Tunicamycin induced endoplasmic reticulum changes in endothelial cells investigated in vitro by confocal Raman imaging.

This paper describes how tunicamycin (Tu), the most widely used pharmacological agent for inducing endoplasmic reticulum (ER) stress, interacts with endothelial cells. Our results show that tunicamycin enters the cells and accumulates within the ER area. ER stress takes place when improperly folded or damaged proteins begin to accumulate; however, spectroscopic markers of these changes have not been identified as yet. In this work, Raman spectroscopy and scanning electron microscopy imaging of individual endothelial cells treated with Tu were performed. The changes in the biochemical composition of endothelial cells induced by Tu attributed to ER stress were studied in detail. A main feature of the Tu impact on the cells was a decrease of the phospholipid content in the area of ER, and the most abundant lipid with phosphorus groups found there, was identified as sphingomyelin.

Preparation of mannan-protein conjugates using high-temperature glycation.

In the present paper, we describe a procedure for the preparation of mannan-protein conjugates. Mannan from Saccharomyces cerevisiae (baker's yeast) was oxidized by NaIO4 and coupled with BSA by means of high-temperature glycation. Depending on the temperature and degree of mannan oxidation, the procedure enabled different levels of substitution to be obtained. For conjugate prepared under selected conditions, the average substitution level was 1.64 molecules of mannan per albumin molecule. The mannan-albumin conjugate was purified by means of ion-exchange chromatography on CM-cellulose at a pH below the pI of albumin. The influence of coupling conditions on protein activity was examined using urease as an example. Urease linked to mannan retained its activity. The mannan-protein conjugates can be used, for example, as carriers of therapeutic substances to macrophages.

Antitumor activity of mannan-methotrexate conjugate in vitro and in vivo.

Conjugation of anticancer drugs with different carriers has been extensively studied recently as a potential method of obtaining improved drug forms. The conjugation often results in the increase of the therapeutic effect, alteration of a toxicity profile, and/or selective targeting of therapeutic agent to the tissue of interest. We have synthesized mannan-methotrexate conjugate by means of methotrexate anhydride and studied its antitumor properties both in vitro and in vivo in comparison with free methotrexate. Mannan-methotrexate conjugate showed significantly improved antitumor activity compared to free methotrexate in the model of P388 mouse leukemia disseminated in the peritoneal cavity treated with intraperitoneally injected chemotherapy. Conversely, the antitumor effects of free methotrexate and mannan-methotrexate conjugate were comparable when leukemia was implanted subcutaneously and chemotherapy agents were administered intravenously. These results suggest that mannan-methotrexate conjugate should be further investigated as a potential therapeutic agent for intraperitoneally disseminated tumors.

[Current status of research on conjugates and related drug delivery systems in the treatment of cancer and other diseases]. / Wspólczesny stan badan nad koniugatami i innymi systemami dostarczania leków w leczeniu schorzen nowotworowych i innych jednostek chorobowych.

Drug delivery systems is an umbrella term describing different types of carrier-drug and carrier-protein conjugates, liposomes, polyplexes, micelles, and hybrid types. Such systems were designed with the aim to enhance delivery and to improve the selectivity and pharmacological properties of both conventional drugs and newly developed agents. In this review the current state-of-the-art in the design of drug delivery systems is described, with particular emphasis on agents developed to deliver antitumor agents. The advantages, limitations, and disadvantages of the particular types of drug delivery systems as well as the theoretical and practical basis of their action are also highlighted here. In addition, results of preclinical and clinical studies on the most developed examples of drug delivery systems to date are summarized.

The effect of the substitution level of some dextran-methotrexate conjugates on their antitumor activity in experimental cancer models.

Methotrexate (MTX) is widely used in the treatment of a number of oncological and hematological diseases. Due to its known limitations, MTX is often conjugated with different carriers to obtain amended forms of the drug. In this study, the potential influence of the substitution level (loading ratio) of the dextran T10- and T40-based MTX conjugates (D-MTX) on their properties were investigated in vitro and in vivo. The clear dependence of the in vitro antiproliferative effect on the substitution level was established only in the case of the dextran T10-based preparations (T10-MTX conjugates). Conjugates with the higher substitution level had the lower antiproliferative effect. For the dextran T40-based (T40-MTX conjugates) set no similar relationship was observed in the tested range of substitution levels, nor was any dependence observed between the biological properties of the D-MTX preparations in vivo and their substitution levels. However, the difference between the two conjugates was well pronounced in a multiple-dose schedule, when the advantage of T40-MTX over T10-MTX was cumulative during the prolonged course of administration.

Antitumor properties and toxicity of dextran-methotrexate conjugates are dependent on the molecular weight of the carrier.

Methotrexate (MTX) is widely utilized in the clinical treatment of many forms of cancer. However, the drug has a short plasma half-life and causes toxic effects on normal proliferating cells. Conjugation with carriers is a possible way to alter these disadvantageous pharmacokinetics. Our aim was to synthesize dextran-MTX (D-MTX) conjugates, using carriers with molecular weights (Mw) ranging from 10 kDa to 500 kDa. Their in vitro and in vivo properties were compared with free MTX. The in vitro studies revealed that D-MTX conjugates had 4- to 10-fold lower antiproliferative effects against neoplastic cell lines compared to free MTX. There was a negative relationship between the Mw of the carrier and the antiproliferative effect of the respective conjugate. The data obtained in a mouse leukemia P388 in vivo model suggested that a lower in vitro antiproliferative effect of the conjugates does not result in diminished antileukemic activity in vivo. The toxicity of the conjugates was greater in comparison with the parent drug and tended to rise with increasing Mw. However, no superiority over free MTX in terms of an antileukemic effect was demonstrated. In particular, the D-MTX conjugate based on the dextran with Mw 10 kDa showed a comparable antileukemic effect with an even lower toxicity than that of free MTX. The data suggest that at least the toxicity of conjugates is dependent on the Mw of the carrier. This fact should be taken into account when designing new anticancer polymer-drug compounds.

Antileukemic activity of glycated fibrinogen-methotrexate conjugates.

BACKGROUND: The aim of the study was to compare the antileukemic activity of methotrexate (MTX) conjugates with native and glycated fibrinogen. We expected that conjugates based on glycated fibrinogen would reveal higher antileukemic activity because of decreased plasmin digestibility and a higher retention rate of glycated fibrinogen in the body. MATERIALS AND METHODS: Fibrinogen was glycated using a high-temperature procedure at 65-85 degrees C. Glycated fibrinogens were examined with respect to their ability to clot and susceptibility to plasmin digestion. Native fibrinogen (F) and fibrinogens glycated at 65 and 73 degrees C (F65 and F73) were conjugated with MTX and tested in mice bearing P388 leukemia, at a dose of 40 mg of MTX per kg of body weight. RESULTS: Glycated fibrinogens retained their ability to clot. Compared to native fibrinogen, they were more resistant to digestion by plasmin. All tested conjugates revealed higher antitumor activity than the free drug. Increases in average lifespan over the control group were 34% for free MTX, 137% for F-MTX, 151% for F65-MTX and 91% for F73-MTX. The differences between the antitumor activities of all conjugates were not statistically significant. CONCLUSION: It seems necessary to compare the antitumor activities of MTX conjugates based on native and glycated fibrinogen in different tumor models, to demonstrate the expected differences.

[Methotrexate for treatment of rheumatoid arthritis]. / Metotreksat w leczeniu reumatoidalnego zapalenia stawów.

Rheumatoid arthritis (RA) is a common chronic inflammatory and destructive arthropathy that cannot be cured and that has substantial personal, social and economic costs. Methotrexate is a well-known folate analogue, and is the most frequently applied drug for the disease to modify antirheumatic therapy in patients with rheumatoid arthritis. Although results of studies have shown the efficacy of such drugs as methotrexate on rheumatoid arthritis, activity measures and their effect on mortality in patients with the disease remain unknown. The current therapeutic approach to rheumatoid arthritis consists of administration of anti-inflammatory, immunomodulating, and immunosuppressive drugs. Immunomodulating drugs, as opposed to non-steroid anti-inflammatory drugs, which only reduce the signs of inflammation, are capable of gradually checking the course of the disease. However, they do not prevent the slow but progressive destruction of joints. The prominent role of proinflammatory cytokines and growth factors in the pathogenesis of RA has been documented. Recent studies have demonstrated the efficacy of anticytokine treatment. Infliximab is a chimeric monoclonal antibody capable of neutralizing human TNF alpha. A number of clinical trials for the treatment of rheumatoid arthritis with infliximab indicated that TNF alpha blockade was effective and well tolerated, with excellent results occurring at 3 and 10 mg/kg in combination with methotrexate. Treatment of RA patients with the combination of infliximab and methotrexate also prevented radiographic evidence of progression of joint damage. If its clinical efficacy is sustainable and its safety confirmed over the long term, infliximab may become an essential agent of choice for the treatment of RA.