Thrombocytopoietic Growth Factors Tpo Pegmgdf Il11 And Promegapoietin

Clearly, the growth factor with the greatest potential for the treatment of thrombo-cytopenia is TPO. The thrombopoietic potential of the two mpl ligands (recombinant human [rHu] TPO, the full-length glycosylated molecule; and megakaryocyte growth and development factor, a nonglycosylated, polyethylene glycol-derived and truncated form of the full-length molecule [PEG-rHuMGDF]) being evaluated in clinical trials was evident early from the consistent and substantial amount of preclinical in vitro and in vivo data in both rodent and nonhuman primate models of myelosuppression (13-18,36-41). Evaluation of the mpl ligands (PEG-rHuMGDF, rHuTPO) in nonhuman primate models of radiation-induced myelosuppression showed that it significantly lessened the degree and duration of thrombocytopenia and also improved myelopoietic and erythropoietic recovery (17,18).

i—Cytokine Administration— i a-a r-metHuG-CSF (10 |xg/kg)

i—Cytokine Administration— i

Time (day) Post Irradiation

Fig. 1. Effects of cytokine administration on peripheral blood platelets (PLT) in irradiated primates. The PLT observed in irradiated rhesus macaques after recombinant human megakaryocyte growth and development factor (rHuMGDF), pegylated (PEG)-rHuMGDF, recombinant human methylated granulocyte colony-stimulating factor (r-metHuG-CSF), PEG-rHuMGDF combined with r-metHuG-CSF, or human serum albumin (HSA) administration. Cytokines or control protein were administered subcutaneously from d 1-18 post exposure. Data represent mean ± SEM of the absolute PLT for the cytokine or HSA-treated animals. (Reprinted with permission from ref. 17.)

Farese et al. (17) first reported the therapeutic efficacy of PEG-rHuMGDF both alone and in combination with rHuG-CSF in a rhesus macaque model of severe radiation-induced myelosuppression. In that study, PEG-rHuMGDF showed an approx 10fold increase in biologic activity relative to the nonpegylated MGDF. Both forms of the truncated MGDF, however, significantly improved all platelet-related parameters indicative of enhanced thrombopoiesis: platelet nadir, duration of thrombocytopenia, recovery time to a platelet count of >20 x 109/L, and number of whole blood transfusions (Fig. 1). It was of interest that PEG-rHuMGDF, co-administered with rHuG-CSF, significantly improved all platelet-related parameters relative to PEG-rHuMGDF monotherapy while significantly enhancing neutrophil recovery relative to rHuG-CSF monotherapy. Wagemaker's group noted comparable responses when evaluating the full-length mpl ligand rHuTPO, in a similar model of sublethally irradiated rhesus macaques (18,38). In another series of studies, the truncated version of mpl ligand was evaluated as monotherapy and in combination with the high-affinity IL-3 receptor agonist called daniplestim in irradiated rhesus macaques (39). Whereas the truncated form of mpl ligand was effective in significantly enhancing recovery of all platelet parameters, the combination of daniplestim plus mpl ligand further improved the stimulatory effect of cytokine monotherapy in this model of severe, radiation-induced myelosup-pression (Fig. 2).

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Cytokine Administration

Cytokine Administration

Time (Days) Post Irradiation

Fig. 2. Effects of cytokine administration on peripheral blood platelets (PLT) in 600-cGy X-irradi-ated nonhuman primates. The PLT were observed prior to and after treatment with control protein (0.1% autologous serum [AS]), daniplestim alone, mpl ligand (Mpl-L) alone, or combined daniples-tim/Mpl-L. Cytokines or control protein were administered subcutaneously from d 1-18 post exposure. Data represent mean values (± SE). (Reprinted with permission from ref. 39. © AlphaMed Press.)

The treatment efficacy of PEG-rHuMGDF and rHuTPO was further underscored by several studies that examined the timing of growth factor administration relative to a cytotoxic insult (15,38,40). Whereas convention dictated the consecutive, daily administration of rHuG-CSF or recombinant human granulocyte colony-stimulating factor (rHuGM-CSF), it was shown that a single injection of PEG-rHuMGDF or rHuTPO at an early time (1-24 h) after irradiation is able to improve recovery from thrombocytopenia effectively in a manner equivalent to conventional daily administration of either mpl ligand (16,41). Shibuya et al. (15) showed that a single injection of PEG-rHuMGDF, 1 h after sublethal irradiation of mice, effectively improved multilineage cell recovery in addition to marrow-derived, committed, and primitive hematopoietic progenitors. Neelis et al. (38) further defined the clinical potential of rHuTPO by investigating the efficacy of single-dose administration with co-administration of either rHuG-CSF or rHuGM-CSF in sublethally irradiated rhesus macaques (Fig. 3). As previously noted, there were no adverse effects of the co-administered cytokines, and a single administration of rHuTPO within hours of irradiation was sufficient to prevent thrombocytopenia (15-18,38). Additionally, the single administration of rHuTPO significantly improved the performance of rHuG-CSF and rHuGM-CSF in alleviating severe neutropenia (38). Remarkably, the single, low dose of rHuTPO or PEG-rHuMGDF administered within 24 h of exposure was as effective as conventional daily administration of the cytokine for 14-21 d in stimulating thrombopoiesis and recovery of platelets.

Fig. 3. Thrombocyte counts after 5 Gy TBI (d 0) for monkeys treated with TPO 5 |ig (filled squares, n = 4) TPO 50 |g (open squares), TPO 0.5 |g (circles, n = 2), and concurrent placebo treatment (triangles, n = 4). The lower shaded area represents the mean (± SD) of four monkeys treated with human TPO for 21 d after irradiation. Data represent the arithmetic mean (± SD) of the various treatment groups. The horizontal line defines the level of thrombocytopenia (40 X 109/L) below which thrombocyte transfusions are given. (Reprinted with permission from ref. 38. © American Society of Hematology.)

The magnitude of the thrombocytopoietic response was significantly affected by the time interval between PEG-rHuMGDF and radiation exposure. Delaying the initiation of PEG-rHuMGDF treatment remarkably reduced its therapeutic effects. A single injection of PEG-rHuMGDF at 60 h after irradiation of mice reduced its effect on platelet recovery to that of the control cohort (15), whereas the initiation of consecutive daily injections of PEG-rHuMGDF 5 d after irradiation in rhesus macaques improved platelet recovery relative to control-treated cohorts but diminished its effect from that noted with initiation of treatment on d 1 (41). It is apparent that treatment, to be effective, must be initiated early (within 24 h) after the cytotoxic insult. These results are consistent with studies demonstrating that treatment efficacy of rHuG-CSF, pegfilgras-tim, IL-6, or IL-11 is most effective when initiated early after irradiation (42-45). These data suggested that although abbreviated single or every-other-day administration schedules may be effective, PEG-rHuMGDF or rHuTPO must be initiated early after a cytotoxic insult.

A singular disappointment in the preclinical development of the mpl ligands was their lack of therapeutic efficacy when administered subsequent to autologous bone marrow transplantation (AuBMT) in nonhuman primates or canines. Neither rHuTPO nor PEG-rHuMGDF, administered daily at a standard dose (2.5-10 |g/kg/d) for several weeks after transplant, prevented thrombocytopenia consequent to myelosuppressive conditioning for AuBMT, nor did it stimulate regeneration of platelets to normal values (Fig. 4) (46-49). These results are consistent with the clinical trial experience (50-54). Conventional doses and schedule of administration of rHuTPO or PEG-rHuMGDF were ineffective in demonstrating meaningful benefit after stem cell transplantation. The preclinical and clinical experience suggests that control of cytotoxic therapy-associated thrombocytopenia would be dependent on defining optimal dose and schedule for specific myelotoxic regimens. Based on mathematical modeling of PEG-rHuMGDF

Fig. 3. Thrombocyte counts after 5 Gy TBI (d 0) for monkeys treated with TPO 5 |ig (filled squares, n = 4) TPO 50 |g (open squares), TPO 0.5 |g (circles, n = 2), and concurrent placebo treatment (triangles, n = 4). The lower shaded area represents the mean (± SD) of four monkeys treated with human TPO for 21 d after irradiation. Data represent the arithmetic mean (± SD) of the various treatment groups. The horizontal line defines the level of thrombocytopenia (40 X 109/L) below which thrombocyte transfusions are given. (Reprinted with permission from ref. 38. © American Society of Hematology.)

Fig. 4. Comparison of the effect of TPO after transplantation of purified stem cells and unfraction-ated bone marrow. In the upper panel the mean thrombocyte counts of four TPO-treated stem cell-transplanted monkeys (closed squares) and three placebo-treated stem cell-transplanted monkeys (closed circles) are plotted. The lower panel displays the mean of two TPO-treated unfractionated marrow-transplanted monkeys (half-closed squares) and the unfractionated marrow-transplanted control monkey (closed triangles). The dose and route of TPO were the same for all monkeys (10 pg/kg/d, sc, d 1-21). The shaded areas represent means ± SDs of thrombocyte levels of 5-Gy irradiated monkeys after TBI: the lower shaded area represents eight placebo-treated controls, and the upper one four TPO-treated monkeys. (Reprinted with permission from ref. 46. © 1997, with permission from Elsevier.)

Fig. 4. Comparison of the effect of TPO after transplantation of purified stem cells and unfraction-ated bone marrow. In the upper panel the mean thrombocyte counts of four TPO-treated stem cell-transplanted monkeys (closed squares) and three placebo-treated stem cell-transplanted monkeys (closed circles) are plotted. The lower panel displays the mean of two TPO-treated unfractionated marrow-transplanted monkeys (half-closed squares) and the unfractionated marrow-transplanted control monkey (closed triangles). The dose and route of TPO were the same for all monkeys (10 pg/kg/d, sc, d 1-21). The shaded areas represent means ± SDs of thrombocyte levels of 5-Gy irradiated monkeys after TBI: the lower shaded area represents eight placebo-treated controls, and the upper one four TPO-treated monkeys. (Reprinted with permission from ref. 46. © 1997, with permission from Elsevier.)

pharmacokinetics and pharmacodynamics, Farese et al. examined the relative efficacy of administering PEG-rHuMGDF in several regimens to rhesus macaques receiving AuBMT (47,55). Conventional, consecutive, daily administration of PEG-rHuMGDF initiated within 24 h of AuBMT failed to enhance platelet recovery. PEG-rHuMGDF when administered either before (d -9 to d -5), or after (daily injections initiated on d 1 after AuBMT) or in an abbreviated schedule with high doses administered at d 1 and d 3 after AuBMT, significantly lessened the duration of thrombocytopenia (Fig. 5). These data confirmed predictions from pharmacokinetic/pharmacodynamic modeling that thrombocytopenia can be prevented after AuBMT using the appropriate dose and schedule of PEG-rHuMGDF.

The preclinical pharmacology associated with rHuIL-11 administration to rodent and nonhuman primate models of modest, chemotherapy-induced thrombocytopenia showed consistent stimulation of platelet recovery relative to control cohorts

Fig. 5. Effect of PEG-rHuMGDF on platelet (PLT) recovery after autologous bone marrow transplantation (AuBMT). Mean peripheral PLT X 103/|L (± SEM) in rhesus monkeys after myeloablation and AuBMT with either no cytokine treatment (AuBMT, n = 9) or pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) administration before and after transplant (2.5 |g/kg/d, from d -9 to d -5 and d 1 post AuBMT until platelet counts reached 200,000/|L (PEG-rHuMGDF pre and post AuBMT, n = 4) or high-dose PEG-rHuMGDF administration (300 |g/kg/d) on d 1 and d 3 only post AuBMT (high-dose PEG-rHuMGDF post AuBMT, n = 5). (Reprinted with permission from ref. 47. © AlphaMed Press.)

Fig. 5. Effect of PEG-rHuMGDF on platelet (PLT) recovery after autologous bone marrow transplantation (AuBMT). Mean peripheral PLT X 103/|L (± SEM) in rhesus monkeys after myeloablation and AuBMT with either no cytokine treatment (AuBMT, n = 9) or pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) administration before and after transplant (2.5 |g/kg/d, from d -9 to d -5 and d 1 post AuBMT until platelet counts reached 200,000/|L (PEG-rHuMGDF pre and post AuBMT, n = 4) or high-dose PEG-rHuMGDF administration (300 |g/kg/d) on d 1 and d 3 only post AuBMT (high-dose PEG-rHuMGDF post AuBMT, n = 5). (Reprinted with permission from ref. 47. © AlphaMed Press.)

(42,56-58). In carboplatin-treated rhesus macaques, rHuIL-11 administered within 24 h after cessation of chemotherapy significantly reduced the depth of the platelet nadir and significantly accelerated platelet recovery compared with the control-treated group (58). Mean platelet counts were <50 x 109/L for 2 d relative to 5.5 d for the rHuIL-11 and control-treated cohorts, respectively. An additional study in cynomolgous macaques subjected to nimustine (ACNU)-induced thrombocytopenia confirmed that rHuIL-11 therapy initiated within 24 h of ACNU and continued for 21 d could successfully improve platelet nadir and recovery time compared with the control cohort (57). Leonard et al. (42) showed that the increased platelet recovery was markedly affected by the interval between chemotherapy and rHuIL-11 administration. Only when rHuIL-11 was injected within 24 h after chemotherapy were the platelet-related parameters improved. Longer delays in platelet recovery were noted with increasing intervals between chemotherapy and rHuIL-11 administration. Furthermore, the most effective administration schedule required 14 d of consecutive administration. A more modest effect of IL-11 therapy was observed after treatment of sublethally irradiated canines (59). Irradiated dogs were treated with rHuIL-11 starting within 2 h of irradiation and continued for 28 d. The rHuIL-11-treated cohort had decreased platelet counts (<150 x 109/L) for a median of 24 d, compared with 28 d for the control group. Although there was a modest trend toward accelerated recovery, the results were not significant.

Collins et al. (60), in an interesting abstract report, described the combination of rHuIL-11 with recombinant murine TPO (rMuTPO) in a murine model of carboplatin/ irradiation-induced myelosuppression. Mice were treated with rHuIL-11 for 10 consecutive days, or rMuTPO every other day, for 10 d as monotherapy or the combination of rHuIL-11 and rMuTPO in their respective regimens. Although the respective monotherapy improved both platelet nadir and recovery, co-administration significantly improved the platelet parameters and completely abrogated the carboplatin/irradiation-induced thrombocytopenia. The combined rHuIL-11/rMuTPO treatment prevented severe anemia and stimulated a marked increase in marrow-derived megakaryocyte progenitors. These data suggested that combined IL-11 and TPO, administered in the schedule described, can significantly improve platelet recovery after myelosuppression. rHuIL-3 has also been combined with rHuIL-11 in a demonstration of hematopoietic synergy in rodent models of severe myelosuppression or bone marrow transplantation (61). The combined therapy enhanced platelet recovery and increased survival of lethally irradiated mice. The pharmacology associated with IL-11 administration suggested that in myelosuppressed patients, treatment with rHuIL-11 must be initiated within 24 h of chemotherapy and maintained for an adequate duration for optimal efficacy in preventing severe thrombocytopenia.

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