pH-sensitive, serum-stable liposomes

Details pH-sensitive, serum-stable liposomes pH-sensitive, serum-stable liposomes

1999-02-02

2002-07-30

Kishore, Gollamudi S. (Department: 1615)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C424S001210, C424S009321, C424S009510, C424S094300, C428S402200

Reexamination Certificate

active

06426086

ABSTRACT:

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
This invention relates to the field of liposomes. In particular, this invention provides novel liposomes that are pH-sensitive, yet are also stable in serum. The invention further relates to methods of conferring pH sensitivity upon liposomes of varying compositions.
BACKGROUND OF THE INVENTION
A number of pharmaceutical agents and potential pharmaceutical agents suffer from poor aqueous solubility, high levels of antigenicity, toxicity, or rapid degradation in serum, which can hamper the development of suitable clinical formulations. One solution to these problems has been to encapsulate the pharmaceutical agent in a delivery vehicle that is soluble in aqueous solutions and that shields the agent from direct contact with tissues and blood. In particular, formulations based on liposome technology are of significant interest. Liposomes are vesicles comprised of concentrically ordered phopholipid bilayers which encapsulate an aqueous phase. They form spontaneously by hydrophobic interactions when phospholipids are exposed to aqueous solutions and can accommodate a variety of bioactive molecules.
Liposomes have proved a valuable tool as an in vivo delivery system for enhancing the efficacy of various pharmacologically active molecules (Ostro et al.,
Liposomes.from Biophysics to Therapeutics,
Dekker, New York, pp. 1-369 (1987)). Animal studies have shown that liposomes can decrease the toxicity of several antitumor and antifungal drugs, leading to clinical trials with promising results (Sculier et al.,
Eur. Cancer Clin. Oncol.,
24: 527-538; Gabizon, et al.,
Eur. J. Cancer Clin. Oncol.,
25: 1795-1803 (1989); Treat et al.,
J. Nati. Cancer Inst.,
82: 1706-1710 (1990); Lopez-Berestein et al.,
J. Infect. Dis.,
151: 704-710 (1985); Present et al.,
Cancer,
62: 905-911 (1988)). In addition, liposomes have been shown to be efficient carriers of antiparasitic drugs for treating intracellular infections of the reticuloendothelial system (RES), in activating macrophage cells to become tumoricidal, in models of metastasis, and in enhancing the immune response to encapsulated antigens, thus facilitating the formulation of artificial vaccines (
Liposomes in the Therapy of Infectious Diseases and Cancer,
Lopez-Berestein & Fidler, eds. Liss, New York (1989); Alving et al.
Immunol. Lett.,
25: 275-280 (1990))
Numerous studies have reported on efforts to develop pH-sensitive liposomes as drug delivery systems (Collins, D. in:
Liposomes as Tools in Basic Research and Industry
(Philippot, J. R. and Schuber, F., Eds.) pp. 201-214, CRC Press, Boca Raton, Fla. (1995) (hereafter, “Collins 1995”); see also, Liu, D. et al.,
Biochim. Biophys. Acta
1022:348-354 (1990); Slepushkin, V. A. et al.,
J. Biol. Chem.
272:2382-2388 (1997)). Since liposomes are internalized by cells mainly via the endocytic pathway (Straubinger, R. M. et al.,
Cell
32:1069-1079 (1983)), whereby the liposomes are internalized and exposed to the lowered pH of an endosome, pH-sensitization of liposomes is an attractive strategy to facilitate the delivery of membrane impermeable drugs to the cytoplasm before lysosomal enzymatic degradation occurs. Unsaturated phosphatidylethanolamine (“PE”) has been widely employed to confer intrinsic pH-sensitivity to liposomes.
At physiological pH in isotonic buffer, the equilibrium phase of unsaturated PE is the inverted hexagonal (Hi
II
) phase (Cullis et al.,
Biochim. Biophys. Acta
559, 399-420 (1979); Tilcock, C. P. S et al.,
Biochim. Biophys. Acta
684,212-218 (1982); Allen et al.,
Biochemistry
29, 2976-2985 (1990)). Under these conditions, PE is protonated and unable to form bilayer (L
a
) vesicles (Papahadjopoulos et al.,
Biochim. Biophys. Acta
135, 624-638 (1967)). The bilayer phase of unsaturated PE can, however, be stabilized by weakly acidic amphiphiles such as oleic acid (OA) (Straubinger et al.,
FEBS Lett.
179, 148-154 (1985)) or cholesterylhemisuccinate (CHEMS) (Ellens et al.,
Biochemistry
23, 1532-1538 (1984)), which confer a negative charge headgroup at pH 7.4. This charge provides electrostatic repulsion to block PE intermolecular interaction/interbilayer contact, thus preventing H
II
phase formation under physiological conditions. Protonation of the amphipile headgroup caused by a reduction of pH, neutralizes the negative charge and the vesicles become destabilized as the PE component reverts to the H
II
phase (Litzinger et al.,
Biochim. Biophys. Acta
11 13, 201-227 (1992). This is generally accompanied by the release of liposomal contents.
Although such liposomes have been shown to be efficient systems for cytoplasmic delivery in cultured cells (Collins 1995, supra), their moderate stability as well as their rapid clearance from the blood have hampered their in vivo use. Despite the fact that small unilamellar PE vesicles (SUV) have been found to be stable in plasma (Liu, et al.,
Biochemistry
28:7700-7707 (1989); Liu, et al.,
Biochemistry
29:3637-3643 (1990)), the extraction of the acidic amphipile by the plasmatic albumin results in the rapid loss of the pH-sensitivity. Leventis et al. (
Biochemistry
26, 3267 (1987); 3276) and Collins et al. (
Biochim. Biophys. Acta
1025, 234-242 (1990) (hereafter “Collins 1990”)) demonstrated that the loss of the pH-sensitive moiety can be slowed down by using titratable double-chain amphiphiles such as 1,2-dipalmitoyl-sn-3-succinylglycerol (1,2-DPSG). It was found that although small dioleoylphosphatidylethanolamine (DOPE) liposomes containing 1,2-DPSG maintained their pH-sensitivity after incubation in plasma, there was a substantial shift of the destabilization pH from 5.3 to 4.2 (Collins 1990). Furthermore, after systemic administration, PE liposomes are rapidly cleared from the blood and accumulate in the lung, liver and spleen (Connor et al.,
Biochim. Biophys. Acta
884:474-481 (1986).
Colloidal stabilization of liposomes can be improved by inclusion of ganglioside (GM
1
) or poly(ethylene glycol)-derivatized lipids (PEG-PE) (Papahadjopoulos, D. et al., in:
Liposomes as Tools in Basic Research and Industry
(Philippot, J. R. and Schuber, F., Eds.) pp. 177-188, CRC Press, Boca Raton, Fla. (1995)) (hereafter, “Papahadjopoulos, 1995”). These so-called “sterically stabilized liposomes” (“SSL,”or Stealth® liposomes) have shown long circulation half-lives, reduced uptake by the mononuclear phagocyte system and accumulation in tumors (Papahadjopoulos, 1995;
Woodle, M. C. et al.,
Biochim. Biophys. Acata
1113:171-199 (1992); Woodle, U.S. Pat. No. 5,356,633). Such coating of PE-based pH-sensitive liposomes increases their stability and circulation time in blood but simultaneously reduces their pH-sensitivity (Liu, D. et al.,
Biochim. Biophys. Acta
1022:348-354 (1990); Slepushkin, V. A. et al.,
J. Biol. Chem.
272:2382-2388 (1997)). To circumvent this drawback, the use of cleavable PEG-coating has recently been proposed (Kirpotin, D. et al.,
FEBS Lett.
388:115‥118 (1997)).
Acid-triggered liposomes destabilization/fusion can be achieved extrinsically by using non-peptidic titratable synthetic polymers (Tirell, D.A. et al.,
Ann. N. Y. Acad. Sci.
446:237-248 (1985); Kono, K. et al.,
Biochim. Biophys. Acta -b
1193
:
1
-
9
(1994)). The advantage of this approach is the potentiality to render different lipid-based formulations sensitive to pH, without the limitations associated with PE-based liposomes. Although fusogenic peptides can also trigger membrane disruption at acidic pH and have been successfully used to enhance the transfection efficiency of plasmid DNA (Wagner, E. et al.,
Proc. Natl. Acad. Sci. USA
89:7934-7938 (1992)), they display several disadvantages in the development of pH-sensitive liposomes including high cost of production, immunogenicity and non-trivial association to the liposome surface.
Several recent studies have shown that liposomes coated with copolymers of N-isopropylacrylamide (NIPA) bearing alkyl chains, acquire thermo-responsive properties

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