Chimeric Growth Factor Receptor Agonists Myelopoietins Leridistem and Peg Leridistim 311 Myelopoietin

Myelopoietins are a family of proteins that are dual receptor agonists of the human IL-3 and G-CSF receptor complexes. Two members of the myelopoietin family, myelopoietin SC-68420 and leridistim, have been evaluated for their ability to enhance hematopoietic recovery in a nonhuman primate model of radiation-induced myelosup-pression (21,68). Myelopoietin (SC-68420) was initially shown to effectively stimulate bone marrow regeneration (clonogenic activity) and neutrophil recovery when administered daily, subcutaneously, in a twice-a-day or once-a-day schedule beginning on d 1 after irradiation and continued for 18-23 d (21). Myelopoietin, regardless of administration schedule, significantly reduced the mean duration of neutropenia and improved the neutrophil nadir and time to recovery of neutrophils to an ANC > 0.5 x 109/L relative to the control-treated cohort. Thus myelopoietin (SC-68240) administered daily was as effective as a twice-a-day schedule for enhancing neu-trophil recovery after high-dose radiation-induced myelosuppression.

3.1.2. Leridistim

These results prompted further evaluation of another structurally distinct member of the myelopoietin family, leridistim.

Leridistim was evaluated in a more abbreviated schedule, lower dose, and intravenous vs subcutaneous administration route (68). Animals were irradiated on d 0 and then received, on d 1, leridistim sc in an abbreviated, every-other-day schedule at 200 pg/kg or daily at 50 pg/kg; or iv, daily or every-other-day schedules at 200 pg/kg/injec-tion. Other experimental cohorts received filgrastim in an every-other-day schedule at 100 pg/kg/injection or autologous serum (daily). Leridistim was administered until an absolute neutrophil count (ANC) > 3 x 109/L was attained (range: 13-18 d). Leridistim administered subcutaneously in an alternate-day schedule significantly enhanced neu-trophil recovery. The abbreviated, every-other-day schedule administered subcuta-neously was as effective as the conventional daily or twice-a-day schedule used to assess myelopoietin (SC-68240), in the same animal model (Table 2) (21). This response was of interest considering the ineffective results of leridistim's component growth factors, IL-3 and G-CSF, when administered in schedules less than twice a day or daily, respectively. Filgrastim (100 pg/kg) administered in an sc, every-other-day schedule did not significantly enhance neutrophil-related parameters (68). On the other hand, filgrastim administered in a conventional daily, schedule either alone or in combination with daniplestim (an IL-3 receptor agonist) effectively enhanced neutrophil recovery in two similar models of radiation-induced myelosuppression (17,18,69). It was hypothesized that since IL-3 receptors are present on CD34+ cell subsets purified from human and rhesus marrow (70,71), the IL-3 component in leridistim could contribute to the enhanced granulopoietic response by priming progenitor cells for subsequent stimulation by endogenous growth factors released within the radiation-damaged marrow microenvironment (72,73) as well as by exerting trophic or survival-promoting effects on responsive progenitors (74-77). These data suggested that if the appropriate dose and schedule could be achieved in clinical trials, leridistim would be an effective therapeutic for drug-induced myelosuppression.

3.1.3. Peg-Leridistim

Leridistim was subsequently pegylated using a 30-kDa polyethylene glycol moiety attached to the Escherichia coli-derived leridistim. Peg-leridistim was subjected to pharmacokinetic and pharmacodynamic analysis before evaluating biologic efficacy in a rhesus model of radiation-induced myelosuppression. The pharmacokinetic parameters assessed after the administration of a single, subcutaneous bolus to normal rhesus monkeys indicated that pegylation effectively enhanced overall relative exposure (area under the concentration curve [AUC]) 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 overall rate of clearance by nearly 20-fold (78). The pharmacokinetic analysis of peg-leridistim in

Table 2

Neutrophil-Related Parameters in Sublethally X-Irradiated Rhesus Monkeys Treated With Leridistim: Duration, Nadir, Time to Recovery, and Clinical Support"

Table 2

Neutrophil-Related Parameters in Sublethally X-Irradiated Rhesus Monkeys Treated With Leridistim: Duration, Nadir, Time to Recovery, and Clinical Support"

Treatment

Duration (d)

ANC nadir

Time to

Antibiotic

(dose, route, schedule)

of neutropenia

(L-1)

recovery (d)

support (d)

Control 0.1% AS,

sc, qd

19.1 ± 2.3

16 ± 6

24.5 ± 2.2

19.6 ± 1.7

Leridistim

50 |g/kg, sc, qd

3.3 ± 1.6*

400 ± 100*

11.0 ± 2.4*

8.0 ± 2.2*

200 |g/kg, sc, qod

1.0 ± 0.6*

601± 100*

4.1 ± 2.2*

5.8 ± 1.0*

200 |g/kg, iv, qd

2.7 ± 1.8*

442 ± 227*

7.0 ± 4.4*

4.3 ± 3.0*

200 |g/kg, iv, qod

9.0 ± 1.7**

63 ± 30**

16.3 ± 1.2**

14.7 ± 0.3

G-CSF

100 |g/kg, sc, qod

14.2 ± 1.9

10 ±4

19.4 ± 1.7

17.8 ± 1.8

SC-64820, myelopoietin

200 |g/kg, sc, qd6

2.8 ± 1.4

435 ± 200

8.3 ± 3.5

8.3 ± 1.3

Abbreviation: ANC, absolute neutrophil count; G-CSF, granulocyte colony-stimulating factor.

a Monkeys received total body irradiation (250 kVp) to 600 cGy with X-radiation and were treated sub-cutaneously (sc) with control protein, autologous serum (AS) (n = 9) or leridistim at 200 pg/kg/d, every other day (qod) (n = 8), or 50 pg/kg/d, daily (qd) (n = 7) or intravenously (iv) with leridistim at 200 pg/kg/d either qd or qod, n = 3 per cohort according to protocol. The duration of neutropenia is defined as days of absolute neutrophil count (ANC) < 0.5 x 109/L and thrombocytopenia is defined as days of platelets < 20 x 109/L. Time to recovery is the number of days required for the ANC to reach >0.5 x 109/L and platelets to reach >20 x 109/L. Data represent mean values ± SEM.

b Previous Dataset: published study showing effects of SC-68420, myelopoietin, administered daily (qd) subcutaneously at 200 pg/d in the same 600 cGy X-irradiation model (21). This study showed that sc, qd, and bid administration were equivalent in bioeffect.

Abbreviation: ANC, absolute neutrophil count; G-CSF, granulocyte colony-stimulating factor.

a Monkeys received total body irradiation (250 kVp) to 600 cGy with X-radiation and were treated sub-cutaneously (sc) with control protein, autologous serum (AS) (n = 9) or leridistim at 200 pg/kg/d, every other day (qod) (n = 8), or 50 pg/kg/d, daily (qd) (n = 7) or intravenously (iv) with leridistim at 200 pg/kg/d either qd or qod, n = 3 per cohort according to protocol. The duration of neutropenia is defined as days of absolute neutrophil count (ANC) < 0.5 x 109/L and thrombocytopenia is defined as days of platelets < 20 x 109/L. Time to recovery is the number of days required for the ANC to reach >0.5 x 109/L and platelets to reach >20 x 109/L. Data represent mean values ± SEM.

b Previous Dataset: published study showing effects of SC-68420, myelopoietin, administered daily (qd) subcutaneously at 200 pg/d in the same 600 cGy X-irradiation model (21). This study showed that sc, qd, and bid administration were equivalent in bioeffect.

* Statistically different from AS-treated controls (p < 0.01).

** Statistically different from AS-treated controls (p < 0.05).

irradiated monkeys suggested that the net effect was a slowing of the receptor-mediated clearance as neutrophil counts decreased and an effective enhancement of the observed elimination half-life (i1/2) from 7-8 h to 33 h.

Peg-leridistim was evaluated as a single- or two-dose regimen separated by 4 or 7 d, to significantly improve neutrophil recovery after radiation-induced myelosup-pression (Fig. 7) (78). Two groups received Peg-leridistim or leridistim at a dose of 600 pg/kg on d 1, and two other groups received Peg-leridistim at 200 pg/kg on d 1 and d 4 or 7. This study showed that all Peg-leridistim treatment schedules significantly improved all neutrophil-related parameters after irradiation compared with the control-treated cohort and were equivalent in effect when compared among themselves. Furthermore, the Peg-leridistim-induced improvement in neutrophil regeneration was equivalent to that noted with leridistim or myelopoietin administered in the conventional daily or every-other-day schedule (21,68). Therefore, the single injection of Peg-leridistim was equivalent in effect to the double injection of Peg-leridistim and leridistim administered twice a day, daily, or every other day over 18-23 d after irradiation.

Time (days)

Fig. 7. Effect of leridistim and peg-leridistim on peripheral blood absolute neutrophil counts (ANC) in 600-cGy X-irradiated rhesus macaques. Animals were administered either leridistim (n = 4) or peg-leridistim (n = 5) at 600 |ig/kg on d 1, or peg-leridistim on d 1 and d 4 (n = 4), or d 1 and d 7 (n = 4) at 200 |g/kg/d, or control autologous serum (AS) (n = 7). Values are mean ± SE. (Reprinted with permission from ref. 78. © AlphaMed Press.)

Time (days)

Fig. 7. Effect of leridistim and peg-leridistim on peripheral blood absolute neutrophil counts (ANC) in 600-cGy X-irradiated rhesus macaques. Animals were administered either leridistim (n = 4) or peg-leridistim (n = 5) at 600 |ig/kg on d 1, or peg-leridistim on d 1 and d 4 (n = 4), or d 1 and d 7 (n = 4) at 200 |g/kg/d, or control autologous serum (AS) (n = 7). Values are mean ± SE. (Reprinted with permission from ref. 78. © AlphaMed Press.)

3.2. Pegfilgrastim

Pegfilgrastim, a new form of filgrastim, has been manufactured by the covalent attachment of a 20-kDa polyethylene glycol moiety to the amino terminus of E. coli-derived filgrastim. Recent studies in a rodent model of 5-fluorouracil (5-FU)-induced myelosuppression showed improved neutrophil recovery after a single dose of pegfil-grastim (79). Hartley et al. (80) extended these studies and evaluated the timing of peg-filgrastim administration relative to chemotherapy. Pegfilgrastim was administered at various times, either before or after treatment of mice with cyclophosphamide. Pread-ministration of pegfilgrastim was relatively ineffective, whereas pegfilgrastim injected early (on d 1 after chemotherapy) appeared to be the most effective in delaying the severity and duration of neutropenia. Further delaying the single-dose administration of pegfilgrastim lessened its effectiveness.

Recent preclinical studies from our laboratory underscored the therapeutic potential of the pegylated form of filgrastim (24,26). A single dose of pegfilgrastim was effective in improving neutrophil recovery in nonhuman primate models of myelosuppres-sion or AuBMT subsequent to high-dose radiation conditioning. Neutrophil recovery, after a single injection of pegfilgrastim (300 |g/kg), was equivalent to conventional daily dosing (10 |g/kg) with filgrastim, whereas when pegfilgrastim was administered as two doses on d 1 and d 7 after irradiation, neutrophil recovery was significantly improved relative to filgrastim after radiation-induced myelosuppression (26). One dose

100.00

10.00

0.10

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 Time (Days) Post Irradiation and AuBMT

Fig. 8. Effect of pegfilgrastim administration on peripheral blood absolute neutrophil counts (ANC) recovery after 920-cGy X-irradiation and autologous bone marrow transplantation (AuBMT) in rhesus macaques. ANC recovery was observed in animals administered pegfilgrstim at 300 |ig/kg iv (n = 4) or sc (n = 3), or filgrastim at 10 |g/kg daily (n = 4) via sc administration until the ANC reached 3,000/|L (range 12-17 d), or control autologous sera on d 1-18 (n = 13) after irradiation and AuBMT. Values are means ± SEM. (Reprinted with permission from ref. 24.)

of pegfilgrastim (300 ^g/kg) also significantly improved neutrophil recovery relative to conventional filgrastim treatment after AuBMT (Fig. 8) (24). These results were consistent with prospective modeling and simulations based on pharmacokinetic and pharmacodynamic analysis in normal animals and early phase 1 clinical trials (81,82).

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