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A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography-tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low microg/L were achieved, whereas linearity was established within 0.5-500 microg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0-102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.

Benzofuroxans are interesting compounds which display several biochemical and pharmacological properties. Recent studies from our laboratory demonstrate that they are reduced by ferrous salts at room temperature and that the principal reaction products are o-nitroanilines. This paper shows that simple benzofuroxan derivatives are also able to oxidise HbO2 2+ to methemoglobin (MetHb3+) (UV detection) and to form o-nitroanilines (HPLC detection). From a toxicological point of view this reaction is interesting, since it indicates that the blood is a site for metabolism of these compounds with consequent methemoglobinemia and formation of toxic compounds.

The synthesis and preliminary biological assays of a new monofunctional reagent to reversible derivatize monoclonal antibodies is described. This compound, comprising a 1,4-polyiol moiety, is cleavable by means of sodium periodate in mild conditions; moreover it also contains a phenolic residue suitable for 125I labelling and a carboxylic group for reaction with epsilon-lysyl amino group of antibodies. These features are suitable to study the monoclonal antibodies cell-internalization process and antigen expression on cell surface. The 125I labelled reagent has been coupled to the monoclonal antibody AR-3, an IgG1 directed toward the CAR-3 antigen widely expressed on human ovarian and colorectal adenocarcinomas. The cleavage capability is tested in different conditions both on reagent and on derivatized MAb. Cell labelling experiments are performed both on target and untarget cell lines.

Immunotoxins have been extensively studied for the treatment of neoplasias; their intracavitary administration could be useful for the therapy of tumors confined to the pleural or peritoneum spaces. To study the feasibility of this "locoregional" treatment, a pharmacokinetic study of immunotoxins delivery is necessary. Ricin, a plant toxin extracted from the seeds of Ricinus communis, has often been used in immunoconjugates for its high activity; nevertheless, appropriate strategies have been necessary to limit the aspecific toxicity. We previously prepared a AR-3-ricin immunotoxin lacking the ability to bind galactosidic cell surface residues, a so-called sterically blocked immunotoxin. The monoclonal antibody AR-3, an IgG1 specific to the CAR-3 antigen, was able to recognize human colorectal adenocarcinomas. Preclinical trials in nude mice, intraperitoneally grafted with the target neoplasia, showed that this immunotoxin suppressed tumor growth without showing any undesirable ricin toxicity. In the present work we report the pharmacokinetic properties of this immunotoxin, showing the in vivo stability and a relatively long blood survival. With a biodistribution study in tumor-bearing mice, we demonstrate that in tumor-invaded tissues, the concentration of the specific AR-3-ricin immunotoxin was higher and progressively increased in a multiple-dose regimen. In contrast, an irrelevant immunotoxin behaved differently because it did not show specific tumor uptake. Moreover the pharmacokinetic data reported in this work improve the potential for "locoregional" treatment of malignancy with blocked immunotoxins.

This study describes the synthesis of a new generation of immunotoxins made by a noncovalent interaction between a monoclonal antibody derivatized with a dichlorotriazinic dye and the ribosomal inhibitor protein gelonin. The scheme of preparation has several advantages with respect to the traditional methods, which used heterobifunctional cross-linkers, such as a higher overall yield of production and the homogeneity of the obtained conjugate. Moreover, because no chemical derivatization of the gelonin was required, the unconjugated ribosome inactivating protein was recovered unaltered and therefore can be reused in other synthetic processes. This immunoconjugate was stable when tested in mouse serum and showed an interesting slow elimination rate when administered intravenously in mice. Although a high dye derivatization degree induced a modification of the specificity of the monoclonal antibody, the native specificity was restored after conjugation with gelonin. Furthermore the noncovalent linkage did not affect the gelonin inhibitory activity; in fact, the specific cytotoxic activity seemed to be similar to that of other disulfide-linked immunotoxins previously prepared in our laboratories.

To obtain more potent immunotoxins for anticancer therapy a gelonin-AR3 antibody immunoconjugate was prepared with different new linkers and coupling procedures. The gelonin was derivatized with the heterobifunctional thioimidate linkers ethyl-acetyl-3-mercaptopropionthioimidate (AMPT) and 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT), and with the succinimidyl type reagents N-succinimidyl-3-(4-carboxamidophenyldithio)propionate (SCDP) and N-succinimidyl-S-acetyl thiolacetate (SATA). The biological activity of gelonin modified with different linkers (AMPT, CDPT, SCDP, SATA) was determined by a rabbit reticulocyte assay. We found that AMPT was the molecule of choice to derivatize the toxin, confirming the preferability of thioimidate linkers. The monoclonal antibody Mab was derivatized with CDPT and SCDP. Then the following immunoconjugates were prepared with different procedures: Mab-CDPT with gelonin-AMPT; Mab-CDPT with gelonin-CDPT; Mab-SCDP with gelonin-SATA. To verify whether selection of the most suitable coupling procedure could affect the antitumoral activity of the gelonin-AR3 immunoconjugate, the three immunotoxins were tested on target HT-29 human colon carcinoma cells versus nontarget MeWo cells. The gelonin immunoconjugate linked via the AMPT-CDPT thioimidate reagents showed highest antitumoral activity as well as best selectivity for the target cells.

Correlation between structure and metabolism was studied within a series of cyclic acetal and thioacetal theophylline derivatives. All the compounds showed marked regioselectivity in in vitro metabolism, the metabolites arising only from 7-cycloalkyl side chain transformation. The 1,3-dioxane derivative, besides N-dealkylation to theophylline, underwent enzymatic ring cleavage, through the oxidation of the acetal carbon and subsequent rearrangement. Thus the acetal group was converted enzymatically to an ester. A similar transformation, catalyzed by cytochrome P450-dependent monooxygenases, was previously found for the 1,3-dioxolane ring of doxophylline. The cyclic thioacetal derivatives (i.e. 1,3-oxathiolane and 1,3-dithiane) were not cleaved during oxidative metabolism. The metabolites arise only from the oxidation of the sulfur atom, the major nucleophilic center in the molecule. No N-dealkylation to theophylline was observed. Enzymatic sulfoxidation proceeded diastereoselectively in both the 1,3-oxathiolane and 1,3-dithiane rings, the trans isomers being the major ones with a ratio trans: cis 75:25 and 60:40 respectively. The sulfoxides were stable to hydrolysis and were not further metabolized. Neither disulfoxides nor sulfones were detected in the incubations.

In an effort to obtain a more potent and specific immunotoxin for cancer therapy, we designed a series of heterobifunctional linkers characterized by a thioimidate group linked to a S-acetyl thiol (4, 5) or substituted aryldithio group (6-10). These ligands were synthesized by a Pinner-type process from the corresponding nitrile derivatives obtained by thiol-disulphide exchange reaction, reaction with substituted benzene-sulphenyl chloride, or other known procedures. To check the reagent of choice for immunoconjugate preparation, we studied thioldisulphide exchange kinetics between the intermediate nitrile derivatives and cysteine. Among the tested aryldithio derivatives (6-10), we selected ethyl 3-(4-carboxamido-phenyldithio)propionthioimidate (CDPT, 9) for further studies. By analyzing the rate of incorporation of the linkers 4, 5, and 9 in a model immunoglobulin G protein, we found similar results with CDPT 9 and ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT, 5) because both reagents showed a linear correlation between the number of introduced thiol groups and factors such as time and protein and reagent concentrations. Comparison of the two acetylthio-derivative ligands 4 and 5 showed that AMPT 5 was more stable toward deacetylation than ethyl S-acetyl 2-mercaptopropionthioimidate ester hydrochloride (AMAT, 4). By comparing the kinetic and biological parameters of seven new thioimidate linkers, we found that two of these (CDPT and AMPT) could be superior ligands for protein-protein conjugation. They offer advantages over the commercially available compounds, such as minimal perturbation of the protein structure, controlled reactivity, and good stability.

A method to produce immunotoxins (conjugates comprising of a monoclonal antibody and toxin) using ribosome inactivating protein anchored on an affinity gel derivatized with triazinic dye is described. The adsorbed toxins were activated with 2-imino-thiolane and then conjugated to monoclonal antibody activated by SPDP. The "heterogeneous phase" system offered several advantages, reducing the usually required purification steps and opening a way to automatize the conjugation procedure.

Various classes of inhibitor of 2,3-oxido squalene cyclase have been synthesized and tested on rat liver and Saccharomyces cerevisiae microsomes, 3T3 fibroblast cultures, and various bacteria, fungi, and yeasts. The compounds include azasqualenes, azasqualanes, bis-azasqualenes, bis-azasqualanes, and N-oxide and ammonium derivatives of squalene. In order to better mimic the transition state involved in the SN2-like opening of 2,3-oxidosqualene, we synthesized squalene N-methyloxaziridine. Other derivatives tested were N-methylimine, aminalic hydroperoxide, and N-methylamide. We also attempted to produce new "suicide" inhibitors of SO cyclase, such as a squalenoid epoxide vinyl ether. Many of the products described inhibited the various cyclases, the best having an IC50 of 0.3 microM on plants and 1.5 microM on rat liver microsomes, and good antibacterial and antifungal activity. In a search for inhibitors of squalene epoxidase, a series of mono- and bifunctional squalenoid acetylenes and allenes were synthesized. Some of them proved to be inhibitors of squalene epoxidase.