Experimental Hematology 2015;43:479–487 Romiplostim promotes recovery in a mouse model of multicycle chemotherapy-induced Patricia L. McElroya, Ping Weia, Keri Bucka, Angus M. Sinclaira, Michael Eschenbergb, Barbra Sasua, and Graham Molineuxa aOncology Research Department, Inc., Thousand Oaks, CA, USA; bMedical Sciences Biostatistics Department, Amgen Inc., Thousand Oaks, CA, USA

(Received 23 April 2014; revised 20 February 2015; accepted 25 February 2015)

Chemotherapy-induced thrombocytopenia can lead to chemotherapy treatment delays or dose reductions. The ability of romiplostim, a (TPO) mimetic, to promote platelet re- covery in a mouse model of multicycle chemotherapy/radiation therapy (CRT)-induced throm- bocytopenia was examined. In humans, an inverse relationship between platelet counts and endogenous TPO (eTPO) concentration exists. In a CRT mouse model, eTPO was not elevated during the first 5 days after CRT treatment (the ‘‘eTPO gap’’), then increased to a peak 10 days after each CRT treatment in an inverse relationship to platelet counts seen in humans. To bridge the eTPO gap, mice were treated with 10–1,000 mg/kg of romiplostim on day 0, 1, or 2 after CRT. In some mice, the romiplostim dose was approximately divided over 3 days. Platelet recovery occurred faster with romiplostim in most conditions tested. Romiplostim doses of $100 mg/kg given on day 0 significantly lessened the platelet nadir. Fractionating the dose over 3 days did not appear to confer a large advantage. These data may provide a rationale for clinical studies of romiplostim in chemotherapy-induced thrombocytopenia. Copyright Ó 2015 ISEH - Inter- national Society for Experimental Hematology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Chemotherapy-induced thrombocytopenia (CIT) is an un- izing antibodies [18]. These findings led to the development met medical need that can lead to delays or reductions in of alternative therapies, including romiplostim. scheduled chemotherapy treatment, which are associated Romiplostim is a TPO-receptor agonist approved for the with poorer outcomes for cancer patients [1,2]. Severe treatment of thrombocytopenia in patients with chronic im- thrombocytopenia occurs in approximately 9% of chemo- mune thrombocytopenia. It is the first United States Food therapy cycles and can necessitate platelet transfusions and Drug Administration–approved member of a new class [1,2]. Recombinant thrombopoietin (TPO) reversed throm- of therapeutics called peptibodies, which are peptides conju- bocytopenia and increased survival in animal models of gated to human Fc and resemble antibodies [19]. Since CIT and chemotherapy/radiation therapy (CRT)-induced chemotherapy-supportive treatments may not be initiated un- thrombocytopenia [3–11]. Specifically, certain dose and til the second or subsequent cycles of chemotherapy, after a timing schedules were evaluated, and it was found that a patient has been identified as being at risk for developing single TPO dose given close to the time of total body irra- thrombocytopenia, we developed a multicycle mouse model diation resulted in better recovery [3,6,7]. Early clinical and administered romiplostim after the first treatment cycle. studies have demonstrated proof of concept for recombi- Using this model, we sought to understand the optimum dose nant TPO/TPO-receptor agonists [12–17]. However, clin- and timing of romiplostim administration that would pro- ical studies using recombinant TPO were halted when mote platelet recovery. A CRT mouse model was used some patients treated with PEGylated recombinant human because mice receiving single chemotherapy agents alone growth and development factor (PEG- did not develop thrombocytopenia severe enough that the ef- rHuMGDF), a truncated form of TPO, developed neutral- fects of adding romiplostim would be easily detectable. Pre- vious studies established that there is an inverse relationship between endogenous TPO (eTPO) concentrations and Offprint requests to: Ms. Patricia L. McElroy, Oncology Discovery platelet counts in patients undergoing chemotherapy Research, Amgen Inc., One Amgen Center Drive, MS 15-2A, Thousand Oaks, CA 91320; E-mail: [email protected] [20,21]. We hypothesized that understanding the relationship

0301-472X/Copyright Ó 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.exphem.2015.02.004 480 P.L. McElroy et al./ Experimental Hematology 2015;43:479–487 between platelet counts and eTPO concentrations in a mouse tion was adapted from work done by Hokom et al. [5], who model of multicycle CRT could inform a rationale for pro- showed that the combination produced a profound thrombocyto- phylactic romiplostim treatment. penia; doses were modified in these studies to make repeated treat- ment feasible. Romiplostim was administered subcutaneously 2, 24, or 48 hours following radiation, in cycle two, cycle three, or both cycles. Romiplostim was not administered in the first cycle Materials and methods to mirror the clinical situation. Untreated or saline-treated mice Mice served as positive controls, and CRT-treated mice not receiving ro- Female B6D2F1 (BDF1) mice, aged 11 to 15 weeks, were used and miplostim served as negative controls. Blood samples were cared for in accordance with the Guide for the Care and Use of Lab- collected by cardiac puncture immediately after euthanasia or oratory Animals, 8th Edition [22]. Animals were group housed at an via the retro-orbital sinus while mice were anesthetized. Association for Assessment and Accreditation of Laboratory Animal Due to the myelosuppressive nature of CRT, developed Care International–accredited facility in sterile, ventilated microiso- in addition to thrombocytopenia. To avoid severely compromising lator housing. All research protocols were approved by the Institu- individual mice by repeated blood sampling, a rotating cohort tional Animal Care and Use Committee. Animals had ad libitum design was used (except for experiment one, in which mice access to pelleted feed, food supplements, and water; were main- were euthanized at each time point). There were 25 mice to a tained on a 12:12 hour :dark cycle; and had access to enrichment group (except for experiment one, which had 75 mice per treat- opportunities. All animals were determined specific pathogen free. ment group to support terminal time points), and results are pre- sented as averages of five mice per time point. Each mouse was Study design bled twice, once early in the cycle and a second time late in the Mice were treated with two or three cycles of CRT (experimental cycle, and was euthanized immediately following the second designs depicted in Fig. 1). Each cycle was 28 days long. For the sampling. first cycle, mice were injected intraperitoneally with 62.5 mg/kg carboplatin on day 0. Four hours after carboplatin treatment, the Analyte measurements mice were exposed to 5 Gy total body radiation from a 137Cs For the experiment comparing eTPO concentrations to platelet source (Gamma Cell 40 Irradiator, Atomic Energy of Canada counts, five mice per group were euthanized by CO2 inhalation Ltd, Chalk River, Ontario, Canada). For cycles two and three, at each time point, and we collected blood by cardiac puncture the carboplatin dose was 56.25 mg/kg, and the radiation dose for complete blood counts (CBCs) and serum eTPO analy- was 4.5 Gy. This multicycle regimen of carboplatin plus irradia- sis. Serum was analyzed for eTPO concentrations using the

Figure 1. Summary of study design. The days and doses of romiplostim administration in seven different experiments are shown here. (Color illustration of figure appears online.) P.L. McElroy et al./ Experimental Hematology 2015;43:479–487 481

Quantikine Mouse TPO Immunoassay (R&D Systems, Minne- apolis, MN). We analyzed CBCs using an automated blood analyzer (Advia 2120, Siemens, Munich, Germany). For experi- ments in which platelet response to romiplostim treatment was monitored, mice were anesthetized with isoflurane, and blood was collected for CBC from five mice per group per time point via the retro-orbital sinus using a heparinized capillary tube.

Statistical analysis An evaluation of treatment effect was made on each study day. An analysis of variance (ANOVA) was performed on the log- transformed platelet counts, with treatment used as a fixed effect in the model. Each treatment group was compared with the group sub- jected to CRT alone, and a Dunnett’s post-hoc test was employed to account for the multiple comparisons. We used SAS version 9.1 (SAS Institute, Cary, NC) software on a Windows Vista (Microsoft, Redmond, WA) operating system for these analyses. Additional eval- uations of treatment effect across CRT with romiplostim-treated groups on other blood cell types (e.g., reticulocytes) were made on each study day using an ANOVA. Since these additional analyses were performed on secondary endpoints, the Bonferroni post-hoc test was performed as a more conservative method to account for the multiple comparisons across the CRT with romiplostim-treated groups. Graphpad Prism 5 (GraphPad Software, Inc., La Jolia, CA) was used for the analysis of these secondary endpoints. Results are presented as means 6 standard error of the mean.

Results

An inverse relationship between platelet counts and Figure 2. Platelet counts demonstrated an inverse relationship with eTPO concentrations during three cycles of CRT-induced thrombocytopenia in endogenous thrombopoietin concentration was observed mice. (A) Platelet counts in mice treated with CRT decreased in each cy- during three successive cycles of chemotherapy/ cle. were measured in saline-treated mice only in the first cycle. radiation therapy (B) Overlay of platelet counts and eTPO concentrations. Data are the The relationship between platelet counts and eTPO concentra- means of five mice per time point, and error bars represent the standard tion was examined during three cycles of CRTin mice to ascer- error of the mean. (Color illustration of figure appears online.) tain when eTPO concentrations were suboptimal and administration of romiplostim might be efficacious. Mice in promoting platelet recovery. This window was targeted for received either CRT treatment or subcutaneous saline as a con- treatment. trol. Data from saline-treated mice were collected in cycle one only. Platelet number decreased after each CRTadministration and gradually recovered to near-baseline levels at the end of Romiplostim administration in the second cycle of each cycle (Fig. 2A). After each administration of CRT, chemotherapy/radiation therapy accelerated platelet eTPO concentration remained at a steady level for approxi- recovery mately 6 days, after which it began to increase until reaching Following the theory that the first 5 days of a CRT cycle a peak on day 10 (Fig. 2B). This eTPO peak corresponded constituted the ideal point for intervention, we examined with the nadir in platelet counts, and as platelet counts gradu- the efficacy of romiplostim in promoting platelet recovery ally recovered, eTPO also decreased to approximately base- in the second cycle of CRT, the time at which, in a clinical line concentration. Endogenous TPO concentration was situation, the need for supportive care would have been lower at the beginning of each successive CRT cycle than in identified. Mice were treated in the second cycle with 10, the previous cycle, and the amount of time required for platelet 30, or 100 mg/kg of romiplostim on day 0, 1, or 2, and counts to recover to baseline was progressively longer. These platelet counts were measured throughout the cycle. In gen- results demonstrated an inverse relationship between platelet eral, romiplostim treatment caused a significant increase in number and eTPO concentration during three cycles of CRT. the rate of platelet recovery after CRT, especially at higher Moreover, there appeared to be a window of time early after doses given on day 0 or day 1 (Fig. 3). Administration CRT administration, before eTPO concentration increased, of romiplostim on day 0 and day 1 significantly lessened during which administration of romiplostim might be effective the platelet nadir (sixfold decrease in nadir for day 482 P.L. McElroy et al./ Experimental Hematology 2015;43:479–487

Figure 3. Treatment with romiplostim in the second cycle accelerated platelet recovery compared with CRT alone. Mice were treated with 10, 30, or 100 mg/ kg of romiplostim on (A) day 0, (B) day 1, or (C) day 2 of the second CRT cycle. Untreated mice were included to show baseline platelet values. Mice with CRT treatment alone were used as a negative control. The data shown are from two different experiments; panel A represents data from one experiment, and panels B and C show the data from a separate experiment, hence the differences seen in the CRT-alone control data. Platelet counts were measured on the indicated days (n 5 5 mice per group, per time point). Error bars represent the standard error of the mean. The symbols above the lines indicate days of statistically significant differences between the following treatment groups and CRT alone: o 5 100 mg/kg; x 5 30 mg/kg; þ 5 10 mg/kg (p ! 0.05, two-way ANOVA with Dunnett’s post-hoc test). (Color illustration of figure appears online.)

0 administration and twofold decrease for day 1 administra- Of note, the platelet nadir generally occurred later with tion for the 100-mg/kg-dose group). During the recovery CRT alone as compared with CRT þ romiplostim. The phase (i.e., the period of time after the platelet nadir), romi- effectiveness of romiplostim appeared to be dose depen- plostim treatment resulted in a dose-dependent increase in dent; mice treated with 100 mg/kg of romiplostim exhibited mean platelet counts compared with CRT alone, reaching more days of significantly higher platelet counts (compared statistical significance at several time points for all admin- with CRT alone) than mice treated with lower doses. istration schedules tested. Taken together, these data indi- Similar to the results in cycle two, it was observed that cate that higher doses of romiplostim administered closer day 0 administration in the second and third cycles lessened to the time of CRT were most effective in preventing the the platelet nadir in cycle three. Romiplostim administered severe decrease in platelets caused by CRT and in acceler- on days 1 or 2 also resulted in increased platelet counts ating platelet recovery. compared with CRT alone but with fewer days of signifi- cant difference from CRT alone than day 0 administration, Responses to romiplostim administration in the third and neither day 1 nor day 2 treatment lessened the platelet cycle of chemotherapy/radiation therapy were similar to nadir compared with CRT alone (Figs. 4B and 4C). those seen in the second cycle We next examined whether treatment with 10, 30, or Higher doses of romiplostim improved platelet recovery 100 mg/kg of romiplostim in both the second and third compared with chemotherapy/radiation therapy alone cycles of CRT on days 0, 1, or 2 resulted in higher platelet To determine if doses of romiplostim that were higher than counts in the third cycle. Administration of romiplostim on previously tested might further accelerate platelet recovery, day 0 resulted in the greatest improvement, with all doses mice were treated with 100, 300, or 1,000 mg/kg of romi- administered on this day resulting in significantly increased plostim on day 0 of cycle two or treated in both cycles platelet counts at several time points compared with CRT two and three. In both these situations, higher doses of alone (Fig. 4A). The exception to this was on day 6, at romiplostim resulted in improved platelet recovery which point platelet counts were higher with CRT alone. compared with CRT alone (Figs. 5A and 5B). When given P.L. McElroy et al./ Experimental Hematology 2015;43:479–487 483

Figure 4. Treatment with romiplostim accelerated third-cycle platelet recovery compared with CRT alone. Mice were treated with 10, 30, or 100 mg/kg of romiplostim on (A) day 0, (B) day 1, or (C) day 2 of the second and third CRT cycles. Untreated mice were included to show baseline platelet values. Mice with CRT treatment alone were used as a negative control. The data shown are from two different experiments; panel (A) represents data from one exper- iment, and panels (B) and (C) show the data from a separate experiment, hence the differences seen in the CRT-alone control data. Platelet counts were measured in the third cycle (n 5 5 mice per group, per time point). Error bars represent the standard error of the mean. The symbols above the lines indicate days of statistically significant differences between the following treatment groups and CRT alone: o 5 100 mg/kg; x 5 30 mg/kg; þ 5 10 mg/kg (p ! 0.05, two-way ANOVA with Dunnett’s post-hoc test). (Color illustration of figure appears online.) in cycle two only, 300 mg/kg was significantly better than effect on reticulocyte counts was observed in mice lower doses but was not different from 1,000 mg/kg in pro- receiving CRT and treated with romiplostim on day 0 of moting second-cycle platelet recovery. When given in cycles cycle three (Fig. 6A). An early decrease in reticulocytes two and three, increasing the dose above 100 mg/kg did not was observed followed by increases on days 12 and 16 in result in significant improvement to cycle-three platelet mice treated with 100 mg/kg of romiplostim. Romiplostim recovery, based on overall platelet counts as measured by treatment prevented the severe drop in hemoglobin in the repeated measures analysis of variance (data not shown). same animals, with higher doses closer to the administra- tion of CRT generally providing greater protection Dividing the dose of romiplostim over 3 consecutive (Fig. 6B). An exception to the protective effect of romi- days also increased platelet counts plostim on hemoglobin values was observed when romi- We tested dividing the total dose of romiplostim over several plostim was not administered until day 2 in the third days to examine whether platelet recovery also occurred in cycle of CRT. In this case, the romiplostim-treated groups this situation. Mice were treated with 30, 100, or 300 mg/kg were not different from CRT alone until the last day of the of romiplostim on each of days 0, 1, and 2 in cycle two or cycle, which may indicate that dosing was too late in this cycles two and three and compared with the previous cycle to provide benefit. responses to a single, large treatment dose. All doses tested In addition to platelet counts, the effects of CRT and improved platelet recovery compared with CRT alone, with romiplostim on other hematologic parameters were exam- the highest dose (300 þ 300 þ 300 mg/kg/day) offering the ined (data not shown). Monocyte counts fell to undetect- greatest benefit (Figs. 5C and 5D). able levels 4 days after CRT and did not begin to recover until late in the cycle (approximately day 24). Romiplostim Romiplostim had a protective effect on hemoglobin treatment did not affect monocyte recovery following CRT values at any of the doses and schedules tested. Neutrophils In addition to its effects on platelet counts, romiplostim decreased to less than 10% of control values by day 4 also had some effects on other cell lineages. A positive post-CRT and did not begin to recover until approximately 484 P.L. McElroy et al./ Experimental Hematology 2015;43:479–487

Figure 5. Increased doses of romiplostim were effective in increasing platelet counts when given as a single dose or as a fractionated dose. Mice were treated with 100, 300, or 1,000 mg/kg of romiplostim on day 0 of CRT (A) cycle two or (B) cycles two and three. (A, B) Untreated mice were included to show baseline platelet values. Mice with CRT treatment alone were used as a negative control. Platelet counts were measured throughout cycles two or three, as indicated (n 5 5 mice per group, per time point). Error bars represent the standard error of the mean. Symbols above the lines indicate days of statistically significant differences between the following treatment groups and CRT alone: v 5 1,000 mg/kg; z 5 300 mg/kg; o 5 100 mg/kg (p ! 0.05, two-way ANOVA with Dunnett’s post-hoc test). Mice were treated with 30, 100, or 300 mg/kg of romiplostim on days 0, 1, and 2 of CRT (C) cycle two, or cycles (D) two and three. (C, D) Untreated mice were included to show baseline platelet values. Mice with CRT treatment alone were used as a negative control. Platelet counts were measured throughout cycles two or three, as indicated (n 5 5 mice per group, per time point). Error bars represent the standard error of the mean. Symbols above the lines indicate days of statistically significant differences between the following treatment groups and CRT alone: z 5 300 mg/kg; o 5 100 mg/kg; x 5 30 mg/kg (p ! 0.05, two-way ANOVA with Dunnett’s post-hoc test). (Color illustration of figure appears online.)

day 20 of the cycle. Romiplostim accelerated neutrophil re- Discussion covery for some treatment regimens, although this was not Chemotherapy-induced thrombocytopenia can lead to consistent. Specifically, in some experiments, neutrophil chemotherapy treatment delays or dose reductions. To counts were higher at some time points late in the cycle understand the potential of romiplostim to treat CIT, we in romiplostim-treated mice compared with mice receiving have used a mouse model of multicycle CRT-induced CRT but no romiplostim. Basophils and eosinophils were thrombocytopenia to explore romiplostim dose levels and also decreased by CRT. Basophils decreased to undetect- schedule. As in humans (reviewed in [23]), studies exam- able levels and did not recover by the end of the cycle. ining eTPO found an inverse relationship between platelet Eosinophils decreased to between 5% and 20% of normal, counts and eTPO concentrations in mice treated with stayed suppressed for the majority of the cycle, and CRT. We hypothesized that the period of time immediately partially recovered to between 35% and 69% of normal after CRT but before the elevation in eTPO might provide a by the end of the cycle. These parameters were generally therapeutic window during which romiplostim administra- unaffected by romiplostim treatment. Chemotherapy/ tion would be effective in enhancing platelet recovery. In radiation therapy decreased lymphocyte counts to approxi- the experiments described here, romiplostim accelerated mately 1% of baseline by day 2 post-CRT. They remained platelet recovery compared with CRT alone in most condi- depressed throughout the cycle and were unaffected by tions tested, with higher doses administered closer to CRT romiplostim. providing the greatest benefit. Doses of $100 mg/kg given P.L. McElroy et al./ Experimental Hematology 2015;43:479–487 485

of total body irradiation resulted in better platelet recovery [3,6,7]. It is worth noting that in mice treated with higher doses of romiplostim ($100 mg/kg) administered on day 0 in cycle three of CRT, we observed that platelet counts decreased more rapidly than in mice receiving CRT alone. The reason for this decrease is unknown and warrants further investigation. The work described here raises some interesting ques- tions. For example, treatment with romiplostim early in the CRT cycle improved platelet recovery. Therefore, it would be of interest to understand the kinetic effects of romiplostim on in the after multicycle CRT. These effects may be similar to the posi- tive impact of recombinant human thrombopoietin (rHuTPO) on megakaryocyte numbers in bone marrow, returning numbers close to normal sooner than controls in a mouse mitomycin C model of myelosuppression [24]. Though romiplostim shortened the platelet nadir, the full recovery to baseline levels did not occur until late in the cycle. It would therefore be interesting to examine whether additional administration of romiplostim later in the cycle would be of benefit. Interestingly, a recent report has found that newly generated platelets from immune thrombocyto- penia patients that were treated with TPO-receptor agonists were less prone to apoptosis after the first week than plate- lets analyzed before treatment [25]. However, this returned to baseline after 2 weeks. This may be due to direct or in- direct action of the TPO-receptor agonists on newly gener- ated platelets to increase prosurvival AKT signaling downstream of the TPO receptor [25]. Although a potential Figure 6. Romiplostim mitigated CRT-induced anemia and promoted direct action of TPO-receptor agonists on platelets could earlier reticulocyte recovery. (A) In the example shown, mice were treated raise a theoretical concern about platelet activation, TPO- m with 10, 30, or 100 g/kg of romiplostim on day 0 of the third CRT cycle. receptor agonists are generally well tolerated. Increases in Similar results were seen with other administration schedules and cycles of treatment. Untreated mice were included to show baseline platelet values. thromboembolic events have been reported in patients, Mice with CRT treatment alone were used as a negative control. Reticulo- though the majority of events reported were in patients cytes were measured throughout cycle three, as indicated (n 5 5 mice per with thromboembolic risk factors (reviewed in [26]). group, per time point). Error bars represent the standard error of the mean. It has been reported that administration of TPO and The symbols above the lines indicate days of statistically significant PEG-rHuMGDF in patients resulted in the generation of differences between the following treatment groups and CRT alone: o 5 100 mg/kg; x 5 30 mg/kg (p ! 0.05, two-way ANOVA with Bonfer- antibodies that cross-react with the endogenous protein roni post-hoc test). (B) In the example shown, mice were treated with 10, [18,27]. We also found that mice generated antibodies in 30, or 100 mg/kg of romiplostim on day 0 of the third CRT cycle. Similar response to romiplostim treatment, likely due to the admin- results were seen with other administration schedules and cycles of treat- istration of a foreign protein (human) to mice (data not ment. Untreated mice were included to show baseline platelet values. Mice shown). Since romiplostim is a TPO-peptide mimetic fused with CRT treatment alone were used as a negative control. Hemoglobin was measured throughout cycle three, as indicated (n 5 5 mice per group, to Fc and is not homologous to TPO, these antibodies did per time point). Error bars represent the standard error of the mean. The not crossreact or neutralize eTPO (data not shown). symbols above the lines indicate days of statistically significant differences In CRT-treated mice, romiplostim had a protective effect on between the following treatment groups and CRT alone: o 5 100 mg/kg; hemoglobin levels but did not affect other hematopoietic line- 5 m þ 5 m ! x 30 g/kg; 10 g/kg (p 0.05, two-way ANOVAwith Bonferroni ages. The lack of romiplostim’s effect on reducing the time of post-hoc test). Hb 5 hemoglobin; Retics 5 reticulocytes. (Color illustra- tion of figure appears online.) neutropenia is in contrast to that observed with rHuTPO in a mouse mitomycin C model of myelosuppression [24],though on day 0 in cycles two and three significantly lessened the in this model a positive impact on reducing anemia was also platelet nadir in both cycle two and cycle three. This finding observed. Positive impacts on platelets and hemoglobin have is in concordance with prior preclinical experiments in also been observed for TPO-mimetic in refrac- mice, in which a single TPO dose given close to the time tory aplastic anemia patients [28]. In the same study, the 486 P.L. McElroy et al./ Experimental Hematology 2015;43:479–487 researchers also observed an improvement in neutropenia and associated with carboplatin and irradiation in mice. Blood. 1995;86: concluded that eltrombopag may have stimulated the TPO re- 4486–4492. ceptor on hematopoietic stem cells, though the precise mech- 6. Neelis KJ, Visser TP, Dimjati W, et al. A single dose of thrombopoie- tin shortly after myelosuppressive total body irradiation prevents anism has not been defined. It is unclear whether romiplostim pancytopenia in mice by promoting short-term multilineage spleen- would have a similar trilineage beneficial effect in aplastic repopulating cells at the transient expense of bone marrow- anemia patients. A positive impact of rHuTPO was observed repopulating cells. Blood. 1998;92:1586–1597. on reticulocytes in a total-body irradiation model in rhesus 7. Shibuya K, Akahori H, Takahashi K, Tahara E, Kato T, Miyazaki H. monkeys, though reticulocyte increases were not seen until af- Multilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in ter 7 days [29]. This is similar to our study, in which we myelosuppressed mice. Blood. 1998;91:37–45. observed an early decrease in reticulocytes in all CRT- 8. Ulich TR, del Castillo J, Senaldi G, et al. The prolonged hematologic treated mice, with subsequent increases above the CRT- effects of a single injection of PEG-rHuMGDF in normal and throm- alone values starting at 12 days and continuing for the latter bocytopenic mice. Exp Hematol. 1999;27:117–130. part of the cycle. 9. Ulich TR, del Castillo J, Senaldi G, Hartley C, Molineux G. PEG-rHuMGDF promotes multilineage hematopoietic recovery in In summary, in a mouse model of CIT, romiplostim was myelosuppressed mice. Exp Hematol. 1999;27:1776–1781. effective at accelerating platelet recovery and reducing the 10. Ulich TR, del Castillo J, Yin S, et al. Megakaryocyte growth and extent of the nadir when dosed in each cycle of CRT tested, development factor ameliorates carboplatin-induced thrombocyto- before eTPO concentrations had increased to stimulate an penia in mice. Blood. 1995;86:971–976. increase in platelets. Though the authors acknowledge the 11. Mouthon MA, Van der Meeren A, Vandamme M, Squiban C, Gaugler MH. 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