The classical definition of lymphohematopoietic stem cells (LHSC), the most primitive progenitors of all blood cells, requires that they have the capacity for self-renewal and for the long-term production of all blood cell lineages. However, other characteristics of LHSC have been debated. Our previous data suggested that mouse LHSC are very slowly proliferating cells that generate delayed multilineage engraftment, while “radioprotection” (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) results from more committed progenitors. Alternatively, some groups have reported that mouse LHSC are responsible for both radioprotection and long-term repopulation of all blood cell lineages. A possible explanation for this difference is that cells with the capacity for long-term production of all blood cell lineages are biologically heterogeneous. We now show that 10 LHSC can generate all blood cell lineages for the lifetime of the animal. However, these cells lacked radioprotection and spleen colony-forming activity. LHSC were identified and isolated by their small size, their lack of expression of antigens characteristic of mature blood cell lineages, and their high expression of aldehyde dehydrogenase. In addition, these cells were found to express undetectable or low levels of many antigens presumed to mark LHSC, including Thy-1, Ly-6A/E (Sca-1), c-kit, and CD34. There appears to be at least two classes of LHSC with the capacity for long-term production of all blood cell lineages: one that generates both radioprotection and long-term engraftment and one that produces delayed but durable engraftment. Our data suggest that this latter class may represent a very primitive class of LHSC.

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The chemistry of the bifunctional alkylating agent phosphoramide mustard and model phosphoramides was probed by multinuclear NMR spectroscopy as a function of pH. Between pH 1 and 11, both the 31P and 15N resonances for phosphoramide mustard displayed a single monobasic titration curve with a pKa of 4.9. The protonation below pH 4.9 correlates with the loss in reactivity of the mustard. The 17O NMR spectrum of 17O-enriched phosphoramide mustard shows little change with pH. The data on the mustard was compared to 15N and 31P NMR data on 15N-enriched phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide. Contrary to the conclusions of previous studies, our combined 31P, 15N, and 17O NMR results are more consistent with N-protonation of phosphoramide mustard rather than an O-protonation. Theoretical calculations on the phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide show O-protonation to be more stable in the gas phase. For the latter two compounds, the calculations suggest that N-protonation may be the most stable protonated form in the aqueous phase. These findings influence our understanding of the structure-activity relationships of phosphoramide mustards.

Most malignant gliomas grow despite treatment by standard chemotherapeutic agents. The authors explored the use of an innovative drug, 4-hydroperoxycyclophosphamide (4HC), delivered via a controlled-release biodegradable polymer to determine whether local delivery would enhance efficacy. This drug is an alkylator-type chemotherapeutic agent derived from cyclophosphamide. Unlike the parent drug, which requires activation by hepatic microsomes, 4HC is active in vitro. Two rat glioma cell lines, 9L and F98, were treated in cell culture with medium containing 4HC. Both cell lines were more sensitive to 4HC than to a nitrosourea, BCNU, an agent of established value in the local therapy of gliomas. Ninety Fischer 344 rats implanted with 9L or F98 gliomas were treated with an intracranial polymer implant containing 0% to 50% loaded 4HC in the polymer, and it was found that 20% 4HC-loaded polymers caused minimum local brain toxicity and maximum survival. These polymers were then used to compare the in vivo efficacy of 4HC to BCNU in rats implanted with 9L glioma. Animals with brain tumors treated with 4HC had a median survival span of 77 days compared to the median survival of 21 days in BCNU-treated animals and median survival of 14 days in untreated animals. Long-term survival for more than 80 days was 40% in the 4HC-treated rats versus 30% in the BCNU-treated rats. The polymer carrier used in this study was a copolyanhydride of dimer erucic acid and sebacic acid 1:1, which was able to maintain the hydrolytically unstable 4HC in a stable state for local delivery. Thus, it is concluded that 4HC-impregnated polymers provide an effective and safe local treatment for rat glioma.

The alkylating agent isophosphoramide mustard (IPM) spontaneously forms a relatively stable aziridine derivative which can be directly observed using NMR spectroscopy. The protonations of IMP and its aziridine were probed using 1H, 31P, 15N, and 17O NMR spectroscopy. The positions of the 31P, 15N, and 17O resonances of IPM between pH 2 and 10 each exhibit a single monobasic titration curve with the same pKa of 4.31 +/- 0.02. On the basis of a comparison with other compounds and our earlier work with phosphoramide mustard, the NMR results for IPM indicate that protonation occurs at nitrogen and not oxygen. Over this same pH range, each of the 1H, 31P, and 15N resonances of IPM-aziridine also show a single monobasic titration with a pKa of 5.30 +/- 0.09. The magnitude of the change in chemical shifts suggests that the protonation of the IPM-aziridine occurs at the ring nitrogen. Theoretical gas-phase calculations of PM, IPM, and IPM-aziridine suggest O-protonation to be more likely; however, aqueous phase calculations predict the N-protonated forms to be most stable. Furthermore, for PM and IPM-aziridine, which contain nonequivalent nitrogens, the theoretical calculations and experimental data both agree as to which nitrogen undergoes protonation. These results suggest that the IMP-aziridine remains unprotonated under physiological conditions and may, in part, explain the lower alkylating activity of IPM as compared to PM.