Frequency and impact of DHODH, ABCG2 and CYP2C19 SNPs on the therapeutic efficacy, tolerability and toxicity of leflunomide.

Introduction: Leflunomide is one of the commonly used drugs in treatment of rheumatoid arthritis (RA), which on administration is converted into its active metabolite teriflunomide. Aim: Our aim is to evaluate the frequencies of dihydrooroate dehydrogenase (DHODH) (rs3213422), ABCG2 (rs2231142) and CYP2C19 (rs4244285) allele distribution among patients receiving leflunomide for RA and their possible impact on leflunomide performance in disease control. Patients & methods: Patients (>18 years) who fulfilled the 2010 ACR classification criteria for RA receiving leflunomide (20 mg/day) were included in the study. Disease activity score 28 was used to assess patients disease activity. Blood samples were collected for full blood count and blood chemistry. Genomic DNA was extracted from peripheral blood. The selection of SNPs was based on the criteria of minor allele frequency among Caucasians. Results: A significant association between the therapeutic outcome of leflunomide and DHODH genotyping was observed but not with CYP2C19 and ABCG2. Importantly, there is a significant association between DHODH (rs3213422) CC genotype and the number of patients with controlled disease. Conclusion: We strongly suggest that polymorphisms in the DHODH are the major factor affecting leflunomide pharmacogenetics and therapeutic efficacy.

Antibiofilm and antivirulence potential of silver nanoparticles against multidrug-resistant Acinetobacter baumannii.

We aimed to isolate Acinetobacter baumannii (A. baumannii) from wound infections, determine their resistance and virulence profile, and assess the impact of Silver nanoparticles (AgNPs) on the bacterial growth, virulence and biofilm-related gene expression. AgNPs were synthesized and characterized using TEM, XRD and FTIR spectroscopy. A. baumannii (n = 200) were isolated and identified. Resistance pattern was determined and virulence genes (afa/draBC, cnf1, cnf2, csgA, cvaC, fimH, fyuA, ibeA, iutA, kpsMT II, PAI, papC, PapG II, III, sfa/focDE and traT) were screened using PCR. Biofilm formation was evaluated using Microtiter plate method. Then, the antimicrobial activity of AgNPs was evaluated by the well-diffusion method, growth kinetics and MIC determination. Inhibition of biofilm formation and the ability to disperse biofilms in exposure to AgNPs were evaluated. The effect of AgNPs on the expression of virulence and biofilm-related genes (bap, OmpA, abaI, csuA/B, A1S_2091, A1S_1510, A1S_0690, A1S_0114) were estimated using QRT-PCR. In vitro infection model for analyzing the antibacterial activity of AgNPs was done using a co-culture infection model of A. baumannii with human fibroblast skin cell line HFF-1 or Vero cell lines. A. baumannii had high level of resistance to antibiotics. Most of the isolates harbored the fimH, afa/draBC, cnf1, csgA and cnf2, and the majority of A. baumannii produced strong biofilms. AgNPs inhibited the growth of A. baumannii efficiently with MIC ranging from 4 to 25 µg/ml. A. baumannii showed a reduced growth rate in the presence of AgNPs. The inhibitory activity and the anti-biofilm activity of AgNPs were more pronounced against the weak biofilm producers. Moreover, AgNPs decreased the expression of kpsMII , afa/draBC,bap, OmpA, and csuA/B genes. The in vitro infection model revealed a significant antibacterial activity of AgNPs against extracellular and intracellular A. baumannii. AgNPs highly interrupted bacterial multiplication and biofilm formation. AgNPs downregulated the transcription level of important virulence and biofilm-related genes. Our findings provide an additional step towards understanding the mechanisms by which sliver nanoparticles interfere with the microbial spread and persistence.

Ameliorative effect of L-carnitine on chronic lead-induced reproductive toxicity in male rats.

Lead toxicity is one of the causative agents of male infertility that raised concern from environmental contamination worldwide. L-carnitine, a biologically active amino acid, present in high concentration in the reproductive organs such as the epididymis, is involved in sperm maturation. The possible protective effect of L-carnitine in experimentally lead-induced male reproductive toxicity in rats was evaluated in this study. Thirty adult male Wistar rats were divided into three groups. Group 1: the negative control group was treated with normal saline; Group 2: exposed to 50 mg/kg lead acetate (2% solution in saline); and Group 3: treated with lead acetate 50 mg/kg (2% solution in saline) + L-carnitine 100 mg/kg. At the end of the experimental period, body and testicular weights were determined, blood samples were withdrawn for hormonal assays of FSH, LH and testosterone. Sperm parameters as sperm count, morphology, viability and motility were measured. Testicular tissue homogenates were prepared for enzymatic assays and for measuring oxidative stress parameters. Lead significantly increased both oxidative stress and the concentration of lactate dehydrogenase-C in the testicular tissues with a decrease in sperm count, motility and viability. Lead acetate treatment, induced alteration in sperms with normal morphology together with reductions in the serum FSH, LH, testosterone, body and testicular weights. The concentration of 17ß-hydroxysteroid dehydrogenase was significantly reduced. Co-administration of L-carnitine significantly reduced testicular oxidative stress, improved sperm parameters, elevated serum FSH, LH and testosterone with an insignificant reduction in the testicular weight. The concentrations of 17ß-hydroxysteroid dehydrogenase and lactate dehydrogenase-C were significantly improved by L-carnitine. The overall results indicate that L-carnitine is expected to improve the lead acetate-induced male reproductive toxicity.

Evaluation of the neuroprotective effect of donepezil in type 2 diabetic rats.

Recent studies raise the possibility that donepezil can delay the progression of Alzheimer's disease (AD). This research evaluated the efficacy of donepezil in an animal model with brain insulin resistance and AD-like alterations. Rats were fed with high-fat/high-fructose (HF/Hfr) diet during the study period (17 weeks) and received one injection of streptozotocin (STZ) (25 mg/kg) after 8 weeks of starting the study. Diabetic (T2D) rats were treated with donepezil (4 mg/kg; p.o.) or vehicle for 8 weeks after STZ injection. The influence of donepezil on AD-related behavioral, biochemical, and neuropathological changes was investigated in T2D rats. Treatment of diabetic rats with donepezil led to a significant decrease in both amyloid-ß deposition and the raised hippocampal activity of cholinesterase (ChE). It significantly increased the suppressed glutamate receptor expression (AMPA GluR1 subunit and NMDA receptor subunits NR1, NR2A, NR2B). It also improved cognitive dysfunction in the passive avoidance and the Morris water maze tests. However, donepezil treatment did not significantly decrease the elevated levels of P-tau, caspase-3, GSK-3ß, MDA, TNF-α, and IL-1ß in the hippocampus of diabetic rats. Also, it did not restore the suppressed levels of glutathione and superoxide dismutase in the brain of these rats. Moreover, donepezil did not alter the elevated serum level of glucose, insulin, and total cholesterol. These findings suggest that donepezil treatment could ameliorate learning and memory impairment in T2D rats through reversal of some of the AD-related alterations, including reduction of amyloid-ß burden and ChE activity as well as restoration of glutamate receptor expression. However, lack of any significant effect on P-tau load, oxidative stress, neuroinflammation, and insulin resistance raises the question about the ability of donepezil to delay the development or arrest the progression of T2D-induced AD and it is still a matter of debate that requires further studies.

Cytotoxic Chemotherapy as an Alternative for Systemic Treatment of Advanced Hepatocellular Carcinoma in Developing Countries.

Systemic therapy options nowadays for advanced hepatocellular carcinoma (HCC) are either immunotherapy with immune checkpoint inhibitors or targeted therapy. As the incidence of liver cancer is much higher in developing countries, these new medications are not readily accessible for most of the patients. Cytotoxic chemotherapy agents are more available and affordable in developing countries. We are trying to explore the effectiveness of the newer cytotoxic agents in the systematic treatment for advanced HCC. This is a systematic review of all randomized controlled trials since 1997 that utilized systemic cytotoxic chemotherapy agents in the systemic treatment for advanced HCC using Scopus, PubMed, and Cochrane library up to February 2020. Six randomized trials were found. Different drugs and dosages were used, so it was statistically inappropriate to conduct a meta-analysis. No Phase III trial showed statistically significant overall survival (OS) benefit for cytotoxic chemotherapy, except subgroup analysis of Chinese patients in one study who had leucovorin, fluorouracil, and oxaliplatin (FOLFOX) regimen. There was no significant progression-free survival (PFS) or response rate in the Phase II trials. There are not enough data to infer the actual benefits of systemic cytotoxic chemotherapy in advanced HCC. However, oxaliplatin-based regimens may give feasible results. Health systems with limited access to targeted therapy and immunotherapy agents may use oxaliplatin-based regimens in clinical trials for advanced HCC. These results should be confirmed in multiple future randomized clinical trials.

Modulation of rifampicin-induced hepatotoxicity using poly(lactic-co-glycolic acid) nanoparticles: a study on rat and cell culture models.

Aim: Hepatotoxicity is the most serious adverse effect of rifampicin (RIF). We aimed to investigate the potential hepatoprotective effect of mannose-functionalized poly(lactic-co-glycolic acid)(PLGA)/RIF nanoparticles (NPs) in rats as a possible promising approach to minimize RIF-induced hepatotoxicity. Materials & methods: Mannose-functionalized PLGA/RIF NPs were fabricated and characterized in vitro, then the hepatoprotective effect of optimized NPs was studied on rat and cell culture models. Results: Following intraperitoneal administration of RIF NPs into rats, highly significant differences in levels of serum transaminases and oxidative stress markers, associated with significant differences in expression of Bax and Bcl-2 genes between NP- and free RIF-treated groups, revealing the hepatoprotective potential of NPs. Conclusion: RIF NPs may represent a promising therapeutic approach for tuberculosis via reducing dose frequency and consequently, RIF-induced hepatotoxicity.

Montelukast modifies simvastatin-induced myopathy and hepatotoxicity.

Montelukast (MNK) has prominent anti-inflammatory and antioxidant activities. It can protect the liver in different hepatotoxic models in animals. Simvastatin (SMV) is one of commonly used lipid lowering drugs for treatment of dyslipidemia in order to reduce cardiovascular disease. It has severe side effects such as myopathy and hepatotoxicity. The aim of the present study is to investigate the possible effect of MNK on SMV-induced myopathy and hepatotoxicity. Four groups of male rats: control group which received saline via stomach tube, MNK treated group (received 10 mg/kg/day MNK via stomach tube), SMV treated group (received 30 mg/kg/day SMV via stomach tube), and MNK + SMV (combination) group which received both MNK and SMV. All animals were treated for 14 days before obtaining blood and tissue samples. SMV has both hepatotoxic effects and myopathy. SMV caused a significant increase in myoglobin, creatinine kinase, ALT, AST, ALP, and bilirubin but, it decreased total proteins, globulin and albumin levels. Co-treatment of SMV and MNK increased the antioxidant activity significantly. MNK modifies partially the myopathic changes and hepatotoxic effect of SMV. Co-administration of MNK and SMV decreased their toxic potentials on the liver, skeletal muscles, and kidney. They have antioxidant activities when given together that produce muscle and hepatic protective effects.

Metoclopramide nanoparticles modulate immune response in a diabetic rat model: association with regulatory T cells and proinflammatory cytokines.

BACKGROUND: The inflammatory basis of diabetes mellitus directed the researchers' attention to the immune system for better management and prevention of complications. Metoclopramide (MCA; the only US Food and Drug Administration-approved for gastroparesis) has the ability to restore immune function through increasing prolactin secretion. This study aimed to test the effect of BSA/MCA nanoparticles (NPs) on modulating immune response. METHODS: BSA/MCA NPs were fabricated by desolvation and evaluated in vitro via measuring loading efficiency, particle size, and surface charge. The selected formula was further evaluated via differential scanning calorimetry and release behavior. Then, NPs were injected into rats (25 mg MCA/kg/week) for 3 weeks to be evaluated histopathologically and immunologically via measuring proinflammatory cytokines, such as IL1ß, IL6, and TNFα, in addition to measuring regulatory T-cell frequency. RESULTS: MCA was successfully loaded on BSA, achieving high encapsulation efficiency reaching 63±2%, particles size of 120-130 nm with good polydispersity, and a negative surface charge indicating that entire positively charged drug was encapsulated inside NPs. Differential scanning calorimetry thermography of selected NPs showed an obvious interaction between components and cross-linking of BSA molecules using glutaraldehyde, resulting in sustained release of MCA (around 50% within 3 days). MCA NPs significantly restored the immune response via decreasing proinflammatory cytokines and increasing regulatory T-cell frequency when compared to control and free MCA (drug not loaded in NPs)-treated groups. Histopathological examination of this MCA NPs-treated group did not show the characteristic lesions of diabetes, and apoptosis nearly disappeared. CONCLUSION: BSA/MCA NPs could be considered a new modality for treatment of gastro-paresis, in addition to management of diabetes itself and preventing its complications via an MCA-immunomodulatory effect.

Alleviation of Simvastatin-Induced Myopathy in Rats by the Standardized Extract of Ginkgo Biloba (EGb761): Insights into the Mechanisms of Action.

Statins are the most widely prescribed cholesterol-lowering drugs to reduce the risk of cardiovascular diseases. Statin-induced myopathy is the major side effect of this class of drugs. Here, we studied whether standardized leaf extracts of ginkgo biloba (EGb761) would improve simvastatin (SIM)-induced muscle changes. Sixty Wistar rats were allotted into six groups: control group, vehicle group receiving 0.5% carboxymethyl cellulose (CMC) for 30 days, SIM group receiving 80 mg/kg/day SIM in 0.5% CMC orally for 30 days, SIM withdrawal group treated with SIM for 16 days and sacrificed 14 days later, and EGb761-100 and EGb761-200 groups posttreated with either 100 or 200 mg/kg/day EGb761 orally. Muscle performance on the rotarod, serum creatine kinase (CK), coenzyme Q10 (CoQ10), serum and muscle nitrite, muscle malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) activities were estimated. Additionally, muscle samples were processed for histopathological evaluation. We found that SIM decreased muscle performance on the rotarod, serum CoQ10, as well as muscle SOD and CAT activities while it increased serum CK, serum and muscle nitrite, as well as muscle MDA levels. SIM also induced sarcoplasmic vacuolation, splitting of myofibers, disorganization of sarcomeres, and disintegration of myofilaments. In contrast, posttreatment with EGb761 increased muscle performance, serum CoQ10, as well as muscle SOD and CAT activities while it reduced serum CK as well as serum and muscle nitrite levels in a dose-dependent manner. Additionally, EGb761 reversed SIM-induced histopathological changes with better results obtained by its higher dose. Interestingly, SIM withdrawal increased muscle performance on the rotarod, reduce serum CK and CoQ10, and reduced serum and muscle nitrite while it reversed SIM-induced histopathological changes. However, SIM withdrawal was not effective enough to restore their normal values. Additionally, SIM withdrawal did not improve SIM-induce muscle MDA, SOD, or CAT activities during the period studied. Our results suggest that EGb761 posttreatment reversed SIM-induces muscle changes possibly through its antioxidant effects, elevation of CoQ10 levels, and antagonizing mitochondrial damage.

Visfatin Serum Levels in Obese Type 2 Diabetic Patients: Relation to Proinflammatory Cytokines and Insulin Resistance.

Visfatin, an adipocytokine with insulin-mimetic activity, has been previously reported to associate with obesity. Herein, we aimed to investigate the serum level of visfatin and association with proinflammatory markers and insulin resistance in obese type 2 diabetic patients. A case control study was carried out among 80 diabetics and 40 non-diabetic healthy controls, after obtaining Anthropometric measurements and blood pressure. Serum level of visfatin and C-reactive protein (CRP) were measured by Enzyme Immunoassay. Interleukin 6 (IL6), tumor necrosis factor α (TNF-) were measured by ELISA and the homeostasis model assessment for insulin resistance was calculated as a marker of insulin resistance. Compared to healthy controls, diabetic patients showed a significant high serum levels of visfatin (40.33±9.98 vs 19.03±8.22), (P= 0.001), IL6 (12.06±2.69 vs 6.02±3.03), (P < 0.0001), TNF- (13.53±2.54 vs 8.70±3.40), P < 0.0001) and CRP (7.77±1.61 vs 6.01±1.99), (P=0.003). Also there was a strong positive correlation between serum level of visfatin, IL6, TNF- and CRP and some anthropometric characteristics including (WC,BMI and insulin resistance). Furthermore, among 80 diabetic patients, serum level of visfatin was positively correlated with IL6 (r=0.47, P < 0.0001), TNF- (r=0.62, P < 0.0001), CRP (r=0.4, P=0.002) respectively. In conclusion, there is a strong positive correlation between visfatin serum level and the inflammatory markers IL6, TNF- and CRP in type 2 diabetic patients. There is also a positive correlation with insulin resistance and BMI which indicates association of visfatin with obesity and type 2 diabetes mellitus.

The immunopotentiator effects of nefopam.

The effects of opiate analgesics and non-steroidal anti-inflammatory drugs on the immune functions have been reported. The effect of the non-opiate analgesic nefopam on the immune functions has not yet been investigated. Male Swiss albino mice were treated with either heat killed E. coli or saline. They were classified into 12 groups. The effects of subacute (15 mg/kg/12 h S.C. daily for one week) and chronic (10 mg/kg/12 h S.C. daily for one month) treatment with nefopam on the levels of interferon-gamma (IFN-gamma) and total immunoglobulins were examined in both normal and immunized mice. Also, the effect of the chronic administration of nefopam on the phagocytic activity of peritoneal macrophage was evaluated in both normal and immunized mice. Subacute and chronic administration of nefopam induced no significant raise in the level of interferon-gamma (IFN-gamma) or in the level of total immunoglobulins in non-immunized animals, while subacute and chronic treatment with nefopam augmented markedly the immunization induced increase of level of interferon-gamma (IFN-gamma). Furthermore, chronic treatment with nefopam potentiated significantly the production of total immunoglobulin induced by heat killed E. coli. Chronic treatment with nefopam also was associated with significant enhancement of innate immune response reflected in the pronounced increase in the phagocytic activity of macrophages in non-immunized and immunized animals. The enhancement of phagocytic activity of macrophages by nefopam in immunized animals was significantly higher than that of non-immunized animals. These findings revealed that nefopam has the ability to trigger the immune response for bacterial antigen. The mechanism behind the immunostimulatory effect of nefopam requires further investigation, but it may be due, at least in part, to the inhibitory effect of nefopam on the serotonin and norepinephrine reuptake at nerve endings. In conclusion, our findings postulated that nefopam stimulated the immune functions and improved the defence mechanism. This information may be of future therapeutic value in diseases that need immunologic enhancement.