Hydroxyethyl starch as an effective methotrexate carrier in anticancer therapy.

At present, effective anticancer therapy remains one of the most challenging tasks facing the scientific community. A major limitation to most conventional low-molecular weight anticancer chemotherapeutics is their unfavourable uptake by healthy tissue, fast metabolism and lack of tumour cell selectivity. One way to solve this problem is the application of hybrid nanoparticles containing widely known therapeutic substances. This study was performed with the aim of investigating the potential of use hydroxyethyl starch (HES) as a high-molecular weight carrier for anticancer drug (methotrexate, MTX). HES-MTX conjugates were characterized in terms of MTX content, hydrodynamic size, zeta potential, and drug release kinetics. In vitro biological characteristics were determined using different cancer cell lines. The antitumor effect in vivo was tested in NOD/SCID mice subcutaneously inoculated with MV-4-11 human leukaemia cells and CDF1 mice intraperitoneally inoculated with P388 murine leukaemia cells. The in vivo experiments revealed the considerably higher antitumor efficacy of HES-MTX conjugates in comparison to unconjugated drug. The results presented in this article demonstrate that the application of HES as an anticancer drug carrier can improve the treatment efficacy and have significant implications for the future design and implementation of drug-carrier conjugates. The study should help create new opportunities in the design of HES-based innovative drug-carrier conjugates.

The relationship between concentrations of vitamin D-binding protein (DBP) in serum and colostrum of mares and in serum of their foals in the neonatal period.

Vitamin D-binding protein (DBP) participates in the actin scavenger system, it is a carrier of vitamin D and its derivatives, it manifests the capacity to bind mainly monounsaturated and saturated fatty acids, it binds to the surface of several cells and enhances chemotactic activity of C5a of the complement. The present study was aimed at answering the question whether serum DBP level in mares is related to levels of this protein in colostrum and in serum of its progeny. For this purpose, sera from 77 mares, colostra from 72 mares and sera from 69 Thoroughbred foals were collected. Mother's age, number of deliveries experienced in the past, month of delivery, feeding of foals with colostra were recorded. Blood of the foals was sampled from the umbilical vein during delivery (0h) and 36-48 h after delivery from the external jugular vein, colostra of the mares were obtained after delivery and blood of the mares was sampled 36-48 h after delivery. Concentration of DBP was estimated by a self-designed ELISA. In the present study, DBP concentrations in newborn's serum were found independent of their concentrations in mother's serum, her age and number of parities experienced in the past. Colostrum DBP level was found to be lower than that in the mare's serum and was not correlated to the concentration of this protein in mare's serum. There was no effect of colostrum feeding on DBP level in the foal serum. These results indicate that serum DBP concentration in newborn foals depends on factors which act directly on the foal. Because of the lack of correlation between plasma and colostrum concentrations of DBP, it can be assumed that DBP is synthesised in the mammary gland and/or specific transport mechanisms exist in the mammary gland.

Antitumor activity of bacteriophages in murine experimental cancer models caused possibly by inhibition of beta3 integrin signaling pathway.

Bacteriophages (phages) as bacterial viruses are generally believed to have no intrinsic tropism for mammalian cells. In this study the interactions between phages and various eukaryotic cells were investigated. Binding of phages to the membranes of cancer and normal blood cells was observed. Moreover, it was shown that the wild-type phage T4 (wtT4) and its substrain HAP1 with enhanced affinity for melanoma cells inhibit markedly and significantly experimental lung metastasis of murine B16 melanoma cells by 47% and 80%, respectively. A possible molecular mechanism of these effects, namely a specific interaction between the Lys-Gly-Asp motif of the phage protein 24 and beta3-integrin receptors on target cells is proposed. It was also shown that anti-beta3 antibodies and synthetic peptides mimicking natural beta3 ligands inhibit the phage binding to cancer cells. This is in line with the well-described beta3 integrin-dependent mechanism of tumor metastasis. It is concluded that the blocking of beta3 integrins by phage preparations results in a significant decrease in tumor invasiveness.

New insights into the possible role of bacteriophages in transplantation.

Due to the increasing prevalence of drug-resistant bacterial infections in the "post-antibiotic era," bacteriophages (bacterial viruses, BP) may be useful to administer to transplant recipients without exposing them to an increased risk of rejection, which occurs consequent to some viral infections. Herein we present evidence that at least some coliphages (T4) do not pose such risk. Interestingly, they may produce immunosuppressive effects extending transplant survival. Our data suggest that BP may be used in clinical transplantation to treat drug-resistant bacterial infections and perhaps as an adjunct to immunosuppressive therapy.

BSE: a consequence of cattle feeding with glycated molecules host-unknown?

Although there is much evidence supporting a prion contribution in the pathogenesis of transmissible spongiform encephalopathies, a novel point of view as to the induction of the diseases can be proposed. It is hypothesized that neurodegenerative diseases, such as scrapie in sheep and goats and bovine spongiform encephalopathy in cattle (BSE), originate from the consumption of glycated proteins contained in their feed. These components are obtained during a high-temperature glycation process.

Antiangiogenic and antitumour effects in vivo of genistein applied alone or combined with cyclophosphamide.

The antitumour and antiangiogenic effects in vivo of genistein, applied alone or in combined therapy with cyclophosphamide, in the Lewis lung carcinoma (LL2) and B16 melanoma mouse tumour models, were analysed. Our own new method, allowing quantification of the volume of blood present in tumour tissue, enabled estimation of the degree of vascularization. Tumour cells entrapped in alginate beads were injected subcutaneously into mice. The quantification of alginate implant vascularization was performed with 125I-labeled mouse albumin injected intravenously. In mice bearing transplantable Lewis lung cancer the additive antiangiogenic, but not cytostatic, effect of genistein combined with cyclophosphamide (CY) was observed, since the treatment with genistein alone reduced tumour blood supply in 35% (tumour weight in 36%), with CY in 38% (tumour weight in 70%) and with both compounds in 61% (tumour weight in 75%). In the B16 melanoma model the respective values were: 60 and 44% for genistein, 83 and 79% for CY and 76 and 74% for combined treatment. These results indicate a higher antiangiogenic rather than cytostatic effect of genistein in both mouse tumour models applied.

Evaluation of high temperature glycation of proteins and peptides by electrospray ionization mass spectrometry.

Recently Boratynski & Roy (Glycoconjugate J., 1998, 15, 131) described a fast and convenient procedure for the synthesis of glycoconjugates. In the present study we used ESI-MS and circular dichroism as tools to analyze non-enzymatic glycation products of proteins and peptides. We discuss influence of reaction conditions on the rate of glycation of lysozyme. We analyze for the first time collision induced dissociation spectra of the obtained peptide conjugates.

[Chemical modifications of macromolecules and their use in preparation of protein antitumor drug conjugates,vaccines and diagnostic reagents]. / Chemiczne modyfikacje makroczasteczek oraz ich wykorzystanie w wytwarzaniu koniugatów bialek z lekami przeciwnowotworowymi, szczepionek i odczynników diagnostycznych.

This article reviews high temperature glycation of proteins, and describes some applications of thus received neoglycoconjugates as vaccines or drug and gene carriers. The use of macromolecules: antibodies, fibrinogen and natural or synthetic polymers as antitumor drug carriers is discussed. Moreover the article presents method for monitoring of proteins modification by glutaraldehyde.

Cytotoxic and antitumor effect of fibrinogen-methotrexate conjugate.

In this paper we describe the chemical procedure of fibrinogen-methotrexate (F-MTX) conjugate preparation and its in vitro and in vivo antitumor activity. F-MTX conjugates were synthesized in reaction of fibrinogen with MTX N-hydroxysuccynimide ester. The conjugates were not cross-linked and were soluble in water. The results of the in vitro and in vivo studies have shown: (1) a lower in vitro cytotoxicity of the F-MTX conjugate as compared with MTX alone; (2) a significantly higher in vivo antitumor activity of the F-MTX conjugate in mice with P388 leukemia as compared with MTX alone; (3) a significantly increased in vivo lethal toxicity of F-MTX as compared with MTX. The results suggest the therapeutic utility of the fibrinogen-methotrexate conjugate and the usefulness of fibrinogen as a chemotherapeutic drug carrier. However, a new effort in the preparation of F-MTX conjugate should be made to decrease its in vivo toxicity.

[Protein glycation--clinical and chemical aspects]. / Glikacja bialek--aspekty kliniczne i chemiczne.

Glucose as well as other reducing sugars react nonenzymatically with amino groups of free amino acids, peptides and proteins. Glycation reaction is responsible for forming inter- and intramolecular cross-links of proteins, known as advanced glycation end-products (AGEs). In old people, patients with diabetes and chronic failure AGEs accumulate in the circulation and tissues, and play a significant role in pathogenesis of cardiovascular and other complications. Glycation reactions has been used for synthesis of artificial glycoconjugates.

Synthesis, and cytotoxic activity of some novel indolo[2,3-b]quinoline derivatives: DNA topoisomerase II inhibitors.

A series of new 5H-indolo[2,3-b]quinoline derivatives bearing methoxy and methyl groups at C-2 and C-9 was synthesized (according to the modified Graebe-Ullmann reaction). These compounds were evaluated for their antimicrobial and cytotoxic activity and tested as inhibitors of DNA topoisomerase II. Lipophilic and calf thymus DNA binding properties of these compounds were also established. In the SAR studies we used quantum-mechanical methodology to analyze the molecular properties of the drugs. All of the 5H-indolo[2,3-b]quinolines tested were found to inhibit the growth of gram-positive bacteria and pathogenic fungi at MIC ranging between 2.0 and 6.0 microM. They showed also cytotoxic activity in vitro against several human cancer cell lines of different origin (ID50 varied from 0.6 to 1.4 microM), and stimulated the formation of topoisomerase-II-mediated pSP65 DNA cleavage at concentration between 0.2 and 0.5 microM. The most active indolo[2,3-b]quinolines which had the greatest contribution to the increase in the Tm of DNA displayed also the highest DNA binding constants and the highest cytotoxic activity. The differences in DNA binding properties and cytotoxic activity seem to be more related to steric than electrostatic effects.

Methoxy- and methyl-, methoxy-5,6,11-trimethyl-6H-indolo [2,3-b]quinolinium derivatives as novel cytotoxic agents and DNA topoisomerase II inhibitors.

New members of the cytotoxic indolo[2,3-b]quinoline family, with a methyl groups at N-5, N-6 (their presence stabilizes the positive charge of the molecule), were prepared using a modified Graebe-Ullmann reaction. The derivatives obtained were well soluble in water in a non-pH-dependent manner. They displayed strong antimicrobial activity against Gram-positive bacteria and pathogenic fungi (the MIC values fall between 0.0025 and 0.12 mM) and highly selective cytotoxicity in vitro against different human cancer cell lines: colon adenocarcinoma SW 707, lung carcinoma A 549, transitional cell carcinoma Hu 1703, and oral epidermoid carcinoma KB, in the range of 0.01 to 3.0 microM. They also stimulated the formation of topoisomerase-II-mediated DNA cleavage at concentration from 0.04 to 0.5 microM. These observations correspond well with the ability of the tested compounds to increase the melting temperature of calf thymus DNA (delta Tm being between 13 degrees C and 22 degrees C).

High temperature conjugation of proteins with carbohydrates.

A new procedure was used to conjugate lactose and dextran with BSA without using coupling or activating reagents. The method is simple, rapid and cheap. Reducing sugars covalently bind to proteins when lyophilized together and briefly heated to a high temperature.

Investigations on human lutropin from pituitary gland and urine.

Two newly isolated monoclonal anti-beta hLH antibodies did not recognize both human chorionic gonadotropin (hCG) and three synthetic peptide fragments corresponding to beta hLH sequence. These antibodies were applied for ELISA of human luteinizing hormone (hLH), however, sensitivities of these assays were much lower than those with two other monoclonal antibodies obtained earlier in our laboratory. No significant differences between assays of hLH from pituitary and urine with 6 different pairs of monoclonal antibodies (4 anti-beta and 1 anti-alpha-subunit) were noticed. The highest hLH concentration in urine samples collected during 53 menstrual cycles of 6 women was demonstrated at different times of the day. In 14 out of 52 "preovulatory urine" portions, preserved for several weeks, over 80% increase of assayed hLH was shown. The highest assays of hLH from pituitary and urine were noted at pH 7.5-9.0. Using affinity chromatography the whole pituitary hLH was bound to ConA-Sepharose column and was eluted with alpha-methyl-D-mannoside as a single peak. However, a small part of hLH from urine was eluted from the column as non retarded peak. When HPLC analysis with DEAE column was performed, pituitary hLH was separated in two fractions while lutropin from urine was eluted as a single peak.

Conjugation of the monoclonal antibody 17-1A with the nitroacridine compound C921 with the poly-L-lysine as an intermediate agent.

Monoclonal antibody 17-1A specific for human gastric carcinoma was coupled directly or indirectly, using poly-L-lysine as an intermediate, with nitroacridine compound C921. The aim of the study was to obtain selectively cytotoxic immunoconjugates for experimental therapy. Directly coupled conjugates retained antibody specificity towards cells of several human adenocarcinoma lines but were non cytotoxic in vitro, whereas conjugates obtained with the use of intermediate poly-L-lysine expressed only low, non-specific cytotoxicity, probably exerted by the poly-L-lysine content.

[The effect of long term erythropoietin treatment on levels of free amino acids in patients with long term hemodialysis]. / Wplyw dlugotrwalego leczenia erytropoetyna na poziom wolnych aminokwasów u chorych przewlekle hemodializowanych.

In order to assess the effect of erythropoietin (Epo) treatment on amino acids profile in hemodialysis patients (HD pts) 2 groups of pts were analyzed: I-12 pts HD for 146 +/- 71 months, Epo treated for 64 +/- 10 months, II-17 pts HD for 120 +/- 39 months, non Epo treated, mean Hb 11.5 +/- 1.2 g/dl. Controls consisted of 11 healthy individuals. Amino acids were estimated by HPLC OPA method. In group I blood levels of leucine (p < 0.03), valine (p < 0.002) were decreased and alanine (p < 0.05) was increased when compared to controls. In this group, blood levels of methionine, tyrosine and asparagine were elevated (p < 0.04) when compared to group II but they were lower (about 30%) then in controls. In group II pts showed reduced levels of valine (p < 0.008), leucine (p < 0.001), methionine (p < 0.0001), tyrosine (p < 0.003), asparagine (p < 0.04), whereas serine, glutamate and alanine (p < 0.04) were increased when compared to controls. Essential to nonessential amino acids ratios in group I, II and controls were as follows: 0.17; 0.12; 0.49 respectively. There were not substantial differences in amino acids in pts with elevated or normal PTH level in both HD groups. Long term EPO treatment only partially corrected changes in amino acids levels in pts with chronic renal failure.

Selective spectrophotometric determination of glucose and fructose in the presence of aldoses using phenol-acetone reagent and cerium(III) chloride.

Aqueous mixtures of glucose and fructose produce red solutions when treated with 2% (w/v) phenol in 5% (v/v) aqueous acetone in the presence of concentrated sulfuric acid. The color is stable for days, and the red chromophore has an absorbance maximum at 568 nm. When the concentration of phenol is raised to 25%, fructose, but not glucose, produces red solutions, allowing for the selective detection of ketoses. Two complementary methods have been developed to remove the interference of ketoses in solutions containing glucose. The first one relies on the selective reduction of ketoses with sodium borohydride in the presence of cerium(III) chloride prior to the addition of the phenol-acetone reagents. The second method is based on the differential specific determination of glucose using 2% versus 25% levels of phenol. The relative sensitivities of different sugars are also presented as well as the applicability of the methods using bacterial polysaccharides for immunochemical analyses. The quantitative determination of glucose or ketoses in the polysaccharides does not require hydrolysis prior to the estimation.

Stability of human lutropin studied with immunoenzymatic technique.

Characteristics of two monoclonal antibodies against human lutropin and their use in enzyme immunoassays of the hormone are presented. Diluted solution of pituitary lutropin was unstable in buffered saline and could be stabilized with some proteins. Concentration of the lutropin in woman urine, collected during "lutropin-peak", was markedly increased during long time storage at 5 degrees C or at room temperature. Using HPLC it was demonstrated that pituitary lutropin, normal urine lutropin and "increased" urinary lutropin were eluted from ion exchange column almost with the same retention time.