Materials and methods

Mice and housing

Female BALB/cAnNCrlBR mice were purchased from Charles River Laboratories (Wilmington, MA, USA), housed in conventional cages and provided food and water ad libitum. Mice were 8–12 weeks of age when used in the study.

Cytokine administration

Flt3L (Immunex Corp., Seattle, WA, USA) and G-CSF (Neupogen Filgrastim, Amgen Inc., Thousand Oaks, CA, USA) were formulated in DPBS and administered once or twice daily (AM/PM). Sterile DPBS was administered as an excipient control. Flt3L was also formulated with ProGelz™ (PG) (22% (w/w) Poloxamer-407 (Pluronic F127NF™, BASF, Mt Olive, NJ, USA) and 5% (w/w) hydroxypropylmethyl cellulose (HPMC, Sigma, St Louis, MO, USA), developed by RxKinetix, Inc. (Louisville, CO, USA). The PG vehicle was used as an excipient control in some studies. Flt3L, G-CSF, PG-Flt3L and the PG vehicle were injected intramuscularly (i.m.) into the hind limb in a volume of 100 l. PG formulations were kept cool (<10°C) prior to injection and administered rapidly to reduce gelation. The injection of 100 l of PG resulted in the injection of 1.5 ng of endotoxin, as measured by the Pyrochrome LAL Chromogenic Test Kit (Associates of Cape Cod, Inc., Fairmouth, MA, USA).

Pharmacokinetic studies

To compare the pharmacokinetic properties of Flt3L formulated in saline vs PG-Flt3L, mice received a single i.m. injection of 5 g Flt3L formulated in saline or a single i.m. injection of PG containing 15 g Flt3L and designed to release its contents over a period of 3 days (approximately 5 g/day). Serum was collected and stored at -20°C for subsequent enzyme-linked immunosorbent assay (ELISA) using the Quantikine Human Flt-3/Flk-2 Ligand Immunoassay Kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. Pharmacokinetic analysis of data was performed for each sample to estimate the maximum serum concentration (C_max), the time to C_max (T_max), bioavailability, as measured by area under serum concentration curve (AUC) estimated from the final time point (AUC_{0 - Tfinal}) and to infinity (AUC_{0 - T}), and the elimination (T) half-life for Flt3L. Calculations were performed using PK Function add-ins (Allergan, Irvine, CA, USA) for Microsoft Excel^® (Microsoft Corp.).

Blood, spleen and bone marrow

Blood was obtained from the retro-orbital sinus of heparinized, anesthetized mice and white blood cell (WBC) numbers determined with a blood analyzer (System 9000 Hematology Series Cell Counter, Serono-Baker Diagnostics Inc., Allentown, PA, USA). Spleen and femoral marrow were removed, single-cell suspensions prepared and cellularities determined using a Careside H-2000 Hematology Analyzer (Culver City, CA, USA).

In vitro colony-forming cell assays

Granulocyte–macrophage colony-forming unit (CFU-GM) and high proliferative potential colony-forming cell (HPP-CFC) assays were performed as previously described.²⁸ Briefly, CFU-GM assays were performed in semisolid agarose containing 200 ng/ml recombinant murine interleukin (rmuIL)-3 (BioSource International, Camarillo, CA, USA) and cultured for 7 days. HPP-CFC assays were performed in semisolid agarose containing 200 ng/ml rmuIL-3 and 35% (v/v) L-cell conditioned medium (a source of macrophage colony-stimulating factor, M-CSF) and cultured for 14 days. Cultures were stained with INT (INT: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride, Sigma, St. Louis, MO, USA) 24 h prior to assay.²⁹ A mean was calculated for the number of CFU-GM and HPP-CFC, and frequencies and total numbers per tissue calculated.

Statistical analysis

Statistical analyses of the data were performed using SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Where appropriate, means were compared using the Student's two-sample t-test. Otherwise data were compared using nonparametric Mann–Whitney analysis. Significance was assumed at p0.05.

Results

Pharmacokinetics

Formulation of Flt3L in PG prolonged (2.3-fold) its elimination (T) half-life compared to formulation in saline (Table 1). The T_max was extended (approximately 2.7-fold) in mice receiving Flt3L formulated in PG compared to formulation in saline. The C_max was increased 1.9-fold following the injection of PG-Flt3L compared to the single injection of 5 g Flt3L formulated in saline. Presumably, the increase in the C_max was due, in part, to the three-fold increased total dose of Flt3L. The T_max and C_max data provide evidence for the slow release of Flt3L from PG. Although the total dose of Flt3L delivered by a single injection of PG-Flt3L was three-fold (15 g) higher than that delivered by a single injection of Flt3L (5 g) formulated in saline, the bioavailability was increased 10-fold as the(AUC_{0 - Tfinal}) and six-fold as the (AUC_{0 - T}). These data suggest that formulation in PG significantly improved the bioavailability of Flt3L.

Table 1 - Pharmacokinetic analysis of Flt3L administered i.m. formulated in DPBS or ProGelz™ (PG).

Full table

Increased mobilization of HPC to the spleen by PG-Flt3L

PG-Flt3L significantly increased the number of CFU-GM (68-fold) (Figure 1a) and HPP-CFC (20-fold) (Figure 1b) per spleen in mice studied on day 4, when compared to control mice. However, injection with PG alone also significantly elevated the numbers of CFU-GM and HPP-CFC (33- and 7-fold, respectively) per spleen on day 4 compared to control, despite the low levels of endotoxin. Nonetheless, the number of CFU-GM and HPP-CFC on day 4 in mice receiving PG-Flt3L were increased 4.6- and 4.8-fold, respectively, compared to mice receiving PG alone. Further, the number of CFU-GM and HPP-CFC in mice receiving PG-Flt3L was significantly greater (CFU-GM: 16-fold and HPP-CFC: 9-fold) compared to mice receiving daily injections of Flt3L formulated in DPBS. In contrast, only on day 5 was a significant increase in CFU-GM and HPP-CFC (16- and 8-fold, respectively) observed in mice injected with Flt3L formulated in DPBS.

Figure 1.

Comparison of bone marrow and spleen hematopoiesis following the short-term administration (days 1–3) of Flt3L (5 g) or the single injection (day 1) of Flt3L (15 g) formulated in ProGelz™ (PG). The figures show the number of CFU-GM and HPP-CFC per spleen (a,b) and per femur (c,d). Flt3L (5 g), formulated in PBS, was injected on days 1-3 and mice assayed on day 4 (Flt3L(d4)) or day 5 (Flt3L(d5)). Flt3L (15 g), formulated in PG, was injected on day 1 and mice were assayed on day 4 (PG-Flt3L(d4)) or day 5 (PG-Flt3L(d5)). PBS was injected on days 1–3 and mice assayed on day 4. PG vehicle alone was injected on day 1 and mice were assayed on day 4. Data are presented as means.e.m. (four mice per group). * – significant difference from control (saline). # – significant difference from PG vehicle. % – significant difference between assay on day 4 and day 5. $ – significant difference between Flt3L formulated in saline and Flt3L formulated in PG.

Full figure and legend (29K)

Increased expansion of HPC in the bone marrow by PG-Flt3L

The injection of PG-Flt3L significantly increased (1.9-fold) the number of HPP-CFC per femur compared to normal animals (Figure 1d). In contrast, the number of HPP-CFC per femur in mice receiving PG alone (no Flt3L) was significantly reduced (62%) at day 4 when compared to DPBS and significantly lower than in the mice receiving PG-Flt3L (5-fold). No significant change was observed in the number of CFU-GM per femur in these mice on day 4, regardless of Flt3L formulation (Figure 1c). Further, the number of CFU-GM and HPP-CFC per femur on day 4 in mice receiving DPBS-formulated Flt3L as a daily injection was not significantly different from control. Only after an additional day (day 5), was there a significant increase (1.8-fold) in the number of HPP-CFC per femur observed in mice given Flt3L formulated in DPBS relative to mice receiving DPBS alone.

Increased mobilization of HPC to the blood by PG-Flt3L

The number of CFU-GM in the blood on day 4 was significantly increased (14.8-fold) in mice receiving PG-Flt3L as compared to DPBS (Figure 2). Mice receiving daily injections of Flt3L, and studied on day 4, showed no increase in the number of CFU-GM in the blood. Only on day 5 was there a significant increase in the number (3.7-fold) of CFU-GM observed in the blood in mice receiving daily Flt3L formulated in DPBS compared to DPBS alone. On day 5, the number of CFU-GM per milliliter of blood from mice receiving PG-Flt3L was 26-fold greater than that observed in control (DPBS) mice, and 7-fold greater than that observed in mice receiving daily Flt3L injections. In contrast to the spleen, PG alone did not significantly increase the number of CFU-GM in the blood. The increase in the number of CFU-GM in the blood was not because of a significant increase in the WBC cellularity, as this parameter did not change in any group (data not shown).

Figure 2.

Comparison of hematopoietic progenitors in the blood following the short-term administration (days 1–3) of Flt3L (5 g) or the single injection (day 1) of Flt3L (15 g) formulated in ProGelz™ (PG). The figure shows the number of CFU-GM in the blood. Flt3L (5 g), formulated in PBS, was injected on days 1–3 and mice assayed on day 4 (Flt3L(d4)) or day 5 (Flt3L(d5)). Flt3L (15 g), formulated in PG, was injected on day 1 and mice were assayed on day 4 (PG-Flt3L(d4)) or day 5 (PG-Flt3L(d5)). PBS was injected on days 1–3 and mice assayed on day 4. PG vehicle alone was injected on day 1 and mice were assayed on day 4. Data are presented as means.e.m. (four mice per group). * – significant difference from control (saline). # – significant difference from PG vehicle. % – significant difference between assay on days 4 and 5. $ – significant difference between Flt3L formulated in saline and Flt3L formulated in PG.

Full figure and legend (33K)

Dose–response for an optimal protocol of Flt3L formulated in DPBS as compared to formulation in PG

Optimal hematopoietic activity is observed only when Flt3L formulated in DPBS is administered daily for 7–10 consecutive days. In contrast, a single injection of PG-Flt3L provided significant biological activity over 3 days, which is the duration of Flt3L availability from the PG formulation as determined by pharmacokinetic analysis. In order to perform a direct comparison with optimally administered Flt3L (daily injection for 10 days), PG-Flt3L was administered once every 3 days (a total of three injections over 10 days), based on the observed pharmacokinetic profile.

In comparison to control (DPBS) animals, increases in the number of CFU-GM and HPP-CFC per spleen of mice receiving either daily Flt3L injections (49- and 122-fold, respectively), or PG-Flt3L (66- and 92-fold, respectively), were significantly increased. However, there was no significant difference between mice injected with Flt3L formulated in either DPBS or PG at the 5 g per day dose (Figure 3a and b, respectively). In contrast, while the delivery of 1 and 2.5 g Flt3L formulated in DPBS did not significantly increase splenic hematopoiesis (Figure 3a), the delivery of 1 or 2.5 g Flt3L per day from PG-Flt3L significantly increased (3- and 20-fold, respectively) the number of CFU-GM per spleen compared to control (DPBS) treated mice. Further, the delivery of Flt3L at 1 g per day formulated in DPBS, or PG, significantly increased (8- and 19-fold, respectively) the number of HPP-CFC per spleen compared to DPBS; the increase was significantly greater (2-fold) in the PG-Flt3L mice compared to mice receiving Flt3L formulated in DPBS (Figure 3b).

Figure 3.

Comparison of hematopoietic progenitors in the bone marrow, spleen and blood following the long-term administration (days 1–10) of Flt3L (1, 2.5 or 5 g) or the administration (days 1, 4 and 7) of Flt3L (3, 7.5 or 15 g) formulated in ProGelz™. The figures show the number of CFU-GM and HPP-CFC per spleen (a,b), per milliliter blood (c,d) and per femur (e,f). Mice received PBS or Flt3L formulated in PBS at 1 , 2.5 or 5 g per day for 10 days. Mice were assayed on day 11. Flt3L formulated in PG (PG-Flt3L) containing 3, 7.5 or 15 g Flt3L was injected on days 1, 4 and 7. Flt3L was released over 3 days at approximately 1 , 2.5 and 5 g per day. Mice were assayed on day 11. Data presented as means.e.m. (four mice per group).* – significant difference from control (saline). # – significant difference between Flt3L formulated in saline and Flt3L formulated in PG.

Full figure and legend (40K)

Studies using the optimal protocol of Flt3L administration (daily injection for 10 days) revealed a significant, similar and dose-dependent increase in CFU-GM (Figure 3c) and HPP-CFC (Figure 3d) per milliliter blood regardless of whether Flt3L was formulated in DPBS, or PG. The greatest increase occurred with the highest dose of Flt3L (5 g per day) (37- and 20-fold, formulated in DPBS or in PG, respectively) compared to control (DPBS) mice. Further, the number of HPP-CFC per milliliter blood was significantly increased in mice receiving Flt3L formulated in DPBS at 5, 2.5, or 1 g per day (92-, 48- and 9-fold, respectively), or PG releasing 5 or 2.5 g of Flt3L per day (31- and 13-fold, respectively) compared to control (DPBS) mice. The increase in the number of HPP-CFC per milliliter blood in mice receiving 2.5 g Flt3 formulated in DPBS as a daily injection was significantly greater (3.7-fold) than that observed in mice receiving an equivalent daily dose of Flt3L formulated in PG. No other comparison was significantly different.

The number of CFU-GM and HPP-CFC per femur (Figures 3e, f) was significantly increased in mice receiving 5 g Flt3L per day as a daily injection, or as PG-Flt3L. At 2.5 g Flt3L per day, a significant increase (3.8-fold) in femur HPP-CFC, but not CFU-GM numbers, was observed only in mice receiving daily Flt3L injections. In contrast, the injection of 1 g Flt3L per day, regardless of whether it was delivered by daily injection or PG-Flt3L, significantly increased the number of HPP-CFC, but not CFU-GM, per femur.

Data presented in Figures 1 and 2 suggest that a single injection of 15 g Flt3L formulated in PG is significantly more active than Flt3L formulated in saline and administered at 5 g per day for 3 days. However, when Flt3L is formulated in saline and administered using an optimal protocol (daily injection for 10 days), differences in hematopoietic progenitor mobilization on day 11 are minor when compared to PG-Flt3L (injected on days 1, 4 and 7).

Increased splenic HPC mobilization with coadministration of G-CSF and PG-Flt3L

Mice receiving daily injections of G-CSF on days 1–4 and Flt3L on days 2–4, and assayed on day 5, had significantly increased numbers of CFU-GM per femur (11-fold), CFU-GM and HPP-CFC per spleen (180- and 51-fold, respectively), and CFU-GM and HPP-CFC per milliliter blood (15.8- and 9.3-fold, respectively) when compared to control (DPBS) animals. Compared to animals receiving either G-CSF or Flt3L alone, mice receiving the combination of G-CSF and Flt3L (formulated in DPBS) showed significantly increased numbers of CFU-GM per femur (3.4- and 3.6-fold, respectively) (Figure 4a), CFU-GM per milliliter blood (4.5- and 6.3-fold, respectively) (Figure 4e) and HPP-CFC per milliliter blood (5.3- and 4.4-fold, respectively) (Figure 4f). Similarly, mice injected with the combination of G-CSF and PG-Flt3L showed a significantly increased number of CFU-GM per femur (5.5-fold), CFU-GM (1037-fold) and HPP-CFC (295-fold) per spleen and HPP-CFC per milliliter blood (9.3-fold) when compared to control. The increases in the number of CFU-GM and HPP-CFC were significantly greater than that observed with either G-CSF or PG-Flt3L alone. In addition, mice receiving both G-CSF and PG-Flt3L had significantly increased numbers of CFU-GM and HPP-CFC per spleen (5.8- and 5.8-fold, respectively) compared to mice receiving daily injections of G-CSF and Flt3L. These increases in CFU-GM and HPP-CFC were not because of increased spleen cellularity, which was only increased 1.4-fold in these mice (data not shown).

Figure 4.

Hematopoietic activity in mice receiving G-CSF and/or Flt3L (formulated in saline or ProGelz™). The figures show the number of CFU-GM and HPP-CFC per femur (a,b), per spleen (c,d) and per milliliter blood (e,f). Mice received PBS on days 1–3 and were assayed on day 4; PG vehicle on day 1 and were assayed on day 4; Flt3L (5 g) in PBS on days 1–3 and were assayed on day 4; PG-Flt3L containing: Flt3L (15 g) on day 1 and were assayed on day 4; G-CSF (6 g) on days 1–4 and were assayed on day 5; 6 g G-CSF on days 1–4 and 5 g Flt3L on days 2–4 (Flt3L+G-CSF) and were assayed on day 5; 6 g G-CSF on days 1–4 and Flt3L (15 g) formulated in PG on day 2 (PG-Flt3L+G-CSF) and were assayed on day 5. Data are presented as means.e.m. (five mice per group). * – significant difference from control (saline). # – significantly different from PG vehicle. % – significant difference between daily Flt3L and PG-Flt3L. & – significant difference between Flt3L+G-CSF and PG-Flt3L+G-CSF. @ – significant difference from Flt3L. $ – significant difference from PG-Flt3L.+–significant difference from G-CSF.

Full figure and legend (72K)

Mice receiving G-CSF at 6 g per day from day 1 for 4 days and studied on day 5 had significantly elevated CFU-GM per femur (3.2-fold) (Figure 4a), and CFU-GM (160-fold) and HPP-CFC (352-fold) per spleen (Figure 4c and d) when compared to control (DPBS) animals. Mice receiving Flt3L formulated in DPBS at 5 g per day for 3 days from day 2 of the study also had significantly elevated numbers of CFU-GM per femur (3-fold), and CFU-GM and HPP-CFC per spleen (27- and 4.2-fold, respectively). In contrast, mice receiving a single injection of PG-Flt3L, releasing the equivalent of 5 g Flt3L per day, on day 2 and assayed on day 5 had significantly increased WBC cellularities (2.1-fold) and numbers of CFU-GM (30-fold) and HPP-CFC (10.4-fold) per spleen as compared to control (DPBS) mice. The hematologic and hematopoietic effects were similar, regardless of whether Flt3L was formulated in DPBS (administered on days 2–4 and assayed on day 5) or PG (administered on day 2 and assayed on day 5), with the exception of the number of HPP-CFC per spleen (increased 2.5-fold).

Discussion

Maximal HPC mobilization with Flt3L (formulated in DPBS) occurs in mice when injected at 5–10 g per day for 7–10 consecutive days.^1,2 Here we report significantly increased biological activity, including mobilization to the spleen, 4 days following a single injection of Flt3L formulated in a sustained release matrix. Pharmacokinetic analysis revealed that the formulation of Flt3L in PG prolonged its elimination (T) half-life, increased its bioavailability (AUC) for a given dose of Flt3L and extended its T_max, compared to Flt3L formulated in DPBS. The poloxamer-associated prolongation of the elimination (T) half-life of Flt3L is consistent with previous reports⁵ and may be a consequence of modified glomerular filtration.⁵ The sustained presence of Flt3L in the serum when formulated with PG may also be a consequence of poloxamer-associated stabilization of the protein³⁰ and/or protection from degradative enzymes. In addition, formulation with poloxamer may affect biodistribution.³¹

These studies demonstrate that a single injection of PG-Flt3L significantly increased its biological activity as compared to Flt3L formulated in DPBS and injected daily for 3 days. In mice receiving PG-Flt3L, the numbers of CFU-GM and HPP-CFC per spleen, and CFU-GM per milliliter blood, were significantly increased approximately 1 day earlier than in mice receiving an equivalent total dose of Flt3L formulated in DPBS. The improved biological activity associated with the delivery of PG-Flt3L may be a consequence of the prolongation of the elimination (T) half-life of Flt3L, or the increased bioavailability and/or the delivery of Flt3L at sustained, potentially more physiologically effective levels. These data suggest that the administration of PG-Flt3L can induce significantly greater HPC expansion and mobilization, with fewer injections, than Flt3L formulated in DPBS. Further, PG-Flt3L elicits a significantly more rapid and greater splenic mobilization than an equivalent dose of Flt3L formulated in DPBS and administered by daily injection.

Consistent with previous studies,^{27,32,33,34,35} we observed that Flt3L increased G-CSF stimulation of HPC mobilization with activity greater than that seen with either cytokine alone. The number of HPP-CFC mobilized per spleen was significantly increased by the coadministration of G-CSF and PG-Flt3L, compared to DPBS (290-fold), G-CSF (8-fold), Flt3L (10-fold), PG-Flt3L (28-fold), or the combination of Flt3L and G-CSF (approximately 6-fold). Similar significant increases (6-fold) of CFU-GM numbers were also observed using the coadministration of G-CSF and PG-Flt3L when compared to the combination of G-CSF and Flt3L injected daily. These data suggest that coadministration of G-CSF and PG-Flt3L may further improve the efficacy of cytokine-induced HSC mobilization by reducing the time needed for optimal HSC mobilization and increasing HSC mobilization.

Injection of the PG vehicle alone resulted in a significant increase in the number of splenic CFU-GM and HPP-CFC compared to control mice receiving DPBS, suggesting that PG alone has hematopoietic activity. This hematopoietic activity is not attributable to endotoxin as the levels of endotoxin present in PG were 1.5 ng per 100 l (unpublished data). Hematopoietic activity associated with the PG vehicle may therefore be because of the stimulation of endogenous cytokine production. PG is composed of Poloxamer-407 and HPMC and both components have been reported to have biological activity. The formulation of wound-care products in Poloxamer-407 has been reported to improve wound healing,^36,37 and the administration of Poloxamer-407 has been shown to improve healing processes after burn injuries.³⁸ In addition, methyl cellulose, a component of HPMC, has also been shown to stimulate hematopoiesis.^39,40,41 These data suggest that the increased hematopoietic activity associated with PG-Flt3L may be due, in part, to a synergy between Flt3L- and PG-induced cytokines.

Another approach to achieving a sustained-release formulation is the use of PEGylation (the addition of poly(ethylene glycol) (PEG) moieties to target molecules). PEG is a water-soluble polymer and, when chemically linked to proteins, improves their pharmacological activity as compared with non-PEGylated molecules. PEGylation can significantly alter the pharmacokinetic properties of proteins by reducing renal clearance³⁸ and protecting against proteolytic cleavage. PEGylated molecules are characterized by a prolonged elimination (T) half-life and sustained biological activity compared to their non-PEGylated counterparts.^{42,43,44,45,46,47} This pattern of bioactivity is similar to our results with the PG-Flt3L sustained-release formulation, although, as discussed above, the PG formulation also augments and accelerates hematopoietic activity, unlike PEGylation.

In conclusion, we demonstrate that Flt3L prepared in a Poloxamer-407-based, sustained-release formulation significantly increased its biological activity and induced more rapid and greater HPC mobilization than an equivalent daily dose of Flt3L formulated in DPBS. This may be a consequence of a PG-associated prolongation of the elimination (T) half-life and an increase in Flt3L bioavailability. Further, we demonstrate significantly increased HPC mobilization and expansion with the coadministration of G-CSF and PG-Flt3L, an effect that is greater than that seen in mice receiving G-CSF coadministered with Flt3L formulated in DPBS. While this study has focused on the hematopoietic activity of Flt3L in the mouse, Flt3L-induced HPC mobilization has also been demonstrated in primates³² and clinical studies,⁴⁸ demonstrating the safety and efficacy of Flt3L-induced HPC mobilization. Our studies suggest that the improved hematopoietic activity associated with PG-Flt3L may have the potential to speed the mobilization of HPC products for transplantation prior to myeloablative treatment in cancer patients. In addition, it may successfully mobilize those patients who are poor mobilizers with the current mobilization strategies.

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Acknowledgements

The authors gratefully acknowledge the gift of Flt3L from Immunex Corp. and the assistance of Lisa Chudomelka, Tina Winekauf and Richard Murcek in the preparation of this manuscript. GJR and JMB are employees of RxKinetix, Inc. and JET is a member of the Scientific Advisory Board of RxKinetix, Inc.