A fluorescein derivative of the lysine analogue of folic acid, N alpha-pteroyl-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (PLF), was synthesized as a probe for dihydrofolate reductase (DHFR) and a membrane folate binding protein (m-FBP). Excitation of PLF at 282 nm and at 497 nm gave a fluorescence emission maximum at 518 nm. Binding of PLF to human DHFR or human placental m-FBP results in approximately a 20-fold enhancement in the magnitude of the fluorescence emission, suggesting that the ligand interacts with a hydrophobic region on these proteins. Additional evidence suggests that an energy transfer may occur between the pteridine and the fluorescein moieties. PLF binds to the active site of human DHFR since methotrexate (MTX) competes stoichiometrically and the denatured enzyme in the presence of PLF did not exhibit fluorescent enhancement. The dissociation constant for the fluorescein derivative with respect to human DHFR is 115 nM as compared to 111 nM for folic acid. The Ki value for the competitive inhibition of human DHFR by the fluorescent analogue of folic acid is 2.0 microM compared to 0.48 microM for folic acid. PLF was reduced to N alpha-(7,8-dihydropteroyl)-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (H2PLF) and assayed by the enzymatic conversion to the tetrahydro derivative. The Km value for human DHFR for the dihydrofolate analogue is 2.0 microM. The KD value for H2PLF to human DHFR is 47 nM as compared to 44 nM for dihydrofolate. The KD values for both H2PLF and PLF indicate that the fluorescein moiety does not significantly affect folate binding in enzyme binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Treosulfan (L-threitol-1,4-bismethanesulfonate) is an alkylating agent with routine clinical application in the treatment of ovarian cancer. In this murine study we show that this drug also has the ability to deplete primitive hematopoietic stem cells in a dose-dependent manner as determined by the cobblestone area-forming cell assay and is similar to its parent compound busulfan. Because busulfan is frequently used as part of the conditioning regimen before stem cell transplantation, we investigated an alternative nonmyeloablative protocol in an allogeneic bone marrow transplantation model in which low-dose treosulfan was added to an immune-suppressive regimen consisting of T cell-depleting antibodies, fludarabine, and thymic irradiation. Although this treatment protocol produced minimal myelosuppression, the addition of treosulfan proved to be important for allowing stable multilineage and mixed chimerism in C57BL/6 recipients of major histocompatibility complex-mismatched B10.A bone marrow without evidence of graft-versus-host disease. Donor lymphocyte infusion performed at 10 weeks after bone marrow transplantation had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence of graft-versus-host disease. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. Thus, low-dose treosulfan may be considered as a useful component of a truly nonmyeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy.

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Summary:The polyfunctional alkylating agent thiotepa (N, NЈ, NЉ-Thiotepa (TT) has long been considered for inclusion triethylene thiophosphoramide) (TT) has now been in cliniin clinical bone marrow transplant (BMT) conditioning cal use for more than 40 years. Myelodepression is well regimens in an attempt to prevent allograft rejection recognized as the major dose-limiting toxicity and there is and leukemia relapse. These studies have been encourcurrent interest in applying escalating doses of TT in conaged by initial murine experiments showing a clear junction with autologous bone marrow transplantation improvement in allogeneic bone marrow engraftment (BMT) (eg Refs 1-3). This agent has also been considered with addition of TT to total body irradiation (TBI)for inclusion in the conditioning regimen prior to allogeneic where it was assumed that TT enhances donor-type BMT 4-7 and there is at least experimental evidence for chimerism via ablation of competing stem cells in the enhanced engraftment of donor stem cells when TT was recipient. The aim of the present study was to re-evaluadded to total body irradiation (TBI). 8 The latter finding in ate the hematological toxicity of TT among different an H-2-incompatible allogeneic BMT model in mice was stem cell subsets that included primitive cells capable attributed to the reduction of stem cell competition through of long-term repopulation and to assess how the combiadditional myeloablation and this effect has been compared nation of TT with TBI influences the development of with host stem cell toxicity afforded by another alkylating donor engraftment in both syngeneic (B6-Gpi-1 a → B6-agent, busulfan. However, it has since been appreciated that Gpi-1 b ) and H-2 compatible allogeneic (BALB.B10 → while the acute myelodepression caused by various cyto-B6) BMT models. At 24 h after TT (20 mg/kg) the femtoxic agents generally reflects depletion of committed prooral content of different stem cell subsets was detergenitor cell populations in the bone marrow, it may not mined from the frequency of transient repopulating, necessarily follow that primitive stem cells capable of longand the more primitive cobblestone area-forming, cells term repopulation are similarly affected. 9-11 The aim of the (CAFCs) growing in stroma-supported cultures. This present study was to re-address the hematological properassay showed a large TT-induced depletion (2% ties of TT with respect to survival of the different progenisurvival) of early clones developing at day 7 in culture tor and stem cell subsets in the mouse bone marrow by but survival recovered towards normal for later appearenumerating cobblestone area-forming cell (CAFC) subsets ing clones developing from more primitive CAFC subafter treatment. We then evaluated how the combination of sets. The sparing of these primitive stem cells was TT with TBI influences the magnitude of short-and longreflected as undetectable levels of donor marrow term engraftment of transplanted marrow cells using conrepopulation in recipients given TT ...

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A series of 7 human squamous carcinoma cell lines of the head and neck (HNSCC), grown in standard medium containing high folate concentrations and in "folate-conditioned" medium containing nanomolar concentrations of folates, were all found to be sensitive (IC50: less than or equal to 50 nM) in growth-inhibition studies to methotrexate (MTX) following drug exposure for 7 days. However, when MTX exposure was limited to 24 hr, only 2 out of 7 HNSCC cell lines were sensitive to MTX (IC50: less than 500 nM), 2 were moderately sensitive (IC50: 1-2 microM), and 3 exhibited inherent resistance to MTX (IC50: greater than 250 microM). In these last 3 cell lines, the mechanism of resistance was not correlated with altered membrane transport of MTX or changes in dihydrofolate reductase activity, but rather was associated with a 3-fold lower activity of intracellular folylpolyglutamate synthase (FPGS) activity compared to MTX-sensitive HNSCC cells. The 3 cell lines exhibiting inherent resistance to a short exposure to MTX, however, did not show inherent cross-resistance after exposure for 24 hr to one or more of 3 novel antifolate compounds. These compounds, which appear to be more efficiently transported and polyglutamylated than MTX, include: 10-ethyl-10-deazaaminopterin (10-EdAM), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI-198,583), and 5,10-dideazatetrahydrofolic acid (DDATHF). These results indicate that antifolate membrane transport and intracellular FPGS activity are important factors in determining sensitivity or resistance of HNSCC cells to short-term antifolate compound exposures.

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