Fabrication of self-healing pectin/chitosan hybrid hydrogel via Diels-Alder reactions for drug delivery with high swelling property, pH-responsiveness, and cytocompatibility.

Self-healing hydrogels with pH-responsiveness could protect loaded drugs from being destroyed till it arrives to the target. The pectin-based hydrogel is a candidate due to the health benefit, anti-inflammation, antineoplastic activity, nontoxicity, and biospecific degradation, et al. However, the abundant existence of water-soluble branched heteropolysaccharide chains influenced its performance resulting in limitation of the potential. In the present study, we prepared a series of self-healing pectin/chitosan hydrogels via the Diels-Alder reaction. Moreover, pectin/chitosan composite hydrogel was prepared as a contrast. By comparison, it can be seen that the Diels-Alder reaction greatly improved the cross-linking density of hydrogels. The self-healing experiments showed excellent self-healing performance. In different swelling mediums, significant transformation in the swelling ratio was shown, indicating well-swelling property, pH- and thermo-responsiveness. The drug loading and release studies presented high loading efficiency and sustained release performance. The cytotoxicity assay that showed a high cell proliferation ratio manifested great cytocompatibility.

Optimised approach to albumin-drug conjugates using monobromomaleimide-C-2 linkers.

Conjugation of therapeutics to human serum albumin (HSA) using bromomaleimides represents a promising platform for half-life extension. We show here that the Cys-34 crevice substantially reduces the rate of serum stabilising maleimide hydrolysis in these conjugates, necessitating reagent optimisation. This improved reagent design is applied to the construction of an HSA-paclitaxel conjugate, preventing drug loss during maleimide hydrolysis.

Cornel iridoid glycoside induces autophagy to protect against tau oligomer neurotoxicity induced by the activation of glycogen synthase kinase-3ß.

Tau oligomers are the etiologic molecules of Alzheimer's disease, and correlate strongly with neuronal loss and exhibit neurotoxicity. Recent evidence indicates that small tau oligomers are the most relevant toxic aggregate species. The aim of the present study was to investigate the mechanisms of cornel iridoid glycoside (CIG) on tau oligomers and cognitive functions. We injected wortmannin and GF-109203X (WM/GFX, 200 µM each) into the lateral ventricles to induce tau oligomer and memory impairment in rats. When orally administered with CIG at 60 and 120 mg/kg/day for 14 days, CIG decreased the escape latency in the Morris water maze test. We also found that CIG restored the expression of presynaptic p-synapsin, synaptophysin, and postsynaptic density-95 (PSD-95) decreased by WM/GFX in rat cortex. CIG reduced the accumulation of tau oligomers in the brain of WM/GFX rats and in cells transfected with wild type glycogen synthase kinase-3ß (wtGSK-3ß). In addition, CIG up-regulated the levels of ATG7, ATG12, Beclin-1, and LC3II in vivo and in vitro, suggesting the restoration of autophagy function. These results suggest that CIG could ameliorate memory deficits and regulate memory-associated synaptic proteins through the clearance of tau oligomers accumulation. Moreover, CIG clears tau oligomers by restoring autophagy function.

In vivo irreversible albumin-binding near-infrared dye conjugate as a naked-eye and fluorescence dual-mode imaging agent for lymph node tumor metastasis diagnosis.

Tumor metastases account for about 90% of cancer-related death, among which lymphatic metastases play a pivotal role. Therefore, high-efficiency sentinel lymph node (SLN) identification is significant for lymph node (LN) metastasis diagnosis in clinic. Herein, a novel in vivo covalent albumin-binding near-infrared (NIR) fluorescent IR820-maleimide conjugate (IR-Mal) is firstly designed as a SLN dual-mode imaging agent. The IR-Mal conjugate exhibits bright blue appearance and its large Stokes shift (over 100 nm) increases the fluorescent imaging resolution effectively. The fluorescence intensity of covalent albumin-binding IR-Mal (BSA-IR-Mal) complex is considerably stronger than that of IR-Mal. In vivo, IR-Mal could rapidly covalently bind the tissue interstitial albumin following subcutaneous administration and BSA-IR-Mal complexes could specifically accumulate on LN, and detect both normal and metastatic SLN through naked-eye and fluorescence imaging with high resolution. Moreover, the light stability and enhanced fluorescence intensity of BSA-IR-Mal complex facilitates its diagnosis accuracy. These findings suggest that such in vivo irreversible albumin-binding fluorescence conjugates could serve as a new agent for dual-mode imaging and have a great potential to be applied in the SLNs imaging and diagnosis.

High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure.

BACKGROUND: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite recovery assays. However, short exposure and assessment may not detect toxicants that affect an early neurogenesis continuum, from a mitotic human neural progenitor (hNP) cell population through the course of neurite outgrowth in differentiating neurons. Therefore, we continuously exposed differentiating hNP cells from DIV 0 through DIV 14 to known toxicants and endocrine active compounds in order to assess at DIV 14 effects of these compounds in a human DNT maturation model for neurogenesis. METHODS: The Human DNT continuum (DIV 0 to DIV 14) was determined using immunocytochemistry for SOX1+ (proliferating hNP) and HuC/D+ (post mitotic neurons). The cumulative effects of five compounds was observed on neurite outgrowth in (ßIII-tubulin+) and (HuC/D+) cells using high content imaging. All data were analyzed using a one-way ANOVA with a significance threshold of p < 0.05. RESULTS: During maturation in vitro, the neural cultures transitioned from uniform hNP cells (DIV 0) to predominantly mature post mitotic neuronal neurons (HuC/D+, 65%; DIV14) but also maintained a smaller population of hNP cells (SOX1+). Using this DNT maturation model system, Bis-1, testosterone, and ß-estradiol inhibited neuronal maturation at micromolar levels but were unaffected by acetaminophen. ß-estradiol also disrupted neurite extension at 10 µM. Treating cells in this window with Bisphenol A (BPA) significantly inhibited neurite outgrowth and branching in these continuum cultures but only at the highest concentrations tested (10 µM). CONCLUSIONS: Cumulative effects of neurotoxicant exposure during a maturation continuum altered human neurogenesis at lower exposure levels than observed in acute exposure of static cryopreserved neurite recovery neurons cultures. Unlike prior acute studies, ß-estradiol was highly toxic when present throughout the continuum and cytotoxicity was manifested starting early in the continuum via a non-estrogen receptor α (ER α) mechanism. Therefore, the effect of neural developmental neurotoxins can and should be determined during the dynamic process of human neural maturation.

Microwave-assisted synthesis and biological evaluation of 3,4-diaryl maleic anhydride/N-substituted maleimide derivatives as combretastatin A-4 analogues.

A series of new CA-4 analogues bearing maleic anhydride/N-substituted maleimide moiety were synthesized via a microwave-assisted process. They were evaluated for the anti-proliferative activities against three tumor cell lines (SGC-7901, HT-1080 and KB). Most compounds showed moderate potencies in micromolar range, with the most promising analogue 6f showing active at submicromolar concentration against HT-1080 cancer cells which was selected to investigate the antitumor mechanisms. In addition, molecular docking studies within the colchicine binding site of tubulin were also in good agreement with the tubulin polymerization inhibitory data and provided a basis for further structure-guided design of novel CA-4 analogues.

Vasculogenic bio-synthetic hydrogel for enhancement of pancreatic islet engraftment and function in type 1 diabetes.

Type 1 diabetes (T1DM) affects one in every 400 children and adolescents in the US. Due to the limitations of exogenous insulin therapy and whole pancreas transplantation, pancreatic islet transplantation has emerged as a promising therapy for T1DM. However, this therapy is severely limited by donor islet availability and poor islet engraftment and function. We engineered an injectable bio-synthetic, polyethylene glycol-maleimide hydrogel to enhance vascularization and engraftment of transplanted pancreatic islets in a mouse model of T1DM. Controlled presentation of VEGF-A and cell-adhesive peptides within this engineered material significantly improved the vascularization and function of islets delivered to the small bowel mesentery, a metabolically relevant site for insulin release. Diabetic mice receiving islets transplanted in proteolytically degradable hydrogels incorporating VEGF-A exhibited complete reversal of diabetic hyperglycemia with a 40% reduction in the number of islets required. Furthermore, hydrogel-delivered islets significantly improved weight gain, regulation of a glucose challenge, and intra-islet vascularization and engraftment compared to the clinical standard of islet infusion through the hepatic portal vein. This study establishes a simple biomaterial strategy for islet transplantation to promote enhanced islet engraftment and function.

N-(1-Pyrenyl) maleimide inhibits telomerase activity in a cell free system and induces apoptosis in Jurkat cells.

Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Agents that interact selectively with telomerase are anticipated to exert specific action on cancer cells. In this study, we evaluated maleimide derivatives for their potency and selectivity of telomerase inhibition. Among the several N-substituted derivatives of maleimide tested, N-(1-Pyrenyl) maleimide was shown to exert the greatest inhibition of telomerase in a cell free system, with an IC50 value of 0.25 µM. Importantly, we demonstrated that N-(1-pyrenyl) maleimide induces apoptosis in Jurkat T cells and displays the greatest differential cytotoxicity against hematopoietic cancer cells. These results suggest that N-(1-pyrenyl) maleimide is an attractive maleimide to be tested and developed as anti-cancer drug.

Development of a cellular tau enzyme-linked immunosorbent assay method for screening GSK-3ß inhibitors.

Glycogen synthase kinase-3ß (GSK-3ß), a serine/threonine kinase also known as tau protein kinase I, has been implicated in the pathogenic conditions of Alzheimer's disease. Many investigators have focused on GSK-3 inhibitor as a therapeutic drug. In this study, we established a cell-based assay for the screening of novel GSK-3ß inhibitors. For this purpose, four-repeat tau cDNAs were stably expressed in human embryonic kidney 293 (HEK293) cells (HEK293-Tau). The proliferation of HEK293-Tau cells was no different from that of HEK293 cells, as measured by the bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). The concentration-dependent reduction of tau phosphorylation by GSK-3 inhibitors, LiCl, Chir98023, and SB415286, was examined by immunoblot analysis and Tau ELISA (in situ ELISA). Highly consistent data were obtained, suggesting that this novel ELISA method is highly reproducible. Using this ELISA strategy, we isolated a few candidate compounds, including compounds 114 and 149, from several hundreds of synthetic agents and demonstrated that such candidates protect nerve growth factor-differentiated PC12 cells against amyloid-ß-induced cell death. These data indicate that this Tau ELISA method in HEK293-Tau cells may be a suitable cell-based assay system to screen for GSK-3ß inhibitors.

Antifungal, cytotoxic and SAR studies of a series of N-alkyl, N-aryl and N-alkylphenyl-1,4-pyrrolediones and related compounds.

The synthesis, in vitro evaluation and SAR studies of 67 maleimides and derivatives acting as antifungal agents are reported. A detailed SAR study supported by theoretical calculations led us to determine that: an intact maleimido ring appears to be necessary for a strong antifungal activity, dissimilarly affected by the substituents in positions 2 and 3. The best activities were shown by 2,3-nonsubstituted followed by 2,3 dichloro- and 2-methyl-substituted maleimides. They all were fungicide rather than fungistatic enhancing the importance of their antifungal activity. 2,3-Dimethyl and 2,3-diphenyl-maleimides possessed marginal or null activity. The presence of a flexible connecting chain in N-phenylalkyl maleimides appears not to be essential for antifungal activity, although its length shows a correlation with the antifungal behavior, displaying maleimides with alkyl chains of n=3 and n=4 the best antifungal activities in most fungi. Different substituents on the benzene ring did not have a clear influence on the activity. Values of chemical potential properties as well as of energy do not sufficiently discriminate between active and inactive compounds. Nevertheless, it was found that, although logP alone is not strong enough to properly predict the antifungal activity, the comparison of its values for compounds within the same sub-type, showed an enhancement of antifungal activity along with an increment of lipophilicity. In addition, the LUMO's electronic clouds of the highly active compounds showed to be concentrated on the imido ring, indicating that their carbon atoms are potential sites for nucleophilic attack. Same results were obtained from MEPs. Most of the active compounds did not show cytotoxic activity against human cancer cell lines and no one possessed hemolytic activity, indicating that their activity is selective to pathogenic fungi and that they are not toxic at MIC concentrations.

Involvement of GSK-3 in regulation of murine embryonic stem cell self-renewal revealed by a series of bisindolylmaleimides.

The ability to propagate embryonic stem cells (ESCs) while maintaining their pluripotency is critical if their potential use in regenerative medicine is to be realized. The mechanisms controlling ESC self-renewal are under intense investigation, and glycogen synthase kinase 3 (GSK-3) has been implicated in regulating both self-renewal and differentiation. To clarify its role in ESCs we have used chemical genetics. We synthesized a series of bisindolylmaleimides, a subset of which inhibit GSK-3 in murine ESCs and robustly enhance self-renewal in the presence of leukemia inhibitory factor (LIF) and serum, but not in the absence of LIF. Importantly, these molecules appear selective for GSK-3 and do not perturb other signaling pathways regulating self-renewal. Our study clarifies the functional importance of GSK-3 in regulation of ESC self-renewal and provides tools for investigating its role further.

Toxicity and hemodynamic effects after single dose administration of MalPEG-hemoglobin (MP4) in rhesus monkeys.

Maleimide-polyethylene glycol-modified (MalPEG) hemoglobin, 4.3 g/dL (MP4; Hemospan), is a hemoglobin-based oxygen carrier consisting of human hemoglobin (Hb) modified with maleimide polyethylene glycol. This study evaluates the potential toxicity and hemodynamic actions of a single dose of MP4 administered by exchange transfusion to rhesus monkeys. Monkeys were administered MP4 (21 mL/kg, or approximately 30% of estimated blood volume) or an equivalent volume of lactated Ringer's solution (LR). In the toxicity study, blood samples were obtained predose and 3, 7, and 13 days after dosing for clinical chemistry and hematology. Animals were euthanized for complete necropsy and histopathology on day 3 or day 13. A separate group of animals was used for evaluation of arterial pressure, core temperature, and electrocardiogram, by telemetry, for 7 days after dosing with MP4. The results demonstrate no significant toxicity, with only modest, transient elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) on day 3. Mild anemia caused by hemodilution was observed at each time point in both groups, but to a slightly greater degree in the MP4-treated animals. Histologic observations included foamy or vacuolated macrophages in the spleen and marrow of the sternum, rib, and femur, representing the accumulation of test article or a metabolite. In the telemetry study, no changes occurred in arterial pressure, heart rate, or electrocardiogram attributable to administration of MP4 at any time for 7 days after administration. These results demonstrate that MP4 is safe and is without hemodynamic effects when administered as an exchange transfusion of 30% of estimated blood volume.

Novel indolylindazolylmaleimides as inhibitors of protein kinase C-beta: synthesis, biological activity, and cardiovascular safety.

Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.

Prevotella intermedia lipopolysaccharide stimulates release of nitric oxide by inducing expression of inducible nitric oxide synthase.

OBJECTIVES: The purpose of this study was to examine the effects of lipopolysaccharide from Prevotella intermedia, a major cause of inflammatory periodontal disease, on the production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. intermedia lipopolysaccharide-induced NO production. MATERIAL AND METHODS: Lipopolysaccharide from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of iNOS and analysis of reverse transcription-polymerase chain reaction (RT-PCR) products were carried out. RESULTS: We found that P. intermedia lipopolysaccharide can induce iNOS expression and stimulate the release of NO without additional stimuli and demonstrated an important role of the transcription factor nuclear factor-kappaB (NF-kappaB) and microtubule polymerization in NO production. The production of NO required l-arginine but not activation of protein kinase C or protein tyrosine kinase. CONCLUSIONS: The present study clearly shows that P. intermedia lipopolysaccharide fully induced iNOS expression and NO production in RAW264.7 cells in the absence of other stimuli. The ability of P. intermedia lipopolysaccharide to promote the production of NO may be important in the pathogenesis of inflammatory periodontal disease.

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

Down-regulation of gap junctional intercellular communication between osteoblastic MC3T3-E1 cells by basic fibroblast growth factor and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate).

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.

Comparison of staurosporine and four analogues: their effects on growth, rhodamine 123 retention and binding to P-glycoprotein in multidrug-resistant MCF-7/Adr cells.

The potent kinase inhibitor staurosporine and its protein kinase C (PKC)-selective analogue CGP 41251 are known to sensitise cells with the multidrug resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) to cytotoxic agents. Here four PKC-selective staurosporine cogeners, CGP 41251, UCN-01, RO 31 8220 and GF 109203X, were compared with staurosporine in terms of their MDR-reversing properties and their susceptibility towards P-gp-mediated drug efflux from MCF-7/Adr cells. Staurosporine was the most potent and the bisindolylmaleimides RO 31 8220 and GF 109203X the least potent cytostatic agents. When compared with MCF-7 wild-type cells, MCF-7/Adr cells were resistant towards the growth-arresting properties of RO 31 8220 and UCN-01, with resistance ratios of 12.6 and 7.0 respectively. This resistance could be substantially reduced by inclusion of the P-gp inhibitor reserpine. The ratios for GF 109203X, staurosporine and CGP 41251 were 1.2, 2.0 and 2.9 respectively, and they were hardly affected by reserpine. These results suggest that RO 31 8220 and UCN-01 are avidly transported by P-gp but that the other compounds are not. Staurosporine and CGP 41251 at 10 and 20 nM, respectively, decreased efflux of the P-gp probe rhodamine 123 (R123) from MCF-7/Adr cells, whereas RO 31 8220 and GF 109203X at 640 nM were inactive. CGP 41251 was the most effective and GF 109203X the least effective inhibitor of equilibrium binding of [3H]vinblastine to its specific binding sites, probably P-gp, in MCF-7/Adr cells. Overall, the results imply that for this class of compound the structural properties that determine susceptibility towards P-gp-mediated substrate transport are complex. Comparison with ability to inhibit PKC suggests that the kinase inhibitors affect P-gp directly and not via inhibition of PKC. Among these compounds CGP 41251 was a very potent MDR-reversing agent with high affinity for P-gp and least affected by P-gp-mediated resistance, rendering it an attractive drug candidate for clinical development.

1,25-dihydroxyvitamin D3 primes acute promyelocytic cells for TPA-induced monocytic differentiation through both PKC and tyrosine phosphorylation cascades.

NB4 cells are the only in vitro model of differentiation in acute promyelocytic leukemia (APL). Although these cells respond to all-trans-retinoic acid to form neutrophils, our group has recently shown that these cells are capable of terminal monocytic differentiation in response to combined treatment with 1,25-dihydroxyvitamin D3 (1,25 D3) and 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that the agents need not be present simultaneously, but may be added sequentially. TPA treatment prior to 1,25 D3 led to the appearance of adherent cells; however, when 1,25 D3 treatment preceded TPA treatment cells expressed all differentiation markers reflective of terminal differentiation. This priming effect of 1,25 D3 was both dose and time dependent. Increasing the interval between 1,25 D3 and TPA treatment caused a decrease in this priming potential indicative of limited commitment inducing capacity of 1,25 D3. In order to characterize the mechanism of action of 1,25 D3 and TPA, chemical inhibitors of phosphorylation were used. Staurosporine and bisindolymaleimide GF 109203X treatment prior to and during 1,25 D3 treatment or TPA treatment caused attenuation of the differentiation response. Experiments utilizing tyrosine kinase and phosphatase inhibitors supported the hypothesis that 1,25 D3 signaling was mediated by both serine/threonine and tyrosine phosphorylation cascades. Results from this study provide evidence to support the hypothesis that 1,25 D3 signaling occurs via nongenomic mechanisms which when combined with the signaling effects of TPA, allow for the terminal differentiation of APL cells. This model should be used to develop new differentiation therapies for APL and other leukemias.

Influence of N-(o-methoxyphenyl)-maleimide on 5-fluorouracil cytotoxicity in Ehrlich (ascites) carcinoma.

N-(o-methoxyphenyl)-maleimide (I), an intermediate obtained during the synthesis of pyrrolidinedione-N-mustards, did not exhibit antitumor activity against Ehrlich (ascites) carcinoma. The effect of co-administration of (I), with established anticancer drugs was studied against P388 leukemia, S180 (ascites) and Ehrlich (ascites) carcinoma. A significant potentiation in the activity of 5-Fluorouracil (5-FU) against Ehrlich (ascites) carcinoma by (I) was observed. The possible mechanisms responsible for potentiation of the activity of 5-FU are presented.

Inhibition of interleukin-2 production in the human T cell line JURKAT by nonpolar maleimides.

The immunosuppressive properties of polar and nonpolar maleimides were studied by measuring their ability to inhibit mitogen-induced interleukin-2 (IL-2) production by JURKAT T cells. The nonpolar maleimides N-ethylmaleimide (NEM) and N-phenylmaleimide (NPM) inhibited IL-2 production by 85-99%, but only when added to JURKAT cells prior to the mitogen. The polar maleimides N-hydroxymaleimide (NHM) and 4-maleimidosalicylic acid (M84) did not suppress IL-2 production significantly, even though NHM reacted with more cellular thiols (12%) than did NPM (8%). Both NEM and NPM suppressed IL-2 production at doses that did not affect proliferation. NEM inhibited IL-2 production induced by PHA, anti-CD3 (alpha CD3) monoclonal antibodies or PMA, and A23187, but did not interfere with the binding of alpha CD3 to the cells. NEM inhibited IL-2 production at concentrations that did not interfere with the PHA-induced increase in intracellular free calcium [( Ca]i). Neither NPM nor NHM inhibited the rise in [Ca]i, even at the highest concentrations tested. Although JURKAT T cells require both PMA and A23187 to induce IL-2 production, we found that cells pretreated with PMA could respond to A23187 added 18 hr later. PMA-treated cells were not resistant to the immunosuppressive effects of NEM or NPM. However, PMA-pretreated cells became resistant to the inhibitory effects of NEM upon the addition of A23187, suggesting that nonpolar maleimides inhibit activation events induced by the rise in [Ca]i.