Experimental Hematology 30 (2002) 1185–1192

0301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(02)00898-6

Marked improvement of thrombocytopeniain a murine model of idiopathic thrombocytopenic purpura by

pegylated recombinant human megakaryocyte growth and development factor

Kazunori Shibuya

a

, Tomoaki Kuwaki

a

, Emiko Tahara

a

,Chizuru Yuki

a

, Hiromichi Akahori

a

, Takashi Kato

b

, and Hiroshi Miyazaki

a

a

Pharmaceutical Development Laboratory and

b

Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., Gunma, Japan

(Received 5 March 2002; revised 11 June 2002; accepted 13 June 2002)

Objective.

We examined the stimulatory effect of pegylated recombinant human megakaryo-cyte growth and development factor (PEG-rHuMGDF) on platelet production in male (NZW

�

BXSB) F

l

(W/B F

1

) mice, a murine model of idiopathic thrombocytopenic purpura.

Materials and Methods.

A cohort of 19- to 25-week-old, severely thrombocytopenic male W/BF

1

mice were given PEG-rHuMGDF at different dosing schedules. Before and at various timesafter therapy, platelet counts, reticulated platelets, platelet lifespan, and levels of platelet-asso-ciated immunoglobulin G were measured. Analysis of megakaryocytic cells was performed.

Results.

Treatment of male W/B F

1

mice with PEG-rHuMGDF (30

�

g/kg/day) three times perweek for several weeks resulted in sustained thrombocytosis, accompanied by increased mega-karyocytopoiesis in both the bone marrow and spleen. The degree of the platelet response toPEG-rHuMGDF varied between individual mice, likely reflecting the heterogeneity of the dis-ease. Production of new platelets in response to PEG-rHuMGDF was manifested by an in-crease in reticulated platelets. Levels of platelet-associated immunoglobulin G decreased in-versely during periods of thrombocytosis. PEG-rHuMGDF therapy also improvedthrombocytopenia in male W/B F

1

mice refractory to splenectomy. Platelet lifespan was not af-fected by PEG-rHuMGDF. Male W/B F

1

mice treated with pegylated murine MGDF, a homo-logue of PEG-rHuMGDF, had persistent thrombocytosis for at least 7 months, suggesting thatantiplatelet antibody production was not enhanced.

Conclusion.

PEG-rHuMGDF therapy potently stimulated platelet production, effectively

ameliorating thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura.

© 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Thrombopoietin (TPO) is the principal physiologic regula-tor of megakaryocytopoiesis and platelet production [1–5].TPO acts primarily on committed megakaryocyte progeni-tor cells (colony-forming unit megakaryocyte [CFU-MK])to generate polyploid mature megakaryocytes that sponta-neously form proplatelets, the initial event leading to plate-let production [6,7]. In vivo administration of pegylated re-combinant human megakaryocyte growth and developmentfactor (PEG-rHuMGDF), a pegylated, amino-terminal do-main of recombinant human TPO expressed in

Escherichia

coli

, dramatically increases circulating platelets in normalanimals [8,9] and markedly accelerates platelet recovery inmyelosuppressed animals [10,11].

PEG-rHuMGDF has been demonstrated to potently stimu-late platelet production in cancer patients before chemotherapy[12] and in healthy volunteers [13]. Further clinical studies ex-amined the therapeutic effect of PEG-rHuMGDF on plateletrecovery in cancer patients receiving myelosuppressive che-motherapy [14,15] as well as in patients with severe throm-bocytopenia associated with myelodysplastic syndromes andaplastic anemia [16]. PEG-rHuMGDF also has been shown toincrease platelet yields by apheresis from healthy volunteers[17]. However, a small but significant proportion of subjects inthese studies developed neutralizing antibodies to endogenousTPO, resulting in thrombocytopenia [18].

Offprint requests to: Hiroshi Miyazaki, Ph.D., Pharmaceutical Devel-opment Laboratory, Kirin Brewery Company, Ltd., 3 Miyahara-cho,Takasaki, Gunma 370-1295, Japan; E-mail: miyazakih@kirin.co.jp

1186

K. Shibuya et al./Experimental Hematology 30 (2002) 1185–1192

Idiopathic thrombocytopenic purpura (ITP) is a disordercharacterized by low platelet counts due to increased plate-let destruction, increased levels of platelet-associated im-munoglobulin, and normal to increased numbers of mega-karyocytes [19,20]. Various therapeutic strategies are usedfor the treatment of patients with ITP, including medical op-tions such as corticosteroids, danazol, immunosuppressiveagents, and splenectomy [21]. High-dose intravenous im-munoglobulin causes a transient increase in platelet countsin the majority of ITP patients [22]. Due to the heterogene-ity of the disease, however, approximately 20% of patientsare refractory to these treatments. Although thrombopoieticcytokines are candidates for a new medical option, recombi-nant human interleukin-11 (IL-11) was not effective in a re-cent pilot trial of patients with refractory ITP [23].

In ITP patients, although serum IL-11 levels are in-versely related to the degree of thrombocytopenia, serumTPO levels are normal or only slightly elevated, despite amarked decrease in platelet counts [24,25]. These observa-tions suggest that PEG-rHuMGDF therapy might be effec-tive in stimulating platelet recovery in ITP patients withnormal or increased numbers of CFU-MK in their bonemarrow.

Male (NZW

�

BXSB) F

l

(W/B F

1

) mice are a murinemodel of human ITP that spontaneously develop chronicthrombocytopenia due to accelerated platelet destruction,probably caused by antiplatelet antibodies [26]. Throm-bocytopenic male W/B F

1

mice have a reduced plateletlifespan and increased levels of platelet-associated antibod-ies (platelet-associated immunoglobulin G [PAIgG]) as wellas of platelet-binding antibodies [27].

To explore the clinical potential of PEG-rHuMGDF inpatients with ITP, we examined the effect of PEG-rHuMGDF in severely thrombocytopenic male W/B F

1

mice. PEG-rHuMGDF therapy potently stimulated plateletproduction, resulting in marked improvement of throm-bocytopenia. The results suggest that treatment with PEG-rHuMGDF might stimulate megakaryocytopoiesis and cor-rect peripheral platelet counts in patients with ITP.

Materials and methods

Mice

Male and female W/B F

1

mice (10 weeks old) were purchased fromJapan SLC Inc. (Shizuoka, Japan). Upon arrival at our laboratory,male W/B F

1

mice had no apparent thrombocytopenia, with a meanplatelet count of 1000

�

10

3

/

�

L. Approximately 25% of the W/B F

1

male mice, however, developed thrombocytopenia as they grew. Inthis study, we used a cohort of 19- to 25-week-old mice with plateletcounts less than 250

�

10

3

/

�

L. Female W/B F

1

mice were used ascontrols. Mice were housed in autoclaved cages and maintained inan air-conditioned, specific pathogen-free animal room regulated ata room temperature of 21°C to 23°C and relative humidity of 50%to 60%. The light cycle was 12/12 hours, beginning at 8:00 A.M.

Sterilized commercial rodent chow and water were available to themice ad libitum. All experiments in this study were approved by theInstitutional Animal Care and Use of Committee of PharmaceuticalDevelopment Laboratory.

Cytokines

rHuMGDF and recombinant murine MGDF (rMuMGDF), a ho-mologue of rHuMGDF, were expressed in

E. coli

using a plasmidthat encodes a truncated amino-terminal domain of human and mu-rine TPO, respectively, and purified to homogeneity. The purifiedmolecules were further derivatized with polyethylene glycol togenerate PEG-rHuMGDF and PEG- rMuMGDF.

Study design

Different doses of PEG-rHuMGDF or vehicle were administeredintravenously in a volume of 0.1 mL per mouse with different ad-ministration schedules. Before and at various times after treatment,peripheral blood samples were obtained from the retro-orbitalplexus using 75-mm heparinized capillary tubes (Funakoshi Phar-maceutical Co., Tokyo, Japan). Complete blood counts were per-formed with a Sysmex automatic microcell counter F-820 (Sys-mex, Kobe, Japan).

Measurement of CFU-MK and megakaryocyte numbers

The number of CFU-MK in the bone marrow and spleen frommice treated with PEG-rHuMGDF or vehicle was determined by amegakaryocyte colony assay, as previously reported [11]. Thenumber of bone marrow megakaryocytes was determined as previ-ously described [11].

Assay of platelet-associated antibodies (PAIgG)

Platelets in 2

�

L of whole blood were washed three times with 200

�

L saline by centrifugation at 1000

g

for 5 minutes to remove freeIgG. The washed platelets were incubated for 15 minutes with phy-coerythrin (PE)-conjugated anti-mouse IgG (Pharmingen, San Di-ego, CA, USA) and fluorescein-5-isothiocyanate (FITC)-conju-gated antiplatelet antibody (Seikagaku-kogyo, Tokyo, Japan).After washing with saline three times, the samples were analyzedon FACSort (Becton-Dickinson, Rutherford, NJ, USA). Plateletswere identified in FITC-positive fraction. Data from at least 2000platelets were obtained. The mean PE fluorescence intensity of theplatelets from five or six normal female mice was used as the con-trol. The PAIgG index was calculated by dividing the mean PE flu-orescence intensity of each sample by the mean PE fluorescenceintensity of the female controls.

Measurement of reticulated platelets

Reticulated platelets were measured as previously described [28].

Measurement of platelet lifespan

Platelet lifespan was determined using an in vivo biotinylationtechnique [29]. Briefly, NHS-biotin was first dissolved in dimeth-ylsulfoxide at a concentration of 40 mg/mL and then diluted withsaline to a final concentration of 4 mg/mL. A volume of 150

�

L ofNHS-biotin solution was injected intravenously into mice twicewith a 1-hour interval. Blood samples were taken 1, 20, 44, 68, 92,116, and 140 hours after the second injection. A 2-

�

L aliquot ofblood was washed with 200

�

L saline three times by centrifuga-tion at 1000

g

for 5 minutes to remove free biotin and biotinylatedplasma proteins. The washed blood cells were incubated with PE-conjugated streptavidin and FITC-conjugated antiplatelet antibody

K. Shibuya et al./Experimental Hematology 30 (2002) 1185–1192

1187

(Seikagaku-kogyo) for 15 minutes. After washing three times, thepercentage of biotin-positive platelets was determined on FAC-Sort. Platelets were identified in the FITC-positive fraction. Datafrom at least 2000 platelets were obtained. The platelet lifespanwas determined by extrapolating the initial slope of the plateletsurvival curve to the time axis.

Splenectomy

Under ether anesthesia, the spleen was exposed via an incision onthe left side. After major blood vessels were ligated with sutures,the spleen was removed.

Statistical analysis

All data are given as mean

�

SEM. The statistical significance ofdifferences was assessed using Student’s

t

-test.

p

value of 0.05 wasconsidered to be statistically significant.

Results

Multiple-injection protocolsof PEG-rHuMGDF significantlyimprove thrombocytopenia in male W/B F

1

mice

Daily intravenous injections of PEG-rHuMGDF at 30

�

g/kg/day for 7 days into thrombocytopenic male W/B F

1

mice resulted in significant transient thrombocytosis, com-pared with vehicle treatment (Fig. 1A). Platelet countspeaked on day 8 after cessation of treatment and graduallyreturned to pretreatment levels. There were apparent differ-ences in the responsiveness to PEG-rHuMGDF betweenPEG-rHuMGDF-treated mice (Fig. 1B), likely due to differ-ent states of the disease. Such individual differences wereobserved throughout the study. Before treatment, thrombocy-topenic male W/B F

1

mice had a much higher percentage ofreticulated platelets, possibly compensatory for antibody-induced platelet destruction. PEG-rHuMGDF treatment alsostimulated a transient increase in reticulated platelets, indi-cating production of new platelets in response to PEG-rHuMGDF (Fig. 1C).

To mimic the chronic use of PEG-rHuMGDF in clinicalsettings, we examined the effect of PEG-rHuMGDF at adosing schedule of three times per week for several weeksin thrombocytopenic male W/B F

1

mice. This intermittenttreatment resulted in dose-dependent, sustained thrombocy-tosis (Fig. 2A). Before administration of PEG-rHuMGDF,thrombocytopenic male W/B F

1

mice had high levels ofPAIgG compared with age-matched female W/B F

1

mice.During PEG-rHuMGDF-induced thrombocytosis, how-ever, the levels of PAIgG decreased below pretreatment val-ues (Fig. 2B). This suggests that antiplatelet antibody mightbe constantly produced and therefore the amount of PAIgGper platelet decreased as platelet counts increased.

We analyzed megakaryocytopoiesis in male W/B F

1

mice treated with 30

�

g/kg/day of PEG-rHuMGDF threetimes per week for 2 weeks. Representative findings areshown in Figure 3. Compared with vehicle-treated controls,

mice treated with PEG-rHuMGDF had a 2.0- and 2.3-foldincrease in bone marrow CFU-MK numbers over pretreat-ment values on days 7 and 14, respectively (Fig. 3A). In thespleen, CFU-MK numbers also increased 8.1- and 17.3-foldon days 7 and 14, respectively (Fig. 3B). At these two timepoints, there was a trend toward increased numbers of bonemarrow megakaryocytes in PEG-rHuMGDF-treated mice(Fig. 3C).

In contrast to the profound effect of PEG-rHuMGDF onthrombocytopenia, white blood cell and red blood cell

Figure 1. Effect of daily administration of PEG-rHuMGDF in male W/BF1 mice. Intravenous PEG-rHuMGDF 30 �g/kg (�) or vehicle (�) wasadministered to mice daily for 7 days. (A) Platelet counts from each groupof mice. (B) Platelet counts from PEG-rHuMGDF-treated, individual mice.(C) Reticulated platelet counts from each group of mice. Each point repre-sents the mean value from five mice. Asterisks indicate significantlygreater than vehicle-treated controls: *p � 0.05.

1188

K. Shibuya et al./Experimental Hematology 30 (2002) 1185–1192

counts were not changed by multiple doses of PEG-rHuMGDF (data not shown).

Platelet response to PEG-rHuMGDFdoes not appear to be related to PAIgG levels

Thrombocytopenic male W/B F

1

mice were heterogeneousin terms of the levels of PAIgG. Therefore, we arbitrarilyclassified these mice into three groups based on the levels ofPAIgG to examine their ability to respond to PEG-rHuMGDF. Before treatment, mice that had higher PAIgGlevels also had lower platelet counts, as previously reported[27]. As shown in Figure 4, treatment with PEG-rHuMGDFthree times per week stimulated platelet recovery on day 7even in mice that had high PAIgG values. The averageplatelet counts before and after therapy were 131

�

10

3

/

�

Land 516

�

10

3

/

�

L in mice with PAIgG index

�

3.5, 175

�

10

3

/

�

L and 499

�

10

3

/

�

L in mice with 3.5

�

PAIgG index

�

6.5, and 87

�

10

3

/

�

L and 293

�

10

3

/

�

L in mice with

PAIgG index

�

6.5. Thus, it appeared that male W/B F

1

mice responded to PEG-rHuMGDF independently of pre-treatment levels of PAIgG. Nevertheless, low or no plateletresponse to PEG-rHuMGDF was observed in some micethat had higher PAIgG levels.

Figure 2. Effect of intermittent treatment with PEG-rHuMGDF in maleW/B F1 mice. Intravenous PEG-rHuMGDF (1 (�), 5 (�), or 30 (�) orvehicle (�) was administered to groups of mice three times per week forseveral weeks (indicated as double-headed arrows). (A) Platelet counts. (B)Levels of PAIgG in platelets from mice treated with 30 �g/kg PEG-rHuMGDF (�). Each point represents the mean value from 5 to 7 mice.Asterisks indicate significantly greater than vehicle-treated controls: **p �0.01; *p � 0.05.

Figure 3. Effect of PEG-rHuMGDF on megakaryocytopoiesis in male W/BF1 mice. Male mice were given 30 �g/kg PEG-rHuMGDF or vehicle intrave-nously three times per week. Female mice were treated with vehicle alone.Cells from the femur and the spleen were prepared at the indicated times beforeand after the start of treatment for a megakaryocyte colony assay. The numberof megakaryocytes in the femur were measured as described in the Materialsand methods section. (A) Number of CFU-MK in the femur. (B) Number ofCFU-MK in the spleen. (C) Number of megakaryocytes in the femur. Eachpoint represents the mean value from five mice. Asterisks indicate significantlygreater than vehicle-treated controls: **p � 0.01; *p � 0.05.

K. Shibuya et al./Experimental Hematology 30 (2002) 1185–1192

1189

PEG-rHuMGDF improves thrombocytopenia in W/B F

1

male mice refractory to splenectomySplenectomy successfully improved thrombocytopenia insome male W/B F1 mice, but other mice relapsed intothrombocytopenia. We examined the effect of PEG-rHuMGDF on thrombocytopenia in these refractory miceapproximately 1 to 4 weeks after surgery. As shown in Fig-ure 5, daily administration of PEG-rHuMGDF (30 �g/kg/day) for 7 days resulted in transient platelet recovery in therefractory mice, indicating that a substantial increase inplatelet counts by PEG-rHuMGDF therapy can occur in theabsence of the spleen.

PEG-rHuMGDF has no influence on the platelet lifespan of male W/B F1 miceWe examined the effect of PEG-rHuMGDF on plateletlifespan using an in vivo biotinylation technique. The meanplatelet lifespan was significantly reduced in thrombocy-topenic male W/B F1 mice compared with control femaleW/B F1 mice. After treatment with PEG-rHuMGDF (30 �g/kg/day) or vehicle three times per week for 3 weeks, plateletlifespan of thrombocytopenic male W/B F1 mice was 3.49 �0.09 days or 3.36 � 0.11 days, respectively (Fig. 6). Thus,platelet lifespan was not significantly affected by PEG-rHuMGDF, suggesting that PEG-rHuMGDF therapy doesnot accelerate platelet destruction.

Long-term treatment withPEG-rMuMGDF, a murine homologue of PEG-rHuMGDFTo examine the effect of long-term treatment on thrombo-cytopenia, a total of 15 male W/B F1 mice were given PEG-rMuMGDF (30 �g/kg/day), a murine homologue of PEG-rHuMGDF, three times per week for approximately 7 months.During the study period, 12 mice died, probably of myocar-dial infarction or lupus nephritis. Long-term treatmentmaintained increased platelet levels in three mice that sur-vived, as well as in other mice while they were alive. Theseobservations suggest that the increase in platelet counts byPEG-rMuMGDF might not lead to augmentation of anti-platelet antibody production (Fig. 7). Throughout the cur-rent study, either PEG-rHuMGDF or PEG-rMuMGDF hadno effect on the survival of male W/B F1 mice.

DiscussionThe present study examined the therapeutic effect of PEG-rHuMGDF on thrombocytopenia in a group of male W/B F1

mice that developed chronic severe thrombocytopenia. Themale W/B F1 mice used in this study had no or slightly in-creased megakaryocytopoiesis in the bone marrow for theirsevere thrombocytopenia, as previously reported [30].These observations suggest that not only increased plateletdestruction but also suppressed megakaryocyte production

Figure 4. Relation between PAIgG levels and platelet response to PEG-rHuMGDF in male W/B F1 mice. Mice were divided into three groups based on thelevels of PAIgG and were given 30 �g/kg PEG-rHuMGDF intravenously on days 1, 3, and 5. Platelet counts were measured before treatment (day 0) and onday 7. (A) Platelet counts from mice having PAIgG index � 3.5 (n � 12). (B) Platelet counts from mice having 3.5 � PAIgG index � 6.5 (n � 20). (C) Plate-let counts from mice having PAIgG index � 6.5 (n � 12).

1190 K. Shibuya et al./Experimental Hematology 30 (2002) 1185–1192

or increased megakaryocyte apoptosis might be involved inpathogenic mechanisms for ITP in male W/B F1 mice.

Previous studies demonstrated that CFU-MK is the pri-mary target population of TPO in platelet production. Invitro studies revealed that TPO acts on CFU-MK to gener-ate mature megakaryocytes that spontaneously form pro-platelets, whereas TPO has little or no effect on maturemegakaryocytes in terms of proplatelet formation [7]. Stud-ies of mice genetically engineered to lack either TPO [31]or the TPO receptor c-Mpl [31,32] indicate that there is amarked decrease in CFU-MK, as well as in megakaryocytesand platelets. Because the number of bone marrow CFU-MK is not reduced in thrombocytopenic male W/B F1 mice,it was thought that PEG-rHuMGDF would have a signifi-cant effect on thrombocytopenia in these mice.

As expected, our results clearly indicate that PEG-rHuMGDF potently stimulates platelet recovery, accompa-nied by increased megakaryocytopoiesis, in thrombocy-topenic male W/B F1 mice. Although platelet destructionwas accelerated in these mice, multiple doses of PEG-rHuMGDF resulted in sustained thrombocytosis, along withan increase in reticulated platelets. These observations sug-gest that PEG-rHuMGDF therapy stimulated production ofnew platelets to such an extent that the increase in plateletcounts induced by PEG-rHuMGDF might overcome plate-let destruction. Our data are in agreement with previousstudies of PEG-rHuMGDF on thrombocytopenia due to ac-celerated platelet destruction in human immunodeficiencyvirus (HIV) infection. PEG-rHuMGDF administrationgreatly improved thrombocytopenia in chimpanzees in-

fected with HIV [33] and in HIV-infected patients in anearly clinical trial [34].

In the present study, pretreatment levels of PAIgG ap-peared to be inversely related to the degree of thrombocy-topenia, as reported previously [27]. Because these datasuggest that platelet destruction is accelerated in male W/BF1 mice with higher PAIgG levels, it was thought that suchmice might have a lower platelet response to PEG-rHuMGDF. However, our results indicate that the plateletresponse by male W/B F1 mice to PEG-rHuMGDF was in-dependent of pretreatment PAIgG levels. This probably re-flects the heterogeneity of the disease. In addition to PAIgGlevels, antiplatelet antibodies might be heterogeneous interms of their affinity and the epitopes that they recognize.Additionally, there might be differences between mice inthe ability of bone marrow CFU-MK to respond to PEG-rHuMGDF.

It is possible that an increase in platelet counts stimulatedby PEG-rHuMGDF therapy might boost production of anti-platelet autoantibodies, but our results indicated that thiswas not the case. During the thrombocytosis elicited by4-week, intermittent treatment with PEG-rHuMGDF, thelevels of PAIgG decreased below pretreatment values.These results suggest that the elevation of platelets inducedby PEG-rHuMGDF resulted in a relative decrease in thelevels of PAIgG per platelet without enhancing antiplateletantibody production. In support of these data, PEG-rHuMGDF treatment led to no further reduction in theplatelet lifespan. Moreover, long-term treatment resulted insustained thrombocytosis in the surviving mice, indicating

Figure 5. Effect of PEG-rHuMGDF on thrombocytopenia in male W/B F1

mice, refractory to splenectomy. Groups of mice whose platelet counts didnot recover 60 days after splenectomy were chosen and given 30 �g/kg ofPEG-rHuMGDF (closed symbols) (n � 4) or vehicle (open symbols) (n �3) intravenously daily for 7 days. Each point represents platelet countsfrom individual mice.

Figure 6. Effect of PEG-rHuMGDF on platelet lifespan. Shown are plate-let lifespan of female control female W/B F1 mice (5.34 days) (�); maleW/B F1 mice given 30 �g/kg of PEG-rHuMGDF intravenously three timesper week for 3 weeks (3.49 days) (�), and male W/B F1 mice given vehicleintravenously three times per week for 3 weeks (3.36 days) (�). Each pointrepresents the mean value from five mice.

K. Shibuya et al./Experimental Hematology 30 (2002) 1185–1192 1191

that there was no enhancement of antiplatelet antibody pro-duction.

Reflecting the heterogeneity of the disease, male W/B F1

mice responded differently to splenectomy, similar to clini-cal observations in patients with ITP. Although some maleW/B F1 mice recovered from thrombocytopenia after sple-nectomy, others did not respond to splenectomy and re-lapsed into thrombocytopenia. Our results indicated thatPEG-rHuMGDF had a profound effect on thrombocytope-nia in the refractory cases. This finding indicates that PEG-rHuMGDF might be effective for treatment of patients withchronic ITP refractory to splenectomy.

In conclusion, our results indicate that PEG-rHuMGDFtherapy potently stimulates megakaryocytopoiesis and plate-let production, thereby effectively improving thrombocy-topenia in a murine model of ITP. These data suggest theclinical usefulness of PEG-rHuMGDF in ameliorating throm-bocytopenia in patients with ITP.

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