Transcytosis vehicles and enchancers for drug delivery

Details Transcytosis vehicles and enchancers for drug delivery Transcytosis vehicles and enchancers for drug delivery

1998-06-25

2001-03-20

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Animal cell, per se, expressing immunoglobulin, antibody, or...

C435S371000, C424S143100, C424S158100, C424S447000

Reexamination Certificate

active

06204054

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to drug delivery. In particular, the invention relates to transcytosis vehicles and enhancers capable of delivering and enhancing passage of drugs across endothelia, epithelia and mesothelia containing the GP60 receptor.
BACKGROUND OF THE INVENTION
For most therapeutic drugs administered by intra-arterial or intravenous routes the intended site of molecular activity lies outside the vasculature. For drugs administered via the airways, the intended site of activity normally is beyond the first cellular barrier of alveolar, bronchiolar or tracheal epithelia. In both cases, there is an endothelial or epithelial barrier which must be crossed before the drug can mediate its effect.
For small lipophilic drugs, there appears to be a paracellular route between the tight junctions of the barrier cells. However, for hydrophilic drugs and larger macromolecular active agents, such as peptides, proteins, genes or anti-sense nucleotides, the only route across the barrier is through the cells. This poses a particular problem for drugs administered intravenously which have exceedingly short half-lives due to rapid degradation or first pass clearance by the liver. In order to maintain therapeutic levels in balance with such excretion and degradation, large doses or infusions are often necessary. Thus, there is clearly a need in the art for more rapid mechanisms for delivering drugs across cellular barriers.
There have been numerous reports of specific receptors which mediate endocytotic events, where a ligand binds to the receptor and is then internalized, complexed to the receptor, by a process similar to pinocytosis. This involves invagination of the cell membrane in the region of the ligand receptor complex and then release of the ligand into the cell by a process which is not fully understood. Numerous endocytotic receptor systems have been reported including LDL, insulin, epidermal growth factor, insulin-like growth factor and tPA-PAI-I (hybrid molecule).
Transcytosis entails invagination and vesicle formation around a ligand receptor complex, followed by transcytotic passage with release by a reverse invagination process at the basolateral membrane. Monoclonal antibodies to the transferrin receptor have been conjugated with toxins, so that they can undergo transcytosis, across blood-brain endothelia. However, there is a continuing need in the art for agents capable of delivering or enhancing passage of drugs by receptor-mediated transcytosis across cellular barriers other than blood-brain endothelia, such as endothelia of the vasculature, alveolar epithelia, and peritoneal mesothelia.
The GP60 receptor, also referred to as albondin, is one of several albumin-binding proteins reported in the literature (Schnitzer and Oh, J. Biol. Chem. 269(8):6072-6082 (1994)). Others include SPARC (serum protein, acidic, rich in cysteine), oesteonectin or basement membrane protein 40, GP30, GP18 and GP60. SPARC and oesteonectin are extra-cellular proteins. GP60 shares some homology with SPARC as determined using anti-SPARC antibodies (Schnitzer and Oh, Am. J. Physiol. 263:H1872-H1879 (1992)).
GP18 and GP30 are membrane glycoproteins found in a variety of cell types but are particularly prevalent in the macrophage (Schnitzer et al, J. Biol. Chem. 267: 24544-24553 (1992)). GP18 and GP30 are the so-called “scavenger receptors” responsible for mediating removal of oxidized, glycated or adduced forms of albumin by endocytosis and are thus believed to play a role in albumin catabolism for a wide variety of organs (Schnitzer and Bravo, J. Biol. Chem. 268(10):7562-7570 (1993)).
In contrast to GP18 and GP30, the GP60 receptor has found to be expressed exclusively in continuous endothelia of the vasculature (Schnitzer, Am. J. Physiol. 262:H246-H254 (1992)), in alveolar epithelia (Kim et al, Am. J. Resp. and Crit. Care Med. 151:A190, (1994) and inferentially in peritoneal mesothelia (Gotloib and Shostak, Kidney International. 47:1274-1284 (1995)). GP60 is particularly abundant in the microvessel endothelia and is, interestingly, absent from the blood-brain barrier, where little albumin flux is observed (Rousseaux et al, Methods in Enzymology 121:163 (1986)). It has been shown that polyclonal antibodies to endothelial GP60 also bind alveolar epithelial GP60 (Kim et al, supra). The GP60 receptor has been implicated in receptor-mediated transcytosis of albumin across epithelia and endothelial cell barriers (Kim et al, supra; Tirrupathi et al, Molecular Biology of the Cell 4 (Supp):338a, Abstract No. 1964 (1993)).
The GP60 amino acid sequence is known in the art (Yamauchi et al, Biochem. Biophys. Res. Comm. 146:1485 (1987)).
SUMMARY OF THE INVENTION
The present invention provides transcytosis vehicles and enhancers capable of transporting physiologically-active agents across epithelia, endothelia and mesothelia containing the GP60 receptor. The GP60 receptor has been implicated in receptor-mediated transcytosis of albumin across cell barriers. By means of the invention, GP60 receptor-mediated transcytosis can be exploited for the transport of not only albumin, but also physiologically-active agents which do not naturally pass through epithelia, endothelia and mesothelia via the GP60 system.
Transcytosis vehicles and enhancers of the invention include albumin, albumin fragments, anti-GP60 polyclonal and monoclonal antibodies, anti-GP60 polyclonal and monoclonal antibody fragments, and GP60 peptide fragments. Further, they include PDI (protein disulphide isomerase) and fragments thereof (any subsequent reference to GP60 fragments may be interpreted as referring also to PDI fragments). A common factor may be a CGMC motif found in PDI and at least the T
1-44
fragment of GP60. If the transcytosis vehicle or enhancer is a GP60 peptide fragment, it is preferably co-administered with other transcytosis vehicles or enhancers of the present invention such as albumin or an albumin fragment. Suitable albumin fragments of 14, 20 and 32 kDa can be generated by cleavage at methionine residues using cyanogen bromide and can be further reduced in size by reduction of disulfide bridges. Anti-GP60 polyclonal and monoclonal antibody fragments useful as transcytosis vehicles and enhancers according to the present invention include Fab, Fab′, F(ab′)
2
, and Fv fragments. Preferred GP60 peptide fragments include the T3118 peptide which corresponds to the N-terminal 18 amino acids of the GP60 protein.
In accordance with the invention, when the above compounds are conjugated to a physiologically-active agent, they are referred to herein as “transcytosis vehicles”. When co-administered with but not conjugated to a physiologically-active agent, the above compounds are referred to herein as “transcytosis enhancers”. In preferred embodiments, the transcytosis vehicles and enhancers of the present invention are useful for delivering or enhancing passage of physiologically-active agents across endothelia of the vasculature, alveolar epithelia and peritoneal mesothelia.
DETAILED DESCRIPTION OF THE INVENTION
As its name indicates, the GP60 protein has been reported in the art as having a molecular weight of about 60 kDa. After a more careful analysis, it has been discovered that the “true” molecular weight for this protein is more probably about 57 kDa. This discrepancy in molecular weight is thought to be due to differences in protein preparation and gel conditions. However, to be consistent with the art, this protein is referred to herein (with the exception of Example 1 below) as the GP60 receptor.
It has been discovered that GP60 receptor-mediated transcytosis can be exploited for the transport of not only albumin, but also for a vast number of therapeutically-important physiologically-active agents which do not naturally pass through epithelia, endothelia and mesothelia via the GP60 system. Thus, the present invention provides an improved method for transporting physiologically-active e.g. those having relatively high molecular weights, e.g. 50, 100, 150 kDa or more, across the cellular b

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