The natural xanthone α-mangostin (αM) exhibits a wide range of pharmacological activities, including antineoplastic and anti-nematode properties, but low water solubility and poor selectivity of the drug prevent its potential clinical use. Therefore, the targeted third-generation poly(amidoamine) dendrimer (PAMAM G3) delivery system was proposed, based on hyperbranched polymer showing good solubility, high biocompatibility and low immunogenicity. A multifunctional nanocarrier was prepared by attaching αM to the surface amine groups of dendrimer via amide bond in the ratio 5 (G3^2B12gh5M) or 17 (G3^2B10gh17M) residues per one dendrimer molecule. Twelve or ten remaining amine groups were modified by conjugation with D-glucoheptono-1,4-lactone (gh) to block the amine groups, and two biotin (B) residues as targeting moieties. The biological activity of the obtained conjugates was studied in vitro on glioma U-118 MG and squamous cell carcinoma SCC-15 cancer cells compared to normal fibroblasts (BJ), and in vivo on a model organism Caenorhabditis elegans. Dendrimer vehicle G3^2B12gh at concentrations up to 20 µM showed no anti-proliferative effect against tested cell lines, with a feeble cytotoxicity of the highest concentration seen only with SCC-15 cells. The attachment of αM to the vehicle significantly increased cytotoxic effect of the drug, even by 4- and 25-fold for G3^2B12gh5M and G3^2B10gh17M, respectively. A stronger inhibition of cells viability and influence on other metabolic parameters (proliferation, adhesion, ATP level and Caspase-3/7 activity) was observed for G3^2B10gh17M than for G3^2B12gh5M. Both bioconjugates were internalized efficiently into the cells. Similarly, the attachment of αM to the dendrimer vehicle increased its toxicity for C. elegans. Thus, the proposed α-mangostin delivery system allowed the drug to be more effective in the dendrimer-bound as compared to free state against both cultured the cancer cells and model organism, suggesting that this treatment is promising for anticancer as well as anti-nematode chemotherapy.

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequencedNucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the Accession number AF162691., allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35 582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Kmapp values describing interactions with dUMP (28·8 μMvs. 3·9 μM) and (6RS, αS)-N5,10-methylenetetrahydrofolate (383 μMvs. 54·7 μM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

α-Mangostin and vadimezan are widely studied potential anticancer agents. Their biological activities may be improved by covalent bonding by amide or ester bonds with the third generation poly(amidoamine) (PAMAM) dendrimer, substituted with α-D-glucoheptono-1,4-lactone and biotin. Thus, conjugates of either ester- (G3^gh4B5V) or amide-linked ( G3^2B12gh5V ) vadimezan, and equivalents of α-mangostin ( G3^gh2B5M and G3^2B12gh5M , respectively), were synthesized, characterized and tested in vitro against cancer cells: U-118 MG glioma, SCC-15 squamous carcinoma, and BJ normal human fibroblasts growth, as well as against C. elegans development. α-Mangostin cytotoxicity, stronger than that of Vadimezan, was increased (by 2.5–9-fold) by conjugation with the PAMAM dendrimer (with the amide-linking being slightly more effective), and the strongest effect was observed with SCC-15 cells. Similar enhancement of toxicity resulting from the drug conjugation was observed with C. elegans. Vadimezan (up to 200 µM), as well as both its dendrimer conjugates, was not toxic against both the studied cells and nematodes. It showed an antiproliferative effect against cancer cells at concentrations ≥100 µM. This effect was significantly enhanced after conjugation of the drug with the dendrimer via the amide, but not the ester bond, with G3^2B12gh5V inhibiting the proliferation of SCC-15 and U-118 MG cells at concentrations ≥4 and ≥12 μM, respectively, without a visible effect in normal BJ cells. Thus, the drug delivery system based on the PAMAM G3 dendrimer containing amide bonds, partially-blocked amino groups on the surface, larger particle diameter and higher zeta potential can be a useful tool to improve the biological properties of transported drug molecules.

Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N⁴-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)₃ beads. The modification, found to concern histidine residues and influence kinetic properties by lowering V_max, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N^5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N⁴-hydroxy-dCMP was found to be strongly dependent on [Mg²⁺], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells.

Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from muscle larvae of both Trichinella spiralis (isolated 1–24 months after infection) and Trichinella pseudospiralis (isolated 5·5–13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N⁴-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.