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Agelastatin A (1), an unusual alkaloid of the axinellid sponge Agelas dendromorpha from the Coral Sea, can be selectively acetylated (7) or methylated at OHC(8a) (4), peracetylated (8) or permethylated at OHC(8a), NH(5), and NH(6) (5), or, finally, subjected to C(9)C(8a) (14) or C(5b)C(8a) β-elimination (11–13), in a regiospecific manner or not, depending on the reaction conditions. Under acidic conditions, compound 12 adds H2O or MeOH, regioselectively though not endo/exo stereoselectively, giving transoid/cisoid mixtures 1/18 or 4/19, respectively. Similarly 11 or 13 add MeOH to give mixtures (−)-2/20 or 15/16, respectively. Compound 13 also adds AcOH giving mixture 8/17. The intermediate cisoid form obtained on treatment of 21 with H3O+ undergoes N(5)N(6) bridging affording pentacyclic 22 which constitutes a proof for the cisoid configuration. From conformational studies, rules are devised that allow assigning the configuration of these compounds from NMR data. In vitro comparative cytotoxicity assays of these compounds show that for high cytotoxic activity, such as of 1in vivo, unsubstituted OHC(8a), HN(5), HN(6) moieties are needed in the natural B/D transoid configuration.

The relationship between different chemical modifications on morpholinylanthracyclines and their ability to overcome multidrug resistance (MDR) has been evaluated testing all compounds in vitro on LoVo and LoVo/DX human colon adenocarcinoma cells and in vivo disseminated P388 and P388/DX murine leukemias. Results obtained led us to the following conclusions: 1) the insertion of the morpholinyl or the methoxymorpholinyl group on position 3' of the sugar moiety confers the ability to overcome MDR in vitro and in vivo; conversely, 4' morpholinyl compounds are effective on MDR cells only in vitro and result inactive in vivo on DX-resistant leukemia; 2) all chemical modifications performed on 3' morpholinyl or methoxymorpholinyl derivatives, that is substitutions on the aglycone or on position 2 of the morpholino ring, do not interfere with the activity of the compounds: all derivatives present in fact the same efficacy on sensitive and resistant models. It is concluded that position 3' in the sugar moiety plays a crucial role in the ability of morpholinyl-anthracyclines to overcome MDR.

The clinical use of Ca++ antagonist agents as modulators of multidrug resistance is limited by their strong vasodilator activity. This study reports data obtained by testing a series of new 1,4 dihydropyridine derivatives (DHPs) for their in vitro resistance modulating activity and their Ca++ antagonist effect. All the tested DHPs are active to increase doxorubicin activity with dose modifying factor values ranging between 2 and 47 on P388/DX cells and 12 and 36 on LoVo/DX cells. Their resistance modulating action is exerted through an increase of DX intracellular level. The Ca++ antagonist activity of DHPs, evaluated as capacity to inhibit the KCl-induced contractions in isolated Guinea pig ileum strips, is not related to their resistance modulating activity. This finding makes it possible to select, for further in vivo evaluations, compounds IX, X and XI, which have strong ability to overcome multidrug resistance and low Ca++ antagonist effect.

The in vivo behavior of the monoclonal antibody (MAb) MOv18, with a restricted specificity for human ovarian carcinoma was analyzed on normal and tumor-bearing animals. The pharmacokinetics of the iodine-labeled MAb carried out in BALB/c mice fits an open two-compartment model. The t 1 2 alpha was found not to be influenced by the different iodine isotopes used (125I vs 131I) and by the time between labeling procedures and administration. The t 1 2 beta were found to be longer after i.p. than i.v. administration and influenced by the time lapse between preparation and administration. A radiolocalization study was carried out in CD1 nu/nu mice bearing i.p. xenotransplant of the human ovarian carcinoma cell line IGROV1. Tumor/non tumor ratios were higher when the time between administration and sacrifice was short and, for 131I-MOv18, with a short interval between labeling and injection. Even if longer half lives were obtained using 125I-MOv18 and i.p. administration a fairly rapid decrease in the values of the percentage of the injected dose per gram of tumor during the time was noted. These data indicate that this MAb could be considered a good candidate for radiotherapeutic approaches.

Three murine monoclonal antibodies (MoAbs), MBrl and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabeled MoAbs were analyzed for their « in vitro » stability in human blood. They were incubated at 37 °C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

Ricin A chain was coupled to murine monoclonal antibodies MBr1 and MOv2 respectively raised against human breast and ovarian carcinomas. Inhibition of protein synthesis only occurred in those cultured human tumor cells bearing the appropriate target antigens, demonstrating that both components of the conjugate were unchanged in regards to specificity and toxicity. Conjugates were 125-200 times more efficient in inhibiting [3H]proline incorporation than the uncoupled ricin A chain. They were however unable to kill the entire population of the appropriate cells even after repeated treatment. Although the two monoclonal antibodies had similar binding kinetics, the conjugates differed in their cytotoxicity kinetics. The MBr1-ricin A chain conjugate had slow kinetics, and about 20 hours were needed to obtain a protein synthesis inhibition above 50% on the appropriate line (mammary carcinoma MCF-7). In contrast, the MOv2-ricin A chain conjugate showed very fast kinetics, reaching 50% inhibition after only 30 minutes of treatment on both appropriate cell lines SW626 and HT-29 from ovarian and colon carcinomas, respectively. Growth conditions of cell lines, i.e., adherent cells versus suspended cells, and plating time were found to greatly influence the conjugates' killing efficiencies. These studies confirm the possibility of preparing ricin A chain-antibody conjugates, which retain specific cytotoxicity against tumor cells; but they also underline the need for further in vitro studies of various parameters before one considers a therapeutic use of such conjugates.

The murine monoclonal antibody (MAb) MOv2 was found to be directed against the carbohydrate moieties of different kinds of molecules expressed on a human ovarian cystoadenocarcinoma. To define further the glycoconjugates carrying the MOv2-defined epitope, different procedures were used to analyze materials from surgical specimens and carcinoma cell lines. SDS-PAGE and immunoblotting showed glycoprotein molecules migrating in the gel as high and intermediate molecular weight components. A low-molecular-weight band, migrating approximately with the dye front, was also immunostained by MOv2. On the other hand, the immunostaining of high-performance thin-layer chromatography (HPTLC) of the total glycolipid extract and its neutral and acid fractions, after DEAE chromatography, showed selective reactivity with a neutral glycolipid. Reanalysis by immunoblotting of this glycolipid band scraped off the HPTLC plate indicated that it corresponds to the low-molecular-weight component. Periodate oxidation and Pronase digestion further demonstrate the saccharide nature of the determinant on both types of glycoconjugates. In conclusion, we report evidence that with a single analytical procedure, i.e., immunoblotting, it is possible to recognize the same carbohydrate determinant carried on both protein and lipid molecules.

The protein toxin restrictocin, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of Mr 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restrictocin toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later. After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites internalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.

In the perspective of therapeutic approaches the monoclonal antibody, MBrl, with a quite restricted spectrum of reactivity for human breast carcinoma, was coupled to restrictocin (Res), a ribosome inactivating protein produced by Aspergillus restrictus. In a cell-free system this toxin was found to have an activity comparable to that of other plant toxins, but its in vitro toxicity was shown to be low on different cell lines. Three batches of MBr1-Res conjugate were prepared and their specificity, efficiency, and maximum level of cytotoxicity were analyzed on the cell line MCF-7 expressing the relevant antigen, on several irrelevant tumor cell lines, and on normal cells. Conjugates were from 600 to 1500 times more efficient than the uncoupled derivatized Res towards MCF-7 cells and were completely ineffective on the other target cells. The antigen-driven cytotoxicity was confirmed by the nontoxicity of an irrelevant conjugate on MCF-7 cells. The cytotoxic efficiency of MBr1-Res was low when compared to the binding level of MBr1 at the same concentration and a portion of treated cells (from 10% to 30%) survived the treatment. The heterogeneity of expression of the relevant antigen, together with its only partial internalization, could account for these limitations. The lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin were tested as potentiating agents but in both cases the cytotoxicity remained unmodified. A neutralization assay performed on a xenogenic model indicated that the MBr1-Res conjugate was capable of reducing the tumor take. These data indicate the possibility of using the Res to prepare a reproducible and highly selective breast cancer conjugate. However, there are still a number of problems which must first be solved before we can consider its clinical application.

In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6-8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBr1, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.

The use of monoclonal antibodies for in vivo therapeutic approaches depends largely on their specificity. During the characterization of ricin A-chain-murine monoclonal antibody conjugates we found that the binding specificity of a monoclonal antibody raised against human ovarian carcinoma (MOv2) seemed altered. Therefore, the binding reactivity of the unmodified antibody (MOv2), the conjugation intermediate (MOv2-PDP) and the conjugate (MOv2-A chain) was titrated by solid-phase radioimmunoassay on 11 human tumor cell lines belonging to seven different histotypes. The three reagents bound with the two reference cell lines (SW626:ovary carcinoma and HT-29:colon carcinoma). The MOv2-PDP and the Mov2-A chain also reacted with seven other cell lines which were unreactive with the unmodified MOv2. In addition MOv2-PDP exhibited reactivity on all normal cells tested (peripheral blood lymphocytes and skin fibroblasts). To elucidate the significance of these findings the following experiments were performed: cross inhibitions between the unmodified and modified monoclonal antibodies; comparative absorption tests with different cell lines; and immunoblotting analysis of the target antigens. The results suggest that after chemical modification with SPDP the monoclonal antibody MOv2 increases its binding activity, so that even a low number of antigenic sites can be detected. This study underlines the need to redefine the specificity of a conjugate before considering therapeutic applications.