A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent currently commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Using the general concept of bioisosteric replacement, variations of the hydroxyglycine central "C" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. By this way, the malonic derivative 13a was discovered as the first example of a new series of potent immunosuppressive agents encompassing a retro-amide bond linked to the hexyl-guanidino moiety. Structure-activity relationships of this series were studied by synthesizing compounds 13g-i and 13k-s. Variation of the "right-amide" of 13a led to the urea 19a and the carbamates 23 and 27a which proved to be equally active as DSG in our GVHD model. Finally 27a was found to be the most potent derivative, being slightly more active than DSG in a heart allotransplantation model in rats. Due to the absence of chiral center in its structure and to its improved chemical stability compared to DSG, 27a was selected as a candidate for clinical evaluation.

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Various substitutions of the spermidine "D" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. Various positions of methylation were first investigated leading to the discovery of the monomethylated malonic derivative 56h in which the pro-R hydrogen of the methylene alpha to the primary amine of the spermidine moiety has been replaced by a methyl group. Synthesis of the similarly methylated analogue of the previously reported glycolic derivative LF 08-0299 afforded 60e which demonstrated a powerful activity at a dose as low as 0.3 mg/kg in the GVHD model and was much more potent than DSG in the demanding heart allotransplantation model in rats. The improvement of in vivo activity was supposed to be related to an increase of the metabolic stability of the methylated analogues compared to the parent molecules. Due to its very low active dose, compatible with a subcutaneous administration in humans, and its favorable pharmacological and toxicological profile, 60e was selected as a candidate for clinical evaluation.

LF 08-0299 is a new immunosuppressive compound. In a fully mismatched rat cardiac allograft model (Dark Agouti [DA]-->Lewis [LEW]), long-term unresponsiveness was observed after LF 08-0299 short-term treatment (20 days). Survival of additional cardiac and skin DA allografts, and rejection of third-party (Brown Norway [BN]) skin allografts demonstrated induction of a donor-specific tolerance state. The aim of this study was to investigate mechanisms of cardiac acceptance in this model. LEW rats with long-term surviving heart grafts (LTS LEW) were examined for their immune proliferative and cytotoxic responses toward donors (DA) and third-party (BN) antigens. Normal proliferative responses were observed and limiting dilution analysis did not reveal a reduction of T cytotoxic cell precursors. In our model, tolerance exists despite the presence of cells reactive with donor alloantigens. In vivo adoptive transfer of serum from LTS LEW failed to transfer unresponsiveness, indicating that serum factors do not seem to be involved in tolerance maintenance. Transfer of spleen cells, obtained from LTS LEW, showed specific prolongation of DA cardiac allografts in syngeneic hosts. Moreover, these cells were able to induce the rejection of third-party BN grafts. These results suggest that although LTS LEW possessed suppressor cells, they remained immunocompetent in recognizing and responding to third-party alloantigens. Purified CD4+ cells transferred unresponsiveness to secondary hosts, but CD8+ cells did not. Taken together, these results suggest that tolerance to donor alloantigens after treatment with LF 08-0299 in the rat cardiac allograft model is most likely due to induction of specific CD4+ suppressor cell activity, rather than induction of suppressive serum factor and selective elimination of antidonor helper or cytotoxic cell precursors (clonal deletion).

We investigated the ability of LF 08--0299, a new immunosuppressive compound, to prevent murine graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). A short term LF 08--0299 treatment at optimal dosage protected more than 75% of recipient mice from lethal GVHD induced either across minor antigens alone or the full H2 barrier. Furthermore, LF 08--0299 still prevented lethal GVHD when treatment was delayed to 10 days post-BMT. Long-term LF 08--0299-treated survivors were free of clinical signs of GVHD, and histopathologic examination of liver, skin, and intestines was normal, demonstrating that recipient mice did not develop chronic GVHD. We assessed the immunocompetence of long-term surviving recipient mice. Results from MLR and CTL assays were weak whereas responses against unrelated H2 antigens were reduced but still preserved. Moreover, in vivo transfer experiments demonstrated that spleen cells from long-term survivors were unable to induce lethal GVHD in irradiated recipients of host origin, while spleen cells injected in irradiated recipients of a host-unrelated H2 were fully competent to induce a lethal GVHD. Together these results indicate that stable chimeric recipient mice were specifically tolerant to host antigens. We further showed that while LF 08--0299 can protect recipient mice from lethal GVHD, it also preserved a graft-versus-leukemia effect when mice were inoculated with P815 tumor cells. These data suggest that LF 08--0299 may be a novel pharmaceutical agent that would prevent GVHD in human unrelated bone marrow transplantation.

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