Nandini Katre

DEPOFOAM TECHNOLOGY: A VEHICLE FOR CONTROLLED DELIVERY OF PROTEIN AND PEPTIDE DRUGS

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 DepoFoam, 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.

Sustained-delivery of an apolipoproteinE–peptidomimetic using multivesicular liposomes lowers serum cholesterol levels

Abstract Receptor-mediated removal of lipoproteins by the liver is predominantly mediated by apolipoproteinE (apoE). Recent data show that a novel dual domain peptide (hE-18A) containing the 10-residue receptor-binding domain of human apoE, and a model class A amphipathic helix (18A), can associate with low density and very low density lipoproteins (LDL and VLDL) and enhance their uptake and degradation by HepG2 cells in vitro, and further, causes a dramatic reduction in plasma cholesterol levels in apoE-null mice. The in vivo cholesterol lowering effect, however, is short-lived because of rapid clearance of the peptide from the circulation. These results indicate that a therapeutic benefit may be achieved by sustained-delivery of this novel peptide as an alternate form of treatment for hypercholesterolemia and hypertriglyceridemia. In this report we describe the encapsulation of this peptide in a multivesicular liposome (MVL) depot-delivery system (DepoFoam™). An in vitro plasma release assay showed sustained release of the peptide from the DepoFoam particles and its subsequent association with LDL and VLDL. Furthermore, a single subcutaneous dose of the DepohE-18A formulation in apoE-null mice, yielded a mean, cumulative 18% decrease in serum cholesterol levels after 6 days, and the cholesterol levels remained low at 8 days. Whereas, an equivalent dose of free peptide showed maximal cholesterol decrease by 4 h, followed by a rapid decline in efficacy by 24–48 h. Fractionation of the different lipoprotein fractions from serum showed that the majority of the serum cholesterol decrease was associated with the VLDL fraction, followed by LDL. These results indicate that the apoE peptidomimetic encapsulated in DepoFoam has potential as an alternative therapeutic treatment of hyperlipidemia.

Modulation of the sustained delivery of myelopoietin (Leridistim) encapsulated in multivesicular liposomes (DepoFoam)

Myelopoietins (MPO) are novel chimeric growth factors containing IL-3 and G-CSF receptor agonists that enhance the biological properties of both cytokines. These cytokines, like many therapeutic proteins, clear rapidly from circulation and must be administered daily to provide efficacy. Therefore, a controlled and sustained delivery system comprised of a biocompatible and biodegradable matrix, would offer important therapeutic advantages in the clinic, such as significantly reducing dose frequency and providing efficacy without toxicity. We report here the encapsulation of Leridistim (a protein from the MPO family) in multivesicular liposomes (DepoFoam) for sustained delivery, and demonstrate that a single injection of DepoFoam-encapsulated Leridistim results in elevated neutrophil counts for 10 days, in contrast to only 2 days for un-encapsulated Leridistim. Moreover, varying the lipid content of the DepoFoam matrix modulated the duration of elevated neutrophils from 2-3 to 9-10 days. The encapsulated Leridistim was released in vivo from the multivesicular liposomes in a uniform manner, consistent with its pharmacodynamic duration. Finally, a reproducible pharmacodynamic effect was observed with several batches of a DepoLeridistim formulation, indicating consistency of the manufacturing process of the DepoFoam delivery system. The capability of altering the release rates by varying the lipid composition provides maximum flexibility for controlled delivery of cytokine therapeutics.

The sustained granulopoietic effect of progenipoietin encapsulated in multivesicular liposomes

Progenipoietin (ProGP), a dual receptor agonist of fetal liver tyrosine kinase-3 (flt3) and granulocyte colony-stimulating factor (G-CSF) receptors, has been shown to significantly enhance production of both polymorphonuclear leukocytes and dendritic cells (DCs) in the peripheral blood and spleen of mice, when administered as daily s.c. injections for about 10 days. Here, we have successfully designed a sustained-delivery formulation for this novel chimeric protein using multivesicular liposomes (DepoFoam), and studied the effects of changing both the triglyceride and phospholipid composition of the lipid matrix to modulate its delivery profile. Encapsulation of ProGP in these particles led to retention of its structural integrity, and maintenance of its biological activity in vivo. Administration of a single s.c. dose of 1mg/kg of an optimized DepoProGP formulation in rats, led to significant elevation of absolute neutrophil counts (ANC) that were maintained at levels >10,000 microliter(-1) for 9-11 days, in a reproducible manner. In contrast, administration of the unencapsulated ProGP at the same dose, resulted in elevation of neutrophils by day 1, followed by a quick decline to base line levels by day 3. These data suggest the possibility of administering a single dose of DepoFoam-encapsulated ProGP to improve hematopoietic recovery time after chemotherapy, and for other indications that require multiple daily doses of ProGP.

Sustained Delivery of an ApolipoproteinE Peptidomimetic Lowers Serum Cholesterol Levels in Dyslipidemic Mice

Receptor-mediated removal of LPs by the liver is predominantly mediated by apolipoproteinE. Recent data show that a novel dual domain peptide (hE-18A) containing the 10-residue receptor-binding domain of human apoE, and a model class A amphipathic helix (18A), can associate with low density and very low density lipoproteins (LDL, VLDL) and enhance their uptake and degradation by HepG2 cells; and further, causes a dramatic reduction in plasma cholesterol levels in apoE-null mice [1,2]. These results provide the basis for developing sustained-release vehicles for this novel peptide as an alternate form of treatment for hypercholesterolemia and hypertriglyceridemia. We encapsulated this peptide into a multivesicular liposome (MVL) depot-delivery system (DepoFoam™) [3], and evaluated its sustained-release properties in vitro and its pharmacodynamic effects in a dyslipidemic mouse model.