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a Glaxo Wellcome Inc., Research Triangle Park, North Carolina, USA;
b Affymax Research Institute, Palo Alto, California, USA
Key Words. Thrombopoietin • Mimetic peptide • GW395058 • Pharmacokinetics • Hematology • Rat • Monkey • PEGylated
Dr. Mark de Serres, Division of Bioanalysis and Drug Metabolism, Department of International Development Support, Glaxo Wellcome Inc., 5 Moore Drive, Research Triangle Park, North Carolina 27709, USA.
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Abstract |
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(half-life) values were >20 h, and the area under the concentration time curve increased proportionally with dose. In cynomolgus monkeys GW395058 plasma t values ranged from 37 to 68 h after s.c. or i.v. dosing, and similar values were observed in rhesus monkeys following s.c. dosing. Rat platelet counts increased following 2 (1.6-fold) or 10 µg/kg (fourfold) s.c. doses. Cynomolgus and rhesus monkey platelet counts did not change significantly at comparable s.c. doses, but did increase slightly (<twofold) in cynomolgus monkeys following a 25 µg/kg s.c. dose and twofold following a 100 µg/kg s.c. dose. Because the plasma t of GW395058 is long in mouse, rat, dog, and monkey, yet the dose required to double platelet levels in monkeys is 50-fold that required in rats, differences in hematological responses may be due to interspecies differences in the interaction of GW395058 with the TPOr.
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Introduction |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
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The lineage-specific cytokine that regulates platelet production, thrombopoietin (TPO), has been described [4]. Two forms of the recombinant human protein have been investigated: full-length glycosylated recombinant human protein (rHuTPO) [5] and PEGylated megakaryocyte growth and development factor (PEG-rHuMGDF) [6]. In mice, dogs, and primates [7-14], rHuTPO and PEG-rHuMGDF promote platelet production and reduce chemotherapy-induced thrombocytopienia. Unfortunately, evidence of TPO-neutralizing antibodies in patients participating in cancer and platelet donor clinical trials forced the discontinuation of PEG-rHuMGDF development [15].
Not long after TPO was first reported, a 28-amino acid TPO mimetic peptide was described that was equipotent to TPO in cell-based assays [16]. This peptide dimer, AF13948, shares no sequence homology with TPO. Studies showed that while AF13948 plasma concentrations were short in mice (plasma t [half-life] ~ 1 h), AF13948 stimulated platelet production following single or multidose regimens of 250 or 1,000 µg/kg (unpublished results).
Subsequent to these studies the sequence of AF13948 was modified with amino acid substitution, and the resulting peptide, AF15705, was PEGylated to produce GW395058 (Fig. 1
). Similar to AF13948 and AF15705, GW395058 binds to and activates the human TPO receptor (HuTPOr) with an affinity similar to that of TPO in vitro [17]. Unlike AF13948, GW395058 has a long t in mice and elevates peripheral platelet counts following a single s.c. dose of as little as 2 µg/kg [18]. In dogs, when coadministered with G-CSF, GW395058 has a long plasma half-life and accelerates platelet recovery in a dog myelosuppression model (unpublished results, manuscript in preparation). Recent studies have shown that GW395058 is not immunogenic in several animal species, and that if an antibody response to GW395058 were elicited, those antibodies would not likely neutralize endogenous HuTPO [19].
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Experimental Procedures |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
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Animal Handling, Dosing, and Sample Collection
All animal procedures described in this report were approved by the Institutional Animal Care and Use Committee and conducted in accordance with Federal guidelines.
Studies in Rats
Single-dose and one-month repeat-dose studies were conducted in Han Wistar rats at Glaxo Wellcome. In the single-dose study, male rats (300 to 350 g, three rats/dose) received single s.c. doses of 2 or 10 µg/kg. Plasma samples for determination of GW395058 concentrations were obtained from each rat predose and at 3, 4, 7, and 10 days postdose. Plasma samples were stored at -70°C until analysis. Blood samples for hematological analysis were obtained from each rat predose and at 3, 7, 10, 14, 17, 21, and 28 days postdosing and analyzed the same day.
In the one-month repeat-dose study, male and female Han Wistar rats (250 to 300 g: 10 rats/sex at the 500 and 1,000 µg/kg/48 h doses, 18 rats/sex at the control and the 2,000 µg/kg/48 h doses) received single i.v. bolus doses every other day for a month. Upon completion of dosing, 10 rats/sex/dose were euthanatized, while remaining control and high-dose rats were allowed to recover for one month and then euthanatized to assess the reversibility of treatment-related effects. Doses of GW395058 in this study were chosen to provide exposures well above those necessary for efficacy in rats and those anticipated in clinical trials. Following doses 1 and 11, serum samples for determination of GW395058 concentration were obtained from 3 rats/sex/time point predose and at 0.8, 0.5, 1, 2, 4, 8, 24 and 48 h after dosing. Following dose 11, additional serum samples were taken at 72 and 96 h. Serum samples were stored at -70°C until analysis. Blood samples for hematological analysis were obtained from each rat predose, on day 24 (near the end of the dosing period), and 13 and 28 days after dosing stopped. Hematologic samples were analyzed the same day they were taken.
Single-dose and one-month repeat-dose studies were conducted in monkeys at Covance (Vienna, VA). In the single-dose study, male cynomolgus monkeys (3 to 5 kg) received single s.c. doses at 2, 10, 25, or 100 µg/kg (three monkeys/dose) or a single i.v. dose at 10 µg/kg (two monkeys). Plasma samples for pharmacokinetic analysis were obtained from each monkey predose and at 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 72, 96, 120, 144, and 168 h postdosing. Plasma samples were stored at -70°C until analysis. Blood samples for hematological analysis were obtained from each monkey at predose (day 1) and 3, 5, 7, 8, 10, 11, 12, 15, 17, 19, and 22 days postdose. Blood samples were stored at room temperature until processed (within 1 h of collection). Because platelet counts increased only slightly at 25 and 100 µg/kg and not at all at 
10 µg/kg, four rhesus monkeys (3 to 5 kg; two/sex) were added to the study to determine if they responded more strongly to GW395058 than did cynomolgus monkeys. Rhesus monkeys received single s.c. doses of GW395058 at 2 or 10 µg/kg. Samples (plasma and blood) were collected as described for cynomolgus monkeys.
In the one-month repeat-dose study, male and female cynomolgus monkeys (3 to 5 kg: three/sex at 500 and 1,000 µg/kg/48 h doses, five/sex at control and at the 2,000 µg/kg/48 h dose) received single i.v. doses every other day for a month. Upon completion of dosing, three monkeys/sex/dose were euthanized, while remaining control and high-dose monkeys were allowed to recover for one month and then euthanized to assess the reversibility of treatment-related effects. Following doses 1 and 14, serum samples were obtained predose and at 0.08, 0.5, 1, 2, 4, 8, and 24 h postdose. Following the first dose, an additional serum sample was collected at 48 h from all animals. Following dose number 14, additional serum samples were taken from animals (two animals/sex) in the vehicle control and 2,000 µg/kg/48 h groups at 48, 72 and 96 h after dosing. Serum samples were stored frozen at or below -20°C until analysis. Blood samples for hematological analysis were obtained from each monkey predose, on days 14 and 26 during the dosing period, and 10 and 24 days after dosing stopped. Hematologic samples were analyzed the same day they were taken.
Determination of GW395058 Plasma Concentrations
Radioimmunoassay (RIA) Concentrations of GW395058-like immunoreactive material in rat and monkey plasma samples were determined by using a competitive binding RIA. A tritiated analog of AF15705 was prepared by derivatizing the two N-termini of AF15705 with succinimidyl propionate,N-[propionate-2,3-3H] obtained from Dupont Company NEN Research Products (Boston, MA). The reaction product was purified by RP-HPLC (2.5 x 150 mm Symmetry® C18 5 µm column, Waters Corp. [Milford, MA]; mobile phase A = 0.1% Trifluoroacetic acid [TFA] in water, mobile phase B = 0.1% TFA in acetonitrile) by using gradient chromatographic conditions (0 to 60% mobile phase B for 30 min followed by 60% to 75% mobile phase B for 5 min). The purified radioligand had a specific activity of ~25Ci/mmol.
In this RIA diluted rabbit antiserum [19] raised to a bovine thyroglobulin conjugate of an AF15705-analog was incubated overnight at 4°C with radioligand (3H-AF15705) and aliquots of study samples or spiked (GW395058) plasma calibration standards. The next day bound and free radioligands were separated by incubating for 1 h at 4°C with antirabbit immunoprecipitation reagent (Sac-Cel®; IDS Ltd.; Boldon, Tyne and Wear, UK), followed by the addition of 2 ml of cold PBS (pH 7.2) containing 0.1% bovine serum albumin and 0.1% Tween-20. Assay tubes were centrifuged for 10 min and the supernatant aspirated to waste. The pellets were resuspended in 2 ml of water and decanted into 20 ml scintillation vials containing 5 ml of scintillation fluid. Bound radioligand was quantified in a liquid scintillation counter, and calibration curves were derived by using the program, Multicalc (Wallac; Turku, Finland), from which control and study sample concentrations were interpolated. All samples were assayed in duplicate. The lower limit of quantitation (LLOQ) was set at 1.6 ng/ml. Because this RIA may also detect metabolic fragments of GW395058, the results here are reported as GW395058 immunoreactivity equivalents.
TPOr Biological Activity Assay Concentrations of GW395058-like biologically active material in monkey plasma samples following a 25 µg/kg s.c. dose of GW395058 were determined by using a reporter assay based on a Baf/3 HuTPOr fos/lux cell line. In these cells activation of the HuTPOr results in the production of luciferase that catalyzes the emission of light upon addition of the substrate luciferin. The cells were grown in Dulbecco's modified Eagle's essential medium/F12; 10% fetal bovine serum; 10% Wehi-3 supernatant and antibiotics/antimycotics.. Approximately 18 h before the assay, cells were starved by transferring them to the same medium made 0.1% Wehi-3 supernatant. On the day of assay, following a washing step in medium without Wehi-3, 1 x 106 cells/ml were combined with study samples or GW395058-spiked plasma calibration standards. Assay plates were incubated for 2 h at 37°C in a 5% CO2 atmosphere, then Luclite reagent (Packard Instrument Co.; Downers Grove, Ill.) was added to each well. Light emission was measured after a five-minute incubation on a Packard Topcount Luminometer (Packard Instrument Co.). The concentrations of GW395058-like biological activity in the plasma samples were interpolated from the linear portion of the standard curve. The LLOQ was set at 0.1 ng/ml. Because this assay does not distinguish GW395058 from other possible agonists, results with this method were reported as GW395058 biological activity equivalents.
Pharmacokinetic Calculations
Area under the concentration time curve (AUC) values for GW395058 were estimated by using the trapezoidal rule, with back-extrapolation of the GW395058 plasma concentrations to zero time. Single day 1 AUC values were calculated from time zero to infinity (0-
), with the extrapolated area from the time of the last measurable sample concentration (Cplast) to infinity time estimate as Cplast/ke, where ke is the terminal phase elimination rate constant. Multidose day last AUC values were calculated from time zero to 
, where is the dosing interval. Cmax values were defined as the highest observed plasma concentration postdose. Mean calculations were performed by using Microsoft® Excel 97 SR-1 (Microsoft Corporation; Redmond, WA). Noncompartmental pharmacokinetic calculations were performed by using WinNonlin, Professional Network Version 1.5 (Scientific Consulting Inc.; Cary, NC) or PKCAL Version 2.0 (Glaxo Wellcome Inc.).
Hematology Clinical hematology and coagulation analyses were performed at Glaxo Wellcome (rats) or at Covance, Vienna, Virginia (monkeys). Clinical hematology parameters measured in all studies were platelet count, total leukocyte count, differential leukocyte counts, total erythrocyte count, reticulocyte count, hematocrit, hemoglobin, and RBC indices (mean cell hemoglobin [MCH], mean cell volume [MCV], mean cell hemoglobin concentration [MCHC]). In one-month studies, mean platelet volume (MPV), platelet distribution width, and coagulation parameters (prothrombin time and activated partial thromboplastin time) also were measured.
Statistical Analysis Mean calculations were performed using Microsoft® Excel 97 SR-1. Statistical analysis (ANOVA) of pharmacokinetic and hematology data was performed by using JMP Statistical Visualization Software Version 3.2.5 (SAS Institute, Inc.; Cary, NC) or SAS System for Windows, Version 6.12, TS Level 0020, (SAS Institute). Comparisons of pharmacokinetic parameter estimates were made between the sexes and days of dosing and among the dose groups by using Tukey's Studentized Range Test. Differences were declared statistically significant when p < 0.05.
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Results |
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. A comparison of individual GW395058 plasma concentrations measured by RIA or TPOr biological activity assay is shown in Figure 2.
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Plasma Concentrations and Pharmacokinetic Parameter Estimates for GW395058 in Han Wistar Rats
Following s.c. dosing with GW395058 at 2 or 10 µg/kg, there were insufficient plasma concentration versus time data available to estimate pharmacokinetic parameters due to the limited amount of blood and plasma that could be collected from each animal over the extended duration of this study. The highest plasma concentrations of GW395058 were observed on day 3 following the 10 µg/kg dose. GW395058 was undetectable in plasma following the 2 µg/kg dose (data not shown).
Pharmacokinetic parameter estimates for GW395058 in male and female Han Wistar rats following i.v. dosing at 500, 1,000, or 2,000 µg/kg/48 h are shown in Table 2.
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values were long at all dose levels. Plasma clearance (CL) and volume of distribution values (Vz) over the fourfold range in i.v. dose were low and generally comparable among dose levels. Dose 1 and dose 11 pharmacokinetic parameter estimates did not differ (data not shown) suggesting that repeated i.v. dosing did not alter GW395058 systemic exposure.
Plasma Concentrations and Pharmacokinetic Parameter Estimates for GW395058 in Cynomolgus Monkeys
Plasma concentrations of GW395058 in cynomolgus monkeys following s.c. dosing at 2, 10, 25, and 100 µg/kg and i.v. at 10 µg/kg are shown in Figure 3. Pharmacokinetic parameter estimates for GW395058 in cynomolgus monkeys following s.c. dosing at 2, 10, 25, and 100 µg/kg and i.v. at 10 µg/kg are shown in Table 3.
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values were long at all dose levels. Plasma clearance (CL/F) values over the 50-fold range in s.c. dose were low and comparable among dose levels. Volume of distribution (Vz/F) values were similar at the 10, 25, and 100 µg/kg dose levels, while the averaged Vz/F value for the 2 µg/kg dose was slightly higher.
Following i.v. dosing with GW395058 at 10 µg/kg, the AUC was slightly lower than the comparable s.c. dose level. The plasma t following i.v. dosing was at least 10 h less than that observed following s.c. dosing. Plasma CL following i.v. dosing was generally low, but slightly higher than that observed following s.c. dosing at the same level. The Vz following i.v. dosing at 10 µg/kg was essentially the same as that observed following s.c. dosing. The volume at steady state (Vss ) was 82 ± 12 ml/kg.
Pharmacokinetic parameter estimates for GW395058 in cynomolgus monkeys following single and repeated i.v. doses at 500, 1,000, 2,000 µg/kg are shown in Table 4. In general for monkeys (male and female) dosed at 500, 1,000, or 2,000 µg/kg; A) Cmax and AUC values increased proportionally with dose; B) t values were long, and C) CL values were low and comparable among dose levels.
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or Vz across the dose groups.
Cmax values increased from dose 1 to dose 14, but these increases were significant only in the 1,000 and 2,000 µg/kg female and the 500 and 1,000 µg/kg male dose groups. The only significant decrease in CL and increase in AUC values following multiple dosing were observed in female monkeys at the 2,000 µg/kg dose. There were no significant changes in t or Vz with multiple dosing.
Cmax, AUC, t, and CL values following doses 1 and 14 were not statistically different between male and female monkeys, except at the 2,000 µg/kg dose group, where Cmax, AUC, and t were greater in males and CL was greater in females. There were no other sex-related trends in any of the other pharmacokinetic parameters examined.
Plasma Concentrations and Pharmacokinetic Parameter Estimates for GW395058 in Rhesus Monkeys
Pharmacokinetic parameter estimates for GW395058 in the rhesus monkey following s.c. dosing at 2 and 10 µg/kg are shown in Table 5. Pharmacokinetic parameter estimates in rhesus monkeys were comparable to those for cynomolgus monkeys following s.c. dosing at 2 and 10 µg/kg. AUC values increased proportionally with increasing dose for rhesus monkeys following s.c. dosing at 2 and 10 µg/kg. Similarly, Cmax values were comparable with those at the same dose levels for cynomolgus monkeys (Table 3), and there was proportionality with increasing dose. Tmax values for rhesus monkeys were slightly shorter than those for cynomolgus monkeys. Plasma t values for rhesus monkeys were long following s.c. dosing, although the plasma t value at the 2 µg/kg dose level was approximately half that observed for cynomolgus monkeys. CL/F and Vz/F values were similar for both rhesus and cynomolgus monkeys at the 2 and 10 µg/kg dose levels.
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). RBC parameters (hematocrit, hemoglobin, RBC count) decreased slightly (10%-15%) at both doses within three days postdose, and did not return to normal until 28 days postdose. Decreases occurred more rapidly and were greater in magnitude at 10 µg/kg. There was a corresponding increase in red cell distribution width, which remained elevated for 28 days postdose, the last time point measured (data not shown).
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Hematological Effects in Rats After One-Month Repeat-Dose Studies
Repeated i.v. administration of GW395058 at 500, 1,000, or 2,000 µg/kg every other day for a month produced much greater changes in platelet, RBC, and WBC parameters than those observed after single low doses. However, at these elevated dose levels, hematologic effects did not differ among dose groups (Table 6). Platelet counts increased about threefold during the dosing period but increased more (about 10-fold) within two weeks after dosing stopped. Platelet counts had returned to normal by a month postdose. Increases in platelet count were accompanied by an increase in MPV.
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Total WBC counts increased markedly (about eightfold) by the end of the dosing period and returned to normal within a month postdose. WBC count increases reflected across-the-board increases in lymphocyte counts (about fourfold) and granulocyte counts (about 20-fold) observed during this study.
Prothrombin time was somewhat prolonged (30%) at the end of the dosing period, but returned to normal within a month postdose. Activated partial thromboplastin time was unaltered (data not shown).
Hematological Effects in Monkeys After Single-Dose Studies
In cynomolgus or rhesus monkeys, no significant changes in platelet counts were observed following s.c. doses at 2 or 10 µg/kg. In cynomolgus monkeys, a 25 µg/kg s.c. dose produced small but significant increases in platelet counts on days 7 and 11 (1.3 times and 1.5 times, respectively) from day 1 values (data not shown). A 100 µg/kg s.c. dose produced significant increases in platelet counts on days 9, 11, and 13 (Fig. 5). However, platelet counts did not increase proportionally with dose from 25 to 100 µg/kg. Additionally, cynomolgus monkeys dosed at 100 µg/kg had significant increases in total WBC counts (1.4-fold) on day 7 (data not shown) and neutrophil counts on day 7 (2.6 times) from day 1 counts (Fig. 6). No significant changes were observed for the other hematological parameters examined.
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). RBC parameters were unaffected during the dosing period, but a mild transient regenerative anemia occurred postdose. This was characterized by slightly decreased RBC parameters (RBC count, hematocrit, hemoglobin) and increased red cell distribution width. Anemia had not completely resolved a month after dosing stopped, although RBC parameters approached normal values.
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Platelet counts increased about sevenfold during the dosing period and returned to approximately normal levels within a month postdose. Increases in platelet counts were accompanied by an increased platelet distribution width and a decreased MPV.
Activated partial thromboplastin time was somewhat prolonged (10%) at the end of the dosing period, but returned to normal within a month postdose. Prothrombin time was unaltered (data not shown).
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Discussion |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
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. The pharmacokinetic behavior of GW395058 is similar in cynomolgus and rhesus monkeys. While repeated administration of GW395058 for a month at high doses stimulated megakaryocytopoiesis, granulocytopoiesis, and lymphocytopoiesis in both rats and cynomolgus monkeys, the nature and magnitude of the responses differed between species. Following repeated administration of GW395058, platelet counts increased more in monkeys than rats, while WBC counts increased more in rats than monkeys. Platelet count increases were accompanied by a decrease in average platelet size in monkeys but an increase in average platelet size in rats. These differences may reflect species differences in receptor sensitivity or inherent responses to receptor stimulation.
Hematologic effects produced by repeated high doses of GW395058 were reversible when dosing stopped, although recovery was prolonged in some cases. This prolonged recovery phase may reflect GW395058’s long t, but it also may reflect the inherent rate at which altered hematopoietic cell counts return to homeostatic levels in the species tested.
The observation that hematological effects in repeat-dose studies did not differ with dose suggests that the dose regimen used was beyond that needed to elicit a maximum response. This in turn suggests that maximum responses could be obtained with lower and/or less frequent doses. The dose regimen chosen made it impossible to characterize the dose-response curves for individual hematologic effects, so it is not possible to address the question here of whether the various effects were mediated by a similar molecular mechanism (i.e., action of GW395058 on a single receptor).
GW395058-induced thrombocytosis was not associated with hemorrhage or thrombosis, suggesting that induced platelets function normally, as has been shown with PEG-rHuMGDF [6, 13].
Because GW395058 produces increased numbers not only of circulating platelets that appear functionally normal, but also of circulating granulocytes and lymphocytes, the data presented here suggest that GW395058 has the potential to ameliorate damage or hasten recovery of multiple hematopoietic cell lines from chemotherapy-induced damage, as has been indicated for rHuTPO [20].
Although GW395058 has a long plasma t in mice [18], rats, dogs and monkeys, the single s.c. dose required to double circulating platelet levels in monkeys is 50-fold that required in rats. In previous studies [17] GW395058 and rHuTPO were shown to bind with similar affinity to murine BaF3 cells transfected with the HuTPOr and equipotent in stimulating megakaryocyte colony formation and megakaryocyte maturation in cells cultured from human bone marrow in vitro. Additional studies will be required to determine if the observed differences in hematological responses between rodents and monkeys are due to a significantly reduced affinity of the monkey TPOr for GW395058, relative to the human and rodent receptors.
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Acknowledgments |
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and 
amino groups of a lysine residue. The sequence of AF13948 (see text) was modified by the replacement of an alanine and a ß-alanine residue with sarcosine (Sar) to remove the chiral centers. Tryptophan residues were replaced with napthylalanine (Npa) for ease of synthesis. The two resulting peptide amino termini of AF15705 were conjugated to 20,000 mw PEG to produce GW395058.
), 25 (
), or 100 (•) µg/kg or an i.v. dose at 10 (
) µg/kg.
