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Abstract
A major challenge in the development of sustained-release formulations for protein and peptide drugs is to achieve high
drug loading sufficient for prolonged therapeutic effect coupled with a high recovery of the protein / peptide. This challenge
has been successfully met in the formulation of several peptide and protein drugs using the DepoFoamE, multivesicular
lipid-based drug delivery system. DepoFoam technology consists of novel multivesicular liposomes characterized by their
unique structure of multiple non-concentric aqueous chambers surrounded by a network of lipid membranes. The objective
of this paper is to demonstrate that DepoFoam technology can be used to develop sustained-release formulations of
therapeutic proteins and peptides with high loading. DepoFoam formulations of a protein such as insulin, and peptides such
as leuprolide, enkephalin and octreotide have been developed and characterized. The data show that these formulations have
high drug loading, high encapsulation efficiency, low content of free drug in the suspension, little chemical change in the
drug caused by the formulation process, narrow particle size distribution, and spherical particle morphology. Drug release
assays conducted in vitro in biological suspending media such as human plasma indicate that these formulations provide
sustained release of encapsulated drug over a period from a few days to several weeks, and that the rate of release can be
modulated. In vivo pharmacodynamic studies in rats also show a sustained therapeutic effect over a prolonged period. These
results demonstrate that the DepoFoam system is capable of efficiently encapsulating therapeutic proteins and peptides and
effectively providing controlled delivery of these biologically active macromolecules. 2000 Elsevier Science B.V. All
rights reserved.
Keywords: Multivesicular liposome; Sustained delivery; High loading liposome; Protein and peptide encapsulation; Controlled release
1. Introduction
Therapeutic proteins and peptides administered
intravenously or subcutaneously are often cleared
*Corresponding author. Tel.: 11-858-625-2424; fax: 11-858625-2439.
E-mail address: nandini katre@skyepharma.com (N.V. Katre)
]
1
Presently SkyePharma, Inc.
0168-3659 / 00 / $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0168-3659( 99 )00146-7
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systems, while a few deal with multivesicular liposomes, or DepoFoamE particles [1013]. A unique
feature of the DepoFoam system is that inside each
DepoFoam particle, discontinuous internal aqueous
chambers, bounded by a continuous, non-concentric
network of lipid membranes render a higher aqueous
volume-to-lipid ratio and much larger particle diameters compared with MLV [10]. Fig. 1 illustrates the
differences between DepoFoam particles, ULV and
MLV. This multivesicular nature also provides for
Fig. 1. Illustrative electron micrographs of (A) unilamellar vesicles (ULV) (adapted from Lasic [14]) which contain a single internal aqueous
compartment and typically have a diameter of 0.020.5 mm; (B) multilamellar vesicles (MLV) (adapted from Lasic [14]) which contain
multiple concentric internal aqueous compartments and typically have a diameter of 0.25 mm; and (C) multivesicular liposomes
(DepoFoamE particles) (adapted from Ref. [15]), which contain multiple non-concentric internal aqueous compartments and typically have
a diameter of 1100 mm. These electron micrographs show that DepoFoam particles are structurally distinct from MLV and ULV.
veloped sustained-release protein and peptide formulations which meet this challenge. In particular,
DepoFoam formulations of the following protein and
peptides are evaluated: (1) recombinant human insulin [16,17], which also has been encapsulated into
traditional liposomes [18]; (2) leuprolide: a nine
amino acid peptide analogue of LHRH [1921]; (3)
met-enkephalin: an endogenous opioid peptide of
five amino acids [22,23]; and (4) octreotide: a cyclic
octapeptide analog of somatostatin [24,25].
2.1. Materials
Recombinant human insulin, produced in E. coli,
FITC-labeled insulin, and met-enkephalin were obtained from Sigma Chemical Co. (St. Louis, MO).
Leuprolide acetate was obtained from Bachem Bioscience Inc. (King of Prussia, PA), and octreotide
was custom-synthesized by Multiple Peptide Systems
Inc. (San Diego, CA). BODIPY 665 / 676 (B-3932)
was obtained from Molecular Probes Inc. (Eugene,
OR). The phospholipids, 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC or DC18:1 PC), 1,2dierucoyl-sn-glycero-3-phosphocholine (DEPC or
DC22:1 PC), 2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC or DC24:1 PC), 1,2-dipalmitoyl-snglycero-3-phosphoglycerol (DPPG) and triolein were
from Avanti Polar Lipids Inc. (Alabaster, AL), and
tricaprylin from Sigma (St. Louis). Cholesterol was
from Spectrum Chemical Manufacturing Corp. (Gardena, CA). L-Lysine was from Degussa Corp. (Marceau, France), normal saline (0.9% sodium chloride)
and 50% glucose from McGaw Inc. (Irvine, CA),
citric acid from Sigma (St. Louis, MO), 1 M
hydrochloric acid from Fisher Chemical (Fair Lawn,
NJ). UV absorbance was determined with a U2000
UV/ VIS spectrophotometer from Hitachi Instruments
Inc. (San Jose, CA). Reverse phase-HPLC was
performed with an Hewlett-Packard Model 1100
HPLC system equipped with a Diode Array Detector
(Hewlett-Packard GmbH, Waldborn, Germany) and a
Waters C18 symmetric column (Millipore, Milford,
MA).
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3. Results
Table 1
Representative DepoFoam encapsulated protein and peptide
formulations a
DepoFoam formulations
Drug
(protein or
peptide)
%Yield
(recovery)
Drug
loading
(mg / ml)
Drug integrity
by HPLC b (% of
standard drug)
Insulin
Leuprolide
Enkephalin
Octreotide
7585
5060
5570
5060
850
818
2535
1626
99.8
99.1
99.5
96.7
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Fig. 2. Confocal images of double-labeled DepoInsulin particle. Confocal microscopy was performed using a BioRad MRC 1024
microscope. DepoInsulin formulation was prepared using FITC-insulin and BODIPY-labeled lipid solution, as described in Section 2. The
far left image shows the red-fluorescent lipid label, the middle image is the green-fluorescent FITC label showing the encapsulated insulin,
and the far right image shows both lipid and protein in the DepoFoam (MVL) particle, illustrating the multivesicular structure of the particle,
and the even distribution of the protein in the DepoFoam particle.
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The rate of drug release from the DepoFoam particles can also be modulated by varying the lipid
composition in the DepoFoam formulation. As
shown for DepoEnkephalin formulations in Fig. 7B,
an increase of tricaprylin:triolein (C8:C18) ratio
from 9:1 to 14:1 resulted in an increase of enkephalin release from about 5% release to 60%
release on day 3. The impact of the triglycerides on
controlling the release rates of DepoFoam-encapsulated proteins, such as GM-CSF, G-CSF and IGF-I,
has been described earlier [28].
It should be noted that the in vitro release profile
obtained may not be quantitatively predictive of the
in vivo behavior. Nevertheless, in vitro release
studies allow for screening and rank-ordering various
formulations by the rates of drug release, and
provide a basis for in vivo studies. The in vitro
release studies shown in Figs. 6 and 7 were performed after dilution of the DepoFoam formulations
in human plasma and incubation at 378C under
dynamic conditions, and therefore do not relate to
storage stability of the formulations (stored undiluted
at 48C). In Figs. 6 and 7, the release at time zero was
normalized to 100%, since there was no drug release
at time zero, and the absolute values corresponded to
the initial drug concentration.
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4. Discussion
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5. Conclusion
In conclusion, the results demonstrate that DepoFoam technology is capable of: (a) encapsulating
therapeutic proteins and peptides with high yields;
(b) accommodating high drug loading; and (c) controlling the sustained delivery of the encapsulated
drugs. In addition to proteins and peptides, the
DepoFoam system is applicable for encapsulation of
a wide range of therapeutic agents, from small
molecule drugs, one of which (cytarabine) has already been demonstrated to be clinically beneficial
[41] and approved for clinical use, to other macromolecule drugs such as nucleic acids and vaccines.
Acknowledgements
We thank Beth Gualdoni and Mario Flores of
DepoTech Corporation for the animal work, Melissa
Langston for the confocal images, Dr Jerrold M.
Olefsky at Department of Endocrinology and Metabolism, UCSD for the insulin receptor assays, and Dr
David Hess at the Oregon Regional Primate Research Center for RIA assay of testosterone.
References
[1] D.D. Lasic, Liposomes: From Physics To Applications,
Elsevier, Amsterdam, 1993.
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