Materials and methods for making improved micelle compositions

Details Materials and methods for making improved micelle compositions Materials and methods for making improved micelle compositions

2000-05-18

2001-11-27

Kishore, Gollamudi S. (Department: 1615)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C424S001210, C424S009321, C424S009510, C424S094300, C424S812000, C436S829000, C514S021800, C514S937000, C428S402200

Reexamination Certificate

active

06322810

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to biologically active compounds and more specifically to compounds and peptides which are amphipathic, i.e., have both hydrophilic and hydrophobic portions. Specifically, the invention relates to improved methods for the delivery and presentation of amphipathic peptides in association with micelles diagnostic, therapeutic, cosmetic and organ, tissue and cell preservative uses.
Of particular interest to the present invention are the biologically active amphipathic peptides which are members of the family of peptide compounds including vasoactive intestinal peptide (VIP), growth hormone releasing factor (GRF), peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), pituitary adenylate cyclase activating peptide (PACAP), gastric inhibitory hormone (GIP), hemodermin, the growth hormone releasing hormone (GHRH), sauvagine and utotensin I, secretin and glucagon. More specifically, the invention relates to improved therapeutic methods for delivering peptides in the VIP/GRF family of peptides to targeted tissues through use of improved micelle compositions comprising a member of the VIP/GRF family of peptides and biologically active analogues thereof.
VIP is a 28-amino acid neuropeptide which is known to display a broad profile of biological actions and to activate multiple signal transducing pathways. See, Said,
Peptides
5 (Suppl. 1):149-150 (1984) and Paul and Ebadi,
Neurochem. Int
. 23:197-214 (1993). A Schiff-Edmundson projection of VIP as a &pgr;-helix reveals segregation of apolar and polar residues onto the opposite faces of the helix and that this amphipathic character is also evident when VIP is modeled as a distorted &agr;-helix, which is reported in Musso, et al.,
Biochemistry
27:8147-8181 (1988). A correlation between the helix-forming tendency of VIP analogues and their biological activity is described in Bodanszky et al.,
Bioorgan. Chem
. 3:133-140 (1974). In pure water, the spectral characteristics of VIP are consistent with those of a random coil. However, organic solvents and anionic lipids induce helical-information in the molecule. See, Robinson et al.,
Biopolymers
21:1217-1228 (1983); Hamed, et al.,
Biopolymers
22:1003-1021 (1983); and Bodanszky, et al.,
Bioorganic Chem
. 3:133-140 (1974).
Short peptides capable of forming amphipathic helices are known to bind and penetrate lipid bilayers. See, Kaiser and Kezdy,
Ann. Rev. Biophys. Biophysical Chem
. 15:561-581 (1987) and Sansom,
Prog. Biophys. Molec. Biol
. 55:139-235 (1991). Examples include model peptides like (LKKLLKL−), which are disclosed in DeGrado and Lear,
J. Am. Chem. Soc
. 107:7684-7689 (1985), and the 26-residue bee venom peptide, melittin, disclosed in Watata and Gwozdzinski,
Chem
-
Biol. Interactions
82:135-149 (1992). Possible mechanisms for the binding include alignment of peptide monomers parallel to the surface of the bilayer mediated by electrostatic interactions between polar amino, acids and phospholipid head groups, and insertion of peptide aggregates into the apolar bilayer core, stabilized in part, by the hydrophobic effect. See, Sansom,
Prog. Biophys. Molec. Biol
. 55:139-235 (1991).
VIP belongs to a family of homologous peptides, other members of which include peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), growth hormone releasing factor (GRF), pituitary adenylate cyclase activating peptide (PACAP), gastric inhibitory hormone (GIP), hemodermin, the growth hormone releasing hormone (GHRH), sauvagine and utotensin I, secretin and glucagon. Like VIP, the other members of the VIP/GRF family of peptides, and biologically active analogues thereof, can form amphipathic helices capable of binding lipid bilayers. The biological action of members of the VIP/GRF family of peptides are believed to be mediated by protein receptors expressed on the cell surface and intracellular receptors and it has recently been demonstrated that calmodulin is likely to be the intracellular receptor for VIP [Staliwood, et al.,
J. Bio. Chem
. 267:19617-19621 (1992); and Stallwood, et al.,
FASEB J
. 7:1054 (1993)].
Bodanszky et al.,
Bioorgan. Chem
. 3:133-140 (1974) were the first to study the conformation of VIP through optical rotary dispersion and circular dichroism spectrum. They showed structural differences in VIP, depending on the hydrophobicity of the solvent in which VIP was dissolved. The VIP-in-water spectrum revealed a mostly random coil structure. (about 80%). However, addition of organic solvents, such as tifluoroethanol (TFE) or methanol, even at low concentration induced a pronounced shift to a helical structure. The authors suggested that these effects of the organic solvents on the structure of the peptide would coincide with receptor conditions, and therefore, the helical conformation of VIP would correspond to an “active architecture” required for its biological activity. These early studies were in agreement with the findings of Robinson et al.,
Biopolymers
21:1217-1228 (1982), who analyzed the conformation of VIP, and two of its family members, secretin and glucagon, in water, anionic detergents, and anionic lipids (PA and phosphatidylglycerol (PG)). They showed an increase in the helix formation probability by arginyl, histidyl, and lysil residues, corresponding in all three peptides to their 13-20 amino acid region. A predominantly disordered structure was again observed for VIP in aqueous solvents, and zwitterionic lipids, suggesting that the charge of the polar head group plays an important role in helix formation. Using circular dichroism (CD) spectra studies with 40% HFIP/H
2
O mixture and
1
H-NMR studies Fournier et al.,
Peptides
5:160-177 (1984), showed that the 15-28 portion of the VIP segment forms an a-helix in the presence of organic solvent. A complete structural study of the native VIP in 40% TFE was performed by Theriault et al.,
Biopolymers
31:459-464 (1991) using two-dimensional
1
H-NMR spectroscopy. Their results were similar to the ones obtained by Fry et al.,
Biochemistry
28:2399-2409 (1989) who investigated VIP in 25% methanol/water. They described two helical segments between the amino acids 7-15 and 19-27 linked by a random coil peptide chain portion that granted mobility to the molecule.
Finally several groups worked on the development of more potent analogs of VIP as potential therapeutic agents, since the native peptide had a very low bioavailability. Interestingly, all of them modified the sequence of VIP to enhance its helicity and amphiphilicity. VIP structure-activity relationship were studied extensively by Bolin and his collaborators (Fry et al.,
Biochemistry
28:2399-2409 (1989); Bolin et al.,
Biopolymers
37:57-66 (1995). Among their results, the enhancement of the helical structure by specific substitutions of amino acid residues was proportionally related to an increase in potency, and the pharmacoactive functional group of the VIP was found to consist of multiple binding sites throughout the entire peptide sequence. Helix based analogs of VIP were also developed by Musso et al.,
Biochemistry
27:8174-8181 (1988) that showed greater interactions with receptors. Stearyl-Norleucine-VIP analog that has a 100-fold greater potency was designed by Gozes et al.,
Endocrinology
134:2121-2125 (1994), for noninvasive impotence treatment and neurodegenerative diseases Gozes et al.
J. Pharmacol. Exp. Ther
. 273:161-167 (1996). The addition of fatty acid moiety and the amino acid substitutions increased the lipophilicity of the peptide, which was believed to improve biological membrane penetration.
In summary, VIP has been shown to adopt a helical conformation in hydrophobic environments, provided by organic solvent, and the helical structure of the VIP increases with an increase in the hydrophobicity of the environment. This helical motif found in the central part of the peptide, which is rich in basic, hydrophobic residues, forms an amphiphilic structure that may facilitate the binding to receptors and promote direct

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