Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-05-22
2004-03-30
Kunz, Gary (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S300000, C424S198100
Reexamination Certificate
active
06713450
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of amyloid &bgr; peptides and a method for inducing an immune response to amyloid &bgr; peptides and amyloid deposits.
2. Description of the Background Art
Alzheimer's disease (AD) is the most common form of late-life dementia in adults (Soto et al., 1994), constituting the fourth leading cause of death in the United States. Approximately 10% of the population over 65 years old is affected by this progressive degenerative disorder that is characterized by memory loss, confusion and a variety of cognitive disabilities. Neuropathologically, AD is characterized by four major lesions: a) intraneuronal, cytoplasmic deposits of neurofibrillary tangles (NFT), b) parenchymal amyloid deposits called neuritic plaques, c) cerebrovascular amyloidosis, and d) synaptic and neuronal loss. One of the key events in AD is the deposition of amyloid as insoluble fibrous masses (amyloidogenesis) resulting in extracellular neuritic plaques and deposits around the walls of cerebral blood vessels. The major constituent of the neuritic plaques and congophilic angiopathy is amyloid &bgr; (A&bgr;), although these deposits also contain other proteins such as glycosaminoglycans and apolipoproteins.
A&bgr; is a 4.1-4.3 kDa hydrophobic peptide that is codified in chromosome 21 as part of a much longer amyloid precursor protein APP (Muller-Hill et al., 1989). The APP starts with a leader sequence (signal peptide), followed by a cysteine-rich region, an acidic-rich domain, a protease inhibitor motif, a putative N-glycosylated region, a transmembrane domain, and finally a small cytoplasmic region. The A&bgr; sequence begins close to the membrane on the extracellular side and ends within the membrane. Two-thirds of A&bgr; faces the extracellular space, and the other third is embedded in the membrane (Kang et al., 1987 and Dyrks et al., 1988). Several lines of evidence suggest that amyloid may play a central role in the early pathogenesis of AD.
Evidence that amyloid may play an important role in the early pathogenesis of AD comes primarily from studies of individuals affected by the familial form of AD (FAD) or by Down's syndrome. Down's syndrome patients have three copies of the APP gene and develop AD neuropathology at an early age (Wisniewski et al., 1985). Genetic analysis of families with hereditary AD revealed mutations in chromosome 21, near or within the A&bgr; sequence (Forsell et al., 1995), in addition to mutations within the presenilin 1 and 2 genes. Moreover, it was reported that transgenic mice expressing high levels of human mutant APP progressively develop amyloidosis in brain (Games et al., 1995). These findings appear to implicate amyloidogenesis in the pathophysiology of AD. In addition, A&bgr; fibrils are toxic in neuronal culture (Yankner et al., 1989) and to some extent when injected into animal brains (Sigurdsson et al., 1996 and 1997).
Furthermore, several other pieces of evidence suggest that the deposition of A&bgr; is a central triggering event in the pathogenesis of AD, which leads subsequently to NFT formation and neuronal loss. The amyloid deposits in AD share a number of properties with all the other cerebral amyloidoses, such as the prion related amyloidoses, as well as the systemic amyloidoses. These characteristics are: 1) being relatively insoluble; 2) having a high &bgr;-sheet secondary structure, which is associated with a tendency to aggregate or polymerize; 3) ultrastructurally, the deposits are mainly fibrillary; 4) presence of certain amyloid-associated proteins such as amyloid P component, proteoglycans and apolipoproteins; 5) deposits show a characteristic apple-green birefringence when viewed under polarized light after Congo red staining.
The same peptide that forms amyloid deposits in AD brain was also found in a soluble form (sA&bgr;) normally circulating in the human body fluids (Seubert et al., 1992 and Shoji et al., 1992). Zlokovic et al. (1994), reported that the blood-brain barrier (BBB) has the capability to control cerebrovascular sequestration and transport of circulating sA&bgr;, and that the transport of the sA&bgr; across the BBB was significantly increased when sA&bgr; was perfused in guinea pigs as a complex with apolipoprotein J (apoJ). The sA&bgr;-apoJ complex was found in normal cerebrospinal fluid (CSF; Ghiso et al., 1994) and in vivo studies indicated that sA&bgr; is transported with apoJ as a component of the high density lipoproteins (HDL) in normal human plasma (Koudinov et al., 1994). It was also reported by Zlokovic et al. (1996), that the transport of sA&bgr; across the BBB was almost abolished when the apoJ receptor gp330 was blocked. It is believed that the conversion of sA&bgr; to insoluble fibrils is initiated by a conformational modification of the 2-3 amino acid longer soluble form. It has been suggested that the amyloid formation is a nucleation-dependent phenomena in which the initial insoluble “seed” allows the selective deposition of amyloid (Jarrett et al., 1993).
Peptides containing the sequence 1-40 or 1-42 of A&bgr; and shorter derivatives can form amyloid-like fibrils in the absence of other protein (Soto et al., 1994), suggesting that the potential to form amyloid resides mainly in the structure of A&bgr;. The relation between the primary structure of A&bgr; and its ability to form amyloid-like fibrils was analyzed by altering the sequence of the peptide. Substitution of hydrophilic residues for hydrophobic ones in the internal A&bgr; hydrophobic regions (amino acids 17-21) impaired fibril formation (Hilbich et al., 1992), suggesting that A&bgr; assembly is partially driven by hydrophobic interactions. Indeed, larger A&bgr; peptides (A&bgr;1-42/43) comprising two or three additional hydrophobic C-terminal residues are more amyloidogenic (Jarrett et al., 1993). Secondly, the conformation adopted by A&bgr; peptides is crucial in amyloid formation. A&bgr; peptides incubated at different pH, concentrations and solvents can have either a mainly &agr;-helical, random coil, or a &bgr;-sheet secondary structure (Barrow et al., 1992; Burdick et al., 1992 and Zagorski et al., 1992). The A&bgr; peptide with &agr;-helical or random coil structure aggregates slowly; A&bgr; with &bgr;-sheet conformation aggregates rapidly (Zagorski et al., 1992; Soto et al., 1995 and Soto et al., 1996). The importance of hydrophobicity and &bgr;-sheet secondary structure on amyloid formation also is suggested by comparison of the sequence of other amyloidogenic proteins.
Analysis of A&bgr; aggregation by turbidity measurements indicates that the length of the C-terminal domain of A&bgr; influences the rate of A&bgr; assembly by accelerating nucleus formation (Jarrett et al., 1993). Thus, the C-terminal domain of A&bgr; may regulate fibrillogenesis. However, in vitro modulators of A&bgr; amyloid formation, such as metal cations (Zn, Al) (Bush et al., 1994 and Exley et al., 1993) heparin sulfate proteoglycans, and apoliprotein E (Strittmatter et al., 1993) interact with the 12-28 region of A&bgr;. Moreover, mutations in the APP gene within the N-terminal A&bgr; domain yield analogs more fibrillogenic (Soto et al., 1995 and Wisniewski et al., 1991). Finally, while the C-terminal domain of A&bgr; invariably adopts a &bgr;-strand structure in aqueous solutions, environmental parameters determine the existence of alternative conformation in the A&bgr; N-terminal domain (Barrow et al., 1992; Soto et al., 1995 and Burdick et al., 1992). Therefore, the N-terminus may be a potential target site for inhibition of the initial random coil to &bgr;-sheet conformational change.
The emerging picture from studies with synthetic peptides is that A&bgr; amyloid formation is dependent on hydrophobic interactions of A&bgr; peptides adopting an antiparallel &bgr;-sheet conformation and that both the N- and C-terminal domains are important for amyloid formation. The basic unit of fibril formation appears to be the conformer adopting an antiparallel &bgr;-sheet compose
Frangione Blas
Sigurdsson Einar M.
Wisniewski Thomas
Darby & Darby
Kunz Gary
New York University
Turner Sharon L.
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