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Under conditions of steady-state hemopoiesis, a small fraction of immature hemopoietic cells, including stem cells, circulates in peripheral blood (PB). In rhesus monkeys, a median number of 1.2 x 10(7)/l CD34+ cells was observed as opposed to a median number of 1.5 x 10(9)/l in aspirated bone marrow (BM). The concentration of circulating CD34+ cells is therefore approximately two logs less than that in BM. Since a 4-kg rhesus monkey has an estimated number of 3 x 10(10) BM cells and approximately 300 ml of blood, the fraction of CD34+ cells that circulates can be estimated at approximately 0.4% of the total pool of CD34+ cells. During hemopoietic reconstitution following a cytotoxic insult such as results from a midlethal dose of TBI, PB CD34+ cell numbers appeared to be correlated to those of BM, suggesting that PB CD34+ cells may reflect reconstitution of BM CD34+ cells. Reconstitution of BM immature cells can be accelerated by treatment with pharmacological doses of growth factors, resulting in largely expanded immature cell populations within a few weeks after TBI. Growth factors observed to exert such an effect included, notably, thrombopoietin. Such an acceleration can be monitored by daily assessment of circulating CD34+ cells. Expansion of immature circulating cells indicates expansion of similar cells in the bone marrow rather than growth factor-induced selective mobilization of immature cells.

Thrombopoietin (TPO) was evaluated for efficacy in a placebo-controlled study in rhesus monkeys with concurrent administration of either granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF, (G-CSF). Rhesus monkeys were subjected to 5 Gy total-body irradiation (TBI), resulting in 3 weeks of profound pancytopenia, and received either TPO 5 microg/kg intravenously (I.V.) at day 1 (n = 4), GM-CSF 25 microg/kg subcutaneously (S.C.) for 14 days (n = 4), TPO and GM-CSF (n = 4), G-CSF 10 microg/kg/d S.C. for 14 days (n = 3), TPO and G-CSF (n = 4), or placebo (carrier, n = 4; historical controls, n = 8). Single-dose I.V. treatment with TPO 1 day after TBI effectively counteracted the need for thrombocyte transfusions (provided whenever thrombocyte levels were <40 x 10(9)/L) and accelerated platelet reconstitution to normal levels 2 weeks earlier than placebo controls. TPO/GM-CSF was more effective than single-dose TPO alone in stimulating thrombocyte regeneration, with a less profound nadir and a further accelerated recovery to normal thrombocyte counts, as well as a slight overshoot to supranormal levels of thrombocytes. Monkeys treated with TPO/GM-CSF uniformly did not require thrombocyte transfusions, whereas those treated with GM-CSF alone needed two to three transfusions, similar to the placebo-treated monkeys, which required, on average, three transfusions. Also, reticulocyte production was stimulated by TPO and further augmented in monkeys treated with TPO/GM-CSF. TPO alone did not stimulate neutrophil regeneration, whereas GM-CSF shortened the period of neutrophil counts less than 0.5 x 10(9)/L by approximately 1 week; TPO/GM-CSF treatment elevated the neutrophil nadir, but did not further accelerate recovery to normal values. TPO also augemented the neturophil response to G-CSF, resulting in similar patterns of reconstitution following TPO/G-CSF and TPO/GM-CSF treatment. TPO/GM-CSF resulted in significantly increased reconstitution of CD34+ bone marrow cells and progenitor cells such as GM-CFU and BFU-E. Adverse effects of combining TPO with the CSFs were not observed. It is concluded that (1) a single I.V. administration of TPO is sufficient to prevent severe thrombocytopenia following myelosuppression, (2) TPO/G-CSF and TPO/GM-CSF treatment result in distinct response patterns, with TPO/GM-CSF being superior to TPO/G-CSF in stimulating thrombocyte and erythrocyte recovery while being equivalent in stimulating neutrophil recovery; and (3) TPO significantly improves the performance of CSFs in alleviating severe neutropenia.

The efficacy of recombinant human thrombopoietin (TPO) and recombinant human granulocyte colony stimulating factor (G-CSF) in stimulating platelet and neutrophil recovery was evaluated in a placebo-controlled study involving transplantation of limited numbers (1-3 x 10(4)/kg) of highly purified autologous stem cells (CD34++/RhLA-DR[dull]) into rhesus monkeys after the animals were subjected to 8 Gy of total body irradiation (TBI) (x-rays). The grafts shortened profound TBI-induced pancytopenia from 5 to 6 weeks to 3 weeks. Daily subcutaneous (sc) injection of TPO (10 microg/kg/day, days 1-21 after TBI) did not stimulate platelet regeneration after transplantation either alone or in combination with G-CSF (5 microg/kg/day sc, days 1-21 after TBI). G-CSF treatment failed to prevent neutropenia in the monkeys and did not stimulate recovery to normal neutrophil levels. Simultaneous administration of TPO and G-CSF did not influence the observed recovery patterns. To test the hypothesis that the limited number of cells transplanted or the subset chosen was responsible for the lack of effectiveness of TPO, three additional monkeys were transplanted with 10(7)/kg unfractionated autologous bone marrow cells. Two of these animals received TPO and the other served as a control. In this setting, as well, TPO treatment did not prevent thrombocytopenia. This study demonstrates that treatment with TPO does not accelerate platelet reconstitution from transplanted stem cells after high-dose TBI. These findings contrast with the rapid TPO-stimulated platelet recovery in myelosuppression induced by 5 Gy of TBI in rhesus monkeys; we conclude from this that the clinical effectiveness of the TPO response depends on the availability of TPO target cells in the first week after TBI, that is, before endogenous TPO levels reach the saturation point. In addition, protracted isolated thrombocytopenia was observed in two G-CSF-treated monkeys, one of which also received TPO. Furthermore, TPO treatment for 7 days in the 6th week after TBI during severe thrombocytopenia in one monkey produced prompt clinical improvement and an increase in platelet counts.

Simultaneous treatment with human thrombopoietin (TPO) and granulocyte colony-stimulating factor (G-CSF) was evaluated in a placebo-controlled rhesus monkey study using 5 Gy total body irradiation (TBI) to induce 3 weeks of pancytopenia. Daily administration of TPO (10 microg/kg/day injected subcutaneously [sc] days 1-21 after TBI) promoted platelet and reticulocyte recovery, resulting in less profound nadirs and a rapid recovery to normal levels. Platelet transfusions were not required in these animals, in contrast to controls, and hemoglobin levels stabilized rapidly. TPO treatment did not influence neutrophil counts. G-CSF (5 microg/kg/day sc days 1-21) stimulated neutrophil regeneration and had no effect on platelet levels. Simultaneous treatment with TPO and G-CSF was as effective as treatment with TPO alone in preventing thrombocytopenia, although with the former regimen platelet levels did not rise to the supranormal levels seen with the latter. Neutrophil recovery was greatly augmented compared with G-CSF treatment alone, resulting in a less profound nadir and a recovery that started much earlier, as did monocyte, CD11b+, CD16+, and CD56+ cell reconstitution. In addition, TPO strongly promoted the recovery of bone marrow cellularity and granulocyte/macrophage and erythroid progenitor cells: The number of bone marrow CD34+ cells was greater by two orders of magnitude in TPO-treated animals than in controls in the second week of treatment, whereas G-CSF by itself had no influence. In the third week after TBI an elevation of LDH1 values was observed in TPO-treated monkeys concurrent with normoblastosis; both of these findings were attributed to rapid erythropoiesis. TPO had no effect on hemostasis parameters. Adverse TPO and/or G-CSF effects were not observed. This study demonstrates that simultaneous TPO and G-CSF treatment after cytoreductive treatment prevents thrombocytopenia, accelerates platelet and red cell reconstitution, alleviates neutropenia, and promotes the recovery of immature bone marrow cells. The effect on CD34+ GM progenitor cells may explain the augmented G-CSF responses in TPO-treated monkeys; it also suggests that TPO may become a key growth factor in the design of treatment regimens to accelerate both immature bone marrow and mature blood cell reconstitution after cytoreductive therapy.