Characterization of lipophilicity and antiproliferative activity of E-2-arylmethylene-1-tetralones and their heteroanalogues.

A molecular library based on E-2-arylmethylene-1-tetralone has been designed and synthesized. A reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed and applied to separate them and to characterize their lipophilicity. The chromatographic method applied here was suitable to separate the structural (ortho and para) isomers of compounds and was sensitive enough to differentiate their lipophilicities. The measured (k') and computer calculated (CLOGP) lipophilicity values has been compared. Good linear correlation has been found in the case of these structurally related molecules. In vitro biological assay has been performed with Methylene blue dying to investigate the antiproliferative potency of the compounds synthesized in this work. The measured (k') and calculated (CLOGP) lipophilicities of the compounds were compared with the antiproliferative activities and an optimum value of lipophilicity has been found for these compounds.

Novel lipoamino acid- and liposaccharide-based system for peptide delivery: application for oral administration of tumor-selective somatostatin analogues.

Lipoamino acid and liposaccharide conjugates of somatostatin analogue TT-232 were synthesized to modify the physicochemical properties of the parent peptide. The relative position, the number, and the nature of the lipid and/or saccharide moieties were varied. Experiments in vitro clearly showed that many compounds modified at the N- and/or C-terminus with lipid or sugar moieties retained the biological activity of the parent compound. An interesting construct was synthesized containing lipid and sugar units at opposite ends of the somatostatin analogue, so that the entire molecule could be considered as an amphipathic surfactant.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Arginine supply for nitric oxide synthesis and arginase is mainly exogenous in elicited murine and rat macrophages.

L-arginine, the precursor of nitric oxide(NO) is provided mainly by extracellular sources in casein-elicited murine and rat peritoneal macrophages. Free extracellular L-arginine(Arg), esters, peptides and proteins containing Arg are the best sources in accordance with the fact that proteolytic activity is high in peritoneal macrophages. The recycling of Arg from citrulline(Cit) was observed but at a low rate. This situation is different from that in endothelial cells where half of Arg is recycled from citrulline. No significant anaplerotic reaction from glutamic acid(Glu) can be demonstrated in peritoneal macrophages.

Cytostatic, cytotoxic and protein tyrosine kinase inhibitory activity of ursolic acid in A431 human tumor cells.

The effect of the tritepene, ursolic acid, on the proliferation of A431 human epidermoid carcinoma cells was studied. According to our investigations, ursolic acid is a potent inhibitor of A431 cell growth. Ursolic acid markedly reduced A431 cell growth in a time- and dose-dependent manner. We found a good correlation between the results of direct cell counting and the MTT test. During long periods of drug exposure, ursolic acid exhibited both cytotoxic and cytostatic activity. The effect was partially reversible on drug removal. The greatest cytotoxicity was observed both in the trypan blue test and in the MTT test at 50 mM. Investigations on tyrosine kinase inhibition were performed by biochemical and cellular assays on A431 cells. Ursolic acid inhibited tyrosine kinase activity of A431 cells in biochemical assay in a dose-dependent manner with an IC50 of 24 mM. In cellular assay, when A431 cells were pretreated with ursolic acid for 24, 48 and 168 hours at various concentrations (5, 10, 20, 30 and 50 mM), lower values of IC50 were measured: 6.8 microM for 24 hours, 5.2 mM for 48 hours and 1.4 mM for 168 hours. The results suggest that ursolic acid exerts an antiproliferative effect through the inhibition of tyrosine kinase enzymes.

Activation of caspase-3 protease during the process of ursolic acid and its derivative-induced apoptosis.

The apoptosis-inducing effect of the triterpene saponins, namely, ursolic acid and its natural derivative, methyl-ursolate beta-D-glucoside on A431 human epidermoid carcinoma cells was studied. The cells treated with 5-50 microg/ml of ursolic acid resulted in a dose- and time-dependent decrease in cell number, due to an increase of apoptotic cells as evidenced by MTT assay together with morphological changes. The highest dose (50 microg/ml) of ursolic acid resulted in approximately 90% inhibition in tumor cell growth after 96 hours of treatment and 60% of apoptosis after 48 hours. To the contrary, when the same treatment was carried out with methyl-ursolate beta-D-glucoside, after 96 hours of treatment the percentage of cell growth inhibition was found to be only 30% at the dose of 50 microg/ml and the value of apoptosis did not exceed 10%. Similarly to these results, ursolic acid effectively induced proteolytic activation of caspase-3 protease in a dose-dependent manner while its derivative showed only weak activity in this enzyme assay. The addition of DEVD-CHO prior to ursolic acid and methyl-ursolate beta-D-glucoside treatment effectively prevented the loss of triterpenes-induced viability. In summary, the triterpene saponins investigated contain an apoptotic-inducing activity in A431 cells and in the case of ursolic acid it is associated with proteolytic activation of caspase-3 and/or other similar caspases. Our results also indicated that methylation of COOH-28 together with the glycosylation of C3 of ursolic acid have a strong impact on its antitumor activity.

Determination of the basicity of Mannich ketones by capillary electrophoresis.

Capillary electrophoretic (CE) method to characterise Mannich ketones (MKs) containing morpholine moiety was developed. Basicity (pKa,exp) of the MKs was characterised by measured data derived from the electrophoretic mobility values obtained in the CE separation. The MKs were found to be weaker bases than the parent morpholine molecule itself and the experimentally determined basicity values proved to be dependent on the chemical structure of the MKs. Since the basicity of the MKs has an influence on their reactivity and biological activity it seems to be useful to determine experimentally the pKa,exp values of the newly synthesised compounds to support rational drug design or screening of the molecule libraries.

Comparison of measured and calculated lipophilicity of substituted aurones and related compounds.

A molecule library containing 55 aurone- and thioaurone-type structures has been designed and synthesised. Reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed to separate these compounds and to characterise their lipophilicity by experimental method (k'). The experimental lipophilicity data have been compared with the computer calculated lipophilicity parameters (CLOGPs) of the same molecules. In general, good correlations between the measured and calculated lipophilicities have been found with the exception of structure isomers and compounds capable for hydrogen bonding. The chromatographic method was suitable to separate the structure (ortho and para) isomers of aurone and thioaurones and was sensitive enough to differentiate their lipophilicities. Our findings suggest the usefulness of the chromatographic method in fast characterisation of the lipophilicity of structurally closely related molecules.

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes.

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.

[Preparation and characterization of potential antineoplastic agents]. / Potenciális daganatellenes hatású vegyületek elóállítása és vizsgálata.

A parallel combinatorial library of over sixteen hundred compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads that is aimed for intervening with the substrate binding site of the pp60c-src enzyme. The new structures were based on known PTK inhibitors having at least two variously substituted aromatic moieties attached by spacer groups of different length and flexibility. Eleven bis-aryl type inhibitory compounds were found in the range of 18-100 micromolar IC50 concentrations from combinations of twelve different substituents. Molecular modeling of the active compounds showed a characteristic distance of 13-14 A between the farthest sp2 carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60c-src PTK [5,6,7] showed that the energy minimized conformers had the same distance between two aromatic moieties. Several compounds in the library not only showed remarkable PTK inhibitory activity but also a significant apoptosis inducing effect on HT-29 human colon tumor cells.

Comparative characterization of experimental and calculated lipophilicity and anti-tumour activity of isochromanone derivatives.

Compound lipophilicity connected to ADME(T)(a) has great importance in drug development and it has to be evaluated by the generally used drug developmental process. In addition to the importance of lipophilicity in ADMET, recently it has been reported that lipophilicity of small molecules correlates with their antiproliferative activity because of certain specific hydrophobic and lipophilic interactions. Due to the complexity of ADME(T) parameters an efficient and fast method is needed to characterize the many promising candidate lead molecules as a preselection in order not to be rejected from the latter phase of drug development. In the present paper we provide an overview of the importance of lipophilicity of drug candidates for biological action and for ADME(T) and describe a novel approach for drug-likeness characterization of a molecular library using correlation study between lipophilicity and biological activity. Lipophilicity and molecular characteristics have been measured, predicted and optimized for a diverse library from which the best members have been selected to describe their biological, chemical and drug-likeness properties. Molecules were selected from the family of alpha,beta-unsaturated ketones and thorough HPLC characterization for lipophilicity and morphological, antiproliferative and flow cytometric studies were carried out on them. Based on the results 17 member isochromanone library including E and Z geometric isomers were selected for further characterization. In this focused library linear correlation has been found between the calculated and measured lipophilicity and significant parabolic correlation was found between the antiproliferative effect and lipophilicity. Using our efficient and fast method, from a diverse library, we identified an outstandingly effective inhibitor of A431 tumour cell growth via a PARP(a) cleavage dependent apoptosis. In summary the optimized HPLC analyses of lipophilicity combined with the cell-culture assay, introduced above, resulted in the determination of an optimal lipophilicity range. This optimized lipophilicity range should be used in designing novel antiproliferative compounds.

Calculation of modified capacity factor in micellar electrokinetic chromatography.

In addition to the expression k' = (tm-t(o))/t(o) (1-tm/tmc), we propose the expression k" = (tm-t(o))/(tmc-t(o)) to calculate the capacity factor in micellar electrokinetic chromatography (MEKC), where tm, t(o), and tmc are the migration time of the analyte, the flow marker, and the micelles, respectively. The k' and k" values that were obtained from simulated data as well as from MEKC analysis of different peptides (in 100 mM sodium dodecyl sulfate/0.1 N sodium borate buffer at pH 11.0) were calculated and compared. The k" value is equal to zero for an analyte remaining in the aqueous phase whereas it is equal to one for an analyte always staying in the micellar phase. By applying k" a finite capacity factor can be obtained for an analyte, indicating its partition between the two moving phases (aqueous and micellar) even in those cases when tm equals tmc. The slope of the curve k" as a function of tm is constant through the whole migration window and therefore peak compression does not occur when applying k" to calculate the capacity factor. A given difference in k" corresponds the same difference in migration times and this value does not depend on the position within the migration window. Since k" is a normalized parameter it is easy to evaluate the significance of a given difference in capacity factor or to estimate the relative position of an analyte with a given capacity factor in the migration window by applying k". Therefore, k" seems to be an adequate parameter to calculate the capacity factor in MEKC and, similar to K', it also refers to the hydrophobicity of the analyte.

Investigation of the chemical stability of a new growth hormone-releasing hormone (GHRH) analogue by HPLC.

The stability of a new active growth hormone-releasing hormone analogue (D-Ala2,Nle27,(gamma-amino-butyric acid)30-GHRH(1-30)-NH2) was investigated during storage at different temperatures in aqueous solution. Samples stored for various periods of time were analysed by HPLC. It is concluded that in aqueous solution D-Ala2, Nle27,(gamma-amino-butyric acid)30-growth hormone-releasing hormone (1-30)-NH2 is stable: at least for 36 days at 4 degrees C; for 28 days at 25 degrees C; and for 10 days at 37 degrees C.

Elevated opioid activity in sera of chronic schizophrenics.

The effect of native serum samples from schizophrenic and control patients on the electrically induced contractions of the isolated mouse vas deferens (MVD) preparations was investigated. It was demonstrated that only the samples of schizophrenic origin elicited a naloxone dependent inhibition on the contractions of the MVD preparations, while sera from healthy individuals and those from non schizophrenic but mentally ill patients proved to be ineffective in this respect. By using ultrafiltration and gel chromatographic techniques, four fractions disclosing MVD related biological activity could be separated from schizophrenic samples. Chemical analysis revealed an elevated quantity of ninhydrin and Lowry positive materials as well as of unidentified carbohydrate components in the active fractions. Molecular mass of the serum ingredients carrying opioid activity was found to range between 0.5 and 5.0 KD. It is speculated that new appearance or the accumulation in the sera of several, and partly at least, unknown peptides and glycopeptides disclosing opioid activity might be characteristic of schizophrenia.

Intermediate molecular weight substances in sera of psoriatic patients.

In the experiments presented here, serum samples were collected both from psoriatic (PS) (n = 8) and non-psoriatic (NPS) patients (n = 8) and were analyzed by the combination of fractional precipitation (80% ethanol, pH 2) and gel filtration techniques as well as by chemical methods. It was demonstrated that, in comparison to the non psoriatic samples, concentration of the free alfa-amino group bearing and the Lowry positive components as well as total carbohydrate content was significantly elevated in the acidic ethanol soluble serum fraction of psoriatic samples. In psoriasis, the quantity of serum components with UV light absorbing capability at 206 nm, and with a molecular mass between 0.3 and 5.0 KD (estimated by chromatographic criteria) also was elevated in one of the fractions obtained by Sephadex SG-25 chromatography. Results presented in this paper indicate, that psoriasis is accompanied by changes in quality and in quantity of middle size molecular weight serum components with chemical properties suggesting their peptide--and/or glycopeptide-like character.

Biological detectors in liquid chromatography.

The applicability of isolated organ preparations as chromatographic detectors was demonstrated. An appropriately designed detector cell (biological detector) applicable in both on-line and off-line modes was developed. Deproteinized sera from healthy volunteers and schizophrenic patients and human amniotic fluid were fractionated by gel-permeation, ion-exchange and reversed-phase liquid chromatography. Ultraviolet absorption and biological activity were compared. The results show that isolated organ preparations selected according to the needs of particular experiments meet the essential criteria of conventional chromatographic detectors. The use of isolated organs allows the detection of biologically active substances in a matrix of physicochemically closely related, but biologically distinct, fluid components. Biological detectors may also provide valuable additional information concerning the chemical structure of biologically active agents in an early stage of isolation.

Comparison of high-performance liquid chromatography and capillary electrophoresis in the analysis of somatostatin analogue peptides.

HPLC and CE methods were developed for analysis of somatostatin analogue (S-analogue) peptides utilizing triethylammonium phosphate-organic solvent modifier solvents as the CE buffer and HPLC eluent. Acetonitrile, methanol, ethanol and 2-propanol were applied as organic modifiers. The applicability of HPLC and CE systems was evaluated and compared. Optimum conditions for the separation were determined for both methods. Retention (migration) time, elution order and selectivity can be influenced by modifying the composition of the eluent (buffer) with organic solvents not only in HPLC but also in CE. Although the HPLC system reacted to changes in the organic solvent concentration in a much more sensitive way than the CE system did (from the point of view of retention time), CE proved to be a more suitable method for separating the peptides investigated. Baseline separation could be achieved within 6-9 min by CE, a result which was impossible to achieve with HPLC working in the isocratic mode. In CE the effect of the alcohols on migration times proved to be opposite to that of acetonitrile. Whereas ACN decreased, the alcohols increased the migration times in a concentration-dependent way. The results suggest that CE can be applied very advantageously in peptide analysis. Its performance regarding selectivity, resolution, theoretical plate number, duration and cost is comparable or sometimes superior to that of HPLC.

Utero-inhibin: a new substance inhibiting uterine contraction, isolated from amniotic fluid.

Amniotic fluid was collected from women in the 2nd trimester of their gravidity by amniocentesis and its effect on the isolated pregnant rat uterine preparation investigated. It was demonstrated that some of the amniotic fluid components partially purified by using the combination of various separation techniques stimulated, while others inhibited the contractions of the isolated rat uterine preparation. The inhibitory component, called utero-inhibin also inhibited the spontaneous contractions as well as the basal tone of the isolated rat intestinal preparation and those of the isolated human pregnant myometrium preparation. It is speculated that the inhibitory factor might play a physiological role in maintaining the resting state of human uterus during pregnancy.

Quantitative and qualitative changes in serum oligopeptide components of patients with end stage malignant tumour disease.

A number of changes in peptide components could be demonstrated in sera of patients with end stage malignant diseases. Total and free alfa amino-N content of such sera was shown to be significantly higher than those in sera of patients with no sign of malignancy. In addition, a significant increase was found in one of the serum fraction obtained by Sephadex G-25 chromatography. This increase was shown to be due to an increase in the amount of one of the isotachophoretically separated serum components of anionic character as well as to the appearance in sera of tumour bearing patients of two additional isotachophoretic components never detected in non tumour bearing patients. Based on their chromatographic behavior as well as on observations made in earlier experiments, the peptide nature of the two isotachophoretic serum components seemingly characteristic of sera of tumour bearing patients is highly probable.

The role of degradation of fetal haemoglobin in the energy supply of very low body weight pre-term babies.

Low body weight premature babies born before the 32nd gestational week were studied to analyse the postnatal fall in plasma haemoglobin and in quantitative changes in amino acid levels. Red blood cells of premature low body weight infants were found to disintegrate more rapidly than those of mature newborns. Thin-layer ion-exchange chromatographic studies showed that amino acids originating from the degrading haemoglobin-F lead to rise in plasma amino acids. These amino acids might play a role as substrates for gluconeogenesis in the energy supply of low body weight premature babies during the special fasting state just after birth.