Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Interferon (IFN) α-2b is an important cytokine and has
been used widely as a therapeutic agent to treat patients
with viral and oncological diseases. It is an essential
component of the treatment of chronic hepatitis B
infection[1]. However, in the recommended dosing regimen, the protein
needs to be administered every other day for 3 months, which
brings about much inconvenience to the patients. The
t1/2 of IFN α-2b, when administered subcutaneously, is only about
4 h[2].The protein was shown to be cleared quickly, therefore
frequent repeated administrations are necessary.
Much effort has been devoted to the development of
IFN α-2b-based products with persistent effects. One
approach, covalent attachment of polyethylene glycol (PEG)
to the protein surface (PEGylation), has been the most
successful. Several PEGylated IFNa products are already
on the market. The half-life of the PEGylated protein is 40 h,
thus it only needs to be administered once a week for similar
therapeutic effects[3]. However, the chemical conjugation
process of PEGylation is rather complex and the PEGylated
products are usually mixtures with different PEG
conjugation sites. In addition, a few studies have suggested that the
chemical modifications can sometimes affect the structure
as well as the bioactivity of the
protein[4].
An alternative approach is to develop sustained-release
depot formulations of IFN α-2b. Liposome formulations of
IFNg have been developed and have been reported to have
prolonged release profiles of up to 160 h. Even so, using
conventional liposome formulations, the drug loading
capacity and encapsulation efficiency are still rather low and
variable[5].
Multivesicular liposomes (MVL), on the other hand, have
a different structure and possess some distinctive properties.
They usually contain a larger internal space, which would
allow more drug to be loaded. Their larger size would also
deter rapid clearance by tissue macrophages so that they
may act as drug depots to enable sustained release of
drugs[6]. The MVL formulation of the anticancer drug cytarabine
(DepocytTM) has been developed successfully and is now
being used widely for the treatment of
leukemia[7].
We took a similar approach in the present study and
evaluated the MVL formulation of IFN α-2b and its
biopharma-ceutical properties. Some of the parameters that affected the
MVL in vivo pharmacokinetic behaviors were further
investigated. Our data suggest that MVL formulation of IFN
α-2b can be developed with satisfactory sustained release
properties in vivo, which may have useful clinical
applica-tions.
Materials and methods
Materials 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC), cholesterol, triolein and
1,2-dipalmitoyl-sn-3-phosphoglycerol (DPPG) were all purchased from Sigma (St
Louis, MO, USA). L-lysine was purchased from Sangon
Biological Engineering Technology and Service Co
(Shang-hai, China). IFN α-2b (recombinant human interferon
α-2b) was kindly provided by Pan Asia Bio (Shanghai, China), and
all other reagents were of analytical grade and purchased
from Shanghai Chemical Reagent Co (Shanghai, China).
Multivesicular liposome
preparation The MVL formulations of IFN
α-2b were prepared based on the typical double-emulsion procedure developed by Kim
et al[8-12]. Briefly, 1 mL chloroform containing the lipids (molar ratio
DOPC:cholesterol:DPPG: triolein, 7:11:1:1; two other molar
ratios were also used: 7:11:1:4 and 7:11:1: 8) was emulsified
at 10 000 r/min for 10 min with 1 mL aqueous solution
containing IFN α-2b in phosphate-buffered solution (PBS) and
various sucrose concentrations to produce a water-in-oil
emulsion. This water-in-oil emulsion was subsequently
emulsified with 4 mL of an aqueous solution containing 4%
glucose (w/v) and 20 mmol/L lysine at 2500 r/min for 10 s, and
then poured into another 4 mL of the same aqueous solution.
Chloroform was removed by flushing nitrogen over the
surface of the double emulsion at 37 °C for approximately 15 min.
The resultant MVL were pelleted at 600
×g and washed twice with PBS to remove unencapsulated IFN
α-2b. The IFN α-2b concentration in MVL was determined by HPLC
quantification and adjusted accordingly.
For preparing MVL samples with narrower size
distribu-tions, the procedures were further modified. For large-sized
MVL, a smaller emulsification force (1000 r/min) was applied
during the second emulsification and the chloroform was
removed slowly (over 30 min). The large MVL were purified
and harvested by centrifugation at
100×g and only the pellet was collected. For small-sized MVL, the second
emulsification step was carried out at 10 000 r/min. Chloroform was
removed over approximately 15 min. The resultant MVL were
then centrifuged twice at 100×g for 10 min, and the
precipitants were discarded. Small-sized MVL were then harvested
in the pellet after centrifugation at
600×g for 10 min. The size distributions were quite reproducible because of the
purification-by-centrifugation step. The IFN α-2b concentration
was determined by HPLC quantification and adjusted
accordingly.
Multivesicular liposome size measurements
The MVL suspensions were diluted in saline. The particle size distri
bution was measured using a CIS100 particle size analyzer
(Ankersmid, the Netherlands).
Encapsulation efficiency
determination IFN α-2b encapsulation efficiency was determined by measuring the amount
of encapsulated protein as compared to the total amount
added[13]. Briefly, the MVL were pelleted by centrifugation
at 600×g for 10 min. The pellet was then treated with
extraction solution (0.2% Triton X-100, 28% ethanol, 71.2% water,
v/v) and quantified using the HPLC assay described below.
IFN α-2b characterization IFN
α-2b was characterized using reverse phase (RP)-HPLC, sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
enzyme-linked immunosorbent assays (ELISA). RP-HPLC was
carried out on a Agilent 1100 liquid chromatography system at
45 °C using a linear gradient of 45%-70% solvent B
[CH3CN, 0.1% Trifluoroacetic Acid (TFA)]
over 11 min, and then a sharp linear gradient of 70%-100% solvent B over 9 min at a
flow rate of 1.0 mL/min. Solvent A was water (0.1% TFA).
IFN α-2b was detected by UV absorbance at 280 nm. The
standard curve showed a linear correlation within the range
of 2.0 µg/mL-100 µg/mL. The intra-day and inter-day assay
precisions were determined to be less than 3% and 2%,
respectively. SDS-PAGE analyses of encapsulated
IFN α-2b were carried out using 12% acrylamide gels under reducing
conditions and stained with silver stain. ELISA were carried
out using the human interferon a ELISA Kit (sandwich
method) from PBL Biomedical Laboratories
(Piscataway,NJ, USA). The protein control and the MVL samples were both
treated with extraction solution (0.2% Triton X-100, 28%
ethanol, 71.2% water) for 30 min and then applied to the
ELISA plate. IFN α-2b concentrations were determined
according to the standard curve supplied with the kit.
In vitro drug release
study Aliquots of IFN α-2b MVL (500 µL) were pipetted into a 50 mL beaker containing 25 mL
of saline solution. The beaker was incubated at 37 °C under
constant rotation at 12 r/min. Three samples were collected
at each time point (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and
168 h) and were centrifuged at
600×g for 10 min. The protein concentrations in the pellets were determined using the
RP-HPLC assay[14].
In vivo pharmacokinetic
studies Free IFN α-2b and
IFN α-2b MVL suspensions were injected subcutaneously
in a single dose in female SD rats. Three rats were included
in each group. Blood samples (0.3 mL) were collected at
specific time points (5 min, 30 min, 2 h, 8 h, 12 h, 24 h, 48 h,
72 h, 96 h, and 120 h after injection) and were placed aside
for 30 min at room temperature. The supernatant (serum)
was collected by centrifugation at
700×g for 10 min. IFN α-2b concentrations were determined using ELISA, which has
a detection limit of 30 pg/mL. Any values lower than
30 pg/mL were considered undetectable.
Results
Interferon α-2b encapsulation in multivesicular
lipo-somes Multivesicular liposomes containing IFN
α-2b were prepared according to the standard double-emulsion method.
The preparations were highly reproducible, usually yielding
MVL with similar size distributions and encapsulation
efficiencies. A representative light microscope image of the
resultant MVL is shown in Figure 1A. The particle size
distribution analysis is shown in Figure 1B. The MVL had a
rather broad size distribution ranging from 2 µm to 50 µm in
diameter. The median size was approximately 18 µm.
The encapsulated proteins were characterized using
SDS-PAGE, ELISA, and HPLC. There was no chemical
degradation in the peptide chain after the preparation. The
structural integrity of the protein is considered crucial to its activity.
We used an ELISA to partially characterize the 3-dimensional
conformational change in the protein. Our data showed that
the antibody binding affinity to the protein was only slightly
reduced, indicating that there was a substantial amount of
native structure remaining in the protein sample after
preparation (Figure 2).
Interferon α-2b encapsulation
efficiencies Several parameters were evaluated for their effects in optimizing IFN
α-2b encapsulation efficiencies. Table 1 lists some of the
representative scenarios. Using the standard lipid MVL
formulation (48.3 mmol/L DOPC, 70.7 mmol/L cholesterol,
6.7 mmol/L DPPG and 6.7 mmol/L triolein), the encapsulation efficiency
was approximately 30%. It can be further increased by
adding more lipids. At a protein-to-lipid ratio of 0.031
(w/w), the encapsulation efficiency was more than 60%. In contrast to
the reported development of MVL formulation of
progeni-poietin, we did not find any evident correlation between the
sucrose concentration in the first aqueous phase and IFN
α-2b encapsulation efficiency[15]. Furthermore, the
encapsulation capacity only seemed to vary slightly with different
triolein contents.
Interferon α-2b release from multivesicular liposomes
in vitro The MVL were stable when stored in saline in small
volumes at 4 °C, with less than 2% protein leaked after
3 months (data not shown). When the MVL were diluted
into a large amount of saline (1:50 dilution) under well-mixed
conditions, the encapsulated protein would gradually leak
out (Figure 3). Approximately 90% of the content was shown
to have been released after 7 d.
In vivo pharmacokinetic
profiles After subcutaneous
injection of a dose of 2.5 mg/kg, free IFN α-2b proteins were
cleared quickly within 1 d (the detection limit of the ELISA
kit was at 30 pg/mL). The MVL sustained release
formula-tions, however, would provide a continuous supply of IFN
α-2b to the systemic circulation, which lasted more than 2 d.
The detailed pharmacokinetic behavior was found to be
related to the triolein content in the MVL formulation (Figure 4).
Increases in triolein content resulted in longer release times.
Effect of multivesicular liposome size on
in vivo protein release
profiles To further optimize the sustained release
profile of IFN α-2b MVL formulations, we specifically
compared the in vivo release properties of MVL with different
sizes. As the typical MVL preparation procedure yielded
MVL with rather broad size distributions (Figure 1B), we
modified some emulsification parameters and added a final
fractionation step to obtain MVL samples in much narrower
size distributions. The lipid formulation remained the same.
Two different-sized populations were obtained and their size
profiles are shown in Figure 5A. The large MVL had sizes of
approximately 40 µm-60 µm diameter, and the small MVL
were approximately 10 µm-25 µm in diameter. The samples
were administered subcutaneously at a dose of 1.2 mg/kg
and the IFN α-2b serum concentrations were determined and
plotted in Figure 5B. It shows that small MVL released the
encapsulated protein content over a longer time compared
with large MVL.
Discussion
Multivesicular liposome formulations have been
developed successfully for the prolonged release of cytarabine,
morphine and other drugs[16,17]. The long-lasting sustained
release properties were most evident when the formulations
were administered in a small confined space, such as the
epidural. We showed here that MVL could also be used to
achieve reasonable prolonged release properties after
subcutaneous administration, and MVL IFN α-2b formulations
may be developed for the treatment of viral infections
requiring less frequent dosing. Our data indicate that MVL can
maintain their structure in the subcutaneous interstitial space
for a few days and slowly release the encapsulated proteins
into the systemic circulation. There was considerable
protein detected in the circulation for more than 5 d, and the
serum half-life was estimated to be approximately 30 h.
The prolonged serum half-life that we achieved is
actually comparable to what has been reported for the PEGylated
IFN α-2b product currently in clinical use, even though their
mechanisms for sustained serum concentration are quite
different. PEGylated IFN α-2b requires chemical
modification of the protein structure, which might affect its bioactivity.
The PEGylated proteins are absorbed into the systemic
circulation quickly after administration but remain there for a
long time by avoiding various clearance mechanisms. In
contrast, the proteins in MVL formulations are unmodified,
which wait inside the subcutaneous MVL depot, slowly leak
out, and enter the circulation. They should maintain their
original structure, and most likely their full bioactivities. Their
distribution and clearance mechanisms should also follow
the same pathway as natural IFN α-2b. Therefore, compared
to the PEGylated product, MVL formulations would have a
more defined safety profile, established manufacture
procedures and drug efficacy, and side effects that are easier to
evaluate. We therefore believe that MVL formulation may
be an attractive candidate for the sustained delivery of
IFN α-2b for the treatment of viral infections.
Another significant advantage of the MVL formulations
is its high drug loading capacity. Compared with
conventional liposomes, which often have limited encapsulation for
hydrophilic proteins, the MVL offer a much larger internal
space and therefore usually have higher encapsulation
efficiencies. For IFN α-2b, the encapsulation efficiency was
usually more than 30%. However, the double-emulsion
preparation method has been shown to cause protein degradation
and denaturation[18]. Also, there might exist
protein-liposomal bilayer interactions that may affect protein conformation
and activity[19,20]. We used three methods to test protein
chemical and structural changes after encapsulation. Both
the SDS-PAGE and HPLC analyses showed that the proteins
were chemically intact. For protein conformational changes,
some studies have used biophysical methods such as
circular dichroism and fluorescence spectroscopy to detect
the secondary structure or local amino acid environment
changes[19]. We adopted a biochemical approach using an
ELISA to probe possible 3-dimensional conformational
changes. The ELISA may have its limitations because it can
only detect changes of structure near binding sites. However,
antibody binding has been shown to be very sensitive to
protein denaturing effects, and ELISA are commonly used in
protein formulation studies to assay protein structure
integrity[21]. Our data showed that the antibody binding affinity
for the protein after encapsulation was only slightly reduced,
indicating that substantial native structure remained after
preparation. Further studies are needed to confirm the
detailed bioactivity of the encapsulated IFN α-2b.
We also tested several parameters that might affect the
release profile of MVL. Triolein is used as a hydrophobic
space filler at lipid membrane intersection points and can
stabilize the junctions[11]. The amount of triolein in the MVL
formulation was suggested to be important for MVL mor
phology and stability[11,22]. We showed that it had a
significant impact on the in vivo release profiles of IFN
α-2b (Figure 4). It is possible that when more triolein is present,
the lipid walls are more stable and therefore the protein is
released more slowly.
With a similar argument, we hypothesized that the size of
the MVL would also be important for the drug release profile,
because the inter-compartmental fusion and diffusion of the
proteins in larger MVL would add another rate-limiting step
and would eventually result in faster protein release into the
environment. However, when we used the typical
preparation procedure, the resultant MVL size distribution was rather
broad, ranging from 2 µm to 50 µm (Figure 1). It is difficult to
differentiate the release profile of different-sized MVL.
Therefore, we developed a modified procedure to make MVL
with much narrower size distributions (Figure 5). Based on
our data, the protein release from MVL with smaller sizes
(10 µm-25 µm) was indeed slower than that from larger MVL
(40 µm-60 µm), which is an important observation. We
therefore suggest that, in further development of control-released
formulations, MVL sizes will need to be optimized and well
controlled.
In summary, we have demonstrated that IFN α-2b MVL
formulation can achieve high encapsulation efficiency, good
stability and sustained release effects. The sustained
release effect can be affected by the triolein content and
particle sizes. Further optimization is needed in order to
develop a clinically valuable sustained release formulation of
IFN α-2b.
Acknowledgement
We would like to thank Pan Asia Bio (Shanghai, China)
for providing IFN α-2b.
References
1 Wai CT, Lok AS. Treatment of hepatitis B. J Gastroenterol
2002; 37: 771-8.
2 Bukowski RM, Tendler C, Cutler D, Rose E, Laughlin MM,
Statkevich P. Treating cancer with PEG intron. Cancer 2002;
95: 389-96.
3 Glue P, Fang JW, Rouzier PR, Raffanel C, Sabo R, Gupta SK,
et al. Pegylated interferon-a2b: pharmacokinetics,
pharmacody-namics, safety, and preliminary efficacy data. Clin Pharmacol
Ther 2000; 68: 556-7.
4 Foser S, Schacher A, Weyer KA, Brugger D, Dietel E, Marti S,
et al. Isolation, structural characterization, and antiviral
activity of positional isomers of monopegylated interferon
a-2a (PEGASYS). Protein Expr Purif 2003; 30: 78-87.
5 Vanslooten ML, Boerman O, Romoren K, Kedar E, Crommelin
DJ, Storm G. Liposomes as a sustained release system for human
interferon-g: Biopharmaceutical aspects. Biochim Biophys Acta
2001; 1530: 134-45.
6 Howell SB. Clinical application of a novel sustained-release
injectable drug delivery system:
DepoFoamTM Technology. Cancer J 2001; 7: 219-25.
7 Kim S, Khatibi S, Howell SB, McCully C, Balis FM, Poplack DG.
Prolongation of drug exposure in cerebrospinal fluid by
encapsulation into DepoFoam. Cancer Res 1993; 53: 1596-8.
8 Kim S, Turker MS, Chi EY, Sela S, Martin GM. Preparation of
multivesicular liposomes. Biochim Biophys Acta 1983; 728:
339-48.
9 Katre NV, Asherman J, Schaefer H, Hora M. Multivesicular
Liposome (DepoFoam) technology for the sustained delivery of
insulin-like growth factor-1 (IGF-1). J Pharm Sci 1997; 87:
1341-6.
10 Ye Q, Asherman J, Stevenson M, Brownson E, Katre NV.
DepoFoamTM technology: a vehicle for controlled delivery of
protein and peptide drugs. J Control Release 2000; 64: 155-66.
11 Mantripragada S. A lipid-based depot (DepoFoam technology)
for sustained release drug delivery. Prog Lipid Res 2002; 41:
392-406.
12 Bilati U, Allemann E, Doelker E. Strategic approaches for
overcoming peptide and protein instability within biodegradable nano-
and microparticles. Eur J Pharm Biopharm 2005; 59: 375-88.
13 Ramprasad MP, Anantharamaiah GM, Garber DW, Katre NV.
Sustained-delivery of an apolipoprotein E peptidomimetic using
multivesicular liposomes lowers serum cholesterol levels. J
Control Release 2002; 79: 207-18.
14 Xiao CJ, Qi XR, Maitani Y, Nagai T. Sustained release of cisplatin
from multivesicular liposomes: potentiation of antitumor
efficacy against S180 murine carcinoma. J Pharm Sci 2004; 93:
1718-24.
15 Ramprasad MP, Amini A, Kararli T, Katre NV. The sustained
granulopoietic effect of progenipoietin encapsulated in
multive-sicular liposomes. Int J Pharm 2003; 261: 93-103.
16 Chamberlain MC, Khatibi S, Kim JC, Howell SB, Chatelut E, Kim
S. Treatment of leptomeningeal metastasis with
intraventricular administration of Depot Cytarabine (DTC101). Arch Neurol
1993; 50: 261-4.
17 Yaksh TL, Provencher JC, Rathbun ML, Myers RR, Powell H,
Richter P, et al. Safety assessment of encapsulated morphine
delivered epidurally in a sustained-release multivesicular
liposome preparation in dogs. Drug Deliv 2000; 7: 27-36.
18 Van de Weert M, Hennink WE, Jiskoot W. Protein instability in
poly(lactic-co-glycolic acid) microparticles. Pharm Res 2000;
17: 1159-67.
19 Van Slooten ML, Visser AJWG, Van Hoek A, Storm G, Crommelin
DJA, Jiskoot W. Conformational stability of human
interferon-gamma on association with and dissociation from liposomes. J
Pharm Sci 2000; 89: 1605-19.
20 Koppenhagen FJ, Visser AJWG, Herron JN, Storm G, Crommelin
DJA. Interaction of recombinant interleukin-2 with liposomal
bilayers. J Pharm Sci 1998; 87: 707-14.
21 Braun A, Alsenz J. Development and use of enzyme-linked
immunosorbent assays (ELISA) for the detection of protein
aggregates in interferon-alpha (IFN-a) formulations. Pharm Res
1997; 14: 1394-400.
22 Langston MV, Ramprasad MP, Kararli TT, Galluppi GR, Katre
NV. Modulation of the sustained delivery of myelopoietin
(Leridistim) encapsulated in multivesicular liposomes
(Depo-Foam). J Control Release 2003; 89: 87-99.
|
