Romiplostim promotes platelet recovery in a mouse model of ...

Experimental Hematology 2015;43:479–487

Romiplostim promotes platelet recovery in a mouse model of multicycle chemotherapy-induced thrombocytopenia Patricia L. McElroya, Ping Weia, Keri Bucka, Angus M. Sinclaira, Michael Eschenbergb, Barbra Sasua, and Graham Molineuxa a

Oncology Research Department, Amgen 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 thrombopoietin (TPO) mimetic, to promote platelet recovery in a mouse model of multicycle chemotherapy/radiation therapy (CRT)-induced thrombocytopenia 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 - 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/).

Chemotherapy-induced thrombocytopenia (CIT) is an unmet medical need that can lead to delays or reductions in scheduled chemotherapy treatment, which are associated with poorer outcomes for cancer patients [1,2]. Severe thrombocytopenia occurs in approximately 9% of chemotherapy cycles and can necessitate platelet transfusions [1,2]. Recombinant thrombopoietin (TPO) reversed thrombocytopenia and increased survival in animal models of CIT and chemotherapy/radiation therapy (CRT)-induced thrombocytopenia [3–11]. Specifically, certain dose and timing schedules were evaluated, and it was found that a single TPO dose given close to the time of total body irradiation resulted in better recovery [3,6,7]. Early clinical studies have demonstrated proof of concept for recombinant TPO/TPO-receptor agonists [12–17]. However, clinical studies using recombinant TPO were halted when some patients treated with PEGylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF), a truncated form of TPO, developed neutralOffprint requests to: Ms. Patricia L. McElroy, Oncology Discovery Research, Amgen Inc., One Amgen Center Drive, MS 15-2A, Thousand Oaks, CA 91320; E-mail: [email protected]

izing antibodies [18]. These findings led to the development of alternative therapies, including romiplostim. Romiplostim is a TPO-receptor agonist approved for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenia. It is the first United States Food and Drug Administration–approved member of a new class of therapeutics called peptibodies, which are peptides conjugated to human Fc and resemble antibodies [19]. Since chemotherapy-supportive treatments may not be initiated until the second or subsequent cycles of chemotherapy, after a patient has been identified as being at risk for developing thrombocytopenia, we developed a multicycle mouse model and administered romiplostim after the first treatment cycle. Using this model, we sought to understand the optimum dose and timing of romiplostim administration that would promote platelet recovery. A CRT mouse model was used because mice receiving single chemotherapy agents alone did not develop thrombocytopenia severe enough that the effects of adding romiplostim would be easily detectable. Previous studies established that there is an inverse relationship between endogenous TPO (eTPO) concentrations and platelet counts in patients undergoing chemotherapy [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

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between platelet counts and eTPO concentrations in a mouse model of multicycle CRT could inform a rationale for prophylactic romiplostim treatment.

Materials and methods Mice Female B6D2F1 (BDF1) mice, aged 11 to 15 weeks, were used and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition [22]. Animals were group housed at an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility in sterile, ventilated microisolator housing. All research protocols were approved by the Institutional Animal Care and Use Committee. Animals had ad libitum access to pelleted feed, food supplements, and water; were maintained on a 12:12 hour light:dark cycle; and had access to enrichment opportunities. All animals were determined specific pathogen free. Study design Mice were treated with two or three cycles of CRT (experimental designs depicted in Fig. 1). Each cycle was 28 days long. For the first cycle, mice were injected intraperitoneally with 62.5 mg/kg carboplatin on day 0. Four hours after carboplatin treatment, the mice were exposed to 5 Gy total body radiation from a 137Cs source (Gamma Cell 40 Irradiator, Atomic Energy of Canada Ltd, Chalk River, Ontario, Canada). For cycles two and three, the carboplatin dose was 56.25 mg/kg, and the radiation dose was 4.5 Gy. This multicycle regimen of carboplatin plus irradia-

tion was adapted from work done by Hokom et al. [5], who showed that the combination produced a profound thrombocytopenia; doses were modified in these studies to make repeated treatment 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 to mirror the clinical situation. Untreated or saline-treated mice served as positive controls, and CRT-treated mice not receiving romiplostim served as negative controls. Blood samples were collected by cardiac puncture immediately after euthanasia or via the retro-orbital sinus while mice were anesthetized. Due to the myelosuppressive nature of CRT, anemia developed in addition to thrombocytopenia. To avoid severely compromising individual mice by repeated blood sampling, a rotating cohort design was used (except for experiment one, in which mice were euthanized at each time point). There were 25 mice to a group (except for experiment one, which had 75 mice per treatment group to support terminal time points), and results are presented as averages of five mice per time point. Each mouse was bled twice, once early in the cycle and a second time late in the cycle, and was euthanized immediately following the second sampling. Analyte measurements For the experiment comparing eTPO concentrations to platelet counts, five mice per group were euthanized by CO2 inhalation at each time point, and we collected blood by cardiac puncture for complete blood counts (CBCs) and serum eTPO analysis. 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.)


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Quantikine Mouse TPO Immunoassay kit (R&D Systems, Minneapolis, MN). We analyzed CBCs using an automated blood analyzer (Advia 2120, Siemens, Munich, Germany). For experiments 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 logtransformed platelet counts, with treatment used as a fixed effect in the model. Each treatment group was compared with the group subjected 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 evaluations 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 endogenous thrombopoietin concentration was observed during three successive cycles of chemotherapy/ radiation therapy The relationship between platelet counts and eTPO concentration was examined during three cycles of CRT in mice to ascertain when eTPO concentrations were suboptimal and administration of romiplostim might be efficacious. Mice received either CRT treatment or subcutaneous saline as a control. 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 each cycle (Fig. 2A). After each administration of CRT, eTPO concentration remained at a steady level for approximately 6 days, after which it began to increase until reaching a peak on day 10 (Fig. 2B). This eTPO peak corresponded with the nadir in platelet counts, and as platelet counts gradually recovered, eTPO also decreased to approximately baseline concentration. Endogenous TPO concentration was lower at the beginning of each successive CRT cycle than in the previous cycle, and the amount of time required for platelet counts to recover to baseline was progressively longer. These results demonstrated an inverse relationship between platelet number and eTPO concentration during three cycles of CRT. Moreover, there appeared to be a window of time early after CRT administration, before eTPO concentration increased, during which administration of romiplostim might be effective

Figure 2. Platelet counts demonstrated an inverse relationship with eTPO concentrations during three cycles of CRT-induced thrombocytopenia in mice. (A) Platelet counts in mice treated with CRT decreased in each cycle. Platelets were measured in saline-treated mice only in the first cycle. (B) Overlay of platelet counts and eTPO concentrations. Data are the means of five mice per time point, and error bars represent the standard error of the mean. (Color illustration of figure appears online.)

in promoting platelet recovery. This window was targeted for treatment. Romiplostim administration in the second cycle of chemotherapy/radiation therapy accelerated platelet recovery Following the theory that the first 5 days of a CRT cycle constituted the ideal point for intervention, we examined the efficacy of romiplostim in promoting platelet recovery in the second cycle of CRT, the time at which, in a clinical situation, the need for supportive care would have been identified. Mice were treated in the second cycle with 10, 30, or 100 mg/kg of romiplostim on day 0, 1, or 2, and platelet counts were measured throughout the cycle. In general, romiplostim treatment caused a significant increase in the rate of platelet recovery after CRT, especially at higher doses given on day 0 or day 1 (Fig. 3). Administration of romiplostim on day 0 and day 1 significantly lessened the platelet nadir (sixfold decrease in nadir for day

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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 administration for the 100-mg/kg-dose group). During the recovery phase (i.e., the period of time after the platelet nadir), romiplostim treatment resulted in a dose-dependent increase in mean platelet counts compared with CRT alone, reaching statistical significance at several time points for all administration schedules tested. Taken together, these data indicate that higher doses of romiplostim administered closer to the time of CRT were most effective in preventing the severe decrease in platelets caused by CRT and in accelerating platelet recovery. Responses to romiplostim administration in the third cycle of chemotherapy/radiation therapy were similar to those seen in the second cycle We next examined whether treatment with 10, 30, or 100 mg/kg of romiplostim in both the second and third cycles of CRT on days 0, 1, or 2 resulted in higher platelet counts in the third cycle. Administration of romiplostim on day 0 resulted in the greatest improvement, with all doses administered on this day resulting in significantly increased platelet counts at several time points compared with CRT alone (Fig. 4A). The exception to this was on day 6, at which point platelet counts were higher with CRT alone.

Of note, the platelet nadir generally occurred later with CRT alone as compared with CRT þ romiplostim. The effectiveness of romiplostim appeared to be dose dependent; mice treated with 100 mg/kg of romiplostim exhibited more days of significantly higher platelet counts (compared with CRT alone) than mice treated with lower doses. Similar to the results in cycle two, it was observed that day 0 administration in the second and third cycles lessened the platelet nadir in cycle three. Romiplostim administered on days 1 or 2 also resulted in increased platelet counts compared with CRT alone but with fewer days of significant difference from CRT alone than day 0 administration, and neither day 1 nor day 2 treatment lessened the platelet nadir compared with CRT alone (Figs. 4B and 4C). Higher doses of romiplostim improved platelet recovery compared with chemotherapy/radiation therapy alone To determine if doses of romiplostim that were higher than previously tested might further accelerate platelet recovery, mice were treated with 100, 300, or 1,000 mg/kg of romiplostim on day 0 of cycle two or treated in both cycles two and three. In both these situations, higher doses of romiplostim resulted in improved platelet recovery compared with CRT alone (Figs. 5A and 5B). When given


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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 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 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 lower doses but was not different from 1,000 mg/kg in promoting second-cycle platelet recovery. When given in cycles two and three, increasing the dose above 100 mg/kg did not result in significant improvement to cycle-three platelet recovery, based on overall platelet counts as measured by repeated measures analysis of variance (data not shown). Dividing the dose of romiplostim over 3 consecutive days also increased platelet counts We tested dividing the total dose of romiplostim over several days to examine whether platelet recovery also occurred in this situation. Mice were treated with 30, 100, or 300 mg/kg of romiplostim on each of days 0, 1, and 2 in cycle two or cycles two and three and compared with the previous responses to a single, large treatment dose. All doses tested improved platelet recovery compared with CRT alone, with the highest dose (300 þ 300 þ 300 mg/kg/day) offering the greatest benefit (Figs. 5C and 5D). Romiplostim had a protective effect on hemoglobin values In addition to its effects on platelet counts, romiplostim also had some effects on other cell lineages. A positive

effect on reticulocyte counts was observed in mice receiving CRT and treated with romiplostim on day 0 of cycle three (Fig. 6A). An early decrease in reticulocytes was observed followed by increases on days 12 and 16 in mice treated with 100 mg/kg of romiplostim. Romiplostim treatment prevented the severe drop in hemoglobin in the same animals, with higher doses closer to the administration of CRT generally providing greater protection (Fig. 6B). An exception to the protective effect of romiplostim on hemoglobin values was observed when romiplostim was not administered until day 2 in the third cycle of CRT. In this case, the romiplostim-treated groups were not different from CRT alone until the last day of the cycle, which may indicate that dosing was too late in this cycle to provide benefit. In addition to platelet counts, the effects of CRT and romiplostim on other hematologic parameters were examined (data not shown). Monocyte counts fell to undetectable levels 4 days after CRT and did not begin to recover until late in the cycle (approximately day 24). Romiplostim treatment did not affect monocyte recovery following CRT at any of the doses and schedules tested. Neutrophils decreased to less than 10% of control values by day 4 post-CRT and did not begin to recover until approximately

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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 recovery for some treatment regimens, although this was not consistent. Specifically, in some experiments, neutrophil counts were higher at some time points late in the cycle in romiplostim-treated mice compared with mice receiving CRT but no romiplostim. Basophils and eosinophils were also decreased by CRT. Basophils decreased to undetectable levels and did not recover by the end of the cycle. Eosinophils decreased to between 5% and 20% of normal, stayed suppressed for the majority of the cycle, and partially recovered to between 35% and 69% of normal by the end of the cycle. These parameters were generally unaffected by romiplostim treatment. Chemotherapy/ radiation therapy decreased lymphocyte counts to approximately 1% of baseline by day 2 post-CRT. They remained depressed throughout the cycle and were unaffected by romiplostim.

Discussion Chemotherapy-induced thrombocytopenia can lead to chemotherapy treatment delays or dose reductions. To understand the potential of romiplostim to treat CIT, we have used a mouse model of multicycle CRT-induced thrombocytopenia to explore romiplostim dose levels and schedule. As in humans (reviewed in [23]), studies examining eTPO found an inverse relationship between platelet counts and eTPO concentrations in mice treated with CRT. We hypothesized that the period of time immediately after CRT but before the elevation in eTPO might provide a therapeutic window during which romiplostim administration would be effective in enhancing platelet recovery. In the experiments described here, romiplostim accelerated platelet recovery compared with CRT alone in most conditions tested, with higher doses administered closer to CRT providing the greatest benefit. Doses of $100 mg/kg given


Figure 6. Romiplostim mitigated CRT-induced anemia and promoted earlier reticulocyte recovery. (A) In the example shown, mice were treated with 10, 30, or 100 mg/kg of romiplostim on day 0 of the third CRT cycle. Similar results were seen with other administration schedules and cycles of treatment. Untreated mice were included to show baseline platelet values. Mice with CRT treatment alone were used as a negative control. Reticulocytes were measured throughout cycle three, as indicated (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 (p ! 0.05, two-way ANOVA with Bonferroni post-hoc test). (B) In the example shown, mice were treated with 10, 30, or 100 mg/kg of romiplostim on day 0 of the third CRT cycle. Similar results were seen with other administration schedules and cycles of treatment. Untreated mice were included to show baseline platelet values. Mice with CRT treatment alone were used as a negative control. Hemoglobin was measured throughout cycle three, as indicated (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 Bonferroni post-hoc test). Hb 5 hemoglobin; Retics 5 reticulocytes. (Color illustration of figure appears online.)

on day 0 in cycles two and three significantly lessened the platelet nadir in both cycle two and cycle three. This finding is in concordance with prior preclinical experiments in mice, in which a single TPO dose given close to the time

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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 questions. 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 megakaryocytes in the bone marrow after multicycle CRT. These effects may be similar to the positive 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 thrombocytopenia patients that were treated with TPO-receptor agonists were less prone to apoptosis after the first week than platelets analyzed before treatment [25]. However, this returned to baseline after 2 weeks. This may be due to direct or indirect action of the TPO-receptor agonists on newly generated platelets to increase prosurvival AKT signaling downstream of the TPO receptor [25]. Although a potential direct action of TPO-receptor agonists on platelets could raise a theoretical concern about platelet activation, TPOreceptor agonists are generally well tolerated. Increases in thromboembolic events have been reported in patients, though the majority of events reported were in patients with thromboembolic risk factors (reviewed in [26]). It has been reported that administration of TPO and PEG-rHuMGDF in patients resulted in the generation of antibodies that cross-react with the endogenous protein [18,27]. We also found that mice generated antibodies in response to romiplostim treatment, likely due to the administration of a foreign protein (human) to mice (data not shown). Since romiplostim is a TPO-peptide mimetic fused to Fc and is not homologous to TPO, these antibodies did not crossreact or neutralize eTPO (data not shown). In CRT-treated mice, romiplostim had a protective effect on hemoglobin levels but did not affect other hematopoietic lineages. The lack of romiplostim’s effect on reducing the time of neutropenia is in contrast to that observed with rHuTPO in a mouse mitomycin C model of myelosuppression [24], though in this model a positive impact on reducing anemia was also observed. Positive impacts on platelets and hemoglobin have also been observed for TPO-mimetic eltrombopag in refractory aplastic anemia patients [28]. In the same study, the

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researchers also observed an improvement in neutropenia and concluded that eltrombopag may have stimulated the TPO receptor on hematopoietic stem cells, though the precise mechanism has not been defined. It is unclear whether romiplostim would have a similar trilineage beneficial effect in aplastic anemia patients. A positive impact of rHuTPO was observed on reticulocytes in a total-body irradiation model in rhesus monkeys, though reticulocyte increases were not seen until after 7 days [29]. This is similar to our study, in which we observed an early decrease in reticulocytes in all CRTtreated mice, with subsequent increases above the CRTalone values starting at 12 days and continuing for the latter part of the cycle. In summary, in a mouse model of CIT, romiplostim was effective at accelerating platelet recovery and reducing the extent of the nadir when dosed in each cycle of CRT tested, before eTPO concentrations had increased to stimulate an increase in platelets. Though the authors acknowledge the limitations of using preclinical models to mimic conditions occurring in patients, we propose that these data may serve as the basis to study the effects of romiplostim in reducing thrombocytopenia in patients receiving high-dose chemotherapy and CRT. Acknowledgments We thank Dr. Molineux, who played an integral part in the design of these experiments, in the interpretation of the data, and in the development of romiplostim. He passed away before publication of this work and is greatly missed. We thank Susanna Mac, employee of Amgen Inc., for medical writing assistance. Funding for the study was provided by Amgen Inc.

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Conflict of interest disclosure All authors are employees or former employees of Amgen Inc. and stockholders in Amgen Inc.

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