Reversible aqueous pH sensitive lipidizing reagents,...

Details Reversible aqueous pH sensitive lipidizing reagents,... Reversible aqueous pH sensitive lipidizing reagents,...

1999-12-09

2003-07-08

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S328000, C530S214000, C530S345000, C530S303000, C514S002600, C514S003100, C549S231000, C549S262000, C435S145000, C552S001000, C562S553000

Reexamination Certificate

active

06590071

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of biology and medicine. More particularly, the present invention is directed to compounds, methods and compositions useful in increasing in mammals the transport and delivery of hydrophilic molecules having an amino group, in particular peptides and proteins.
2. Related Art
Advances in biochemistry have made possible the production of large amounts of therapeutically active and pure proteins and peptides. Currently, the therapeutic effects of most of these agents can be achieved only when they are administered via invasive routes, such as by injection. Since most proteins have very short half lives, effective concentrations of these agents can be maintained only when administered by frequent injections.
Although the administration of protein by injection is the most effective means of their delivery in vivo, patient tolerance of multiple injections is very poor. In addition, drug injection requires training and skill that may not always be transferable to patients. In cases where protein drugs have a life-saving role, the administration by injection can be acceptable by the patients. However, in cases where protein drugs are just one of several possible therapies, injections of proteins and peptides are unlikely to be accepted by the patients. Therefore, alternative routes of protein and peptide delivery need to be developed.
Such alternative routes may include the buccal, nasal, oral, pulmonary, rectal and ocular routes. Without exception, these routes are less effective than the parenteral routes of administration, but are still far more attractive than the parenteral routes because they offer convenience and control to the patients. The oral route is particularly attractive because it is the most convenient and patient-compliant.
Mucosal barriers, which separate the inside of the body from the outside (e.g., gastrointestinal, ocular, pulmonary, rectal and nasal mucosa), comprise a layer of tightly joined cell monolayers which strictly regulate the transport of molecules. Individual cells in barriers are joined by tight junctions which regulate entry into the intercellular space. Hence, the mucosa is at the first level a physical barrier, transport through which depends on either the transcellular or the paracellular pathways (Lee, V. H. L.,
Critical Rev. Ther. Drug Delivery Sys
. 5:69-97 (1988)).
Paracellular transport through water filled tight junctions is restricted to small molecules (MW<1 kDa) and is essentially a diffusion process driven by a concentration gradient across the mucosa (Lee, V. H. L.,
Critical Rev. Ther. Drug Delivery Sys
. 5:69-97 (1988); Artursson, P. and Magnusson, C., J. Pharm. Sci. 79:595-600 (1990)). The tight junctions comprise less than 0.5% of the total surface area of the mucosa (Gonzalez-Mariscal, L. M., et al.,
J. Membrane Biol
. 86:113-125 (1985); Vetvicka, V. and Lubor, F.,
Critical Rev. Ther. Drug Deliv. Sys
. 5:141-170 (1988)); therefore, they play only a minor role in the transport of protein drugs across the mucosa.
The transcellular transport of small drugs occurs efficiently provided the physicochemical properties of the drug are suited to transport across hydrophobic cell barriers. However, the transcellular transport of proteins and peptides is restricted to the process of transcytosis (Shen, W. C., et al.,
Adv. Drug. Deliv. Rev
. 8:93-113 (1992)). Transcytosis is a complex process in which proteins and peptides are taken up into vesicles from one side of a cell, and are subsequently shuttled through the cell to the other side of the cell, where they are discharged from the endocytic vesicles (Mostov, K. E. and Semister, N. E.,
Cell
43:389-390 (1985)). The cell membrane of mucosa barriers is a hydrophobic lipid bilayer which has no affinity for hydrophilic, charged macromolecules like proteins and peptides. In addition, mucosa cells may secrete mucin which can act as a barrier to the transport of many macromolecules (Edwards, P.,
British Med. Bull
. 34:55-56 (1978)). Therefore, unless specific transport mechanisms exist for proteins and peptides, their inherent transport across mucosa barriers is almost negligible.
In addition to providing a tight physical barrier to the transport of proteins and peptides, mucosa barriers possesses enzymes which can degrade proteins and peptides before, after, and during their passage across the mucosa. This barrier is referred to as the enzymatic barrier. The enzymatic barrier consists of endo- and exopeptidase enzymes which cleave proteins and peptides at their terminals or within their structure. Enzymatic activity of several mucosa have been studied and the results demonstrated that substantial protease activity exists in the homogenate of buccal, nasal, rectal and vaginal mucosa of albino rabbits and that these activities are comparable to those present in the ilium (Lee, V. H. L.,
Critical Rev. Ther. Drug Delivery Sys
. 5:69-97 (1988)). Therefore, regardless of the mucosa being considered, the enzymatic barrier present will feature strongly in the degradation of the protein and peptide molecules.
The N and the C termini of peptides are charged and the presence of charged side chains imparts highly hydrophilic characteristics on these macromolecules. In addition, the presence of charged side chains means that proteins and peptides have strong hydrogen bonding capacities; this H-bonding capacity has been demonstrated to play a major role in inhibiting the transport of even small peptides across cell membranes (Conradi, R. A., et al.,
Pharm. Res
. 8:1453-1460 (1991)). Therefore, the size and the hydrophilic nature of proteins and peptides combine to severely restrict their transport across mucosa barriers.
One approach that has been used to alter the physical nature of the mucosa barriers is the use of penetration enhancers. The use of penetration enhancers is based on the disruption of the cell barriers by low molecular weight agents which can fluidize cell membranes (Kaji, H., et al.,
Life Sci
. 37:523-530 (1985)), open tight junctions (Inagaki, M., et al.,
Rhinology
23:213-221 (1985)), and create pores in the cell membrane (Gordon, S., et al.,
Proc. Natl. Acad. Sci. USA
82:7419-7423 (1985); Lee, V. H. L., et al.,
Crit. Rev. Ther. Drug. Carrier Syst
. 8:91-192 (1991)). The use of these agents leads to a non-specific loss of barrier integrity and can lead to the absorption of a variety of large molecules which can be toxic to cells in vivo.
Protease inhibitors have been co-administered with proteins and peptides and have shown some limited activity in enhancing the absorption of these macromolecules in vivo (Kidron, M., et al.,
Life Sci
. 31:2837-2841 (1982); Takaroi, K., et al.,
Biochem. Biophys. Res. Comm
. 137:682-687 (1986)). The safety and the long-term effects of this approach have yet to be thoroughly investigated.
The prodrug approach is based on the modification of peptides in a manner that will protect them from enzyme degradation and recognition. This has been achieved by the blockage of vulnerable groups on peptides by amidation and acylation. The prodrug approach has thus far proven useful only for small peptides which have easily identifiable domains of activity.
Reduction in size is another feasible approach to increasing the transport potential of proteins. However, the active sites of proteins need to be mapped before size reduction can be attempted. In general, this approach is difficult to apply to the majority of proteins.
Carrier ligands, by virtue of their properties, can alter the cell uptake and transport characteristics of proteins and peptides. The essence of this approach is that a cell-impermeant protein or peptide is covalently attached to a carrier which is highly transported into cells. The mechanisms through which carrier ligands became endocytosed and transcytosed are important in deciding the suitability of the carrier for enhancing the transport of proteins and peptides. Macromolecular carriers are hydrophilic and do not partition i

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