E L S E V I E R Journal of Controlled Release 35 (1995) 67-72

journal of control led

re lease

Sustained in vivo activity of recombinant human granulocyte colony stimulating factor (rHG-CSF) incorporated

into hyaluronan

James Meyer, Lane Whitcomb, Michael Treuheit, David Collins * Department of Pharmaceutics and Drug Delivery, Mail Stop 8-1-A-215, Amgen, Inc., 1840 DeHavilland Drive,

Thousand Oaks, CA 91320, USA

Received 16 November 1994; accepted 31 January 1995

Abstract

Recombinant human granulocyte colony stimulating factor (rHG-CSF), was incorporated into viscous solutions of hyaluronan (HA) for subcutaneous injection into hamsters. Incorporation of rHG-CSF into HA caused no apparent aggregation or degradation of the protein after storage for up to 6 weeks at either 4°C or 37°C. Subcutaneous (s.c.) injection of rHG-CSF alone caused an elevation of white blood cells (WBC) which peaked at 24 h and returned to baseline 48 h after injection. By contrast, s.c. injection of rHG-CSF formulated in 2% (w/v) HA (rHG-CSF/2% HA) yielded elevated WBC at 4-5 days after injection. rHG-CSF/2% HA exhibited prolonged elevation of plasma rHG-CSF levels for up to 4 days after s.c. injection as compared to rHG-CSF alone. Both the molecular weight and concentration of HA affected the viscosity and release of rHG-CSF after injection. At equal viscosity, incorporation of rHG-CSF into higher Mr HA exhibited a more prolonged WBC elevation than rHG-CSF incorporated into lower Mr HA; at equal Mr, 2% (w/v) and 4% (w/v) solutions of HA exhibited more prolonged elevation of WBC than did 1% (w/v) HA. However, no significant difference in release was noted between the 2% (w/v) and 4% HA (w/v) formulations. These results are discussed in relation to the non-ideal colloid osmotic properties of HA.

Keywords: Granulocyte colony stimulating factor; Hyaluronic acid; Sustained release

1. Introduct ion

Granulocyte colony stimulating factor (G-CSF) stimulates neutrophil proliferation and activity [ 1-3 ]. A recombinant form of the protein ( rHG-CSF) , a non- glycosylated single chain polypeptide of Mr 1.85 x 104,

has become an important therapeutic for the treatment of neutropenia following chemotherapy.

Due to rapid clearance from the bloodstream, typical dosage regimens for therapeutic proteins such as rHG-

* Corresponding author. Tel.: (805)-447-2074; Fax: (805)-498- 8674.

0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDIO 1 6 8 - 3 6 5 9 ( 9 5 ) 0 0 0 2 0 - 8

CSF involve daily intravenous (i .v.) or subcutaneous (s.c.) injections. Sustained release formulations of

these proteins are desirable to improve drug efficacy

and patient compliance. To date, several systems have been examined [4] .

Hyaluronan ( H A ) is a naturally occuring linear pol- ymer of repeating disaccharide units of/3-D-glucuronic

acid and fl-N-acetyl-D-glucosamine, coupled via/3 [ 1- 4] l inkages [5 -7 ] . Depending on the source and method of preparation, the molecular weight of HA can vary from 50 000 to several million. HA exhibits inter- esting and useful rheological properties [ 6--9[. In par-

68 J. Meyer et al. /Journal of Controlled Release 35 (1995) 67-72

ticular, HA exhibits shear-dependent viscosity [6-9] which allows even very viscous solutions ( 10--40 mg/ ml) of high molecular weight HA to be pushed through small bore needles. HA also exhibits molecular weight- and concentration-dependent viscosity [ 8 ]. HA occurs naturally in the body in synovial fluid, skin and corpus vitreum [5-7], is biocompatible and non-irritating [ 10,11 ] and has been used in drug delivery systems to provide sustained release of human insulin-like growth factor [ 12] and interferon [ 13].

In the present study, we have incorporated rHG-CSF into viscous HA solutions and examined the in vivo release and activity of rHG-CSF after subcutaneous injection into hamsters. We find that HA can be used to prepare a biocompatible sustained release formula- tion for rHG-CSF.

in PBS at room temperature and degassed prior to use. All measurements were carried out at room tempera- ture.

2.3. Preparation of rHG-CSF/HA solutions

Various concentrations of HA were prepared in PBS from lyophilized material. The syringe containing HA was attached by means of a stopcock to a syringe con- taining rHG-CSF in 7.5 mM NaAcetate (pH 4.5). The solutions in the two syringes were mixed by injecting the rHG-CSF solution into the HA solution and trans- ferring the contents back and forth through the stop- cock.

2.4. Treatment of the rHG-CSF/HA solutions with hyaluronidase

2. Materials and methods

2.1. Materials

rHG-CSF was prepared by Amgen, Inc. as described [3]. HA, from rooster comb of M r > 4 X l 0 6 was obtained from Biomatrix, Inc. (Ridgefield, NJ). HA of Mr< 1.8X 106 was obtained from Genzyme (Cam- bridge, MA) and HA of Mr< 1X 106 was obtained from Sigma (St. Louis, MO). Male golden Syrian ham- sters were obtained from Charles River Laboratories (Willmington, MA). Phosphate-buffered saline (PBS) was obtained from Gibco-BRL (Gaithersburg, MD). Hyaluronidase from Hirudinaria manillenis, was purchased from Calbiochem (San Diego, CA). Waters Chromatography System (Milford, MA) was utilized for all HPLC analyses. The size exclusion column used was the Phenomenex BioSep SEC2000 (Torrance, CA) and the cation exchange column was the Toso- Haas SP-5PW (Montgomeryville, PA). The 10-20% Daiichi Miniplus gels were obtained through Integrated Separation Systems (Nattick, MA).

2.2. Viscosity measurements

The viscosities of different HA solutions, varying in concentration from 0.5 to 3% (w/v ) , were measured on a Brookfield R V D V I I + C P digital viscometer (Stoughton, MA) using a cone and plate device (CP 52). The lyophilized HA preparations were hydrated

Syringes containing rHG-CSF (1 mg/ml) alone, rHG-CSF(1 mg/ml) in 1% HA (w/v) (rHG-CSF/ 1% HA) and 1% HA (w/v) alone were placed at both 4 and 37°C and analyzed for protein aggregation and degradation after 2, 4 and 6 weeks of incubation. The Mr of the HA used in these studies was > 4 X 106. Due to the viscosity of the 1% HA (w/v) solutions, the HA was digested in order to analyze the protein for any potential aggregation or degradation. After incubation for the time indicated, 300/.d of the rHG-CSF/1% HA and 1% HA samples were diluted with 300/xl of 20 mM sodium acetate, pH 4.0, 20 /xl of hyaluronidase (5000 units dissolved in 500 /xl of 50 mM sodium acetate, pH 4.0) was added and incubated at 37°C for 15 h. For the rHG-CSF samples, 300/zl was diluted as above and treated with either hyaluronidase (20/~1) or 20/~1 of buffer only (50 mM sodium acetate, pH 4.0) and incubated at 37°C for 15 h. For the enzyme blanks, 300/xl of 20 mM sodium acetate, pH 4.0 was diluted with 300/xl of deionized H20 and 20/zl of enzyme and incubated for 15 h at 37°C. After incubation, all samples were analyzed by size exclusion and cation exchange chromatography and SDS-PAGE.

2.5. Size exclusion chromatography

All high performance liquid chromatography was performed on a Waters liquid chromatography system equipped with a WISP 712 autosampler refrigerated at 4°C, and a 490E multiwavelength detector. The sam-

J. Meyer et al. /Journal o f Controlled Release 35 (1995) 67-72 69

pies for size exclusion chromatography were analyzed with an isocratic mobile phase of 0.1 M sodium phos- phate, pH 6.9, on a Phenomenex BioSep 2000 column eluted at 0.8 ml/min. The absorbance of the eluent was monitored at 280 nm and the data was collected by Waters Maxima software.

2.6. Cation exchange chromatography

All samples were analyzed on a TosoHaas SP-5PW cation exchange colunm initially equilibrated in 20 mM sodium acetate, pH 5.4 (Buffer A), and eluted with a linear gradient of 20 mM sodium acetate, pH 5.4, con- taining 0.5 M NaC1 (Buffer B) at a rate of 2% B/min over 35 min at 1.0 ml/min. The absorbance of the eluent was monitored at 230 nm and the data was col- lected by Waters Maxima software.

2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed according to Laemmli [ 14 ] under both reducing and non-reducing conditions. The analytical gels consisted of 1-mm-thick 10-20% Daiichi Miniplus gels which were run on an apparatus from Integrated Separation Systems.

2.8. In vivo activity of rHG-CSF/HA solutions

A total volume of 0.2 ml of rHG-CSF or rHG-CSF/ HA was injected subcutaneously into hamsters between the shoulder blades. At various times after injection, animals were killed and blood samples obtained by cardiac puncture. White blood cells (WBC) were counted using a Sysmex F-800 Microcell counter. Part of the blood sample was centrifuged at 10 000 rpm for 10 min in an Eppendorf refrigerated centrifuge and the plasma collected, frozen and stored at - 80°C until ELISA analysis could be performed, rHG-CSF ELISA was performed using microtiter plates coated with 0.5 ug/ml of rabbit anti-rHG-CSF polyclonal antisera in 50 mM Tris buffer pH 7.4. Non-specific sites on the wells were blocked using 2% bovine serum albumin. After incubation of rHG-CSF standards and plasma samples, the plates were washed five times and bound rhG-CSF was detected using horseradish peroxidase- conjugated mouse anti-rhG-CSF monoclonal antibody 231A.

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Fig. 1. Incorporation into HA does not cause rHG-CSF aggregation upon storage for 6 weeks at 37°C. Size exclusion chromatographs are shown for (A) rHG-CSF incubated in buffer only (for details see Materials and Methods); (B) rHG-CSF/ 1% (w/v) HA treated with hyaluronidase prior to chromatography (broken line) versus 1% (w/v) HA alone treated with hyaluronidase (solid line) and (C) rHG-CSF treated with hyaluronidase prior to chromatography (broken line) versus hyaluronidase alone (solid line).

3. Results and discussion

Solution stability studies for rHG-CSF in the absence of HA, indicated that no physical (Fig. 1, A) or chem- ical degradation occurred after 6 weeks storage at 37°C (or 4°C). Incorporation into HA (Mr__>4X 106) did not lead to aggregation of rHG-CSF under these con- ditions of storage (Fig. 1B and data not shown). The higher molecular weight material detected by size exclusion chromatography represents non-monomeric HA (Fig. 1B) and the enzyme, hyaluronidase (Fig. 1C). Analysis of the solutions by cation exchange chro- matography and SDS-PAGE yielded similar results (data not shown).

70 J. Meyer et al. /Journal of Controlled Release 35 (1995) 67-72

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Fig. 2. HA prolongs the in vivo activity of rHG-CSF. Hamsters were injected subcutaneously with either rHG-CSF alone ( 0 ) , 2% (w/ v) HA ( M r > 4 × 106) alone (D) or rHG-CSF/2% (w/v) HA (O) and WBC measured over time. The injected dose of rHG-CSF was 200/xg/kg. The data represent the mean + S.D. for n = 8 hamsters/ point.

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Fig. 3. Incorporation into HA leads to prolonged elevation of plasma rHG-CSF levels. Hamsters were injected subcutaneously with 200 p.g/kg of either rHG-CSF alone ( 0 ) or rHG-CSF/2% (w/v) HA (O). At various times hamsters were killed and blood levels of rHG- CSF determined by ELISA as described in methods. The data rep- resent the mean + S.D. for n = 8 hamsters/point.

As shown in Fig. 2, s.c. injection of rHG-CSF alone leads to an elevation in WBC within the first 24 h after injection. WBC return to baseline within 48 h under these conditions. By contrast, s.c. injection of rHG- CSF/2% HA (HA M r > 4 × 106) leads to prolonged elevation of WBC starting within 24 h and continuing until 5 days after injection. Baseline levels of WBC are achieved 7 days after injection. Injection of HA in the absence of rHG-CSF caused no elevation in WBC (Fig. 2).

Incorporation of rHG-CSF into 2% HA (mr>__4X 10 6) leads to prolonged elevation of the

plasma levels for rHG-CSF after s.c. injection as com- pared to rHG-CSF alone (Fig. 3 ). For rHG-CSF alone, plasma levels decline rapidly in the first 24-48 h after injection (Fig. 3). By contrast, s.c. injection of rHG- CSF/2% HA provides a 9-fold increase in plasma rHG- CSF at 2 days and at least a 2-fold enhancement of rHG-CSF plasma is maintained for up to 4 days after injection (Fig. 3). The shape of the plot of rHG-CSF plasma concentration vs. time for rHG-CSF/2% HA suggests that release of rHG-CSF from the injection site is a first order process, in aggreement with earlier studies using interferon incorporated into HA [ 13 ].

Elevation of WBC after injection of rHG-CSF/2% HA (HA M r ~ 4 X l 0 6 Da) exhibits a curvelinear response to rHG-CSF dose (Fig. 4). Fig. 4 shows the WBC levels obtained at 3, 4 and 5 days after injection of rHG-CSF/2% HA. The data suggest that low doses (below 60 ~g/kg rHG-CSF) of rHG-CSF/2% HA do not significantly elevate WBC at these later time points, as compared to rHG-CSF alone (Fig. 2). This is con- sistent with the fact that rHG-CSF is released slowly over a long period of time when incorporated into HA (Fig. 3), at lower doses, sub-threshold plasma levels of rHG-CSF are achieved.

The properties of HA depend upon both the molec- ular weight and concentration [6-9]. Doubling the concentration of HA in solution leads to a 10-fold increase in bulk viscosity [8]. It was of interest to examine the effects of concentration and molecular weight of HA on the in vivo release of rHG-CSF.

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0 200 400

Dose (p.g rHG-CSF/kg)

Fig. 4. The dose-response relationship to rHG-CSF/2% (w/v) HA. rHG-CSF was incorporated into 2% (w/v) HA (M~>_4 X 106) and various doses injected subcutaneously into hamsters. WBC were measured at 3 (O) 4 ( 0 ) and 5 (D) days after injection. The data represent the mean + S.D. for n = 4 hamsters/point.

J. Meyer et al. /Journal of Controlled Release 35 (1995) 67-72 71

rHG-CSF/HA mixtures were prepared using HA of different molecular weights and the viscosity of all solutions adjusted to 6.4 X 107 centipoise. In order to achieve equivalent viscosities, higher concentrations of lower molecular weight HA were required. As shown in Fig. 5, higher molecular weight HA exhibited more prolonged release of rHG-CSF than did lower molec- ular weight HA. As described [ 8 ], the colloid osmotic pressure of HA solutions increases very rapidly with concentration. Injection of these solutions into the sub- cutaneous compartment is envisioned to cause rapid water flow into the HA network resulting in a dilution of HA concentration and a decrease in viscosity. This would lead to a more rapid release of rHG-CSF.

Fig. 6 shows the effect of HA concentration, at con- stant molecular weight (Mr>___4xl06), o n WBC elevation at 4 days after injection of various rHG-CSF/ HA mixtures. As shown in Fig. 6, increasing HA con- centration from 1% (w/v) to 2% (w/v) has a greater effect than increasing concentration from 2% (w/v) to 4% (w/v) . Again, this is most likely due to the non- ideal colloid osmotic behaviour of HA [8]. Bothner and Wik [8] have predicted that s.c. injection of very highly concentrated HA solutions would also promote water flow into the HA network to balance the osmotic pressure of the HA with that of the surroundings. The result is a dilution of the HA concentration and viscos-

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HA concentration (%) Fig. 6. HA concentration at constant molecular weight affects the in vivo release of rHG-CSF. HA of Mr > 4 × 106 was used to prepare rHG-CSF/HA mixtures with different concentrations (%, w/v) of HA which were injected subcutaneously into hamsters. The injected dose of rHG-CSF was 200/xg/kg. Four days after injection, hamsters were killed and WBC measured as described in methods. The data represent the mean + S.D. for n = 4 hamsters.

ity. Our data suggests that for this HA (Mr > 4 x 106),

the 4% (w/v) sample is diluted by interstitial water upon injection to around 2% (w/v) , thus leading to very similar release kinetics.

4. Conclusions

Our data suggest that incorporation of protein ther- apeutics, such as rHG-CSF, into viscous HA solutions is one way to achieve sustained in vivo activity. Prep- aration of the rHG-CSF/HA solutions was simple and led to no detectable aggregation or denaturation of rHG-CSF. The duration of protein release was limited by the osmotic behavior of HA. In particular, our data suggest that the use of high molecular weight HA ( > 4 X 10 6) at relatively low concentrations ( > 2% (w/v) ) is preferred to achieve sustained activity for 4-5 days in vivo.

Days

Fig. 5. HA molecular weight at constant viscosity affects the in vivo release of rHG-CSF. HA preparations of Mr < 1 X 106 ([ ] ) , 1.8 × 106 ( I ) and > 4 X 106 (O) were used to prepare rHG-CSF/HA for- mulations and the in vivo activity compared to rHG-CSF alone ( • ) . The HA preparations were adjusted to achieve a viscosity of 6.4 x 107 centipoise before injecting into hamsters. The injected dose of rHG- CSF was 200/xg/kg. The concentrations of HA used were 2% (w/ v) (O) , 3% (w/v) ( I ) and 4.4% (w/v) (D) . The data represent the mean + S.D. for n = 4 hamsters/point.

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