Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients.

Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy.

Reduction of heat-induced haemotoxicity in a hyperthermic purging protocol of murine acute myeloid leukaemic stem cells by AcSDKP.

The tetrapeptide AcSDKP (Goralatide) is a cytokine with known inhibitory effects on cell proliferation. Many purging agents used in autologous bone marrow transplantation protocols, including hyperthermia, preferentially kill cycling cells. A pretreatment with Goralatide offers a possibility to reduce the haemotoxicity in many purging settings. The impact of Goralatide on the hyperthermic purging protocol was investigated in normal and myeloid leukaemic (SA8) murine cells. The median survival time after transplantation (i.e. leukaemia incidences) was used as an in vivo parameter to determine the effects on leukaemic cells. The hyperthermic effect on normal and leukaemic cells was also investigated in vitro using the cobblestone area-forming cell (CAFC) assay. A heat treatment of 90 min at 43 degrees C resulted in a 4-log depletion of leukaemic stem cells. For normal progenitor cells (CFU-GM) a 2-log cell kill was shown. The reduction in proliferative activity of the CFU-GM after an 8 h incubation with 10(-9) M Goralatide resulted in a decrease in the heat sensitivity of the progenitor subset to approximately a 1-log cell kill. The leukaemic precursor cells seem insensitive to Goralatide inhibition, implicating an increase in the therapeutic window of the hyperthermic purging protocol. Finally, simulated remission bone marrow (5% leukaemic blasts) was incubated with Goralatide followed by a heat treatment of 90 min at 43 degrees C. Lethally irradiated (10 Gy) mice transplanted with heat-treated remission bone marrow (10(6) normal bone marrow cells versus 5 x 10(4) leukaemic cells) died of aplasia while Goralatide-pretreated remission bone marrow could rescue the irradiated mice without revealing leukaemic engraftment. These findings confirmed the enhanced protection against hyperthermia of the normal haemopoietic subsets by Goralatide and thus increased the success of the hyperthermic purging protocol.

Increased rejection of murine allogeneic bone marrow in presensitized recipients.

The role of presensitizing murine recipients with donor spleen cells prior to T cell-depleted or -repleted H-2 compatible allogeneic bone marrow transplantation (BMT) was investigated at two different doses of total body irradiation (TBI). Recipients that were presensitized with 2 x 10(7) irradiated donor spleen cells at 1 week before a sublethal dose of 6 Gy TBI and BMT showed no evidence of donor blood chimerism while unsensitized recipients showed about 80% donor engraftment as determined by blood Gpi phenotyping. After raising the TBI dose to 9.5 Gy an increase in mortality from marrow failure was observed in presensitized animals. No significant engraftment-promoting effect of up to 2 x 10(6) T cells (20% of total marrow dose) was seen either in presensitized or unsensitized mice. It can be concluded that presensitized recipients are more susceptible to acute marrow rejection and that T cells added to the bone marrow did not influence the level of donor engraftment in these recipients.

Stem cell factor has contrasting effects in combination with 5-fluorouracil or total-body irradiation on frequencies of different hemopoietic cell subsets and engraftment of transplanted bone marrow.

The effect of stem cell factor (SCF) given at 24, 12 and 2 h before either 5-fluorouracil (5-FU) or total-body irradiation (TBI) was investigated on a range of bone marrow hemopoietic cell subsets that included primitive stem cells capable of long-term repopulation in bone marrow transplant (BMT) recipients. At 24 h after treatment, the femoral content of transient and permanent repopulating stem cell subsets was assessed from the frequency of early- and late-developing cobblestone area-forming cells (CAFCs) growing in stroma-associated cultures. At this time untreated 3 x 10(6) congenically marked donor bone marrow cells (B6-Gpi-Ia-->B6-Gpi-Ib) were transplanted and the level of erythroid engraftment was followed over 1 year. Analysis of the frequencies of CAFCs in host bone marrow after treatment with SCF demonstrated a remarkable increase in the number of early-developing CAFC subsets by about 10-fold. At the same time SCF conferred a sensitization of these subsets after treatment with 5-FU, which indicated an enhanced proliferative activity. The SCF-induced increase in the number of progenitor cells, however, was the more dominant process in the irradiated animals, resulting in less overall depletion of CAFCs. These contrasting results provide an explanation for the sensitization by SCF of 5-FU-induced lethality and its converse protection against radiation-induced lethality as reported by others. Nevertheless, the number of the more primitive CAFC subsets appearing at 28 and 35 days in culture and their sensitivity to 5-FU or radiation remained unaffected by this short SCF treatment. The number of CAFCs that remained in the bone marrow largely predicted the subsequent patterns of donor marrow engraftment in the treated BMT recipients: SCF enhanced short-term engraftment after treatment with 5-FU while it reduced the need for short-term engraftment after irradiation. Only irradiation afforded long-term engraftment through depletion of primitive host stem cells, and this was moderately improved by prior treatment with SCF.

Relationships between ablation of distinct haematopoietic cell subsets and the development of donor bone marrow engraftment following recipient pretreatment with different alkylating drugs.

A number of different alkylating chemotherapeutic agents--busulphan, dimethylbusulphan (DMB), isopropylmethane sulphonate (IMS), melphalan, cyclophosphamide (CY) and bischloroethylnitrosourea (BCNU)--were investigated for their cytotoxic effects on different haemopoietic cell populations in host mice and for their ability to induce short- and long-term engraftment of transplanted bone marrow. At 24 h after drug treatment the femoral content of transient and permanent repopulating stem cell subsets was assessed, respectively, from the frequency of early- (day 5-15) and late- (day 25-35) developing cobblestone area-forming cells (CAFCs), growing in vitro in long-term bone marrow cultures (LTBMCs). At this time a fixed complement of 10(7) congenically marked donor bone marrow cells (B6-Gpi-1a-->B6-Gpi-1b) was infused in the drug-treated mice and erythroid engraftment was followed over 36 weeks. Diverse effects on early- and late-developing CAFC frequencies were found for the different drugs; these were generally related to the pattern of donor bone marrow engraftment in treated recipients. Melphalan was more toxic to early-developing than to late-developing CAFC subsets, and the transplant only offered an early wave of blood chimerism followed by return of host cells. CY and BCNU had minimal to moderate effects on CAFC content and engraftment with no apparent preference for any particular haemopoietic cell subset. IMS also had a relatively low toxic effect on host marrow CAFC frequencies but appeared exceptional in its ability to allow for more donor-type engraftment. The dimethane sulphonate compounds busulphan and DMB were especially potent at depleting late CAFC subsets and ensured high and lasting levels of donor bone marrow engraftment. These studies support the value of CAFC measurements for predicting the fate and growth of transplanted bone marrow cells in recipients pretreated with a variety of cytotoxic agents.