day of metformin (STZ + M100) and Diabetic +200 mg/kg/day of metformin. Diabetes was induced by streptozotocin (90 mg/kg, i.p.). Only animals with glycemia ≥270 mg/dl were considered diabetics. Metformin prevented atrophy of myelinated axons, and reduced expression of inflammatory mediators (interleukin-1ß, inducible nitric oxide synthase and nitric oxide). However, treatment with 200 mg of metformin was more effective in increasing neurotrophic (myelin basic protein and neural growth factor), angiogenic (vascular endothelial growth factor) and anti-inflammatory (inhibitor kappa B-alpha and interleukin 10) factors. Thus, metformin treatment, especially at the dose of 200 mg, protected the nerve from damages related to chronic hyperglycemia.

A new diethylcarbamazine formulation (NANO-DEC) as a therapeutic tool for hepatic fibrosis.

The aim of the present study was to assess if the uninterrupted and prolonged administration of nanoparticles containing diethylcarbamazine (NANO-DEC) would cause liver, kidney and heart toxicity and then analyze for the first time its action in model of liver fibrosis. Thus, NANO-DEC was administered in C57BL/6 mice daily for 48 days, and at the end the blood was collected for biochemical analyzes. In the long-term administration assay, the evaluation of serological parameters (CK-MB, creatinine, ALT, AST and urea) allowed the conclusion that NANO-DEC prolonged administration did not cause hepatic, renal and cardiac damage. For fibrosis assays, C57BL/6 mice were divided into six groups: 1) control (Cont); 2) carbon tetrachloride (CCl4); 3) CCl4 + DEC 25 mg/kg; 4) CCl4 + DEC 50 mg/kg; 5) CCl4 + NANO-DEC 5 mg/kg and 6) CCl4 + NANO-DEC 12.5 mg/kg. Carbon tetrachloride induced hepatic fibrosis observed through increased inflammatory (TNF-α, IL-1ß, COX-2, NO and iNOS) and fibrotic markers (TGF-ß and TIMP-1), changes in the hepatic morphology, high presence of collagen fibers and elevated serum levels of AST, ALT and ALP. Treatment with NANO-DEC exhibited a superior anti-inflammatory and anti-fibrotic effects compared to the DEC traditional formulation, restoring liver morphology, reducing the content of collagen fibers and serological parameters, besides decreasing the expression of inflammatory and fibrotic markers. The present formulation of nanoencapsulated DEC is a well tolerated anti-inflammatory and anti-fibrotic drug and therefore could be a potential therapeutic tool for the treatment of chronic liver disorders.

New thiazolidinedione LPSF/GQ-2 inhibits NFκB and MAPK activation in LPS-induced acute lung inflammation.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are responsible for high mortality rates in critical patients. Despite >50 years of intensive research, there is no pharmacologically effective treatment to treat ALI. PPARs agonists, chemically named thiazolidinediones (TZDs) have emerged as potential drugs for the treatment of ALI and ARDS due to their anti-inflammatory efficacy. The present study aims to evaluate the potential anti-inflammatory effects of new TZDs derivatives, LPSF/GQ-2 and LPSF/RA-4, on ALI induced by LPS. BALB/c mice were divided into five groups: 1) Control; 2) LPS intranasal 25 µg; 3) LPSF/GQ-2 30 mg/kg + LPS; 4) LPSF/RA-4 20 mg/kg + LPS; and 5) DEXA 1 mg/Kg + LPS. BALF analyses revealed that LPSF/GQ-2 and LPSF/RA-4 reduced NO levels in BALF and inflammatory cell infiltration induced by LPS. MPO levels were also reduced by the LPSF/GQ-2 and LPSF/RA-4 pre-treatments. In contrast, histopathological analyses showed better tissue protection with LPSF/GQ-2 than DEXA and LPSF/RA-4 groups. Similarly, LPSF/GQ-2 reduced inflammatory markers (IL-1, iNOS, TNFα, IL-1ß, IL-6) better than LPSF/RA-4. The LPSF/GQ-2 anti-inflammatory action could be attributed to the inhibition of NFκB, ERK, p38, and PARP pathways. In contrast, LPSF/RA-4 had no effect on the expression of p38, JNK, NFκB. The present study indicates that LPSF/GQ-2 presents a potential therapeutic role as an anti-inflammatory drug for ALI.

Diethylcarbamazine attenuates the expression of pro-fibrogenic markers and hepatic stellate cells activation in carbon tetrachloride-induced liver fibrosis.

BACKGROUND AND AIM: While diethylcarbamazine citrate (DEC) displays important anti-inflammatory effects in experimental models of liver injury, the mechanisms of its action remain poorly understood. The aim of the present study was to investigate the fibrolytic potential of DEC. METHODS: Mice receive two injections of carbon tetrachloride (CCl4) per week for 8 weeks. DEC 50 mg/kg body weight was administered through drinking water during the last 12 days of liver injury. RESULTS: The expression of hepatic stellate cells (HSCs) activation markers, including smooth muscle α-actin (α-SMA), collagen I, transforming growth factor-ß 1 (TGF-ß1), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) was assessed. The influence of DEC on the intracellular MAPK pathways of the HSCs (JNK and p38 MAPK) was also estimated. DEC inhibited HSCs activation measured as the production of α-SMA and collagen I. In addition, it down regulated the production of TGF-ß1 and TIMP-1, and concomitantly increased MMP-2 activity. Furthermore, DEC significantly inhibited the activation of the JNK and p38 MAPK signaling pathways. CONCLUSIONS: In conclusion, DEC significantly attenuated the severity of CCl4-induced liver injury and the progression of liver fibrosis, exerting a potential fibrolytic effect in the CCl4-induced fibrosis model.

Characterization and evaluation of nanoencapsulated diethylcarbamazine in model of acute hepatic inflammation.

Previous studies from our laboratory have demonstrated that Diethylcarbamazine (DEC) is a potent anti-inflammatory drug. The aim of the present study was to characterize the nanoencapsulation of DEC and to evaluate its effectiveness in a model of inflammation for the first time. C57BL/6 mice were divided into six groups: 1) Control; 2) Carbon tetrachloride (CCl4); 3) DEC 25mg/kg+CCl4; 4) DEC 50mg/kg+CCl4; 5) DEC-NANO 05mg/kg+CCl4 and 6) DEC-NANO 12.5mg/kg+CCl4. Liver fragments were stained with hematoxylin-eosin, and processed for Western blot, ELISA and immunohistochemistry. Serum was also collected for biochemical measurements. Carbon tetrachloride induced hepatic injury, observed through increased inflammatory markers (TNF-α, IL-1ß, PGE2, COX-2 and iNOS), changes in liver morphology, and increased serum levels of total cholesterol, triglycerides, TGO and TGP, LDL, as well as reduced HDL levels. Nanoparticles containing DEC were characterized by diameter, polydispersity index and zeta potential. Treatment with 12.5 nanoencapsulated DEC exhibited a superior anti-inflammatory action to the DEC traditional dose (50mg/kg) used in murine assays, restoring liver morphology, improving serological parameters and reducing the expression of inflammatory markers. The present formulation of nanoencapsulated DEC is therefore a potential therapeutic tool for the treatment of inflammatory hepatic disorders, permitting the use of smaller doses and reducing treatment time, while maintaining high efficacy.

Effects of metformin on inflammation and short-term memory in streptozotocin-induced diabetic mice.

The aim of the present study was to analyze the action of metformin on short-term memory, glial cell activation and neuroinflammation caused by experimental diabetic encephalopathy in C57BL/6 mice. Diabetes was induced by the intraperitoneal injection of a dose of 90mg/kg of streptozotocin on two successive days. Mice with blood glucose levels ≥200dl/ml were considered diabetic and were given metformin hydrochloride at doses of 100mg/kg and 200mg/kg (by gavage, twice daily) for 21 days. On the final day of treatment, the mice underwent a T-maze test. On the 22nd day of treatment all the animals were anesthetized and euthanized. Diabetic animals treated with metformin had a higher spatial memory score. The hippocampus of the diabetic animals presented reactive gliosis, neuronal loss, NF-kB signaling activation, and high levels of IL-1 and VEGF. In addition, the T-maze test scores of these animals were low. Treatment with metformin reduced the expression of GFAP, Iba-1 (astrocyte and microglial markers) and the inflammation markers (p-IKB, IL-1 and VEGF), while enhancing p-AMPK and eNOS levels and increasing neuronal survival (Fox-1 and NeuN). Treatment with metformin also improved the spatial memory scores of diabetic animals. In conclusion, the present study showed that metformin can significantly reduce neuroinflammation and can decrease the loss of neurons in the hippocampus of diabetic animals, which can subsequently promote improvements in spatial memory.

Diethylcarbamazine: possible therapeutic alternative in the treatment of alcoholic liver disease in C57BL/6 mice.

Alcoholic liver disease is a major cause of chronic liver disease worldwide. Diethylcarbamazine (DEC) is a drug that has anti-inflammatory properties due to its effects on the metabolism of arachidonic acid. The present study examined the anti-inflammatory effects of DEC on the mechanisms of alcoholic liver disease. C57BL/6 mice were divided into seven groups: (i) control; (ii) DEC 50 mg/kg; (iii) alcohol; (iv) alcohol + DEC 50 mg/kg; (v) alcohol + celecoxib 50 mg/kg; (vi) alcohol + pyrrolidine dithiocarbamate 100 mg/kg; and (vii) alcohol + pyrrolidine dithiocarbamate 100 mg/kg + DEC 50 mg/kg. Liver fragments were stained with haemotoxylin-eosin and Sirius red, and processed for immunofluorescence, western blot, and immunohistochemistry. Serum was also collected for biochemical measurements. Alcohol induced liver damage, elevated collagen content, and increased expression of nuclear factor kappa-light-chain-enhancer of activated B cells and inflammatory markers (tumour necrosis factor-α, interferon-γ, interleukin-1ß, inducible nitric oxide synthase, cyclooxygenases-2, and transforming growth factor-ß). Treatment with DEC was able to reduce liver damage, collagen content, the expression of nuclear factor kappa-light-chain-enhancer of activated B cells and inflammatory markers; it also ameliorated biochemistry parameters (total cholesterol, high-density lipoprotein cholesterol, triglyceride content and aspartate aminotransferase) and increased the expression of anti-inflammatory markers (p-5' adenosine monophosphate-activated protein kinase and interleukin-10). Future clinical trials may demonstrate that oral administration of DEC may be suitable for the treatment of alcoholic liver disease and other liver diseases.

Diethylcarbamazine reduces chronic inflammation and fibrosis in carbon tetrachloride- (CCl4-) induced liver injury in mice.

This study investigated the anti-inflammatory effects of DEC on the CCl4-induced hepatotoxicity in C57BL/6 mice. Chronic inflammation was induced by i.p. administration of CCl4 0.5 µL/g of body weight through two injections a week for 6 weeks. DEC (50 mg/kg) was administered by gavage for 12 days before finishing the CCl4 induction. Histological analyses of the DEC-treated group exhibited reduced inflammatory process and prevented liver necrosis and fibrosis. Immunohistochemical and immunofluorescence analyses of the DEC-treated group showed reduced COX-2, IL1ß, MDA, TGF-ß, and αSMA immunopositivity, besides exhibiting decreased IL1ß, COX-2, NFκB, IFNγ, and TGFß expressions in the western blot analysis. The DEC group enhanced significantly the IL-10 expression. The reduction of hepatic injury in the DEC-treated group was confirmed by the COX-2 and iNOS mRNA expression levels. Based on the results of the present study, DEC can be used as a potential anti-inflammatory drug for chronic hepatic inflammation.

Diethylcarbamazine inhibits NF-κB activation in acute lung injury induced by carrageenan in mice.

Diethylcarbamazine citrate (DEC) is widely used to treat lymphatic filariasis and Tropical Pulmonary Eosinophilia. A number of studies have reported a possible role in the host immune system, but exactly how DEC exerts this effect is still unknown. The present study reports the effects of DEC pretreatment on NF-κB regulation using the pleurisy model induced by carrageenan. Swiss male mice (Mus musculus) were divided into four experimental groups: control (SAL); carrageenan (CAR); diethylcarbamazine (DEC) and curcumin (CUR). The animals were pretreated with DEC (50mg/kg, v.o), CUR (50mg/kg, i.p) or distilled water for three consecutive days before pleurisy. One way analysis of variance (ANOVA) was performed by Tukey post-hoc test, and values were considered statistically significant when p<0.05. DEC pretreatment reduced tissue damage and the production of inflammatory markers, such as NO, iNOS, PGE2, COX-2, and PARP induced by carrageenan. Similarly, a known inhibitor of NF-κB pathway (curcumin) was also able to reduce these parameters. Like curcumin, DEC prevents NF-κB activation by reducing NF-κB p65 phosphorylation and IκBα degradation. DEC prevented NF-κB activation via p38 MAPK, but did not interfere in the ERK pathway in this experimental model. However, further studies should be developed to confirm this hypothesis. These findings suggest that DEC could be a promising drug for inflammatory disorders, especially in pulmonary diseases such as Acute Lung Inflammation, due its high anti-inflammatory potential which prevents NF-κB activation.

Inhibition of NF-κB activation by diethylcarbamazine prevents alcohol-induced liver injury in C57BL/6 mice.

Induction of NF-κB-mediated gene expression has been identified in the pathogenesis of alcoholic liver disease (ALD). Diethylcarbamazine (DEC) is a piperazine derivative drug with anti-inflammatory properties. The present study was designed to evaluate the effect of DEC on NF-κB pathways in mice undergoing alcoholism induced hepatic inflammation. Forty male C57BL/6 mice were divided equally into four groups: control group (C); DEC-treated group, which received 50mg/kg (DEC50); alcoholic group (EtOH), submitted to chronic alcohol consumption and the alcohol-DEC treated group (EtOH50), submitted to chronic alcoholism consumption plus DEC treatment. Histological analysis of the alcoholic group showed evident hepatocellular damage which was reduced in EtOH50 group. Immunohistochemistry and western blot results showed elevated expression of inflammatory markers such as MDA, TNF-α, IL-1ß, COX-2 and iNOS in hepatocytes of EtOH group. However, low immunopositivity for these markers was detected following DEC treatment. In the EtOH group the activation of NF-κB was observed by an increase in the expression of both NF-κB and pNF-κB in hepatocytes. This expression was significantly reduced in livers of EtOH50 group. Protein expression of Iκßα was measured to determine whether activation of NF-κB might be the result of Iκßα degradation. It was observed that expression of this protein was low in EtOH group, while animals treated with DEC had a high expression of Iκßα. The results of the present study indicate that DEC alleviates alcoholic liver injury, in part by the inhibiting activation of NF-κB and by suppressing the induction of NF-κB-dependent genes.