Transdermal delivery of selegiline from alginate-Pluronic composite thermogels

The present work was carried out to design a practical, controlled-release transdermal system for selegiline using thermosensitive hydrogels. The copolymers of alginate and Pluronic F127 (PF127) were used to design thermogels by either physical blending (A+P) or chemical grafting (AP). The thermogels were characterized in terms of the sol-gel temperature, scanning electron microscopy (SEM), degradation ratio, and skin permeation behavior. The chemical grafting of alginate to PF127 could delay the sol-gel temperature from 24.1 to 30.4°C, which is near the temperature of the skin surface. The gelling temperature of the physical mixture of alginate and PF127 (A+P) did not significantly differ. The porosity of the A+P structure was greater compared to that of the AP structure. AP thermogels were regularly degraded, with 60% of the gel matrix remaining after a 48-h incubation. PF127 and A+P hydrogels showed almost no degradation. The results of skin permeation across porcine skin and nude mouse skin suggested that the thermogels could produce sustained selegiline release, with AP showing the most-sustained permeation. AP hydrogels exhibited linear permeation properties for the transdermal delivery of selegiline. Inter-subject variations in skin permeation were reduced by incorporation of the thermogel. Such a thermosensitive hydrogel can be advantageous as a topical therapeutic formulation for selegiline.     

Invasive fungal infection in systemic lupus erythematosus: an analysis of 15 cases and a literature review

OBJECTIVE: To analyse 15 cases of invasive fungal infection and mortality parameters in the largest series in the last 35 yrs of patients with systemic lupus erythematosus (SLE) at a single medical centre. METHODS: Fifteen patients with SLE and invasive fungal infections were retrospectively enrolled. Clinical and laboratory data, fungal species and infected sites, corticosteroid and immunosuppressant doses and SLE disease activity index were assessed retrospectively. Comparison and correlation analyses utilized Fisher's exact test, the chi-square test, Mann-Whitney U-test or the Wilcoxon signed-rank test where appropriate. RESULTS: In contrast to other review reports, Cryptococcus neoformans was the most commonly identified fungus in this Taiwanese series. Notably, the prevalence of autoimmune haemolytic anaemia and positive results for the anti-cardiolipin antibody in this study were significantly higher than those in SLE patients in general (P < 0.0001 and P < 0.0001, respectively). Fungal infection contributed to cause of death in 7 of 15 (46.7%) patients, of which Cryptococcus neoformans accounted for six of these infections. Low-dose prednisolone (<1 or <0.5 mg/kg/day based on arbitrary division) prior to fungal infection tended to correlate with 1 yr mortality after diagnosis of SLE (P = 0.077 or P = 0.080). However, following fungal infection, patients who died from infection itself had been prescribed with higher prednisolone dose or equivalent than surviving patients (P = 0.016). All SLE patients with fungal infections had active SLE (SLEDAI >7). CONCLUSIONS: Cryptococcus neoformans infection accounted for most fatalities in SLE patients with fungal infections in this series. Active lupus disease is probably a risk factor for fungal infection in SLE patients. Notably, low prednisolone doses prior to fungal infection or high prednisolone doses following fungal infection tended to associate with or correlated to fatality, respectively. Therefore, we suggest that different prednisolone doses prescribed at various times impact the incidence of fungal infection and its associated mortality.     

Do circulating leucocytes and lymphocyte subtypes increase in response to brief exercise in children with and without asthma?

Background

Exercise can alter health in children in both beneficial (eg reduced long‐term risk of atherosclerosis) and adverse (eg exercise‐induced asthma) ways. The mechanisms linking exercise and health are not known, but may rest, partly, on the ability of exercise to increase circulating immune cells. Little is known about the effect of brief exercise, more reflective of naturally occurring patterns of physical activity in children, on immune cell responses.

Objectives

To determine whether (1) a 6‐min bout of exercise can increase circulating inflammatory cells in healthy children and (2) the effect of brief exercise is greater in children with a history of asthma.

Methods

Children with mild–moderate persistent asthma and age‐matched controls (n = 14 in each group, mean age 13.6 years) performed a 6‐min bout of cycle‐ergometer exercise. Spirometry was performed at baseline and after exercise. Blood was drawn before and after exercise, leucocytes were quantified and key lymphocyte cell surface markers were assessed by flow cytometry.

Results

Exercise decreased spirometry only in children with asthma, but increased (p<0.001) most types of leucocytes (eg lymphocytes (controls, mean (SD) 1210 (208) cells/μl; children with asthma, 1119 (147) cells/μl) and eosinophils (controls, 104 (22) cells/μl; children with asthma, 88 (20) cells/μl)) to the same degree in both groups. Similarly, exercise increased T helper cells (controls, 248 (60) cells/μl; children with asthma, 232 (53) cells/μl) and most other lymphocyte subtypes tested. By contrast, although basophils (16 (5) cells/μl) and CD4+ CD45RO+ RA+ lymphocytes (19 (4) cells/μl) increased in controls, no increase in these cell types was found in children with asthma.

Conclusions

Exercise increased many circulating inflammatory cells in both children with asthma and controls. Circulating inflammatory cells did increase in children with asthma, but not to a greater degree than in controls. In fact, basophils and T helper lymphocyte memory transition cells did not increase in children with asthma, whereas they did increase in controls. Even brief exercise in children and adolescents robustly mobilises circulating immune cells.Although it is well recognised that physical activity can influence health in children, the mechanisms that link exercise and health remain enigmatic. Immune responses to exercise could explain some of the adverse (eg bronchoconstriction, urticaria, anaphylaxis^1,2,3) or beneficial (reduction of cardiovascular disease risk^4,5) health effects of physical activity, and, indeed, growing evidence in children suggests that exercise is associated with a substantial increase in circulating cytokines (such as interleukin 6) and leucocytes.^6,7 However, the vast majority of studies to date designed to explore immune and stress responses to exercise in adults and children have used relatively long exercise protocols,^8,9 which do not reflect natural patterns of physical activity in children.Real‐life patterns of exercise in children are brief.¹⁰ Whether or not short episodes of exercise in children and adolescents cause an increase in circulating inflammatory cells has not been well studied. Consequently, the goal of this study was to test the hypothesis that a standard, brief (6‐min) exercise protocol—used commonly to elicit bronchoconstriction in children—would lead to large increases in circulating inflammatory cells, some of which are associated with specific diseases. As habitual levels of physical activity and related factors, such as body composition and nutritional status, can alter peripheral leucocyte function,^11,12 testing this hypothesis would be a necessary first step in understanding the potential mechanistic link between exercise and diseases, such as asthma, in which exercise is a potent trigger of airway inflammation.We chose to measure the levels of the standard circulating leucocytes (ie neutrophils, eosinophils, basophils, lymphocytes and monocytes). In addition, we used flow cytometry to focus on several lymphocyte cell surface markers that have been found, albeit mostly from studies in adults, to have a variety of roles in inflammatory diseases ranging from asthma to atherosclerosis (table 1​1).). Finally, we elected to study this phenomenon in healthy control children and adolescents, as well as in children and adolescents with a known history of mild‐to‐moderate asthma. Exercise‐induced bronchoconstriction (EIB) is a common and well‐described adverse effect of exercise,^13,14 and is related to airway dehydration leading to an abnormal airway inflammatory response.² Consequently, we hypothesised that the effect of brief exercise on circulating immune cells would be greater than in controls without asthma.Table 1 Cell surface markers evaluated

Direct effect of statins on homocysteine-induced endothelial adhesiveness: potential impact to human atherosclerosis

Background Although homocysteine (HCY) is a risk factor for cardiovascular diseases, recent clinical trials failed to show the benefits by reducing plasma HCY. Alternative strategy with 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors, statins, might be feasible. This study investigated HCY‐induced endothelial adhesiveness with mononuclear cells (MNCs) from patients with coronary artery disease (CAD). The direct endothelial protective effects of statins were also examined. Materials and methods Circulating MNCs were isolated from 14 stable CAD patients and 7 age‐ and gender‐matched healthy subjects. Superoxide production of MNCs was determined by Ultra‐weak and luminol‐enhanced chemiluminescence. Human aortic endothelial cells (HAECs) were used for endothelial adhesiveness to MNCs or U937 human monocytic cells. Endothelial expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) were examined by Western blot. Results Superoxide production of MNCs and plasma HCY and high‐sensitive CRP levels were significantly increased in CAD patients than in healthy subjects. Stimulation with HCY enhanced the endothelial adhesiveness to MNCs from CAD patients or to U937 cells in a dose‐dependent manner, whereas it was obscure with MNCs from healthy subjects. HCY stimulated endothelial VCAM‐1 but not ICAM‐1 expression in a dose‐dependent manner. Monoclonal antibodies to VCAM‐1 attenuated HCY‐induced endothelial adhesiveness. Simvastatin or pravastatin significantly reduced HCY‐induced VCAM‐1 expression and endothelial adhesiveness to MNCs from CAD patients. Conclusion Circulating MNCs were activated in CAD patients, which was critical to HCY‐induced endothelial adhesiveness. Statins could directly reduce HCY‐induced endothelial‐MNC adhesion via VCAM‐1 inhibition, suggesting its potential implication in HCY‐related atherosclerosis disease.     

Effect of pH and human serum albumin on the cytotoxicity of a glucuronide prodrug of 9-aminocamptothecin

Purpose 9-aminocamptothecin glucuronide (9ACG) is a prodrug of 9-aminocamptothecin (9AC) that displays potent antitumor activity against human tumor xenografts in nude mice. Camptothecins exist in a pH dependent equilibrium between active lactone and inactive carboxy forms that can be altered by binding to human serum albumin (HSA). Here we investigated the influence of pH and HSA on the lactone-carboxy equilibrium, HSA binding, and cytotoxicity of 9ACG. Methods Microfiltration and HPLC were used to measure the influence of pH on lactone to carboxy conversion and HSA binding of 9ACG as compared to other camptothecins. In vitro cytotoxicity of drugs was determined against EJ human bladder carcinoma cells and CL1-5 human lung cancer cells. Results The rate of lactone to carboxy conversion was similar for 9ACG and 9AC. Decreasing the pH from 7.6 to 6.0 increased the equilibrium levels of the lactone forms of the drugs from 20 to almost 95% of total drug. HSA moderately diminished the amount of free 9ACG lactone but did not change the ratio of 9ACG lactone to 9ACG carboxy. Consistent with the effect of pH on lactone levels, lowering the pH of EJ human bladder carcinoma cells from 7.6 to 6.8 decreased the IC₅₀ of 9ACG from 480 to 98 nM and 9AC from 33 to 12 nM. Activation of 9ACG by human β-glucuronidase anchored on the surface of EJ cells further decreased its IC₅₀ value to 26 nM. Although HSA significantly decreased the cytotoxicity of 9AC and 9ACG, activation of 9ACG at cancer cells with an antibody-β-glucuronidase immunoconjugate produced greater cytotoxicity than 9AC. Conclusions Acidification and targeted delivery of β-glucuronidase can enhance 9ACG cytotoxicity even in the presence of HSA.     

Eps8 decreases chemosensitivity and affects survival of cervical cancer patients

The oncoprotein Eps8 facilitates proliferation in fibroblasts and colon cancer cells. However, its role in human cervical cancer is unclear. By immunohistochemical staining and Western blotting, aberrant Eps8 expression was observed in cervical carcinoma compared with normal cervical epithelial cells. Clinicopathologic analysis of 45 patients indicated that Eps8 expression was associated with parametrium invasion and lymph node metastasis, two major poor prognostic factors for early-stage cervical cancer. Kaplan-Meier analysis of cervical cancer specimens also indicated an inverse relationship between the level of Eps8 and the patients' survival rate. Using small interfering RNA of eps8, we observed reduced proliferation and tumorigenesis in Eps8-attenuated HeLa and SiHa cells cultured in dishes or inoculated in mice. Furthermore, diminished Eps8 impeded G(1)-phase progression in HeLa and SiHa cells that might be attributable to reduced expression of cyclins D1, D3, and E, elevated accumulation of p53 and its downstream target p21(Waf1/Cip1), and suppressed hyperphosphorylation of retinoblastoma. Alteration of these cell cycle-related proteins could be reversed by ectopic Eps8, implicating that the effect of Eps8 on the mentioned cell cycle modulators was specific. Notably, the augmented expression of p53 by diminished Eps8 was at least due to its decreased turnover rate. Concurrent with p53 up-regulation and the decrement of Src and AKT activity, Eps8-attenuated HeLa and SiHa cells exhibited increased chemosensitivity to cisplatin and paclitaxel. Together, our findings implicate the involvement of Eps8 in chemoresistance and show its importance in prognosis of cervical cancer patients.     

Assessment of ifosfamide pharmacokinetics, toxicity, and relation to CYP3A4 activity as measured by the erythromycin breath test in patients with sarcoma

BACKGROUND: Ifosfamide is a chemotherapeutic agent that requires cytochrome P450 3A (CYP3A) for bioactivation and metabolism. To the authors' knowledge, the correlation between dose, pharmacokinetics, CYP3A, and toxicity has not been fully evaluated. A randomized Phase II trial was performed on 22 soft tissue sarcoma patients treated with doxorubicin (60 mg/m(2)/cycle) and either high-dose ifosfamide (12 g/m(2)/cycle) or standard-dose ifosfamide (6 g/m(2)/cycle). The pharmacokinetics of ifosfamide and CYP3A measurements observed are reported. METHODS: Pharmacokinetic parameters for ifosfamide, 2-dichloroethylifosfamide (2-DCE), and 3-dichloroethylifosfamide (3-DCE) were collected after the first ifosfamide infusion in 13 patients. Bayesian designed limited pharmacokinetic data were collected from an additional 41 patients. The erythromycin breath test (ERMBT) was performed on 81 patients as an in vivo phenotypic assessment of CYP3A activity. RESULTS: Fourteen-hour (peak) plasma levels of ifosfamide, 2-DCE, and 3-DCE were found to correlate strongly with the respective area under the curve (AUC) 0-24 values (r=0.97, 0.94, and 0.95; P<.0001). Patients who experienced a grade 3-4 absolute neutrophil count (ANC), platelet, or creatinine toxicity (using the National Cancer Institute Common Toxicity Criteria [version 2]) were found to have statistically significantly higher median 14-hour plasma levels of ifosfamide, 2-DCE, and 3-DCE compared with patients with grade 0-2 toxicity. ERMBT was not found to correlate with pharmacokinetic parameters of ifosfamide and metabolites or toxicity. CONCLUSIONS: The 14-hour plasma level of ifosfamide, 2-DCE, and 3-DCE is a simple and appropriate substitute for describing the AUC of ifosfamide after 1 day of a 1-hour to 2-hour infusion of drug. Fourteen-hour plasma levels of ifosfamide and metabolites are useful predictors of neutropenia, thrombocytopenia, and creatinine toxicity. ERMBT was not found to accurately correlate with ifosfamide pharmacokinetics or clinical toxicity.