Treatment method by pegfilgrastim and romiplostim

Abstract

To provide a method for treating a disease and pathological condition characterized by a low neutrophil level (neutropenia) and / or a low platelet level (thrombocytopenia).SOLUTION: Provided is a method including simultaneous administration of pegfilgrastim and romiplostim. This method enhances effect to a neutrophil level and platelet level. Furthermore, provided is a method including administering romiplostim at a dose of approximately 1 μg / kg to approximately 10 μg / kg as a method for treating patient exposed to radiation. Furthermore, provided is a method for administering a single-dose of romiplostim to a patient, and a method for administering romiplostim within approximately 24 hours after radiation exposure. Furthermore, provided is a therapy by romiplostim and pegfilgrastim for radiation exposure. The method relates to a treatment of acute radiation syndrome, radiotherapy, and a treatment for cancelling effects of the other radiation exposure source.SELECTED DRAWING: Figure 8A

Description

[Technical field]

[0001] Government support acknowledged This invention was made with Government support under HHSN272201500013I awarded by the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Department of Health and Human Services. The Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 734,899, filed September 21, 2018, U.S. Provisional Patent Application No. 62 / 657,594, filed April 13, 2018, and U.S. Provisional Patent Application No. 62 / 641,224, filed March 9, 2018, all of which are incorporated by reference in their entireties. [Background technology]

[0003] Romiplostim (Nplate®) is a platelet-producing promoting peptibody that binds to the thrombopoietin receptor (TPO-R) on platelets and platelet precursor cells. Romiplostim binds to and activates TPO-R in a similar mechanism to endogenous TPO, promoting megakaryocyte proliferation and maturation and increasing circulating platelets (PLTs). Early publications reported the PK and PD of romiplostim in mice, non-human primates, and healthy and diseased humans. This led to its approval in 2008 and later worldwide for use as a treatment for chronic primary immune thrombocytopenic purpura (ITP), an autoimmune disease characterized by persistently reduced platelet counts. Romiplostim is approved for use in adults in the United States for the treatment of chronic ITP that has had an inadequate response to corticosteroids, immunoglobulin, or splenectomy, and in Europe for people who have had splenectomy and are refractory to other therapies, or as second-line treatment in non-splenectomized patients in whom surgery is contraindicated. Romiplostim is administered at a starting dose of 1 μg / kg and then once platelet counts reach 50 × 10 9 / L or more 400×10 9 Doses of 1 μg / kg are administered weekly to achieve and maintain a serum serum creatinine concentration below 1 / L.

[0004] Pegfilgrastim (Neulasta®) is a form of the recombinant granulocyte colony-stimulating factor (GCSF) analog filgrastim (Neupogen®) conjugated to monomethoxypolyethylene glycol. Thus, pegfilgrastim mimics the activity of GCSF to bind and activate specific cell surface receptors, stimulating the proliferation and differentiation of neutrophil precursor cells and selected neutrophil functions. Conjugation of the GCSF analog to a polyethylene glycol molecule greatly increases its therapeutic half-life. In the United States, pegfilgrastim is approved to reduce the incidence of febrile neutropenia-emergent infections in patients with nonmyeloid malignancies receiving myelosuppressive anticancer agents that are associated with a clinically significant incidence of febrile neutropenia. The recommended dosage of pegfilgrastim is a single subcutaneous injection of 6 mg administered once per chemotherapy cycle. Pegfilgrastim has also been shown to increase survival in patients acutely exposed to myelosuppressive doses of radiation. For this indication, two doses of 6 mg subcutaneously given one week apart are recommended.

[0005] A search of the medical and scientific literature did not reveal any reports of coadministration of pegfilgrastim and romiplostim; however, the literature did contain reports of coadministration of filgrastim and romiplostim.

[0006] Vandrass et al. reported such co-administration in a patient who had been treated with temozolomide and developed bone marrow failure (Non-Patent Document 1). These authors found only a transient increase in granulocyte levels, discontinued filgrastim after 1 month, and discontinued romiplostim after 2 months of treatment, with little effect on platelets despite increasing the dose.

[0007] Bosch-Vilaseca et al. reported on such coadministration in a retrospective study on the usefulness of the thrombopoietin (TPO) agonists eltrombopag and romiplostim for persistent clinically relevant thrombocytopenia after allogeneic stem cell transplantation (Non-Patent Document 2). They identified as possible variables associated with a higher response to TPO agonists under age 40, the presence of megakaryocytes in bone marrow aspirates, and / or prior response to other hematopoietic growth factors. They found that 6 of 13 patients who showed a response to filgrastim (i.e., increased white blood cell levels) also showed a response to TPO agonists (i.e., increased platelet levels).

[0008] Amiriana et al. have reported such co-administration in the treatment of patients with multiorgan involvement of Langerhans cell histiocytosis. (Non-Patent Document 3) They reported that when patients were administered filgrastim and romiplostim to stimulate bone marrow production of white blood cells and platelets, the white blood cell count responded but the platelet count did not.

[0009] Kovtonyuk et al. reported the co-administration of filgrastim and romiplostim in a study on the induction of hematopoietic stem cells (HSCs). (Non-Patent Document 4). Based on in vivo high-resolution single HSC division tracking, they concluded that they demonstrated that clinically applied TPO receptor (but not G-CSF receptor) agonists induce HSCs to undergo self-renewal divisions and result in quantitative expansion of functional HSCs, as defined by their in vivo continuous multilineage and long-term repopulation potential. They concluded that these results suggest that thrombopoietin mimetics may be applicable to expand HSCs in vivo and increase the sensitivity of thrombopoietin receptor-expressing HSCs to cell cycle-dependent cytotoxic drugs.

[0010] An indication of interest for both pegfilgrastim and Proplastim is acute hematopoietic syndrome of acute radiation syndrome (HS-ARS). In HS-ARS, the rapid reduction in white blood cells, platelets and reticulocytes leads to infection, bleeding and death. Given the possibility of radiation accidents or acts of terrorism and the severity of diseases resulting from radiation exposure, there is an urgent need to develop and approve effective medical countermeasures (MCM) for radiation syndrome. HS-ARS is characterized by neutropenia, thrombocytopenia and anemia. To date, no pharmacological therapy has been shown to affect the platelet levels associated with HS-ARS. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Vandraas et al.(2016),BMJ Case Reports 2016;2016:bcr2016215797 [Non-Patent Document 2] Bosch-Vilaseca et al.(2018),Eur.J.Hematol.101(3):407-14 [Non-Patent Document 3] Amiriana et al. (2015), J.Med.Cases 6(8):376-7 [Non-Patent Document 4] Kovtonyuk et al.(2016),Blood.127(25):3175-3179 Summary of the Invention [Means for solving the problem]

[0012] The present invention relates to a method comprising the co-administration of pegfilgrastim and romiplostim for the treatment of diseases and conditions characterized by low neutrophil levels (neutropenia) and / or low platelet levels (thrombocytopenia). The present invention relates to the enhanced effect on neutrophil and platelet levels resulting from the co-administration of pegfilgrastim and romiplostim. The method is. As shown herein in non-human primate studies of ARS, such studies support the treatment of other conditions characterized by low levels of platelets and / or neutrophils. Preferred doses for such co-administration are about 1-10 μg / kg of romiplostim and about 6 mg of pegfilgrastim for HS-ARS. Preferred dosing frequency is two doses of 6 mg of pegfilgrastim, given at intervals of one week. Other doses are suitable for other indications, including those approved by the US Food and Drug Administration for pegfilgrastim and romiplostim.

[0013] According to the present invention, there is provided a method of administering pegfilgrastim and romiplostim, wherein romiplostim is administered as follows: a. An initial dose of 1 mcg / kg administered weekly; B. Platelet count is 50 x 10 9 Adjust dose in 1 mcg / kg increments weekly to achieve and maintain ≥K.

[0014] Further, the present invention provides a method of administering pegfilgrastim and romiplostim, wherein 6 mg of pegfilgrastim is administered. When platelet levels are affected by chemotherapy, romiplostim and pegfilgrastim are administered, and pegfilgrastim is preferably administered once per chemotherapy cycle, preferably at a dose of 6 mg.

[0015] Further, the present invention provides a method for treating a condition characterized by decreased platelet levels or increased platelet degradation.Accordingly, the present invention relates to a method of administering romiplostim and pegfilgrastim to a mammal, where the mammal has been exposed to radiation (e.g., acute radiation syndrome, radiotherapy for cancer), has undergone or is undergoing chemotherapy, or has a condition such as idiopathic thrombocytopenic purpura (ITP), a viral infection, a bacterial infection, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, or anemia (e.g., aplastic anemia).

[0016] Further, the present invention provides a method for treating a condition characterized by reduced neutrophil levels or neutrophil degradation.Accordingly, the present invention relates to a method for administering romiplostim and pegfilgrastim to a mammal, the mammal having been exposed to radiation (e.g., acute radiation syndrome, radiation therapy), has undergone chemotherapy, or is undergoing chemotherapy, or has a condition such as chronic idiopathic neutropenia, Kostmann syndrome, leukemia, myelodysplastic syndrome, myelofibrosis, myeloid cell pooling, vitamin deficiency, hepatitis A, hepatitis B, hepatitis C, HIV infection, AIDS, Lyme disease, malaria, viral infection of bone marrow, Salmonella infection, sepsis, hypersplenism, or rheumatoid arthritis.

[0017] According to the present invention, romiplostim is an effective medical countermeasure (MCM) for the treatment of HS-ARS and radiation exposure based on its ability to enhance platelet production. According to the present invention, a method for treating a patient exposed to radiation is provided, comprising administering romiplostim. Further, according to the present invention, such a method is provided, wherein a single dose of romiplostim is administered to the patient. Further, according to the present invention, such a method is provided, wherein the patient so treated has acute radiation syndrome. Further, according to the present invention, such a method is provided, wherein the radiation exposure is due to radiotherapy, such as that included in cancer treatment. Treatment of other sources of radiation exposure is also within the scope of the present invention.

[0018] In a method carried out in accordance with the present invention, male and female C57BL / 6J mice were administered immunization therapy with a 30% 30-day survival rate (LD 70 / 30 Patients were treated with total body irradiation (TBI) with 680 cGy of X-rays reducing survival to 100 s. Romiplostim was administered subcutaneously (sc) at ≥24 h after TBI at doses of 3, 30, or 100 μg / kg as a single dose, or at 30 μg / kg (up to 5 doses) to assess the survival benefit of treatment. Pharmacokinetic (PK) and hematological parameters were also evaluated.

[0019] Results of the aforementioned methods showed that a single dose of romiplostim (30 or 100 μg / kg) 24 hours after TBI increased survival by approximately 40% in mouse studies. Repeated administration of 30 μg / kg romiplostim once daily for up to 3 or 5 days after TBI was equally effective. Platelet levels declined rapidly in romiplostim-treated TBI mice, nadiring at day 8 before robust recovery. Conversely, PLT levels in vehicle-treated TBI mice were delayed, nadiring at approximately day 10 before the onset of recovery. The kinetic profiles of other hematological parameters were similar between TBI romiplostim-treated and vehicle control mice. Peak blood concentrations of romiplostim in TBI mice occurred 4 to 24 hours after injection (T max ) and t 1 / 2 It was about 24 hours. max The values ​​were approximately 6 ng / ml after 30 μg / kg + / - TBI and approximately 200 ng / ml after 300 μg / kg. A 10-fold increase in the AUC last The value increased by approximately 35-fold.

[0020] From these results, it was concluded that single or repeated administration of romiplostim injection in mice administered 24 hours after TBI increased survival rate and accelerated PLT recovery. PK results + / - TBI showed that romiplostim was absorbed and distributed efficiently in mice.

[0021] Furthermore, the present invention provides a method for treating acute radiation syndrome, which comprises administering romiplostim at a dose of about 1 to about 10 μg / kg.

[0022] Furthermore, according to the present invention, there is provided a method for treating acute radiation syndrome, which includes administering romiplostim together with pegfilgrastim, whereby a greater effect is obtained than with either drug alone. The combined use of both drugs was associated with the lowest platelet and neutrophil nadirs compared to the data for each drug alone in tests on non-human primates. The combination treatment also resulted in the earliest platelet and neutrophil recovery compared to each drug alone.

Brief Description of the Drawings

[0023] [Figure 1A] Figures 1A and 1B show the serum concentrations of romiplostim after single subcutaneous administration 24 - 26 hours after irradiation in male and female mice, respectively. [Figure 1B] Figures 1A and 1B show the serum concentrations of romiplostim after single subcutaneous administration 24 - 26 hours after irradiation in male and female mice, respectively. [Diagram 2] Figure 2 is a table showing the pharmacokinetics of romiplostim in irradiated mice versus non-irradiated mice. The serum concentration of romiplostim was <LLOQ in all samples of the second group that received 600 cGy and 3 μg / kg of romiplostim (see the detailed description of the present invention below). "NC" in the table means "not calculated" due to insufficient final-stage data points for accurate value calculation. The values in the table are n = 3 / group / sex / time point. Figures 1A, 1B, and 2 show that (a) romiplostim is rapidly and efficiently absorbed after subcutaneous injection, (b) at a dose of 30 μg / kg, it has a similar pharmacokinetic (PK) profile in both sexes regardless of radiation exposure, (c) Tmax is 4 - 24 hours and T1 / 2 is approximately 24 hours, (d) Cmax and area under the curve (AUC) show an increase exceeding the dose ratio, and (e) a 10-fold higher romiplostim dose increased the exposure values of Cmax and AUClast by approximately 35-fold. [Figure 3A]Figures 3A-3C show the pharmacodynamics (PD) of romiplostim in irradiated and non-irradiated mice after a single dose. In Figure 3A, the number of surviving irradiated animals from which blood could be collected was dramatically reduced in vehicle control (black) and low dose (blue) mice after day 14. In Figure 3B, platelet levels were similar in all dose groups on day 4. Figure 3B shows that the 30 and 300 μg / kg dose groups exhibit a faster, but milder, decline in platelet levels after irradiation compared to the saline and 3 μg / kg groups (nadir at day 8 vs. day 10). A faster recovery to near baseline is observed at day 22 vs. day 29. In Figure 3C, platelet volume increased in the mid- and high-dose groups on days 6-8. This finding suggests that new platelets are released more rapidly from the bone marrow after romiplostim administration in irradiated mice. [Figure 3B] Figures 3A-3C show the pharmacodynamics (PD) of romiplostim in irradiated and non-irradiated mice after a single dose. In Figure 3A, the number of surviving irradiated animals from which blood could be collected was dramatically reduced in vehicle control (black) and low dose (blue) mice after day 14. In Figure 3B, platelet levels were similar in all dose groups on day 4. Figure 3B shows that the 30 and 300 μg / kg dose groups exhibit a faster, but milder, decline in platelet levels after irradiation compared to the saline and 3 μg / kg groups (nadir at day 8 vs. day 10). A faster recovery to near baseline is observed at day 22 vs. day 29. In Figure 3C, platelet volume increased in the mid- and high-dose groups on days 6-8. This finding suggests that new platelets are released more rapidly from the bone marrow after romiplostim administration in irradiated mice. [Figure 3C]Figures 3A-3C show the pharmacodynamics (PD) of romiplostim in irradiated and non-irradiated mice after a single dose. In Figure 3A, the number of surviving irradiated animals from which blood could be collected was dramatically reduced in vehicle control (black) and low dose (blue) mice after day 14. In Figure 3B, platelet levels were similar in all dose groups on day 4. Figure 3B shows that the 30 and 300 μg / kg dose groups exhibit a faster, but milder, decline in platelet levels after irradiation compared to the saline and 3 μg / kg groups (nadir at day 8 vs. day 10). A faster recovery to near baseline is observed at day 22 vs. day 29. In Figure 3C, platelet volume increased in the mid- and high-dose groups on days 6-8. This finding suggests that new platelets are released more rapidly from the bone marrow after romiplostim administration in irradiated mice. [Figure 4A] 4A-4D show other key hematological parameters that are affected by radiation but not by romiplostim. The parameters shown are white blood cell count (FIG. 4A), red blood cell count (FIG. 4B), neutrophil count (FIG. 4C), and reticulocyte count (FIG. 4D). [Figure 4B] 4A-4D show other key hematological parameters that are affected by radiation but not by romiplostim. The parameters shown are white blood cell count (FIG. 4A), red blood cell count (FIG. 4B), neutrophil count (FIG. 4C), and reticulocyte count (FIG. 4D). [Figure 4C] 4A-4D show other key hematological parameters that are affected by radiation but not by romiplostim. The parameters shown are white blood cell count (FIG. 4A), red blood cell count (FIG. 4B), neutrophil count (FIG. 4C), and reticulocyte count (FIG. 4D). [Figure 4D] 4A-4D show other key hematological parameters that are affected by radiation but not by romiplostim. The parameters shown are white blood cell count (FIG. 4A), red blood cell count (FIG. 4B), neutrophil count (FIG. 4C), and reticulocyte count (FIG. 4D). [Figure 5A]Figures 5A and 5B show the survival benefit of romiplostim in the mouse HS-ARS efficacy model. Figure 5A shows that a single dose of 30 or 100 μg / kg of romiplostim administered 24 hours after total body irradiation (TBI) resulted in a statistically significant 30-day survival benefit of about 40%. Figure 5A further shows that a survival benefit of about 24% was observed with doses of 3 or 10 μg / kg (not statistically significant). Figure 5B shows a statistically significant 30-day survival benefit (about 33%) with doses of 30 μg / kg of romiplostim administered once, three or five times a day starting 24 hours after TBI. Repeated doses of 30 μg / kg of romiplostim did not improve survival compared to single doses of romiplostim. Figure 5C shows a statistically significant increase in survival with romiplostim (30 μg / kg), pegfilgrastim (300 μg / kg), and their combination. No difference in survival benefit was observed when comparing the individual agents with their combination. [Figure 5B] Figures 5A and 5B show the survival benefit of romiplostim in the mouse HS-ARS efficacy model. Figure 5A shows that a single dose of 30 or 100 μg / kg of romiplostim administered 24 hours after total body irradiation (TBI) resulted in a statistically significant 30-day survival benefit of about 40%. Figure 5A further shows that a survival benefit of about 24% was observed with doses of 3 or 10 μg / kg (not statistically significant). Figure 5B shows a statistically significant 30-day survival benefit (about 33%) with doses of 30 μg / kg of romiplostim administered once, three or five times a day starting 24 hours after TBI. Repeated doses of 30 μg / kg of romiplostim did not improve survival compared to single doses of romiplostim. Figure 5C shows a statistically significant increase in survival with romiplostim (30 μg / kg), pegfilgrastim (300 μg / kg), and their combination. No difference in survival benefit was observed when comparing the individual agents with their combination. [Figure 5C]Figures 5A and 5B show the survival benefit of romiplostim in the mouse HS-ARS efficacy model. Figure 5A shows that a single dose of 30 or 100 μg / kg of romiplostim administered 24 hours after total body irradiation (TBI) resulted in a statistically significant 30-day survival benefit of about 40%. Figure 5A further shows that a survival benefit of about 24% was observed with doses of 3 or 10 μg / kg (not statistically significant). Figure 5B shows a statistically significant 30-day survival benefit (about 33%) with doses of 30 μg / kg of romiplostim administered once, three or five times a day starting 24 hours after TBI. Repeated doses of 30 μg / kg of romiplostim did not improve survival compared to single doses of romiplostim. Figure 5C shows a statistically significant increase in survival with romiplostim (30 μg / kg), pegfilgrastim (300 μg / kg), and their combination. No difference in survival benefit was observed when comparing the individual agents with their combination. [Figure 6A] Figures 6A-6C show survival curves (by sex and combined sex) for the non-human primate pharmacokinetic / pharmacodynamic study detailed below. Figure 6A shows the male only survival curve, 6B shows the female only survival curve, and 6C shows the combined sex survival curve. Survival (combined sex) was 40% for the irradiated control group and 100% for each of the romiplostim or pegfilgrastim treatment groups. [Figure 6B] Figures 6A-6C show survival curves (by sex and combined sex) for the non-human primate pharmacokinetic / pharmacodynamic study detailed below. Figure 6A shows the male only survival curve, 6B shows the female only survival curve, and 6C shows the combined sex survival curve. Survival (combined sex) was 40% for the irradiated control group and 100% for each of the romiplostim or pegfilgrastim treatment groups. [Figure 6C]Figures 6A-6C show survival curves (by sex and combined sex) for the non-human primate pharmacokinetic / pharmacodynamic study detailed below. Figure 6A shows the male only survival curve, 6B shows the female only survival curve, and 6C shows the combined sex survival curve. Survival (combined sex) was 40% for the irradiated control group and 100% for each of the romiplostim or pegfilgrastim treatment groups. [Figure 7] FIG. 7 shows absolute neutrophil counts (combined males and females) in the non-human primate pharmacokinetic / pharmacodynamic study detailed below. [Figure 8A] Figure 8A shows absolute platelet counts (combined sexes) in the non-human primate pharmacokinetic / pharmacodynamic study detailed below. Figure 8B shows mean platelet volume over time in rhesus monkeys following radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Error bars + / - standard error. Lower error bars have been omitted for clarity. Legend for Figure 8A is the same as for Figure 8B (Group 1, n=4-10; Groups 2-6, n=8). [Figure 8B] Figure 8A shows absolute platelet counts (combined sexes) in the non-human primate pharmacokinetic / pharmacodynamic study detailed below. Figure 8B shows mean platelet volume over time in rhesus monkeys following radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Error bars + / - standard error. Lower error bars have been omitted for clarity. Legend for Figure 8A is the same as for Figure 8B (Group 1, n=4-10; Groups 2-6, n=8). [Figure 9] Figure 9 shows the pharmacokinetics of romiplostim in combined male and female non-human primates. Mean serum concentrations of romiplostim over time in rhesus monkeys. RP: romiplostim; PF: pegfilgrastim. (Group 1, n=10; Groups 2-6, n=8). [Figure 10]Figure 10 shows the mean absolute body weight of rhesus monkeys over time after radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Error bars + / - standard error. Lower error bars omitted for clarity (control, n=4-10; groups 2-6, n=8). [Figure 11A] Figures 11A-11E show selected hematological parameters over time in rhesus monkeys after radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Mean concentrations + / - standard error are shown. Lower error bars have been omitted for clarity. RBC: red blood cells; WBC: white blood cells; (Group 1, n=4-10; Groups 2-6, n=8). [Figure 11B] Figures 11A-11E show selected hematological parameters over time in rhesus monkeys after radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Mean concentrations + / - standard error are shown. Lower error bars have been omitted for clarity. RBC: red blood cells; WBC: white blood cells; (Group 1, n=4-10; Groups 2-6, n=8). [Figure 11C] Figures 11A-11E show selected hematological parameters over time in rhesus monkeys after radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Mean concentrations + / - standard error are shown. Lower error bars have been omitted for clarity. RBC: red blood cells; WBC: white blood cells; (Group 1, n=4-10; Groups 2-6, n=8). [Figure 11D] Figures 11A-11E show selected hematological parameters over time in rhesus monkeys after radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Mean concentrations + / - standard error are shown. Lower error bars have been omitted for clarity. RBC: red blood cells; WBC: white blood cells; (Group 1, n=4-10; Groups 2-6, n=8). [Figure 11E]Figures 11A-11E show selected hematological parameters over time in rhesus monkeys after radiation and treatment with romiplostim (RP), pegfilgrastim (PF), or a combination of both. Mean concentrations + / - standard error are shown. Lower error bars have been omitted for clarity. RBC: red blood cells; WBC: white blood cells; (Group 1, n=4-10; Groups 2-6, n=8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] This specification reports the results of experiments in mouse models of ARS and rhesus monkeys. Those skilled in the art will appreciate that the results demonstrate that romiplostim is effective in treating exposure to many sources. Those skilled in the art will further appreciate that the enhanced efficacy observed with pegfilgrastim and romiplostim in the non-human primate HS-ARS study demonstrates that such enhancement is also present in other conditions characterized by low platelet and / or neutrophil levels. EXAMPLES

[0025] Example 1 Evaluation of romiplostim (Nplate®) in mice as a potential countermeasure to acute radiation syndrome method Mice: Male and female C57BL / 6J (Jackson Laboratories), 11-12 weeks old on day 0. Housed ≤3 per cage. Food and water were provided ad libitum. Beginning 4 days after radiation, all mice received chow and hydrogel softened with supplemental water. Study duration ≤31 days.

[0026] Animals were housed in an internationally accredited facility, the American Association for Accreditation of Laboratory Animal Care, and all study protocols were approved by the Institutional Animal Care and Use Committee.

[0027] Radiation: Conscious mice in individual plastic box holders were exposed to X-rays on day 0 using a Pantak (HF320) unit. ≦6 mice were exposed simultaneously. The LD70 / 30 (70% lethal dose after 30 days) and LD30 / 30 TBI exposures were 680 cGy and 650 cGy, respectively. Dose measurements were performed with a Universal field-class dosimeter controlled by a PTW-UNIDOS microprocessor. Power level (230 kVp or 250 kVp, 5 mA, 2 mm aluminum filtered), distance from the exposure beam, and dose rate of approximately 1 Gy / min were kept constant. Mice were exposed in the morning on a rotating platform (approximately 1 rpm) to ensure uniform dose distribution.

[0028] Subcutaneous (sc) injections of saline (vehicle control) or 3, 10, 30, 100 or 300 μg / kg romiplostim were started 24 hours after irradiation (5 ml / kg at a single site) at romiplostim formulation concentrations of 0, 0.6, 2, 6, 20 or 60 ng / μl. Repeat doses were administered once daily approximately 24 hours thereafter, if applicable.

[0029] For blood analysis (Siemens Advia 2120), blood was collected from the retro-orbital plexus of mice anesthetized with isoflurane using K3EDTA anticoagulant. Hematological examinations at the indicated times, data from both sexes combined, n = 1–11 / group / time point (n varied due to the occurrence of moribundity).

[0030] Serum romiplostim levels were quantified by ELISA (inVentiv Health, Princeton, NJ) with an LLOQ of 0.270-0.401 ng / ml and a ULOQ of 10.9 ng / ml; n = 3 mice / sex / time point / dose group; time points = 1, 4, 12, 24, 48, 72, and 96 hours after romiplostim administration.

[0031] PK analysis: Non-compartmental analysis with Phoenix® WinNonlin® v6.3.

[0032] Kaplan-Meier survival analysis: GraphPad Prism 6 and StrataSE 14.1; data from both sexes combined (21 mice / sex / group).

[0033] Unplanned euthanasia: Consistent criteria based on severity scoring of adverse clinical signs. Euthanasia was performed at 30% weight loss, moribundity, or ≥2 criteria scored as moderate or severe. Adverse signs included: Abnormal appearance (e.g., furring, eye or nasal discharge, vertigo, swelling) Abnormalities in posture or behavior (e.g., hunched back, ataxia) ·Dehydration, hypothermia, decreased activity Weight loss compared to pre-study weight (20-29% is considered moderate, ≥30% is considered severe) Abnormalities in physiological functions (urination, defecation, eating, drinking, breathing).

[0034] conclusion Survival benefit after irradiation: A single dose of 30 μg / kg or 100 μg / kg romiplostim delivered 24 hours after irradiation provided a survival benefit of approximately 40%. Repeated doses of 30 μg / kg romiplostim did not improve survival compared to a single dose.

[0035] Pharmacokinetics (PK): Radiation did not affect romiplostim PK in mice. A 10-fold increase in dose resulted in C max and AUC lastincreased approximately 35-fold. PK increased over a dose-ratio, consistent with nonlinear PK of romiplostim.

[0036] Pharmacodynamics (PD): In irradiated mice treated with romiplostim, platelet levels fell faster compared to controls, but not as low as controls (nadir on day 8 vs. day 10). Platelet levels recovered to near baseline more quickly after romiplostim treatment (day 22 vs. day 29). Platelet volume increased after romiplostim treatment, suggesting a more rapid release of platelets from the bone marrow. Other major hematological parameters were affected by radiation, but the effects of romiplostim were not significant.

[0037] Example 2 Evaluation of romiplostim (Nplate®) and pegfilgrastim (Neulasta®) in mice as potential countermeasures to acute radiation syndrome Rapid reductions in white blood cells, platelets (PLTs), and reticulocytes are characteristic of acute radiation syndrome-hematopoietic syndrome (HS-ARS) and, if left untreated, can lead to serious health consequences, including death. Pegfilgrastim (Neulasta®) has been shown to increase survival in patients exposed to myelosuppressive doses of radiation, but is not currently available as a treatment for thrombocytopenia. Romiplostim (Nplate®), a thrombopoietin receptor agonist, is the first platelet production-stimulating protein approved by the FDA for the treatment of low PLT counts in adults with chronic immune thrombocytopenia. The objective of our study was to evaluate romiplostim as a medical treatment to improve survival and PLT recovery after acute radiation exposure in mice.

[0038] Single or repeated subcutaneous injections of romiplostim administered 24 hours after a LD70 / 30 dose of total body X-irradiation (TBI) in female and male C57BL / 6J mice (n=21M / 21F) increased survival and accelerated PLT recovery. A single dose of 30 μg / kg of romiplostim increased survival by approximately 40%, resulting in a full or maximal benefit. Higher doses of romiplostim (30 or 100 μg / kg) or more frequent administration (3 or 5 days of administration), treatment with pegfilgrastim (300 μg / kg), or combination treatment with pegfilgrastim and romiplostim did not result in any further survival benefit. In conclusion, a single injection of romiplostim administered 24 h after TBI, with or without pegfilgrastim, dramatically increased mouse survival and is a promising radiation countermeasure that promotes PLT recovery.

[0039] Example 3 Efficacy of romiplostim and pegfilgastim on acute radiation-induced thrombocytopenia and neutropenia in non-human primates. Abstract: Neutropenia and thrombocytopenia are recognized as major pathological events resulting from acute radiation exposure and leading to death. 60 Rats were exposed to total body irradiation from a single source and 24 hours later received subcutaneous injections of control, 2.5 mg / kg or 5 mg / kg romiplostim (a thrombopoietin receptor agonist), 0.3 mg / kg pegfilgastim (polyethylene glycol linked to granulocyte colony-stimulating factor (G-CSF) to stimulate neutrophil production), or both romiplostim (5 mg / kg) and pegfilgrastim (0.3 mg / kg). Results: Blood was monitored for 45 days after irradiation. Treatment with pegfilgastim or pegfilgastim / romiplostim resulted in nadir absolute neutrophil counts (ANCs) of 0.43 x 10 for pegfilgastim and 0.43 x 10 for pegfilgastim and 0.43 x 10 for pegfilgastim / romiplostim, respectively. 9 / L, and 0.57 x 10 9 / L, which is 0.09 to 0.13 × 10 9This was a clinically significant improvement compared to the nadir ANC in control irradiated animals, which was 0.01 × 10 / L. Animals receiving pegfilgastim / romiplostim also showed a faster recovery with higher neutrophil counts compared to the pegfilgastim alone group. Severe thrombocytopenia was observed in control animals after irradiation, with a neutrophil count of 20.6 × 10 12 All groups receiving romiplostim or pegfilgastim experienced relatively mild thrombocytopenia, ranging from 47.63 to 69.13 × 10 12 Animals receiving romiplostim / pegfilgastim had the mildest nadir in platelet counts (92.13 × 10 12 10 / L) and subsequently recovered more quickly with higher PLT counts. The effect was enhanced by the combination of romiplostim and pegfilgrastim, with the combination of both drugs producing the smallest platelet and neutrophil nadirs and the most rapid recovery. Although the study was not designed to show a survival benefit, all treated animals survived to 45 days compared with 4 of 10 animals treated with the control.

[0040] The objective of this study was to provide data of adequate quality and completeness to support submissions to the US Food and Drug Administration (FDA) and other regulatory authorities. Therefore, this study was in accordance with the OECD Principles of Good Laboratory Practice (ENV / MC / CHEM(98)17) as endorsed by the US Food and Drug Administration.

[0041] The purpose of this study was to evaluate the efficacy and safety of irradiated patients with or without subcutaneous administration of Neulasta® (pegfilgrastim) (target LD 30 / 45) The objective of this study was to characterize the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the test article Nplate® (romiplostim) after one or two subcutaneous doses in rhesus non-human primates (NHPs). Animals received supportive care including antibiotics, fluids, antiulcer drugs, antiemetics, analgesics, nutritional supplementation, and wound disinfection according to predefined criteria. No blood products were provided.

[0042] Target mortality rate of 30% at 45 days (LD 30 / 45 To achieve this, all animals were 60 550cGy (LD) from a source (Theratron 1000) 30 / 45 ) at a dose rate of approximately 50 cGy / min for a total exposure time of 10 minutes 45 seconds to 11 minutes 13 seconds. On the dosing day, each animal received vehicle, romiplostim, and / or pegfilgrastim by subcutaneous injection. Administration of romiplostim, pegfilgrastim, and / or vehicle began 24 hours ± 2 hours after irradiation. A table showing the irradiation scheme and number of animals in each dose group is shown in Table 1, and the dose administered to each group on each dosing day is shown in Table 2.

[0043] [Table 1]

[0044] [Table 2]

[0045] The animals were aged 3-6 years and weighed 3.7-6.4 kg at the start of treatment.

[0046] Evaluations during the study included mortality checks, clinical observations, body weights and temperatures. Blood samples were taken from all animals for clinical pathology (hematology and coagulation) twice during the pretreatment period and on days 1, 3, 5, pre-dose, 8 (pre-dose), 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 24, 28, 35, 42 and 45 (for hematology) and on days 3, 8 (pre-dose), 10, 14, 17, 21, 24, 28, 35, 42 and 45 (for coagulation). Blood samples for pharmacokinetic evaluation were taken before irradiation and on days 1 and 8 (1, 2, 4, 8, 12, 24, 48 and 72 hours after dosing) and on days 14, 21, 28, 35 and 45. Blood cultures were taken at non-terminal times (if febrile neutropenia was confirmed or infection was suspected) or at unscheduled termination. After the final blood draw on day 45, surviving animals were released from the study.

[0047] During the study, six animals (three males, three females) died, all from Group 1 (vehicle). Clinical signs leading to discontinuation or death included severe hemorrhage, severe hypoactivity, and hypothermia. Three animals (two males, one female) were found dead on days 5, 14, and 18. The male found dead on day 5 had moderate decreased appetite, mild tremors, and bloody stools the day before death. The remaining three animals were euthanized on days 15 and 17 prior to scheduled discontinuation.

[0048] In control animals, subcutaneous administration of vehicle after total body irradiation of 550 cGy resulted in 60% mortality (LD 60 / 45 ), but no deaths were reported in either the romiplostim or pegfilgrastim groups. This mortality rate was within the target LD 30 / 45 It was higher.

[0049] Clinical signs, weight changes, and temperature changes expected in a radiation model were observed to be less severe and / or occurring in the treatment groups compared to the vehicle control group, with Group 6 showing earlier recovery and better weight recovery compared to the other treatment groups. As expected, there were no significant changes in coagulation parameters in the control and treatment groups following irradiation.

[0050] Hematological parameters (RBC, HCT, WBC, LYMPH, MONO) showed the expected decrease after irradiation, with all treatment groups showing a faster and better recovery compared to the control group. In general, Group 6 (receiving both romiplostim and pegfilgrastim) showed a faster recovery and milder radiation-induced effects compared to the other treatment groups. NEUT (neutrophils) and PLT (platelet) levels decreased as expected after irradiation. Groups 4 and 6 (both receiving pegfilgrastim) showed a faster recovery of NEUT compared to the remaining treatment groups (as would be expected since pegfilgrastim stimulates neutrophil production), whereas Group 6 (receiving both romiplostim and pegfilgrastim) showed a faster recovery of NEUT compared to the remaining treatment groups (pegfilgrastim alone, 0.51 × 10 9 / L, day 13) compared with a faster 13 day count (1.25 × 10 9 / L). The nadir PLT values ​​were not significantly worse in the treatment groups, and in group 6, the cell count was 92.1 × 10 9 / L (minimum on day 11) and showed the mildest PLT nadir and the earliest recovery (105.5 × 10 9 / L, day 12). These results were significantly different from groups 1 and 4 (p ≤ 0.01). Group 5 animals received two doses of romiplostim, but did not show significant differences in NEUT or PLT counts at nadir or onset of recovery when compared to group 3 (one dose of romiplostim). Based on these results, coadministration of romiplostim and pegfilgrastim appears to have a synergistic effect on NEUT and PLT.

[0051] There was considerable variability in serum romiplostim concentrations between animals in some dose groups, which resulted in considerable variability in PK parameters. max and AUC last Romiplostim serum exposure based on the mean C was increased by over 80% in females and decreased by approximately 15% in males in Group 6 compared to Group 3. Increasing the romiplostim dose from 2.5 mg / kg to 5 mg / kg resulted in a disproportionate increase in romiplostim serum exposure. There was a tendency for romiplostim to accumulate with repeated dosing, with a higher mean C in females receiving Dose 2 compared to Dose 1. max is about 43% larger, and AUC (0~72時間) The trend was also more evident in females, as PLT was approximately 36% greater in females. A significant increase (p≦0.01) in PLT was observed only on days 14 and 15 when compared to group 3.

[0052] Rationale for dose level setting of romiplostim and pegfilgrastim The dose level selected for romiplostim was based on previous single and repeat dose toxicity studies in normal healthy NHPs. The dose range selected was expected to achieve a clinically relevant increase in platelets. The highest dose selected was the maximum dose that could be administered based on the constraints of the dose volume and dosage formulation. The dose level selected for pegfilgrastim was based on previous NHP studies showing clinically relevant increases in neutrophils supporting the licensure of this product for acute radiation syndrome (ARS).

[0053] Appropriateness of exposure level The 550 cGy dose level was chosen to be approximately LD 100, based on available data from studies conducted at Citoxlab North America and clinical history data. 30 / 45 This dose was chosen to provide an assessment of PK / PD in the setting of radiation, but also to provide a sufficient level of survival to provide a meaningful data set.

[0054] Reasons for choosing the route of administration The subcutaneous route is the intended route of administration of the test article in humans.

[0055] Reasons for choosing the test system The rhesus monkey (Macaca mulatta) was chosen because it is an acceptable species for PD and PK profiling. Extensive post-irradiation clinical history data is also available for rhesus monkeys, supporting the use of this species for this study.

[0056] Appropriateness of the number of animals selected The number of animals used in the study was considered the minimum necessary to achieve the study objectives based on regulatory requirements, statistical power, and / or availability of clinical history data and was within IACUC-approved guidelines. Characteristics of test substances, diluents and vehicle / reference substances Test substance 1 Identification: Romiplostim (lyophilized powder in vials, 600 μg / vial; 0.5 mg / mL when reconstituted with 1.2 mL of sterile water for injection; also known as Romiplostim and AMG531) Batch number: 0010294965 Retest date: October 31, 2018 Storage conditions: Refrigerate, 2-8℃, and protect from light. Test substance 2 Identification: Pegfilgrastim (also known as pegfilgrastim) Lot number: 0010236097 Expiration / Retest Date: March 2018 Storage conditions: Refrigerate, 2-8℃, and protect from light. Diluent for romiplostim Identification: Sterile Water for Injection, USP Lot: W7J18A0 Expiration date: October 2018 Vehicle / reference substance for romiplostim Identification: 0.9% Sodium Chloride for Injection, USP Lot: W7B10C1 Expiration date: May 2018

[0057] Preparation of dosage forms Romiplostim test article formulations were prepared using sterile reagents and clean techniques in a laminar flow hood. Dosing solutions were protected from light and kept on wet ice or refrigerated until administration, which was administered within 20 hours after preparation.

[0058] On the day of use, romiplostim was reconstituted to a concentration of 500 μg / mL (0.5 mg / mL) by adding 1.2 mL of sterile water for injection to each vial via syringe. The vials were gently swirled and inverted to reconstitute, then inspected to ensure the solution was clear and colorless, after which the volumes of all reconstituted vials were pooled into a sterile container. The analytical chemistry sample was then removed, after which the sterile container was capped with a rubber septum.

[0059] The pegfilgrastim test substance formulation was allowed to come to room temperature (protected from light) at least 30 minutes prior to administration, pooled in a sterile container with a rubber septum, and used as is.

[0060] Dosage analysis of formulated romiplostim A concentration analysis of the reconstituted test article romiplostim was performed for this study. On the day of dosing, samples were taken from the formulated romiplostim dosing solution (1 mL in duplicate) and a concentration analysis was performed. Samples were kept refrigerated (target 4°C) prior to analysis. Homogeneity analysis was not performed as the formulated product is an aqueous solution. A comparable sample was taken from the vehicle formulation. Dosage formulation concentrations were confirmed to be within ±10% acceptance criteria using a validated method. Pegfilgrastim was used as supplied and therefore no dose analysis of the formulation was performed.

[0061] Administration of dosage formulation Romiplostim and vehicle / reference article were administered by subcutaneous injection in the upper back and / or lumbar region using a 25 gauge needle attached to a syringe as detailed above in the study design tables (Tables 1 and 2). The actual volume administered to each animal was calculated and adjusted based on each animal's most recent actual body weight.

[0062] Injections were given at multiple sites in the upper back and / or lower back. Romiplostim and / or vehicle / reference substance were administered separately by subcutaneous injection over six sites on days 1 and 8. Pegfilgrastim was administered at a separate seventh site (above the level of sites 1 and 2) on the upper back on both days 1 and 8.

[0063] Prior to dosing, injection sites were shaved and tattooed (approximately 1.5 cm x 1.5 cm square per site). Only animals receiving pegfilgrastim had the seventh dosing site tattooed. Dosing syringes were filled from a sterile container and warmed immediately prior to injection into each animal.

[0064] Occasionally during the dosing period, individual animals had small amounts of dosing formulation leaking from the dosing site or were administered intradermally as shown in Table 3.

[0065] [Table 3]

[0066] On day 1, animal 4602 (Group 4, pegfilgrastim) received vehicle and pegfilgrastim in amounts calculated based on the animal's weight (51 mL vehicle instead of 37 mL, and 0.15 mL pegfilgrastim instead of 0.11 mL, +36% of nominal dose). Subsequent clinical evaluation and hematological analysis of the animal after dosing showed no adverse effects from the increased volume of fluid administered subcutaneously, and the animal's neutrophil count was comparable to other animals in the group. The animal's condition and hematological results were comparable to other animals in the group, and the increased dose had minimal or no effect on the animal's condition or study objectives, as the volumes were considered to be very small compared to the doses administered.

[0067] Test system Fifty rhesus monkeys (25 males, 25 females), including two spare animals / sex, were received from Kunming Biomed International Ltd (China) and transferred to the laboratory on January 15, 2018. The age of the animals at the start of treatment ranged from 3 to 6 years. Body weights ranged from 3.8 to 6.4 kg and 3.7 to 5.5 kg for males and females, respectively.

[0068] Total body irradiation Animals in groups 1–6 were treated with Co at a dose rate of approximately 50 cGy / min. 60 Animals were exposed to a single uniform total-body dose of radiation from a source (Theratron 1000). A nominal dose of 550 cGy was provided to a radiation physicist to calculate exposure times. To ensure accuracy of the radiation treatment plan, exposure times and assigned radiation doses were verified by two different reviewers prior to animal irradiation. Dosimetry analysis showed that mean dosimetry was consistent across groups, ranging from 514 to 562 cGy for the Nanodot and 541 to 546 cGy for the Farmer ionization chamber.

[0069] Prior to the day of irradiation, the radiation dose was calibrated using an acrylic phantom placed in the same experimental setup used for the animal irradiation. Animals were irradiated in random replicates. Exposure time for each animal was calculated individually based on body dimensions.

[0070] Two dosimeters (Landauer, Inc. Model Nanodot) were placed on each animal prior to total body irradiation to quantify the dose. On the day of irradiation, one dosimeter was placed on the midplane at approximately the xiphoid level and the second was placed at the corresponding level in the dorsal region below the interscapular region. The dosimeter was placed under the gel bolus approximately 5 mm (superflav) and secured with a bandage. The dosimeter was returned to Landauer for estimation of exposure. Phantom dosimetry, dose rate, duration of irradiation and actual irradiation time were recorded for each animal.

[0071] To quantify radiation dose, a Farmer ionization chamber was connected to an electrometer and included in each radiation treatment field. The energy recorded by the electrometer was converted to radiation dose and included in the study report. Ambient temperature and atmospheric pressure were recorded after each exposure for real-time dosimetry calculations.

[0072] Animals were fasted overnight prior to whole body irradiation, fed after irradiation in the irradiation facility (ie, each animal was given a banana after irradiation), and fed again immediately after returning to the facility.

[0073] Prior to irradiation, each animal was anesthetized with ketamine (10 mg / kg, IM) and transferred to the irradiation treatment room. Additional anesthetic drugs were administered if necessary. Soft music was provided in the treatment room to reduce animal stress. The animals were placed in a horizontal position for total body irradiation. The animal's position was confirmed by a linear marker installed in the treatment room.

[0074] To generate a uniform dose distribution, the treatment was divided into two parts. First, animals received half the dose via anterior-posterior (AP) irradiation. The second half of the dose was delivered via posteroanterior (PA) irradiation. Each irradiated animal received fluid therapy (10 mL / kg lactated Ringer's solution; IV) after irradiation to help manage hypotension.

[0075] In-life observations mortality rate Mortality checks were recorded simultaneously with cage-side observations for clinical signs during all phases of the study. Some animals with irreversible pain or dysfunction were euthanized when possible. Clinical pathology blood samples, blood cultures, and PK samples were taken from animals euthanized early when possible. Data collected at unscheduled euthanasia were excluded from any group mean summaries.

[0076] Hematology / Pharmacodynamics (PD) and Coagulation Clinical pathology evaluation (hematology and coagulation tests) was performed on all animals as described below. Blood samples were taken from the femoral vein (cephalic or saphenous vein was also used if necessary) from non-fasting animals. The femoral vein was used to obtain blood samples from animals with platelet counts >80×10 9 Except for 100 / L, none were used between days 6 and 24. Prior to all blood collections (starting after irradiation), the blood collection site was wiped with 70% isopropyl alcohol followed by a 4% chlorhexidine gluconate wipe. After each blood collection, a small amount of gauze soaked in 70% isopropyl alcohol was held in place and manual pressure was applied to the collection site. Once complete hemostasis was achieved, the area around the bleeding site was again wiped with 70% isopropyl alcohol.

[0077] As part of the test facility health and safety standard operating procedures, in addition to scheduled collections, blood was drawn (5 mL each) from animals 6102 and 6501 in group 6 on days 10 and 14, respectively, due to injuries sustained by technicians to these animals.

[0078] a) Hematology / PD Hematological evaluations were performed on all surviving animals twice during the pretreatment period (days -12 to -5), on days 1, 3, and 5 (pre-dose), and on days 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 24, 28, 35, 42, and 45 after exposure to radiation.

[0079] Except for pre-procedure sampling, no hematology or coagulation tests were repeated to avoid potential influence on outcome. Animals permitted for unscheduled euthanasia were attempted to have blood drawn for hematology and coagulation tests only if a blood draw had not already been performed that day.

[0080] The following parameters were measured on blood samples collected in tubes containing K3EDTA as the anticoagulant: The target volume of blood collected for each sample was 0.5 mL. Hematocrit Platelet Distribution Width Hemoglobin Platelet count Hemoglobin distribution width Plateletcrit / Thrombocrit Mean corpuscular hemoglobin Red blood cell count Mean corpuscular hemoglobin concentration Red blood cell distribution width Reticulocyte count (absolute and relative) Mean corpuscular volume White blood cell count (WBC) Mean platelet volume White blood cell differential (absolute value + relative value)

[0081] b) coagulation Samples (1.0 mL in citrate tubes) were taken once during the pretreatment period, on day 3 (pre-dose), and on days 8, 10, 14, 17, 21, 24, 28, 35, 42 and 45. The following parameters were recorded: Activated partial thromboplastin time Prothrombin time Fibrinogen Appearance of the sample (abnormal)

[0082] Remaining blood or plasma after hematology or coagulation analysis, or unanalyzed samples (depending on sample quality), were stored at -70°C until shipment to the sponsor for possible future analysis or discarded after completion of the report.

[0083] c) Blood culture When febrile neutropenia was confirmed (absolute neutrophil count <0.5 × 10 9 Blood was drawn from an appropriate vein for blood cultures (target volume of 1 ml during the study and 10 ml at euthanasia) at the time of study (≥ 100 / L, rectal temperature ≥ 104°F / 40.0°C) or at the time of euthanasia, but not more than three times per week from animals (except at the time of euthanasia). Blood was also drawn and blood cultures were obtained if clinical signs and physical examination on other days suggested sepsis to the veterinarian.

[0084] Blood samples were inoculated directly from the syringe into blood culture vials. The volume of the blood sample, determined from the syringe graduations, was recorded. Inoculated blood vials were stored at room temperature and shipped to IDEXX as soon as possible after collection, and were received by IDEXX within 3 days. Each sample was labeled with the animal number, collection date, specimen type, storage conditions, blood volume, and study number. Aerobic and anaerobic cultures were performed by IDEXX. Bacterial positive blood cultures were subjected to antibiotic susceptibility evaluation.

[0085] For non-terminally performed blood cultures, blood samples (target volume 1 mL) were collected from the cephalic vein (or another suitable vessel). For terminally performed blood cultures, blood samples (target volume 10 mL) were collected from the femoral vein (or another suitable vessel). For planned and unplanned euthanasia, the skin was shaved and prepared aseptically with chlorhexidine gluconate 4% and alcohol 70%. Samples were collected using aseptic procedures and transferred to culture medium at IDEXX. Aerobic and anaerobic cultures were performed at IDEXX according to their procedures.

[0086] Pharmacokinetics (PK) Blood samples for romiplostim serum concentration measurements (target of 1.0 mL each) were collected from all animals using a uniform procedure described below, with precise records of each collection time, and provided to SRI International for subsequent pharmacokinetic analysis. Samples collected from animals administered romiplostim were then analyzed for romiplostim serum concentrations. Representative samples from animals in groups 1 and 4, which received only the reference substance / vehicle and pegfilgrastim, respectively, were analyzed as reference controls.

[0087] Before irradiation (day -2) Day 1: For all groups, before injection and 1, 2, 4, 8, and 12 hours after injection Day 2 (24±1 hours after administration on Day 1) Day 3 (48±1 hour after administration on Day 1) Day 4 (72±1 hours after administration on Day 1) Day 8: in all groups, before injection and 1, 2, 4, 8, and 12 hours after injection Day 9 (24 ± 1 hour after administration on day 8) Day 10 (48±1 hour after administration on day 8) Day 11 (72±1 hours after administration on day 8) Approximately every week thereafter (14th, 21st, 28th, and 35th days) Day 45

[0088] Blood samples for PK were collected via the femoral vein (except days 6-24), cephalic vein, or saphenous vein. An appropriate amount of blood was placed in serum separator tubes (SST), gently inverted 5 times, and allowed to clot for 30-60 minutes at room temperature. Samples were then centrifuged for 15 minutes at target (4°C, 1500g settings). Serum was removed, aliquoted into two cryovials, placed on dry ice, and transferred to a freezer set at -70°C within 2 hours of blood collection. Serum samples were sent weekly on dry ice to Raquica Butler at Syneos Health for analysis. Aliquots from the same sample were not sent in the same shipment. Samples were stored in a freezer set to maintain temperatures below -60°C.

[0089] Serum samples were analyzed for romiplostim concentrations by Syneos Health using their validated sandwich immunoassay assay. Analysis of pre-irradiation (day -2) samples was not performed because analysis of the day 1 pre-injection samples was sufficient to show that no romiplostim was present in each animal prior to injection on day 1.

[0090] Samples from groups 1 and 4 were analyzed only at the following time points and served as representative reference control samples. Day 1: 4 hours after injection Day 2: 24 hours after injection Day 8: 4 hours after injection Samples from Group 1 at days 1 and 8 and other time points (not listed above) were used in bioanalytical methods for post-irradiation selectivity testing.

[0091] A portion of the pharmacokinetic samples was reanalyzed to assess the reproducibility of the bioanalytical method (incurred sample re-analysis). The incurred sample re-analysis values ​​were not used for the pharmacokinetic analysis.

[0092] Serum drug concentration data were analyzed using Phoenix® WinNonlin® software version 6.3 to perform non-compartmental modeling. Administered doses were entered into the program as mg / kg, so no additional correction for the individual body weights of the animals was required.

[0093] Parameters and constants determined using noncompartmental analysis were included at maximum serum concentration (C max ), time to reach maximum serum concentration (T max ), area under the serum concentration-time curve (AUC), and elimination half-life (t 1 / 2 ) was included.

[0094] Data Handling and Reporting Data Capture Data were collected for at least 2 weeks prior to the start of treatment in this study. Data from 1 week prior to the start of treatment were considered sufficient for baseline comparison. Any data that may have been collected prior to this period were retained in the study files.

[0095] The following data capture systems were used during the conduct of this study: Collecting data during life: Provantis In-Life Module 9.3.0.0 Clinical Pathology: Provantis Clinical Pathology Module 9.3.0.0 Pathology:Provantis Pathology Module 9.3.0.0 Analysis:EZChrom Elite 3.3.1 Biological analysis: Softmax Pro 5.2 Pharmacokinetics: Phoenix® WinNonlin 6.3 Statistical analysis: SAS 9.2 and 9.3 Indoor environment:Siemens Insight 3.13

[0096] Data analysis Data recorded in unscheduled cases were reported separately but are not included in the analyses below.

[0097] Numerical data obtained during the conduct of the study were subjected to calculation and reported as group means, medians and standard deviations. Non-numerical data obtained during the conduct of the study were reported as individual results and / or group occurrences.

[0098] Each pairwise comparison of interest was performed two-sided with a significance level of 5%, and significant results were reported as either p≦0.001, p≦0.01, or p≦0.05, where p represents the observed probability.

[0099] Survival data Statistical intergroup comparisons of mortality data were performed using Fisher's exact test for sex pooled across all groups. Overall intergroup comparisons showed significant differences in mortality (p ≤ 0.05). Mortality in group 1 was compared with each of the other groups using Fisher's exact test. Because there was no mortality outside group 1, Fisher's exact test could not be performed for other pairwise group comparisons of interest.

[0100] Numerical Data Plots of absolute values ​​of body weight, body weight change, temperature, hematological parameters [white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), platelets (PLT), hematocrit, red blood cells (RBC) and reticulocytes (RETIC)] for males, females and pooled sexes by treatment mean [± standard error (SEM)] and median over time are shown.

[0101] Numerical dataset submitted for statistical group comparison: Weight and weight changes Body temperature Clinical Pathology (Haematology: all parameters including WBC, NEUT, LYMPH, PLT, RBC, RETIC, Coagulation: Activated Partial Thromboplastin Time, Prothrombin Time, Fibrinogen)

[0102] The following statistical group comparisons were performed on the numerical data sets listed above, optionally in two phases. In the first phase, the treatment effect of romiplostim only was evaluated. In the second phase, the treatment effects of romiplostim, pegfilgrastim, and their possible interactions were evaluated.

[0103] Phase 1 Comparison Romiplostim treatment effects were also evaluated using the following statistical comparisons: Comparisons included only results from Groups 1, 2, 3, and 5.

[0104] In each case, a univariate two-way analysis of variance (ANOVA) was performed. The model included group, sex, and the interaction group x sex as fixed effects.

[0105] To assess the significance of variance heterogeneity among the levels of the fixed effects considered, three heteroscedastic ANOVA models were fitted by modeling different covariance matrices for each level of 1-group, 2-sex, and 3-group and sex combinations, respectively. If the final Hessian was not positively determined for the model, or if computational limitations / convergence problems were encountered when fitting the model, the model results were not considered in the following steps. Among the well-fitting heteroscedastic models, the one with the lowest corrected Akaike information criterion (AICC) was selected and compared with the reduced homoscedastic ANOVA model (a model with a common covariance matrix for all levels of group and sex).

[0106] A likelihood ratio test based on the REML estimation results was used to test whether the selected heteroscedastic model (full model) provided a significantly better fit than the homoscedastic model (reduced model). If the likelihood ratio test was not significant (p>0.05), the results from the reduced model were retained; otherwise, the results from the full model were retained. The Kenward-Roger method was used to calculate the denominator degrees of freedom for tests involving fixed effects. The alternative Satterthwaite method was used when the Kenward-Roger method was not applicable.

[0107] If the retained model indicated that the fixed effects interactions were not significant from the retained ANOVA model (p>0.10), group effects were assessed with least squares means pooled across sexes using ANOVA F-tests. If the interactions were significant (p≦0.10), group effects were assessed for each sex with least squares means using ANOVA F-tests.

[0108] Where group effects were significant (p≦0.05), t-tests were used to make objective pairwise comparisons with least squares means: vehicle control Group 1 vs. each of the other groups, and Group 3 vs. Group 5.

[0109] Phase 2 Comparison The following statistical comparisons were used to evaluate the treatment effects of romiplostim, pegfilgrastim, and their possible interactions, and were therefore performed including only the results of Groups 1, 3, 4, and 6.

[0110] A three-way analysis of variance (ANOVA) was performed, which included as fixed effects romiplostim (two levels: 0 and 5 mg / kg on day 1), pegfilgrastim (two levels: 0 and 0.3 mg / kg on days 1 and 8), sex, and their two-way and three-way interactions.

[0111] To assess the significance of variance heterogeneity among the levels of the fixed effects considered, heteroscedastic ANOVA models were fitted, modeling a different covariance matrix for each level of each fixed effect and each level of the combination of fixed effects. If the final Hessian was not positively determined for the model, or if computational limitations / convergence problems were encountered when fitting the model, the model results were considered in the following steps. Among the heteroscedastic models considered, the one with the lowest corrected Akaike Information Criterion (AICC) was selected and compared with a reduced homoscedastic ANOVA model (a model with a common covariance matrix for all levels of the fixed effects).

[0112] A likelihood ratio test based on the REML estimation results was used to test whether the selected heteroscedastic model (full model) provided a significantly better fit than the homoscedastic model (reduced model). If the likelihood ratio test was not significant (p>0.05), the results from the reduced model were retained; otherwise, the results from the full model were retained. The Kenward-Roger method was used to calculate the denominator degrees of freedom for tests involving fixed effects. The alternative Satterthwaite method was used when the Kenward-Roger method was not applicable.

[0113] If the results of the retained three-way ANOVA indicated that the triple interaction was significant (p ≤ 0.10), the romiplostim effect was assessed within each combination of pegfilgrastim and sex, and the pegfilgrastim effect was assessed within each combination of romiplostim and sex, using least squares means t tests.

[0114] If no significant triple interactions were found (p>0.10), the table below (Table 4) details how the effects of romiplostim and pegfilgrastim were evaluated depending on which interactions were significant (p≦0.10).

[0115] [Table 4]

[0116] As described in the table above, the effects of romiplostim and pegfilgrastim were evaluated using t-tests with the respective least squares means from the ANOVA model. The significance of the effects of romiplostim and pegfilgrastim was evaluated at the 5% alpha level.

[0117] result Analysis of test substance concentrations The absence of test article concentrations (romiplostim) in vehicle samples (Groups 1, 2, 4) was demonstrated. Agreement with the nominal concentrations of the test article was within ±10% from the nominal concentration (-7.4% to -0.2% from the nominal concentration) as detailed in the table below.

[0118] [Table 5]

[0119] mortality rate During the study, six animals (three males and three females) died, all from group 1 (vehicle). Figures 6A-6C show the survival curves (by sex and combined males and females), indicating that the irradiation level in this study was approximately LD 60 / 45 This shows that.

[0120] Loss of appetite, hunching over, signs of bleeding (petechia), and diarrhea were seen in both sexes after irradiation.

[0121] Anorexia was observed in all groups, with no general differences in incidence or severity between groups. Diarrhea (liquid stool) was observed on days 2-17 during the expected period of radiation-induced diarrhea. Curved back was observed in all groups, with the highest incidence in group 1. Males appeared to be less affected than females (only one male in group 3 was observed in this case on day 1). Petechia was observed in all groups, with a slightly lower incidence in group 6 animals.

[0122] Febrile neutropenia (absolute neutrophil count <0.5 × 10 9 Three animals had blood cultures performed because of suspected infection with bronchitis (< 40°C; P < 0.05; 1 / L; P < 0.05), and the results were negative.

[0123] Other clinical signs (including but not limited to skin wounds with or without discharge, thin hair coat, red liquid material, red discharge from the vulva) were related to the experimental procedures (prophylactic subcutaneous injection of preparations that may cause abscesses and wounds, thin hair coat due to shaving at dosimeter placement or blood sampling sites) or were seen in laboratory-bred members during estrus and / or were not considered related to dose level in incidence or severity.

[0124] Hematology (Pharmacodynamics) RBC and HCT values ​​declined after irradiation and began to recover in all treatment groups by day 17. Group 6 remained slightly higher and Group 4 slightly lower compared to the other treatment groups.

[0125] RETIC (reticulocyte) levels were all decreased after irradiation with no differences between groups, including the control group. Recovery of RETIC counts was improved in the treatment groups (i.e., romiplostim and / or pegfilgrastim) when compared to the control group, but there were no significant differences between treatments.

[0126] WBC, LYMPH and MONO values ​​decreased after irradiation and started to recover gradually from about day 17. LYMPH showed higher counts in all treatment groups (i.e., groups 2-6) compared to group 1 and were generally comparable to each other. WBC and MONO had higher counts in all treatment groups (i.e., group 6 showed the fastest recovery) compared to group 1.

[0127] The NEUT (neutrophil) level decreased after irradiation, reaching a nadir (0.13 × 10 9 / L), and recovery began on the 15th day (0.15 × 10 9 / L), the 4th and 6th groups were 0.09 to 0.13 × 10 9 / L range, respectively. 9 / L and 0.56×10 9 / L, showing milder nadir values. The nadir values ​​of Groups 4 and 6 were significantly different only from Groups 1 and 3 (p ≦ 0.05). Group 6 (pegfilgrastim and romiplostim) showed a higher cell count (1.25 × 10 9 / L) showed the fastest recovery, followed by Group 4 (pegfilgrastim) at 0.51 × 10 on day 13. 9 / L. Interestingly, Groups 4 and 6 were also seen to exhibit a transient increase in NEUT on days 8-9 prior to the nadir. Animals in Group 5 received two doses of romiplostim and were not significantly different in either neutrophil nadir or onset of recovery when compared to Group 3 (which received a single dose of romiplostim). The median neutrophil counts for each group (males and females combined) are shown in Figure 7, and the neutrophil nadir and recovery days are shown in Table 7.

[0128] [Table 6]

[0129] A: Statistically significant difference from group 1. B: Statistically significant difference from group 3. C: Statistically significant difference from group 4.

[0130] In group 1, severe thrombocytopenia was observed after irradiation, with a nadir PLT value of 20.6 × 10 9 The groups receiving either romiplostim or pegfilgrastim (i.e., groups 2 to 5) had relatively little thrombocytopenia, ranging from 47.6 to 71.0 × 10 9 / L, whereas the group receiving both romiplostim and pegfilgrastim (i.e., group 6) had the mildest platelet count nadir on day 11 (92.1 × 10 9 / L), which was significantly different compared to groups 1 and 4 (p≦0.01). Group 6 also subsequently showed a higher PLT count (105.5×10 9 / L, day 12) showed a faster recovery. Regarding the nadir, the cell counts observed in groups 3 (one dose of romiplostim) and 5 (two doses of romiplostim) were very similar (71 × 10 9 / L and 69.1×10 9 / L), which was significantly different from Group 1 only (p ≤ 0.05). A similar profile was observed at the onset of recovery in Groups 3 and 5 (81.8 × 10 9 / L and 74.1×10 9 / L). No significant differences were observed between groups 3 and 5. The median platelet counts (combined males and females) are shown in Figure 8, and the platelet nadir and recovery (combined males and females) are shown in Table 8.

[0131] [Table 7]

[0132] A: Statistically significant difference from group 1. B: Statistically significant difference from group 3. C: Statistically significant difference from group 4.

[0133] Some variation was observed during the remainder of the observation period, but by day 45 all groups were similar to baseline values. There appeared to be a synergistic effect on neutrophil and platelet recovery when romiplostim and pegfilgrastim were administered together. No changes in PT and aPTT were observed during the study. Although there were occasional increases in individual results, there were no correlating changes in other coagulation parameters, and this increase may be due to sample collection and handling rather than biological changes.

[0134] coagulation Neither romiplostim nor pegfilgrastim administration, nor their combination, affected secondary coagulation endpoints, as no statistically significant treatment-related differences were observed in prothrombin time (PT) and activated partial thromboplastin time (aPTT) in the treatment groups compared to the control group. Fibrinogen concentrations were slightly elevated in all groups (including control) and were significantly higher in the groups receiving pegfilgrastim (i.e., groups 4 and 6) than in the control group (p<0.05) on day 3. Values ​​returned to baseline by day 8, after which there was a second increase, peaking between days 14 and 17. Vehicle-treated animals (group 1) had the highest fibrinogen concentrations, with peak values ​​on day 17, suggesting greater inflammation in this group.

[0135] Pharmacokinetics Table 9 shows the pharmacokinetics of romiplostim in rhesus monkeys. Means (+ / - standard error) of selected pharmacokinetic parameters are shown. max :Maximum blood concentration;T max : Time to reach maximum blood concentration; AUC: Area under curve / exposure; T 1 / 2 : Elimination half-life. RP: romiplostim; PF: pegfilgrastim. (Group 1, n=10; Groups 2-6, n=8).

[0136] [Table 8]

[0137] There was considerable variability in serum romiplostim concentrations among animals in several treatment groups, resulting in considerable variability in the resulting PK parameters.

[0138] Romiplostim significantly increased mean T after a single dose in groups 2, 3, and 6. max The mean terminal phase t was absorbed in about 5 to 12 hours. In Groups 2, 3, and 6, it was absorbed in about 37 to 74 hours after the single dose, and in Group 5, it was absorbed in about 52 to 63 hours after the second dose. 1 / 2 Romiplostim disappeared.

[0139] Serum romiplostim exposure increased disproportionately with increasing dose from 2.5 mg / kg (Group 2) to 5 mg / kg (Group 3). max and AUC last (Group 3) with a two-fold dose increase from 2.5 mg / kg to 5 mg / kg was approximately 2.3-fold and 2.5-fold, respectively, in males and 1.3-fold and 1.4-fold, respectively, in females, greater than those measured at 2.5 mg / kg (Group 2). max were 990 ± 402 ng / ml (males) and 1,425 ± 1,001 ng / ml (females) at 2.5 mg / kg (group 2) and 2,263 ± 1,790 ng / ml (males) and 1,782 ± 1,421 ng / ml (females) at 5 mg / kg (group 3). last The mean plasma concentrations were 30,147 ± 11,164 hr·ng / ml (males) and 33,501 ± 21,232 hr·ng / ml (females) at 2.5 mg / kg (group 2) and 74,957 ± 44,396 hr·ng / ml (males) and 45,260 ± 28,652 hr·ng / ml (females) at 5.0 mg / kg (group 3).

[0140] Serum romiplostim exposure was compared between the group administered 5 mg / kg romiplostim with concomitant administration of pegfilgrastim and the group administered 5 mg / kg romiplostim without concomitant administration of pegfilgrastim (Groups 3 and 6, respectively). Mean C max and AUC last were decreased by approximately 15% in males and increased by more than 80% in females in Group 6 with pegfilgrastim compared to these parameters without pegfilgrastim in Group 3. In Group 6 with pegfilgrastim, the mean C max was 1,921 ± 981 ng / ml (males) and 3,255 ± 850 ng / ml (females), and the mean AUC lastThe serum exposure of romiplostim was 64,150 ± 22,719 hr·ng / ml (males) and 85,233 ± 18,387 hr·ng / ml (females). This suggests that the exposure of romiplostim was greater (>80%) when coadministered with pegfilgrastim in females. In addition, the serum exposure of romiplostim in females in group 3 was approximately 21% (C max ) and about 40% (AUC last ) was low and therefore may have contributed to the greater difference in serum exposure in females when pegfilgrastim was added to the regimen versus when it was not (i.e., Group 6 vs. Group 3).

[0141] For the two doses of romiplostim (days 1 and 8, group 5), the overall mean T max The overall mean C after both doses was 8.0 hours in two of the males, 180.0 and 176.0 hours in the other two males, and 176.0 ± 3.3 hours in the females. max The overall mean AUC last The mean C was 153,103 ± 53,375 hr·ng / ml (males) and 180,394 ± 33,936 hr·ng / ml (females). max(投与1) was 3,186±1,951ng / ml (males) and 3,345±291ng / ml (females), with mean C max(投与2) The mean AUC (0~72時間、投与1) were 71,789 ± 41,898 hr ng / ml (males) and 66,065 ± 8,732 hr ng / ml (females), and the mean AUC (0~72時間、投与2) The mean plasma concentrations were 55,593 ± 36,237 hr·ng / ml (males) and 90,113 ± 24,604 hr·ng / ml (females). There was a tendency for accumulation of romiplostim with repeated dosing, which was more evident in females. max and AUC (0~72時間) ) were approximately 43% and 36% greater in females after dose 2 compared with dose 1, respectively. In males, the mean C maxand AUC (0~72時間) were approximately 11% higher and 23% lower, respectively. However, the variability in serum romiplostim concentrations was more pronounced in males than in females in Group 5. Measurable serum concentrations of romiplostim were observed before dose 2 in most animals in Group 5, which may have contributed to the greater serum exposure after dose 2 in this group, especially in females.

[0142] Consideration Romiplostim (at various dose levels or time points), pegfilgrastim, or a combination of romiplostim and pegfilgrastim administered subcutaneously after total body irradiation of 550 cGy resulted in 60% mortality in control animals, but no mortality was observed in either the romiplostim or pegfilgrastim groups.

[0143] Clinical signs, weight changes, and temperature changes predicted by the radiation model were less severe and / or less prevalent in the treatment groups, with Group 6 showing earlier recovery and greater body weight compared to the other treatment groups. There were no changes in coagulation parameters.

[0144] Hematological parameters (RBC, HCT, WBC, LYMPH, MONO) showed the expected decrease after irradiation, with all treatment groups showing a faster and better recovery compared to the control group (Figures 11A-11E). In general, Group 6 (administered both romiplostim and pegfilgrastim) was found to show a faster recovery with better outcomes compared to the other treatment groups. NEUT and PLT levels decreased as expected after irradiation. Groups 4 and 6 (both administered pegfilgrastim) showed a faster recovery compared to the remaining treatment groups (as expected since pegfilgrastim stimulates myeloid lineages), while Group 6 (administered both romiplostim and pegfilgrastim) showed a slower recovery of 0.51 x 10 NEUT compared to the remaining treatment groups. 9 At day 13, a higher cell count (1.25 × 10 9Although PLT nadirs were less severe in the treatment groups, the group with the mildest PLT nadir and fastest recovery was group 6 (treated with both romiplostim and pegfilgrastim), with a cell count of 92.1 × 10 9 / L (minimum value on day 11) and 105.5 × 10 9 / L (start of recovery on day 12), which was significantly different from groups 1 and 4 (p ≤ 0.01). Animals in group 5 received two doses of romiplostim, but there were no significant differences in nadir or start of recovery neutrophil or platelet counts when compared to group 3 (single dose of romiplostim).

[0145] Romiplostim and pegfilgrastim given together appeared to have a greater effect on neutrophil and platelet recovery than either drug given alone.

[0146] There was considerable variability in serum romiplostim concentrations between animals in the treatment groups, which resulted in considerable variability in PK parameters. max Value and AUC last Romiplostim serum exposure based on C values ​​was increased by more than 80% in females and decreased by approximately 15% in males when pegfilgrastim was coadministered, suggesting that pegfilgrastim affects the pharmacokinetics of romiplostim and that this effect is more pronounced in females. A dose increase from 2.5 mg / kg to 5 mg / kg resulted in a disproportionate change in serum exposure of romiplostim when administered alone. There was a tendency for romiplostim to accumulate with repeated dosing, with a higher mean C in females receiving dose 2 compared to dose 1. max is about 43% larger, and AUC (0~72時間) The trend was also more pronounced in females, as their puberty scores were approximately 36% greater in males.

[0147] The NHP effects of romiplostim presented herein are consistent with the study of romiplostim administered to irradiated mice reported herein, and also with previous studies demonstrating that thrombopoietin (TPO) administration improves platelet levels and survival in irradiated NHP (Neelis et al. (1997), Blood 90(7):2565-2573; Neelis et al. (1997), Exp Hematol. 25(10):1084-1093). As with the NHP studies reported herein, a single dose of TPO was as effective as repeated doses to reduce thrombocytopenia. However, neutralizing antibodies develop following TPO administration, and research into its use has been largely abandoned. Although romiplostim acts as a TPO receptor agonist, it has no sequence homology to TPO, and therefore the risk of developing neutralizing antibodies is fairly low.

[0148] In addition to the expected effects on neutrophil counts, pegfilgrastim also appears to improve platelet counts, a finding that has been reported previously (Hankey et al. (2015), Radiat Res. 183(6):643-655). However, the results reported here indicate that romiplostim is more effective in treating radiation-induced thrombocytopenia than pegfilgrastim alone.

[0149] The combination of romiplostim and pegfilgrastim improved platelet and neutrophil counts to levels above those observed with either agent alone. These results suggest that the two agents likely act on different hematopoietic pathways and lineages but have beneficial effects on both neutrophil and thrombopoiesis. This finding is important because severe thrombocytopenia and an inverse correlation between days of neutropenia and survival have been reported in irradiated rhesus monkeys (Stickney et al. (2007), Int Immunopharmacol. 7(4):500-505). Administration of TPO and G-CSF to irradiated rhesus monkeys (Neelis et al. (1997), Blood 90(7):2565-2573; Neelis et al. (1997), Exp Hematol. 25(10):1084-1093) and mice (Grossmann et al. (1996) Blood, 88(9):3363-3370) has been observed to have increased efficacy in ARS. In these studies, the apparent increase in neutrophil responses to G-CSF was attributed to TPO-mediated expansion of bone marrow progenitor cells, including granulocyte / monocyte colony-forming units (GM-CFUs) (Farese et al. (1996), J Clin Invest. 97(9):2145-2151; Ku et al. (1996), Blood, 87(11):4544-4551; Sitnicka et al. (1996), Blood, 87(12):4998-5005). Furthermore, the effects of romiplostim and pegfilgrastim may be due to a complex interaction of TPO with G-CSF, along with their effects on megakaryocytes and neutrophils. In mice, injection of G-CSF increases TPO concentrations in the bone marrow, releases neutrophil-attracting cytokines from megakaryocytes, and recruits neutrophils (Kohler et al. (2011), Blood, 117(16):4349-4357). Thus, administration of pegfilgrastim may also stimulate TPO production to increase platelet production, and romiplostim (and TPO) may in turn stimulate neutrophil recruitment.

[0150] In addition to improving platelet levels, we report for the first time that romiplostim and pegfilgrastim contribute to maintaining higher mean platelet volume (MPV) than control levels after radiation (Figure 8B), which has long been recognized as a marker of platelet activity and turnover (Thompson et al. (1984), Blood, 63(6): 1372-1375). This is another indication that both of these agents, especially the combination, stimulate platelet production, potentially impacting the incidence of bleeding and ultimately improving survival outcomes. Decreased MPV has been associated with shorter survival in patients with non-small lung cancer (Inagaki et al. (2014), Lung Cancer, 83(1): 97-101, Kumagai et al. (2015), Mol Clin Oncol. 3(1): 197-201).

[0151] Each reference cited herein is incorporated by reference in its entirety. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0152] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method of treating thrombocytopenia in a patient comprising administering romiplostim and pegfilgrastim.

2. A method of treating neutropenia in a patient comprising administering romiplostim and pegfilgrastim.

3. The method of claim 1 , wherein the patient is exposed to radiation.

4. The method of claim 3, wherein the patient has acute radiation syndrome.

5. The method of claim 3 , wherein the patient has undergone or is undergoing radiation therapy.

6. 10. The method of claim 1, wherein the patient has undergone or is undergoing chemotherapy.

7. 2. The method of claim 1, wherein the patient has idiopathic thrombocytopenic purpura (ITP).

8. 10. The method of claim 1, wherein the patient has a condition selected from a viral infection, a bacterial infection, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and anemia.

9. The method of claim 8, wherein the patient has aplastic anemia.

10. The method of claim 2 , wherein the patient is exposed to radiation.

11. The method of claim 10, wherein the patient has acute radiation syndrome.

12. The method of claim 10, wherein the patient has undergone or is undergoing radiation therapy.

13. 3. The method of claim 2, wherein the patient has undergone or is undergoing chemotherapy.

14. 3. The method of claim 2, wherein the patient has a condition selected from chronic idiopathic neutropenia, Kostmann syndrome, leukemia, myelodysplastic syndrome, myelofibrosis, myeloid cellular pool, vitamin deficiency, hepatitis A, hepatitis B, hepatitis C, HIV infection, AIDS, Lyme disease, malaria, viral infection of the bone marrow, Salmonella infection, sepsis, hypersplenism, and rheumatoid arthritis.

15. a. administering romiplostim at an initial dose of 1 mcg / kg once weekly; and b. Platelet count is 50 x 10 ９ Adjust the dose of romiplostim by 1 mcg / kg weekly to achieve and maintain a blood glucose level of ≥ 1.5K. The method according to any one of claims 1, 2, 6 to 9, 13 and 14, comprising:

16. 15. The method of any one of claims 1 to 14, comprising administering pegfilgrastim at a dose of 6 mg.

17. 14. The method of claim 6 or 13, comprising administering pegfilgrastim once per chemotherapy cycle.

18. 1. A method of treating a patient exposed to radiation, comprising administering romiplostim at a dose of about 1 μg / kg to about 10 μg / kg.

19. 20. The method of claim 18, wherein a single dose of romiplostim is administered to the patient.

20. 20. The method of claim 19, wherein the romiplostim is administered within about 24 hours after the radiation exposure.

21. 20. The method of claim 18, wherein the patient has acute radiation syndrome.

22. 20. The method of claim 18, wherein said radiation exposure is included in a cancer treatment.

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