Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Farese, A. M.
Right arrow Articles by MacVittie, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Farese, A. M.
Right arrow Articles by MacVittie, T. J.
Stem Cells, Vol. 19, No. 6, 514-521, November 2001
© 2001 AlphaMed Press

A Single Dose of Pegylated Leridistim Significantly Improves Neutrophil Recovery in Sublethally Irradiated Rhesus Macaques

Ann M. Faresea, Daniel B. Caseya, Roy M. Vigneullea, Ned R. Siegelb, Rory F. Finnb, Jon A. Kloverb, Walter G. Smithb, John P. McKearnb, Thomas J. MacVittiea

a University of Maryland, Greenebaum Cancer Center, Baltimore, Maryland, USA;
b Pharmacia Corp., St. Louis, Missouri, USA

Key Words. Leridistim • Myelopoietin • Myelosuppression • Rhesus • Neutrophil

Ann M. Farese, M.S., University of Maryland, Greenebaum Cancer Center, 655 West Baltimore Street, BRB 7-049, Baltimore, Maryland USA, 21201; Telephone: 410-328-5347; Fax: 410-328-5488; e-mail: afarese{at}umaryland.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leridistim, a member of the myelopoietin family of dual receptor agonists that binds interleukin-3 and G-CSF receptors, has been shown to enhance hematopoietic activity in rhesus monkeys above that observed with either cytokine alone or in combination. This study demonstrated the ability of a pegylated form of leridistim (peg-leridistim), administered s.c., as a single- or two-dose regimen separated by 4 or 7 days, to significantly improve neutrophil recovery following radiation-induced myelosuppression. Rhesus macaques were total body x-irradiated (250 kVp, TBI) to 600 cGy. Following TBI, two groups received peg-leridistim (n = 5) or leridistim (n = 4) at a dose of 600 µg/kg on day 1, while two other groups (both n = 4) received peg-leridistim at 200 µg/kg on day 1 and day 4, or day 1 and day 7. The irradiation controls (n = 7) received 0.1% autologous serum for 18 days. All peg-leridistim treatment schedules significantly improved all neutrophil-related parameters following TBI as compared with nontreated controls and were equivalent in effect when compared among themselves. Administration of a single high dose or two separate lower doses of peg-leridistim significantly improved neutrophil regeneration, in a manner equal to that of conventional daily or abbreviated every-other-day administration of leridistim in this nonhuman primate model of severe myelosuppression.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leridistim, a member of the myelopoietin family of dual receptor agonists that binds interleukin-3 (IL-3) and G-CSF receptors, has been shown to enhance hematopoietic activity in myelosuppressed nonhuman primates. Multilineage hematopoiesis was stimulated above that observed with either cytokine alone or in combination when leridistim was administered in daily (qd) or every other day (qod) schedules for 18 days [1,2]. The favorable pharmacodynamic profile exhibited by the abbreviated, qod administration of leridistim suggested that further alteration in its pharmacokinetic properties by addition of polyethylene glycol may significantly enhance its therapeutic utility from both standpoints of patient compliance and administration schedule. Recently, a new pegylated form (SD-01) of filgrastim (rmet-HuG-CSF) has been shown to have a sustained concentration in vivo [3–5]. SD-01 has demonstrated enhanced ability to mobilize peripheral blood progenitors and increase neutrophil recovery in chemotherapy-treated mice or rhesus macaques following radiation-induced myelosuppression or bone marrow transplantation when administered as a single or two separate injections relative to standard, daily injections of filgrastim [3–5]. The potential to reduce the effective administration schedule of therapeutic cytokines will offer significant advantages in treatment scheduling as well as allow for further insights into stimulating hematopoietic regeneration subsequent to cytotoxic therapy or stem cell transplantation. We report herein on the relative therapeutic efficacy of peg-leridistim administered as a single or two separate injections to stimulate hematopoietic recovery in rhesus macaques following high-dose sublethal irradiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male rhesus monkeys, Macaca mulatta, mean weight 4.35 ± 0.32 kg, were housed in individual stainless steel cages in conventional holding rooms at the Veterinary Resources Department at the Greenebaum Cancer Center in animal facilities accredited by the American Association for Accreditation of Laboratory Animal Care (http://www.aaalac.org). Monkeys were provided 10 air changes/hour of 100% fresh air, conditioned to 72° ± 2°F with a relative humidity of 50% ± 20% and maintained on a 12-hour light/dark full spectrum light cycle, with no twilight. Monkeys were provided with commercial primate chow, supplemented with fresh fruit and tap water ad libitum. Research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals [6], prepared by the Institute of Laboratory Animal Resources, National Research Council (http://www.nas.edu/nrc).

Irradiation
Monkeys, following a prehabituation period, were unilaterally irradiated in Lucite® restraining chairs with 250 kVp x-radiation at 13 cGy/minute in the posterior-anterior position, rotated 180° at the mid-dose (300 cGy) to the anterior-posterior position for completion of the total 600 cGy midline tissue exposure. Dosimetry was performed using paired 0.5 cm3 ionization chambers, with calibration factors traceable to the National Institute of Standards and Technology.

Clinical Support
All animals received clinical support that consisted of antibiotics, fresh irradiated whole blood, and fluids as needed. Gentamicin (Elkin Sinn, an AH Robbins subsidiary; Cherry Hill, NJ) (10 mg/day, i.m., qd) was administered during the first 7 days of treatment, and Baytril® (Bayer Corporation; Shawnee Mission, KS; http://www.bayerus.com) (10 mg/day i.m., qd) was administered for the entire period of antimicrobial treatment. The administration of antibiotics continued until the animal maintained a WBC >=1,000/µl for 3 consecutive days and had attained an ANC >=500/µl [7,8]. Fresh, irradiated (1,500 cGy Co-60) whole blood (approximately 30 ml/transfusion) from a random donor pool (monkeys of >10 kg) was administered when the platelet (PLT) count was <20,000/µl and the hematocrit was <18%.

Recombinant Cytokines
Leridistim is from the myelopoietin family of engineered, chimeric hematopoietic growth factors that bind and activate the IL-3 and G-CSF receptors [9]. It was produced in E. coli through the use of a plasmid-based expression vector and expressed in insoluble inclusion bodies within the E. coli cells. Washed inclusion bodies were solubilized in urea buffer and disulfide bonds formed through air oxidation after lowering the urea concentration. Leridistim was purified by ion exchange chromatography and filtration. Purified protein was stored as a frozen solution in 10 mM Tris buffer, ph 8.0.

Synthesis of Pegylated Leridistim
Leridistim (4.5 mg/ml in 10-20 mM sodium acetate, pH 4.5) was reacted with 30,000 molecular weight Methoxy-peg-propionaldehyde (M-peg-ALD; Shearwater Polymers Inc.; Huntsville, AL; http://www.shearwatercorp.com) by addition of solid M-peg-ALD to yield a relative peg:leridistim molar ratio of 6.5:1. Reactions were catalyzed by addition of stock 1 M NaCNBH4 dissolved in H20 to a final concentration of 20 mM. Reactions were carried out at 4°C for 18-96 hours and, subsequently, were stopped by lowering pH to 4.0 with 0.1 N acetic acid or by adding a 5x molar excess of Tris HCl. Peg-leridistim was subsequently purified using cation exchange chromatography carried out on an SP Sepharose high performance column (Pharmacia XK 26/20, 70 ml bed volume) equilibrated in 10 mM sodium acetate pH 4.5 (buffer C). The reaction mixture was diluted 10x with buffer C and loaded onto the column at a flow rate of 5 ml/min. Next the column was washed with five column volumes of buffer C, followed by five column volumes of 12% buffer D (10 mM acetate pH 4.5, 1 M NaCl). Subsequently, the peg-leridistim was eluted from the column with a linear gradient of 12% to 27% buffer D in 20 column volumes. Fractions were pooled, buffer-exchanged into 10 mM acetate pH 4.5 buffer, and concentrated to 1-5 mg/ml in a stirred cell fitted with an Amicon YM10 membrane.

Study Design

Normal Animals   Normal animals were treated with either A) leridistim administered at 200 µg/kg, s.c., (n = 3), or B) peg-leridistim at 200 µg/kg, s.c. (n = 2) in order to determine pharmacokinetic data.

Irradiated Animals   In each experimental group, animals were irradiated at day 0 and randomly assigned to a treatment protocol: A) controls (n = 7) received autologous serum (AS), s.c. for 18 days (d); B) leridistim administered at 600 µg/kg, s.c., on day 1 only (n = 4); C) peg-leridistim at 600 µg/kg/d, s.c., on day 1 only (n = 5); D) peg-leridistim at 200 µg/kg/d, s.c., on day 1 and day 4 (n = 4), and E) peg-leridistim at 200 µg/kg, s.c., on day 1 and day 7 (n = 4) post 600 cGy of x-irradiation. Complete blood counts were monitored for 60 days following irradiation and the durations of neutropenia (ANC <500/µl) and thrombocytopenia (PLT <20,000/µl) were assessed. Bone marrow-derived clonogenic activity was examined prior to irradiation (baseline) and on days 7, 14, 21, and 46 post-total body iradiation (TBI).

Bone Marrow Aspirations
Animals were sedated with Ketaset® (10 mg/kg, i.m.; Fort Dodge Laboratories; Fort Dodge, IA) plus buprenorphine (Buprenex® Injectable 10 µg/kg, i.m.; Rickett & Coleman Pharmaceuticals; Richmond, VA) and approximately 2 mls of heparinized bone marrow (BM) were aspirated from the humerus. Low-density (<1.077 g/cm3) mononuclear cells (MNC) were separated using Histopaque (Sigma; St. Louis, MO; http://www.sigma-aldrich.com) and resuspended in Iscove's modified Dulbecco's medium (IMDM) (GIBCO; Grand Island, NY; http://www.tmc.tulane.edu/sif/tulgib.htm).

Hematologic Evaluations

CBCs   Peripheral blood was obtained from the saphenous vein to assay complete blood (Sysmex K-4500; Long Grove, IL) and differential counts (Wright-Giemsa Stain, Ames Automated Slide Stainer; Elkhart, IN) for 60 days post-TBI.

Bone Marrow-Derived Clonogenics   Culture medium contained 0.9% methylcellulose (MethoCult H4230, Stem Cell Technologies; Vancouver, BC; http://www.stemcell.com) in IMDM with 30% fetal calf serum (Hyclone Laboratories; Logan, UT; http://www.hyclone.com). In addition, a combination of recombinant human (rHu) cytokines, G-CSF (5 ng/ml), stem cell factor (50 ng/ml), erythropoietin (2 U/ml), megakaryocyte growth and development factor (20 ng/ml; Amgen; Thousand Oaks, CA; http://www.amgen.com), IL-3 (20 ng/ml), GM-CSF (5 ng/ml), and IL-6 (40 ng/ml; Sandoz Pharmaceuticals; East Hanover, NJ; http://www.pharma.novartis.com) were added to each culture dish. BM-derived MNC were cultured at a plating density of 3.2 to 5 x 104 cells/ml (days 0, 21, 46, and 60 post-TBI) or 1 x 105 cells/ml (days 7 and 14 post-TBI). Cells were incubated for 10 days at 37°C with 5% CO2 in air in a fully humidified incubator. GM-colony-forming cells (GM-CFC) derived colonies (>50 cells) were expressed as the number of CFC/105 MNC.

Pharmacokinetic Analysis
Leridistim and peg-leridistim levels in rhesus plasma samples were determined using a sandwich enzyme-linked immunosorbent assay. Microtiter plates (Dynatech-Immulon II, VWR; St. Louis, MO) were coated overnight at room temperature with affinity purified goat-anti-rHuG-CSF (C17S) polyclonal antibody at 1 mg/ml in 100 mM NaHCO3, pH 8.2. Plates were blocked for 1 hour at 37°C with 3% bovine serum albumin (Sigma) in Dulbecco's, pH 7.4 (GIBCO BRL; Grand Island, NY) containing 0.05% Tween 20 (Sigma). Plates were washed four times with 150 mM NaCl (J. T. Baker; Phillipsburg, NJ; http://www.jtbaker.com ) containing 0.05% Tween 20 (wash buffer). Standards and plasma samples were diluted in rhesus pooled plasma matrix and incubated for 2.5 hours at 37°C. Unbound protein was washed away and plates were incubated with horseradish peroxidase-conjugated, affinity-purified goat-anti-rHuIL-3 (15-125, E50D) polyclonal antibody, for 1.5 hours at 37°C in a humidified incubator. Plates were washed and developed with 3, 3', 5, 5' tetramethylbenzidine peroxidase substrate (Kirkegaard and Perry; Gaithersburg, MD; http://www.kpl.com). Absorbance was measured at 650 nm using a microtiter plate reader (Molecular Devices; Sunnyvale, CA; http://www.moleculardevices.com) and concentrations of immunoreactive leridistim or peg-leridistim were calculated based upon standard curves determined for each compound, using software supplied with the plate reader. Pharmacokinetic analyses (one compartment) were performed using WinNonlin software (Pharsight; Mountain View, CA; http://www.pharsight.com).

Statistical Analysis
The Normal Scores Test was used to make pairwise comparisons of the durations of neutropenia and thrombocytopenia and to evaluate the statistical significance between the nadirs. The exact p values were obtained. The test was carried out using the software package StatXact (Cytel Software Corp.; Cambridge, MA; http://www.cytel.com). BM-derived clonogenic activities were analyzed for each endpoint at each day across the control and leridistim and peg-leridistim treatment groups using the Kruskal-Wallis Test. Post hoc tests were made if the p value for the Kruskal-Wallis Test was <=0.05 and were made using a Dunn's Test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pharmacokinetic Parameters of Peg-Leridistim and Leridistim

Pharmacokinetics in Normal Animals   The effect of PEGylation upon the clearance of leridistim can be observed following single, s.c. dosing to normal (untreated) rhesus monkeys. The pharmacokinetic parameters determined (Fig. 1Go, Table 1Go) following the administration of a single 200 µg/kg bolus dose of leridistim, or peg-leridistim, indicate that PEGylation effectively enhanced overall relative exposure (area under the concentration time curve [AUCI]) by delaying the time (Tmax) to, as well as the level of, the observed maximum plasma concentration (Cmax), while also effectively doubling the elimination half-life (t1/2). The net result was a reduction in the overall rate of clearance (Cl/F) by nearly 20-fold.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. The pharmacokinetics of leridistim and peg-leridistim in normal and irradiated rhesus macaques. Normal and 600 cGy-irradiated rhesus macaques were administered leridistim or peg-leridistim s.c.: leridistim at 200 µg/kg (n = 3), peg-leridistim at 200 µg/kg (n = 2); Irradiated macaques: leridistim at 600 µg/kg (n = 4), peg-leridistim at 200 µg/kg (n = 3), or 600 µg/kg (n = 4). Plasma samples were taken before and at selected times after injection of the growth factors (Materials and Methods). Values are mean ± standard error.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Pharmacokinetic parameters for leridistim and peg-leridistim in normal and irradiated rhesus macaques following s.c. dosing
 

Pharmacokinetics in Irradiated Animals   The pharmacokinetic parameters were also measured after leridistim and peg-leridistim were administered to cohorts of 600 cGy-irradiated animals. In this case, while the dosage level was increased to 600 µg/kg and the drug administered 24 hours after irradiation (as a single, s.c. bolus), the data (Fig. 1Go, Table 1Go) indicate that leridistim was cleared with essentially the same relative kinetics (i.e., t1/2 and Cl/F) in both the normal and the irradiated monkeys. In fact, when dose adjusted, the relative Cmax and overall exposure (AUCI) were very similar for all animals treated with the leridistim. For peg-leridistim, while the irradiated monkey cohorts treated with a single bolus of drug at 200 µg/kg cleared the drug at roughly the same rate as did their normal counterparts (i.e., similar overall exposure (AUCI) and average Cl/F values), the net effect of pegylation upon the relative rate of clearance (versus unconjugated leridistim) presumably allowed drug to remain in circulation until the circulating pool of neutrophils began to drop consequent to irradiation (Fig. 2Go). The net effect was a slowing of the receptor-mediated clearance as the ANC fell and an effective enhancement of the observed elimination half-life (t1/2) from 7.8 hours to 33 hours. This effect was even more dramatic when the level of the peg-leridistim dose was raised to 600 µg/kg. In these animals, the higher dose of drug resulted in a further protraction in the relative elimination t1/2, an enhanced net overall exposure, and a drop in the average rate of clearance, presumably due to the fact that more drug was present in the circulation during the period of irradiation-induced neutropenia.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Effect of leridistim and peg-leridistim on peripheral blood ANC in 600 cGy x-irradiated rhesus macaques. Animals were administered either leridistim (n = 4) or peg-leridistim (n = 5) at 600 µg/kg on day 1 or peg-leridistim on day 1 and day 4 (n = 4), or day 1 and day 7 (n = 4) at 200 µg/kg/d, or control AS (n = 7) as described in Materials and Methods. Values are mean ± standard error.

 
Neutrophil Recovery

Peg-Leridistim Administered on Day 1, Day 1 and Day 4, or Day 1 and Day 7 Schedules Versus Control   All neutrophil-related parameters were significantly improved relative to the control-treated cohort by administration of peg-leridistim at a single dose of 600 µg/kg on day 1 or two separate doses of 200 µg/kg on day 1 and day 4 or day 1 and day 7 post 600 cGy x-irradiation (Table 2Go, Fig. 2Go). Peg-leridistim, administered in either dose or schedule, effected a shorter duration of neutropenia (ANC < 500/µl) from 14.8 days in controls to 2.6, 3.0, and 2.0 days with the 600 µg/kg dose administered on day 1 or the 200 µg/kg dose on day 1 and either day 4 or day 7 (p <= 0.005), respectively. In a similar fashion, the respective ANC nadirs were significantly higher at 837/µl, 244/µl, and 377/µl relative to 8/µl for the control cohort (p < 0.005). As expected from these results, the time to recovery to an ANC >= 500/µl was also significantly improved (p <= 0.005) from the control value of 20.8 days to 7.0 days, 8.8 days, and 6.8 days for the day 1, or days 1 and day 4, or day 1 and day 7 administration schedules, respectively (Table 2Go, Fig. 2Go). Consequently, the days on antibiotics were significantly reduced (p <= 0.01) from 16.8 days for the controls to 6.2 days, 6.8 days, and 6.3 days for the respective cohorts treated with peg-leridistim.


View this table:
[in this window]
[in a new window]
  		Table 2. Neutrophil-related parameters in sublethally x-irradiated rhesus macaques treated with leridistim or peg-leridistim: duration, nadir, time to recovery, and clinical support
 
Leridistim Administered in the High-Dose, Day 1 Schedule Versus Control
In contrast, leridistim, administered as a single, high dose (600 µg/kg) on day 1 post-irradiation neither significantly improved the duration of neutropenia (11.8 days), nor the time to recovery to ANC >500/µl (19.3 days) nor antibiotic requirements (18.0 days) relative to the AS-treated controls (14.8 days, 20.8 days, and 16.8 days, respectively). Only the neutrophil nadir was improved relative to the controls (Table 2Go, Fig. 2Go).

Peg-Leridistim Versus Single, High-Dose Leridistim Administration Schedule
Peg-leridistim administered in either the day 1, day 1 and day 4, or day 1 and day 7 schedules significantly improved the duration of neutropenia (2.6 days, 3.0 days, 2.0 days), time to recovery to ANC >500/µl (7.0 days, 8.8 days, 6.8 days), and antibiotic requirements (6.2 days, 6.8 days, 6.3 days) respectively, relative to the single, high-dose leridistim injection (11.8 days, 19.3 days, and 18.0 days, respectively) on day 1 post-irradiation. The ANC nadirs following peg-leridistim administration at day 1 or day 1 and 7 were significantly higher than that of the single high-dose leridistim cohort.

Comparison of Peg-Leridistim-Induced Recovery to that of Leridistim Administered in a qod Schedule
We previously investigated the relative efficacy of the myelopoietin chimeric factors in bid, qd, and qod administration schedules in the same 600 cGy TBI model used herein [1,2]. We have added the key neutrophil-related parameters for leridistim administered in an abbreviated qod schedule, as well as its control cohort in Table 2Go for ready comparison to those generated herein.

In that study, the qod administration of leridistim at 200 µg/kg (nine injections over 17 days), significantly improved (p < 0.005) all neutrophil-related parameters relative to its time-matched control cohort (Table 2Go).

Herein, we showed that peg-leridistim administered in day 1, day 1 and day 4, or day 1 and day 7 schedules stimulated neutrophil recovery in a manner equivalent to that noted with the qod schedule (Table 2Go).

Peg-Leridistim Administered on Day 1 and Day 4 Versus Day 1 and Day 7
The more proximate day 1 and day 4 administration of peg-leridistim relative to the day 1 and day 7 schedule was used to enhance proliferation and concentration of BM-derived myeloid progenitors (GM-CFC) with an intended consequent increase in production of neutrophils. Although GM-CFC activity was significantly increased (p = 0.01) at day 14 post-exposure in the day 1 and day 4 cohort versus the time-matched control cohort (Table 3Go) the schedule did not result in a greater increase in neutrophil production (Table 2Go, Fig. 2Go) relative to the other peg-leridistim treatment groups. In fact, there was no significant difference in neutrophil-related parameters among the peg-leridistim-treated cohorts. All schedules were equivalent in stimulating neutrophil recovery in the myelosuppressed animal.


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of leridistim or peg-leridistim on the concentration of bone marrow-derived GM-CFC (reported as percent of baseline values) in 600 cGy x-irradiated rhesus macaques
 
BM-Derived Clonogenic Activity
BM-derived GM-CFC (CFC/105 MNCs, presented as percent of baseline), were evaluated before (baseline) and at day 7, 14, 21, 46, and 60 after TBI in control, leridistim-, and peg-leridistim-treated cohorts (Table 3Go). In the control cohort, GM-CFC activity was significantly diminished through 14 days (5% of baseline) and had recovered to values approximately 50% of baseline by 21 days post-exposure. Administration of peg-leridistim in either the day 1 and day 4 or day 1 and day 7 schedule significantly improved (p = 0.01 and 0.05, respectively) recovery of GM-CFC within 14 days post-irradiation to values that are, respectively, 62% and 31% of their time-matched control cohort. All of the peg-leridistim treated cohorts recovered to within or above their baseline, preirradiation values by 21 days post-exposure whereas the single dose (600 µg/kg), day 1 leridistim cohort, recovered in a manner similar to the controls.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Modification of the chimeric growth factor receptor agonist leridistim with poly (ethylene glycol) significantly enhanced its pharmacokinetic and pharmacodynamic profile such that a single high dose, or two separate lower doses administered at day 1, day 1 and day 4, or day 1 and day 7, respectively, stimulated all neutrophil-related parameters in a manner equivalent to that of conventional, qd or abbreviated, qod, administration of nonpegylated leridistim [1,2]. The pharmacokinetic parameters following one or two doses of peg-leridistim in irradiated animals suggested that the sustained in vivo availability would translate into equivalent pharmacodynamics to that noted with conventional, daily injection schedules of leridistim. In fact, Molineux et al. recently demonstrated that a new sustained-duration form of filgrastim (SD-01), modified by association with poly (ethylene glycol), was effective as a single dose in reducing 5-fluorouracil-induced neutropenia in mice as well as mobilizing CD34+ cells in normal human volunteers relative to respective conventional dosing [3]. Furthermore, SD-01 as a single or double injection separated by 7 days has significantly improved neutrophil recovery following radiation-induced myelosuppression or autologous bone marrow transplantation in rhesus macaques [4,5]. In the myelosuppression study, a single dose of SD-01 appeared to be as effective as daily filgrastim administration while two doses of SD-01 at a weekly interval further improved all neutrophil-related parameters [5]. Herein, the administration of peg-leridistim regardless of schedule, that is, day 1, and either day 1 and day 4, or day 1 and day 7, was equivalent to conventional dosing of myelopoietin or abbreviated (qod) dosing of leridistim in stimulating recovery of neutrophils consequent to radiation-induced myelosuppression [1,2].

As mentioned earlier, the biological effects of peg-leridistim are most likely related to its improved pharmacokinetics. A reasonable assumption is that peg-leridistim or leridistim is cleared from the circulation by a G-CSF receptor-dominant process and therefore similar to the mechanisms involved for G-CSF and SD-01 [10–13]. Peg-leridistim, similar to SD-01, may be more efficiently regulated by circulating levels of neutrophils than by renal clearance, since the pegylation of the cytokine may reduce renal clearance [10,14]. In the study presented herein, the efficacy of peg-leridistim in stimulating early production of neutrophils would effectively "self-regulate" its circulating concentration [10]. The pharmacokinetic analysis performed in the peg-leridistim-treated, irradiated animals suggested increased G-CSF receptor-mediated clearance due to improved neutrophil levels.

These studies suggest that a single injection of peg-leridistim is equivalent to more conventional bid, qd, or qod administration of myelopoietin/leridistim in stimulating neutrophil recovery in a nonhuman primate model of severe radiation-induced myelosuppression [1,2]. These results further suggest that a prolonged neutrophil-stimulating effect will be obtained from a single injection of peg-leridistim. The potential to reduce the effective administration schedule of therapeutic growth factors will offer a significant advantage in treatment scheduling subsequent to cytotoxic therapy.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors wish to thank Michael Flynn, Carrie Redinger, Scott Perry, Jennifer Schumaker, Allison Carlen, James Bloss, Jerry Galluppi, Karen Walters, Peter Nicastro, and Doreen Villani-Price for their superb technical assistance and William Jackson for assistance with the statistical analysis.

Research supported by contract provided by Pharmacia Corporation. The views presented herein are those of the authors.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. MacVittie TJ, Farese AM, Smith WG et al. Myelopoietin, an engineered chimeric IL-3 and G-CSF receptor agonist, stimulates multilineage hematopoietic recovery in a nonhuman primate model of radiation-induced myelosuppression. Blood 2000;95:837–845.[Abstract/Free Full Text]

  2. Farese AM, Casey DB, Smith WG et al. Leridistim, a chimeric dual G-CSF and IL-3 receptor agonist, enhances multilineage hematopoietic recovery in a nonhuman primate model of radiation-induced myelosuppression: effect of schedule, dose, and route of administration. STEM CELLS 2001;6:522–533.

  3. Molineux G, Kinstler O, Briddell B et al. A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans. Exp Hematol 1999;27:1724–1734.[CrossRef][Medline]

  4. Farese AM, Roskos L, Cheung E et al. A single administration of r-metHuG-SD/01 (SD01) significantly improves neutrophil recovery following autologous bone marrow transplantation. Blood 1998;92:112a.

  5. Farese AM, Roskos L, Stead RB et al. r-metHuG-CSF-SD/01 (SD/01) significantly improves neutrophil recovery in myelosuppressed non human primates. Blood 1999;94:49a.

  6. The Institute of Laboratory Animal Resources, National Research Council: Guide for the Care and Use of Laboratory Animals. Washington: National Institutes of Health, 1996:86.

  7. Farese AM, Herodin F, McKearn JP et al. Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced marrow aplasia. Blood 1996;87:581–591.[Abstract/Free Full Text]

  8. Farese AM, Hunt P, Grab LB et al. Combined administration of recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor enhances multilineage hematopoietic reconstitution in nonhuman primates after radiation-induced marrow aplasia. J Clin Invest 1996;97:2145–2151.[Medline]

  9. Smith WG, Giri JG, Abegg A et al. Preclinical pharmacology of the myelopoietin leridistim, a multifunctional agonist of IL-3 and G-CSF receptors. Blood 1998;92:380a.

  10. Roskos LK, Yank B, Schwab G et al. Cytokinetic model of rmethHuG-CSF-SD/01 (SD/01) mediated granulopoiesis and the "self-regulation" of SD/01 elimination in non-small cell lung cancer. Blood 1998;92:507a.

  11. Tanaka H, Kaneko T. Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in the rat. Single and multiple dosing studies. Drug Metab Dispos 1991;19:200–204.[Abstract]

  12. Roskos L, Cheung EN, Vincent M et al. Pharmacology of filgrastim (r-metHuG-CSF). In: Morstyn G, Dexter TM, Foote MA, eds. Filgrastim in Clinical Practice. New York: Marcel Dekker, 1998:51-71.

  13. Tanaka H, Satake-Ishikawa R, Ishikawa M et al. Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to poly (ethylene glycol) in rats. Cancer Res 1991;51:3710–3714.[Abstract/Free Full Text]

  14. Tanaka H, Tokiwa T. Influence of renal and hepatic failure on the pharmacokinetics of recombinant human granulocyte colony-stimulating factor (KRN8601) in the rat. Cancer Res 1990;50:6615–6619.[Abstract/Free Full Text]

Received July 3, 2001; accepted for publication July 10, 2001.



This article has been cited by other articles:


Home page
 JCOHome page
T. J. Smith, J. Khatcheressian, G. H. Lyman, H. Ozer, J. O. Armitage, L. Balducci, C. L. Bennett, S. B. Cantor, J. Crawford, S. J. Cross, et al.
2006 Update of Recommendations for the Use of White Blood Cell Growth Factors: An Evidence-Based Clinical Practice Guideline
J. Clin. Oncol., July 1, 2006; 24(19): 3187 - 3205.
[Abstract] [Full Text] [PDF]

Home page
 ANN INTERN MEDHome page
J. K. Waselenko, T. J. MacVittie, W. F. Blakely, N. Pesik, A. L. Wiley, W. E. Dickerson, H. Tsu, D. L. Confer, C. N. Coleman, T. Seed, et al.
Medical Management of the Acute Radiation Syndrome: Recommendations of the Strategic National Stockpile Radiation Working Group
Ann Intern Med, June 15, 2004; 140(12): 1037 - 1051.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
A. M. Farese, T. J. MacVittie, L. Roskos, and R. B. Stead
Hematopoietic Recovery Following Autologous Bone Marrow Transplantation in a Nonhuman Primate: Effect of Variation in Treatment Schedule with PEG-rHuMGDF
Stem Cells, January 1, 2003; 21(1): 79 - 89.
[Abstract] [Full Text] [PDF]

Home page
 ASH Education BookHome page
N. Dainiak, J. K. Waselenko, J. O. Armitage, T. J. MacVittie, and A. M. Farese
The Hematologist and Radiation Casualties
Hematology, January 1, 2003; 2003(1): 473 - 496.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Farese, A. M.
Right arrow Articles by MacVittie, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Farese, A. M.
Right arrow Articles by MacVittie, T. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS