Murine Studies Delivery of Flt3 Ligand (Flt3l) Using a Poloxamer-Based Formulation Increases Biological Activity in Mice

Bone Marrow Transplantation (2003) 31, 361–369 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00 www.nature.com/bmt Murine studies Delivery of Flt3 ligand (Flt3L) using a poloxamer-based formulation increases biological activity in mice

SN Robinson1, JM Chavez1, VM Pisarev1, RL Mosley1, GJ Rosenthal2, JM Blonder2 and JE Talmadge1

1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska; and 2RxKinetix, Inc., Louisville, CO, USA

Summary: manner may provide a more effective delivery method and potentially improve its biological efficacy. In a murine Fms-like tyrosine kinase (Flt3L) is a potent stimulator of model, we compared the hematopoietic activity of Flt3L hematopoietic progenitor cell (HPC) expansion and when formulated in a sustained-release Poloxamer-407- mobilization; however, this requires 7–10 days of admin- based matrix (PG) with the hematopoietic activity of Flt3L istration. We investigated whether sustained delivery of formulated in DPBS and delivered by daily injection. Flt3L using a poloxamer-based matrix (PG) could Poloxamers are amphiphilic, nonionic block polymers of accelerate and/or improve the hematopoietic activity of hydrophobic propylene oxide and hydrophilic ethylene Flt3L in mice. A single injection of PG-Flt3L stimulated oxide.3 Poloxamer-407 (Pluronic F-127t) is characterized significantly more rapid and greater HPC mobilization to by the property of reversible thermal gelation, being liquid the spleen and peripheral blood than the daily injection of when cold (o101C) and forming a semisolid gel at Flt3L formulated in saline. Pharmacokinetic analysis physiologic temperatures. Proteins formulated with Polox- demonstrated that the formulation of Flt3L in PG amer-407 produce a matrix that when injected provides a prolonged its elimination (Tb) half-life (2.3-fold) and depot fromwhich the protein is released in a sustained increased its bioavailability (4two fold) and the time to manner.4–16 The release of the protein can be modulated by 17–24 maximum serum concentration (Tmax) (2.7-fold). Further, varying the concentration of the poloxamer and by the coadministration of G-CSF and PG-Flt3L allowed lower addition of polymeric stabilizers such as hydropropyl- doses of Flt3L to be active, withsignificantly greater methyl cellulose (HPMC). HPMC is another matrix that hematopoietic and mobilization activity, compared to the also has slow release properties.9 The combination of same total dose of G-CSF, Flt3L or G-CSF and Flt3L Poloxamer-407 and HPMC provides a drug delivery matrix formulated in saline. These data demonstrate that with slower drug release compared to either component formulation of Flt3L in PG significantly accelerates and alone.10,21,25,26 increases HPC expansion and mobilization. The observa- In the present studies, we examined the mobilization tion of increased bioactivity by PG-Flt3L in rodents properties of Flt3L when formulated in a sustained release suggests the potential for improved clinical efficacy matrix composed of Poloxamer-407 and HPMC. Our of Flt3L by reducing the time required for HPC pharmacokinetic analyses demonstrated that the adminis- mobilization. tration of Flt3L formulated with PG (PG-Flt3L) resulted in Bone Marrow Transplantation (2003) 31, 361–369. sustained delivery. In addition, the biological activity of doi:10.1038/sj.bmt.1703816 Flt3L was significantly increased, resulting in more rapid Keywords: Flt3 ligand (Flt3L); Sustained delivery HPC mobilization at lower doses than with Flt3L Poloxamer-based matrix; Hematopoiesis; Mobilization formulated in DPBS. Consistent with previous findings,27 we also found that the concurrent injection of recombinant human granulocyte colony-stimulating factor (G-CSF) with Flt3L significantly increased HPC mobilization Administration of recombinant human fms-like tyrosine compared to either factor alone. Further, HPC mobiliza- kinase (Flt)-3 ligand (Flt3L) stimulates the expansion and tion observed with the concurrent injection of G-CSF mobilization of hematopoietic progenitor cells (HPC) in and PG-Flt3L matched, or was significantly greater than mice.1,2 Optimal hematopoietic expansion and mobilization that observed with G-CSF and Flt3L formulated in currently requires daily administration of Flt3L for 7–10 saline. consecutive days. The requirement for repeated daily injections suggests that the delivery of Flt3L in a sustained Materials and methods

Correspondence: Dr SN Robinson, Department of Genetics, Cell Mice and housing Biology and Anatomy, 986395 University of Nebraska Medical Center, Omaha, NE 68198-6395, USA Female BALB/cAnNCrlBR mice were purchased from Received 5 February 2002; accepted 30 September 2002 Charles River Laboratories (Wilmington, MA, USA), housed Sustained delivery improves activity of Flt3L SN Robinson et al 362 in conventional cages and provided food and water ad libitum. Brieﬂy, CFU-GM assays were performed in semisolid Mice were 8–12 weeks of age when used in the study. agarose containing 200 ng/ml recombinant murine interleukin (rmuIL)-3 (BioSource International, Camarillo, CA, Cytokine administration USA) and cultured for 7 days. HPP-CFC assays were performed in semisolid agarose containing 200 ng/ml Flt3L (Immunex Corp., Seattle, WA, USA) and G-CSF rmuIL-3 and 35% (v/v) L-cell conditioned medium (a (Neupogen Filgrastim, Amgen Inc., Thousand Oaks, CA, source of macrophage colony-stimulating factor, M-CSF) USA) were formulated in DPBS and administered once or and cultured for 14 days. Cultures were stained with INT twice daily (AM/PM). Sterile DPBS was administered as an (INT: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazo- excipient control. Flt3L was also formulated with ProGelzt liumchloride, Sigma,St. Louis, MO, USA) 24 h prior to (PG) (22% (w/w) Poloxamer-407 (Pluronic F127NFt, assay.29 A mean was calculated for the number of CFU- BASF, Mt Olive, NJ, USA) and 5% (w/w) hydroxypro- GM and HPP-CFC, and frequencies and total numbers per pylmethyl cellulose (HPMC, Sigma, St Louis, MO, USA), tissue calculated. developed by RxKinetix, Inc. (Louisville, CO, USA). The PG vehicle was used as an excipient control in some studies. Statistical analysis Flt3L, G-CSF, PG-Flt3L and the PG vehicle were injected Statistical analyses of the data were performed using SPSS intramuscularly (i.m.) into the hind limb in a volume of 10.0 for Windows (SPSS Inc., Chicago, IL, USA). Where 100 ml. PG formulations were kept cool (o101C) prior to appropriate, means were compared using the Student’s injection and administered rapidly to reduce gelation. The two-sample t-test. Otherwise data were compared using injection of 100 ml of PG resulted in the injection of p1.5 ng nonparametric Mann–Whitney analysis. Signiﬁcance was of endotoxin, as measured by the Pyrochrome LAL assumed at pp0.05. Chromogenic Test Kit (Associates of Cape Cod, Inc., Fairmouth, MA, USA). Results

Pharmacokinetic studies Pharmacokinetics To compare the pharmacokinetic properties of Flt3L Formulation of Flt3L in PG prolonged (2.3-fold) its formulated in saline vs PG-Flt3L, mice received a single elimination (Tb) half-life compared to formulation in saline i.m. injection of 5 mg Flt3L formulated in saline or a single (Table 1). The Tmax was extended (approximately 2.7-fold) i.m. injection of PG containing 15 mg Flt3L and designed to in mice receiving Flt3L formulated in PG compared to release its contents over a period of 3 days (approximately formulation in saline. The Cmax was increased 1.9-fold 5 mg/day). Serumwas collected and stored at À201C for following the injection of PG-Flt3L compared to the single subsequent enzyme-linked immunosorbent assay (ELISA) injection of 5 mg Flt3L formulated in saline. Presumably, using the Quantikine Human Flt-3/Flk-2 Ligand Immu- the increase in the Cmax was due, in part, to the three-fold noassay Kit (R&D Systems, Inc., Minneapolis, MN, USA) increased total dose of Flt3L. The Tmax and Cmax data according to the manufacturer’s protocol. Pharmacokinetic provide evidence for the slow release of Flt3L fromPG. analysis of data was performed for each sample to estimate Although the total dose of Flt3L delivered by a single the maximum serum concentration (Cmax), the time to Cmax injection of PG-Flt3L was three-fold (15 mg) higher than (Tmax), bioavailability, as measured by area under serum that delivered by a single injection of Flt3L (5 mg) concentration curve (AUC) estimated from the final time formulated in saline, the bioavailability was increased 10- point ðAUC Þ and to infinity ðAUC Þ, and the 0ÀTfinal 0ÀT1 fold as the AUC0ÀTfinal and six-fold as the AUC0ÀT1 . These elimination (Tb) half-life for Flt3L. Calculations were data suggest that formulation in PG significantly improved performed using PK Function add-ins (Allergan, Irvine, the bioavailability of Flt3L. CA, USA) for Microsoft Excels (Microsoft Corp.). Increased mobilization of HPC to the spleen by PG-Flt3L Blood, spleen and bone marrow PG-Flt3L significantly increased the number of CFU-GM Blood was obtained fromthe retro-orbital sinus of (68-fold) (Figure 1a) and HPP-CFC (20-fold) (Figure 1b) heparinized, anesthetized mice and white blood cell (WBC) numbers determined with a blood analyzer (System 9000 Hematology Series Cell Counter, Serono-Baker Table 1 Pharmacokinetic analysis of Flt3L administered i.m. Diagnostics Inc., Allentown, PA, USA). Spleen and formulated in DPBS or ProGelzt (PG) femoral marrow were removed, single-cell suspensions prepared and cellularities determined using a Careside H- Pharmacokinetic measure Flt3L PG-Flt3L

2000 Hematology Analyzer (Culver City, CA, USA). Cmax (ng/ml) 5697121 1079772

Tmax (h) 1.570.5 4.070.0 7 7 ðAUC0ÀT Þ (ng h/ml) 1869 85 18 649 1997 In vitro colony-forming cell assays final 7 7 AUC0ÀT1 (ng h/ml) 3262 14 20 182 2928 Elimination (Tb) Half-life (h) 5.270.4 11.772.0 Granulocyte–macrophage colony-forming unit (CFU-GM) and high proliferative potential colony-forming cell (HPP- Flt3L (5 mg) formulated in DPBS. PG-Flt3L containing 15 mg Flt3L. Data CFC) assays were performed as previously described.28 shown as mean7s.e. (s.e.m.).

Bone Marrow Transplantation Sustained delivery improves activity of Flt3L SN Robinson et al 363 a CFU-GM b HPP-CFC the number of CFU-GM per femur in these mice on day 4,

5 #$ regardless of Flt3L formulation (Figure 1c). Further, the 4 * *#$ 8 number of CFU-GM and HPP-CFC per femur on day 4 in mice receiving DPBS-formulated Flt3L as a daily injection 3 6 was not signiﬁcantly different fromcontrol. Only after an

spleen (x10 ) spleen (x10 $ 2 *# additional day (day 5), was there a signiﬁcant increase (1.8-

per 4 fold) in the number of HPP-CFC per femur observed in (Mean±SEM) 1 % # $ 2 * mice given Flt3L formulated in DPBS relative to mice * * Number * * receiving DPBS alone. 0 0 c d

) # Increased mobilization of HPC to the blood by PG-Flt3L 3 # # * * 40 30 * The number of CFU-GM in the blood on day 4 was 30 # $ signiﬁcantly increased (14.8-fold) in mice receiving PG-

femur (x10 20 Flt3L as compared to DPBS (Figure 2). Mice receiving

per 20 *#$ daily injections of Flt3L, and studied on day 4, showed no (Mean±SEM) 10 increase in the number of CFU-GM in the blood. Only on 10 * % day 5 was there a signiﬁcant increase in the number (3.7-

Number * 0 0 * fold) of CFU-GM observed in the blood in mice receiving 5) 5) PBS PG PBS PG daily Flt3L formulated in DPBS compared to DPBS alone. 3L(d4) L(d5) 3L(d5) Flt3L(d4) Flt3L(d t3 Flt3L(d4)t3L(d4)Flt3L(d PG Flt PG Fl PG Fl PG Flt On day 5, the number of CFU-GM per milliliter of blood frommicereceiving PG-Flt3L was 26-fold greater than that Figure 1 Comparison of bone marrow and spleen hematopoiesis following the short-termadministration(days 1–3) of Flt3L (5 mg) or the single observed in control (DPBS) mice, and 7-fold greater than injection (day 1) of Flt3L (15 mg) formulated in ProGelzt (PG). The figures that observed in mice receiving daily Flt3L injections. In show the number of CFU-GM and HPP-CFC per spleen (a,b) and per contrast to the spleen, PG alone did not significantly femur (c,d). Flt3L (5 mg), formulated in PBS, was injected on days 1-3 and increase the number of CFU-GM in the blood. The mice assayed on day 4 (Flt3L(d4)) or day 5 (Flt3L(d5)). Flt3L (15 mg), increase in the number of CFU-GM in the blood was not 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 because of a significant increase in the WBC cellularity, as mice assayed on day 4. PG vehicle alone was injected on day 1 and mice were assayed on day 4. Data are presented as mean7s.e.m. (four mice per group). * – significant difference fromcontrol (saline). # – significant difference fromPG vehicle. % – significant difference between assay on day CFU-GM per ml blood 4 and day 5. $ – significant difference between Flt3L formulated in saline 8 and Flt3L formulated in PG. #$

) * 3 per spleen in mice studied on day 4, when compared to 6 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. ml blood (x10 4

Nonetheless, the number of CFU-GM and HPP-CFC on per #$

(Mean±SEM) * day 4 in mice receiving PG-Flt3L were increased 4.6- and 4.8-fold, respectively, compared to mice receiving PG 2 alone. Further, the number of CFU-GM and HPP-CFC in mice receiving PG-Flt3L was significantly greater (CFU- CFU-GM * GM: 16-fold and HPP-CFC: 9-fold) compared to mice 0 receiving daily injections of Flt3L formulated in DPBS. In contrast, only on day 5 was a significant increase in CFU- PBS PG d5) GM and HPP-CFC (16- and 8-fold, respectively) observed Flt3L(d4) Flt3L( PG-Flt3L(d4) in mice injected with Flt3L formulated in DPBS. PG-Flt3L(d5) Figure 2 Comparison of hematopoietic progenitors in the blood following the short-termadministration(days 1–3) of Flt3L (5 mg) or the single Increased expansion of HPC in the bone marrow by injection (day 1) of Flt3L (15 mg) formulated in ProGelzt (PG). The figure PG-Flt3L shows the number of CFU-GM in the blood. Flt3L (5 mg), formulated in PBS, was injected on days 1–3 and mice assayed on day 4 (Flt3L(d4)) or The injection of PG-Flt3L significantly increased (1.9-fold) day 5 (Flt3L(d5)). Flt3L (15 mg), formulated in PG, was injected on day 1 the number of HPP-CFC per femur compared to normal and mice were assayed on day 4 (PG-Flt3L(d4)) or day 5 (PG-Flt3L(d5)). animals (Figure 1d). In contrast, the number of HPP-CFC PBS was injected on days 1–3 and mice assayed on day 4. PG vehicle alone per femur in mice receiving PG alone (no Flt3L) was was injected on day 1 and mice were assayed on day 4. Data are presented as mean7s.e.m. (four mice per group). * – significant difference from significantly reduced (62%) at day 4 when compared to control (saline). # – significant difference fromPG vehicle. % – significant DPBS and significantly lower than in the mice receiving difference between assay on days 4 and 5. $ – significant difference between PG-Flt3L (5-fold). No significant change was observed in Flt3L formulated in saline and Flt3L formulated in PG.

Bone Marrow Transplantation Sustained delivery improves activity of Flt3L SN Robinson et al 364 this parameter did not change in any group (data not observed only in mice receiving daily Flt3L injections. In shown). contrast, the injection of 1 mg Flt3L per day, regardless of whether it was delivered by daily injection or PG-Flt3L, Dose–response for an optimal protocol of Flt3L formulated significantly increased the number of HPP-CFC, but not in DPBS as compared to formulation in PG CFU-GM, per femur. Data presented in Figures 1 and 2 suggest that a single Optimal hematopoietic activity is observed only when injection of 15 mg Flt3L formulated in PG is significantly Flt3L formulated in DPBS is administered daily for 7–10 more active than Flt3L formulated in saline and adminis- consecutive days. In contrast, a single injection of PG- tered at 5 mg per day for 3 days. However, when Flt3L is Flt3L provided significant biological activity over 3 days, formulated in saline and administered using an optimal which is the duration of Flt3L availability fromthe PG protocol (daily injection for 10 days), differences in formulation as determined by pharmacokinetic analysis. In hematopoietic progenitor mobilization on day 11 are order to perform a direct comparison with optimally minor when compared to PG-Flt3L (injected on days 1, 4 administered Flt3L (daily injection for 10 days), PG-Flt3L and 7). was administered once every 3 days (a total of three injections over 10 days), based on the observed pharmaco- Increased splenic HPC mobilization with coadministration kinetic profile. of G-CSF and PG-Flt3L In comparison to control (DPBS) animals, increases in the number of CFU-GM and HPP-CFC per spleen of mice Mice receiving daily injections of G-CSF on days 1–4 and receiving either daily Flt3L injections (49- and 122-fold, Flt3L on days 2–4, and assayed on day 5, had significantly respectively), or PG-Flt3L (66- and 92-fold, respectively), increased numbers of CFU-GM per femur (11-fold), CFU- were significantly increased. However, there was no GM and HPP-CFC per spleen (180- and 51-fold, respec- significant difference between mice injected with Flt3L tively), and CFU-GM and HPP-CFC per milliliter blood formulated in either DPBS or PG at the 5 mg per day dose (15.8- and 9.3-fold, respectively) when compared to control (Figure 3a and b, respectively). In contrast, while the (DPBS) animals. Compared to animals receiving either G- delivery of 1 and 2.5 mg Flt3L formulated in DPBS did not CSF or Flt3L alone, mice receiving the combination of G- significantly increase splenic hematopoiesis (Figure 3a), the CSF and Flt3L (formulated in DPBS) showed significantly delivery of 1 or 2.5 mg Flt3L per day fromPG-Flt3L increased numbers of CFU-GM per femur (3.4- and 3.6- significantly increased (3- and 20-fold, respectively) fold, respectively) (Figure 4a), CFU-GM per milliliter the number of CFU-GM per spleen compared to blood (4.5- and 6.3-fold, respectively) (Figure 4e) and HPP- control (DPBS) treated mice. Further, the delivery of CFC per milliliter blood (5.3- and 4.4-fold, respectively) Flt3L at 1 mg per day formulated in DPBS, or PG, (Figure 4f). Similarly, mice injected with the combination significantly increased (8- and 19-fold, respectively) the of G-CSF and PG-Flt3L showed a significantly increased number of HPP-CFC per spleen compared to DPBS; the number of CFU-GM per femur (5.5-fold), CFU-GM increase was significantly greater (2-fold) in the PG-Flt3L (1037-fold) and HPP-CFC (295-fold) per spleen and mice compared to mice receiving Flt3L formulated in HPP-CFC per milliliter blood (9.3-fold) when compared DPBS (Figure 3b). to control. The increases in the number of CFU-GM and Studies using the optimal protocol of Flt3L administra- HPP-CFC were significantly greater than that observed tion (daily injection for 10 days) revealed a significant, with either G-CSF or PG-Flt3L alone. In addition, mice similar and dose-dependent increase in CFU-GM receiving both G-CSF and PG-Flt3L had significantly (Figure 3c) and HPP-CFC (Figure 3d) per milliliter blood increased numbers of CFU-GM and HPP-CFC per spleen regardless of whether Flt3L was formulated in DPBS, or (5.8- and 5.8-fold, respectively) compared to mice receiving PG. The greatest increase occurred with the highest dose of daily injections of G-CSF and Flt3L. These increases in Flt3L (5 mg per day) (37- and 20-fold, formulated in DPBS CFU-GM and HPP-CFC were not because of increased or in PG, respectively) compared to control (DPBS) mice. spleen cellularity, which was only increased 1.4-fold in these Further, the number of HPP-CFC per milliliter blood was mice (data not shown). significantly increased in mice receiving Flt3L formulated Mice receiving G-CSF at 6 mg per day fromday 1 for 4 in DPBS at 5, 2.5, or 1 mg per day (92-, 48- and 9-fold, days and studied on day 5 had significantly elevated CFU- respectively), or PG releasing 5 or 2.5 mg of Flt3L per day GM per femur (3.2-fold) (Figure 4a), and CFU-GM (160- (31- and 13-fold, respectively) compared to control (DPBS) fold) and HPP-CFC (352-fold) per spleen (Figure 4c and d) mice. The increase in the number of HPP-CFC per milliliter when compared to control (DPBS) animals. Mice receiving blood in mice receiving 2.5 mg Flt3 formulated in DPBS as a Flt3L formulated in DPBS at 5 mg per day for 3 days from daily injection was significantly greater (3.7-fold) than that day 2 of the study also had significantly elevated numbers observed in mice receiving an equivalent daily dose of of CFU-GM per femur (3-fold), and CFU-GM and HPP- Flt3L formulated in PG. No other comparison was CFC per spleen (27- and 4.2-fold, respectively). In contrast, significantly different. mice receiving a single injection of PG-Flt3L, releasing the The number of CFU-GM and HPP-CFC per femur equivalent of 5 mg Flt3L per day, on day 2 and assayed on (Figures 3e, f) was significantly increased in mice receiving day 5 had significantly increased WBC cellularities (2.1- 5 mg Flt3L per day as a daily injection, or as PG-Flt3L. At fold) and numbers of CFU-GM (30-fold) and HPP-CFC 2.5 mg Flt3L per day, a significant increase (3.8-fold) in (10.4-fold) per spleen as compared to control (DPBS) mice. femur HPP-CFC, but not CFU-GM numbers, was The hematologic and hematopoietic effects were similar,

Bone Marrow Transplantation Sustained delivery improves activity of Flt3L SN Robinson et al 365 a b ) CFU-GM HPP-CFC 3 * 200 30 * * * spleen (x10 20 100 per

(Mean±SEM) *# * 10 * *# * Number * 0 0 c d ) 3

* 2 10

ml blood (x10 5 * 1 per

(Mean±SEM) * * # * * *

Number 0 0 e f

) 10 3 * 10 * 8 * 6 * * * femur (x10 5

per 4 #

(Mean±SEM) * 2 Number 0 0 Flt3L PG-Flt3L Flt3L PG-Flt3L

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 mg) or the administration (days 1, 4 and 7) of Flt3L (3, 7.5 or 15 mg) formulated in ProGelzt. 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 mg per day for 10 days. Mice were assayed on day 11. Flt3L formulated in PG (PG-Flt3L) containing 3, 7.5 or 15 mg Flt3L was injected on days 1, 4 and 7. Flt3L was released over 3 days at approximately 1 , 2.5 ’ and 5 mg per day. Mice were assayed on day 11. Data presented as mean7s.e.m. (four mice per group). * – significant difference fromcontrol (saline). # – significant difference between Flt3L formulatedin saline and Flt3L formulatedin PG. regardless of whether Flt3L was formulated in DPBS spleen, 4 days following a single injection of Flt3L (administered on days 2–4 and assayed on day 5) or PG formulated in a sustained release matrix. Pharmacokinetic (administered on day 2 and assayed on day 5), with the analysis revealed that the formulation of Flt3L in PG exception of the number of HPP-CFC per spleen (increased prolonged its elimination (Tb) half-life, increased its 2.5-fold). bioavailability (AUC) for a given dose of Flt3L and

extended its Tmax, compared to Flt3L formulated in DPBS. The poloxamer-associated prolongation of the Discussion elimination (Tb) half-life of Flt3L is consistent with previous reports5 and may be a consequence of modified Maximal HPC mobilization with Flt3L (formulated in glomerular filtration.5 The sustained presence of Flt3L in DPBS) occurs in mice when injected at 5–10 mg per day for the serum when formulated with PG may also be a 7–10 consecutive days.1,2 Here we report significantly consequence of poloxamer-associated stabilization of the increased biological activity, including mobilization to the protein30 and/or protection fromdegradative enzymes.In

Bone Marrow Transplantation Sustained delivery improves activity of Flt3L SN Robinson et al 366 a b CFU-GM HPP-CFC

3 50 40 #$ 40 @ + * 30

30 femur (x10 ) 20 *# $ per 20 (Mean±SEM) * * 10 10 Number 0 0 c d PBS PG Flt3L PG Flt3L G-CSFFlt3L + G-CSF PBS PG Flt3L PG Flt3L G-CSFFlt3L + G-CSF 3 *#&$+ 40 *#&$+ 150 30 100 spleen (x10 ) 20 * @ per

(Mean±SEM) 20 *

10 @ * * * *%

Number * * * 0 * 0 e f 3 PBS PG Flt3LPG Flt3LG-CSFFlt3L + G-CS*#F 3 PBS PG Flt3LPG Flt3LG-CSFFlt3L + G-CS*#F$+ 1.5 *@+

2 1.0 @ + ml blood (x10 ) * per (Mean±SEM) 1 0.5 # #

0.0 0 Number F F G 3L L F PBS PG CS PBS P Flt CS Flt3L - Flt3G-CSF CSF PG Flt3LG-CS G PG L + G-CSF Flt3L Flt3+ Flt3L + G- PG PG Flt3L + G-

Figure 4 Hematopoietic activity in mice receiving G-CSF and/or Flt3L (formulated in saline or ProGelzt). 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 mg) in PBS on days 1–3 and were assayed on day 4; PG-Flt3L containing: Flt3L (15 mg) on day 1 and were assayed on day 4; G-CSF (6 mg) on days 1–4 and were assayed on day 5; 6 mg G-CSF on days 1–4 and 5 mg Flt3L on days 2–4 (Flt3L+G-CSF) and were assayed on day 5; 6 mg G-CSF on days 1–4 and Flt3L (15 mg) formulated in PG on day 2 (PG-Flt3L+G-CSF) and were assayed on day 5. Data are presented as mean7s.e.m. (five miceper group). * – significant difference fromcontrol (saline). # – significantly different fromPG vehicle. % – significant difference between daily Flt3L and PG-Flt3L. & – significant difference between Flt3L+G-CSF and PG-Flt3L+G-CSF. @ – significant difference fromFlt3L. $ – significant difference from PG-Flt3L.+–significant difference fromG-CSF.

addition, formulation with poloxamer may affect biodis- be a consequence of the prolongation of the elimination tribution.31 (Tb) half-life of Flt3L, or the increased bioavailability These studies demonstrate that a single injection of PG- and/or the delivery of Flt3L at sustained, potentially Flt3L significantly increased its biological activity as more physiologically effective levels. These data suggest compared to Flt3L formulated in DPBS and injected daily that the administration of PG-Flt3L can induce for 3 days. In mice receiving PG-Flt3L, the numbers of significantly greater HPC expansion and mobilization, CFU-GM and HPP-CFC per spleen, and CFU-GM per with fewer injections, than Flt3L formulated in DPBS. milliliter blood, were significantly increased approximately Further, PG-Flt3L elicits a significantly more rapid and 1 day earlier than in mice receiving an equivalent total dose greater splenic mobilization than an equivalent dose of of Flt3L formulated in DPBS. The improved biological Flt3L formulated in DPBS and administered by daily activity associated with the delivery of PG-Flt3L may injection.

Bone Marrow Transplantation Sustained delivery improves activity of Flt3L SN Robinson et al 367 Consistent with previous studies,27,32–35 we observed that creased HPC mobilization and expansion with the coadmi- Flt3L increased G-CSF stimulation of HPC mobilization nistration of G-CSF and PG-Flt3L, an effect that is greater with activity greater than that seen with either cytokine than that seen in mice receiving G-CSF coadministered alone. The number of HPP-CFC mobilized per spleen was with Flt3L formulated in DPBS. While this study has significantly increased by the coadministration of G-CSF focused on the hematopoietic activity of Flt3L in the and PG-Flt3L, compared to DPBS (290-fold), G-CSF mouse, Flt3L-induced HPC mobilization has also been (8-fold), Flt3L (10-fold), PG-Flt3L (28-fold), demonstrated in primates32 and clinical studies,48 demon- or the combination of Flt3L and G-CSF (approximately strating the safety and efficacy of Flt3L-induced HPC 6-fold). Similar significant increases (6-fold) of mobilization. Our studies suggest that the improved CFU-GM numbers were also observed using the coadmi- hematopoietic activity associated with PG-Flt3L may have nistration of G-CSF and PG-Flt3L when compared the potential to speed the mobilization of HPC products for to the combination of G-CSF and Flt3L injected daily. transplantation prior to myeloablative treatment in cancer These data suggest that coadministration of G-CSF and patients. In addition, it may successfully mobilize those PG-Flt3L may further improve the efficacy of cytokine- patients who are poor mobilizers with the current induced HSC mobilization by reducing the time needed mobilization strategies. for optimal HSC mobilization and increasing HSC mobilization. Injection of the PG vehicle alone resulted in a significant Acknowledgements increase in the number of splenic CFU-GM and HPP-CFC compared to control mice receiving DPBS, suggesting that The authors gratefully acknowledge the gift of Flt3L from PG alone has hematopoietic activity. This hematopoietic Immunex Corp. and the assistance of Lisa Chudomelka, Tina activity is not attributable to endotoxin as the levels of Winekauf and Richard Murcek in the preparation of this endotoxin present in PG were p1.5 ng per 100 ml (unpub- manuscript. GJR and JMB are employees of RxKinetix, Inc. lished data). Hematopoietic activity associated with the PG and JET is a member of the Scientific Advisory Board of vehicle may therefore be because of the stimulation of RxKinetix, Inc. endogenous cytokine production. PG is composed of Poloxamer-407 and HPMC and both components have been reported to have biological activity. The formulation References of wound-care products in Poloxamer-407 has been reported to improve wound healing,36,37 and the adminis- 1 Brasel K, McKenna HJ, Morrissey P et al. Hematologic effects tration of Poloxamer-407 has been shown to improve of Flt3 ligand in vivo in mice. Blood 1996; 88: 2004– healing processes after burn injuries.38 In addition, methyl 2012. cellulose, a component of HPMC, has also been shown to 2 Robinson S, Mosley RL, Parajuli P et al. Comparison of the stimulate hematopoiesis.39–41 These data suggest that the hematopoietic activity of Flt-3 ligand and granulocyte– increased hematopoietic activity associated with PG-Flt3L macrophage colony-stimulating factor acting alone or in J Hematother Stem Cell Res may be due, in part, to a synergy between Flt3L- and PG- combination. 2000; 9: 711– 720. induced cytokines. 3 Moghimi SM, Hunter AC. Poloxamers and poloxamines in Another approach to achieving a sustained-release nanoparticle engineering and experimental medicine. Trends formulation is the use of PEGylation (the addition of Biotechnol 2000; 18: 412–420. poly(ethylene glycol) (PEG) moieties to target molecules). 4 Morikawa K, Okada F, Hosokawa M, Kobayashi H. PEG is a water-soluble polymer and, when chemically Enhancement of therapeutic effects of recombinant interleukin linked to proteins, improves their pharmacological 2 on a transplantable rat fibrosarcoma by the use of a activity as compared with non-PEGylated molecules. sustained release vehicle, Pluronic Gel. Cancer Res 1987; 47: PEGylation can significantly alter the pharmacokinetic 37–41. properties of proteins by reducing renal clearance38 5 Johnston TP, Miller SC. Inulin disposition following intra- J and protecting against proteolytic cleavage. PEGylated muscular administration of an inulin/Poloxamer gel matrix. Parenter Sci Technol 1989; 43: 279–286. molecules are characterized by a prolonged elimination 6 Johnston TP, Punjabi MA, Froelich CJ. Sustained delivery of (Tb) half-life and sustained biological activity compared interleukin-2 from a Poloxamer 407 gel matrix following 42–47 to their non-PEGylated counterparts. This pattern of intraperitoneal injection in mice. Pharm Res 1992; 9: bioactivity is similar to our results with the PG-Flt3L 425–434. sustained-release formulation, although, as discussed 7 Pec EA, Wout ZG, Johnston TP. Biological activity of urease above, the PG formulation also augments and accelerates formulated in Poloxamer 407 after intraperitoneal injection in hematopoietic activity, unlike PEGylation. the rat. J Pharm Sci 1992; 81: 626–630. In conclusion, we demonstrate that Flt3L prepared in a 8 Lu GW, Jun HW, Dzimianski MT et al. Pharmacokinetic Poloxamer-407-based, sustained-release formulation signif- studies of methotrexate in plasma and synovial fluid following Pharm icantly increased its biological activity and induced more i.v. bolus and topical routes of administration in dogs. Res 1995; 12: 1474–1477. rapid and greater HPC mobilization than an equivalent 9 Paavola A, Yliruusi J, Kajimoto Y et al. Controlled release of daily dose of Flt3L formulated in DPBS. This may be a lidocaine frominjectable gels and efficacy in rat sciatic nerve consequence of a PG-associated prolongation of the block. Pharm Res 1995; 12: 1997–2002. elimination (Tb) half-life and an increase in Flt3L 10 Paavola A, Yliruusi J, Rosenberg P. Controlled release and bioavailability. Further, we demonstrate significantly in- dura mater permeability of lidocaine and ibuprofen from

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