Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Nitrogen containing other than solely as a nitrogen in an...
Reexamination Certificate
2001-09-06
2004-09-21
Padmanabhan, Sreeni (Department: 1617)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Nitrogen containing other than solely as a nitrogen in an...
C514S506000, C514S513000, C514S557000, C514S561000, C514S579000, C514S592000, C514S603000, C424S630000
Reexamination Certificate
active
06794414
ABSTRACT:
FIELD OF INVENTION
The present invention is directed to methods and compositions for treating diseases mediated by transglutaminase activity, by inhibiting the activity of transglutaminase.
BACKGROUND OF THE INVENTION
Protein cross-linking resulting in the formation of aggregates is a common feature of a number of neurodegenerative diseases, including Alzheimer's disease and the family of diseases exemplified by Huntington's Disease, caused by expansion of CAG trinucleotides encoding polyglutamine (Green, 1993; Prosiner et al, 1983; Davies et al, 1997; Scherzinger et al, 1997; DiFiglia et al, 1997).
Several neurodegenerative diseases, including Huntington's Disease, spinobulbar atrophy (Kennedy's disease), various spinocerebellar ataxias (SCA 1, 2, 3, 6, 7), and dentatorubralpallidoluysian atrophy (DRPLA), involve proteins with long stretches of polyglutamines in their N-terminus (Ross, 1995). Cross-linking of these polyglutamine containing proteins may be critical in the neurologic dysfunction and pathology characteristic of these disorders (Ross, 1995). Recently, nuclear inclusions containing ubiquinated aggregates of huntingtin (htt), DRPLA protein, ataxin 1 and ataxin 3, respectively, have been observed in the affected brain areas of patients with Huntington's Disease, DRPLA, SCA-1 and SCA-3 (DiFiglia et al, 1997; Igarashi et al, 1998; Skinner et al, 1997; Paulson et al, 1997). Interestingly, both htt and ataxin 3 are primarily cytoplasmic proteins in healthy individuals.
In Huntington's Disease, as well as in spinobulbar atrophy, various spinocerebellar ataxias (SCA 1, 2, 3, 6, 7) and dentatorubralpallidoluysian atrophy, the gene encoding the mutant protein contains expanding trinucleotide repeats of the codon CAG. These repeats encode glutamine (Q). With each ensuing generation, because of the expansion of these repeats, disease onset is earlier, a phenomenon known as genetic anticipation. There is no genetic anticipation, however, when the disease is transmitted through the female line in Huntington's Disease. The importance of the polyglutamine domain is further emphasized by the observation that CAG repeats, ectopically introduced into an unrelated gene encoding hypoxanthine phosphoribosyltransferase (hrpt), produce a phenotype similar to that seen in the human neurologic disorders related to abnormal polyglutamine domains (Ordway et al, 1997). The length of the polyglutamine domain is absolutely critical for the appearance of Huntington's Disease, as well as the other neurologic diseases involving mutations in genes involving expansion of CAG repeats. In Huntington's disease, for example, if the polyglutamine domain exceeds 36 Q repeats, the fatal neurologic disease ensues. In other CAG trinucleotide repeat diseases, there is a pathologic threshold, although the length varies from disease to disease, with the shortest threshold (21Q) in SCA-6, and longer thresholds in SCA-3 (61Q) and dentatorubralpallidoluysian atrophy (49Q) (Lunkes et al, 1997).
Huntingtin is expressed at similar levels in patients with Huntington's Disease and controls, regardless of the number of glutamine repeats. Huntingtin is also expressed throughout all tissues of the body and is expressed in equal amounts in all regions of the normal brain. In affected areas of Huntington's disease brain, mutant huntingtin is much less abundant than wild-type huntingtin (Schilling et al, 1995; Trottier et al, 1995; Strong et al, 1993). Although the Huntington's Disease gene is widely expressed (Huntington's Disease Collaborative Research Group, 1993; Sharp et al, 1995), the pathology of Huntington's Disease is restricted to the brain, and to specific regions within the brain, for reasons that remain poorly understood. At death the brain is small and often weighs less than one kilogram, as compared to the brain of a normal young adult, which weighs 1.4 kg. The frontal and parietal lobes are smaller than normal, but the most distinctive damage is visible in the head of the caudate nucleus, which is shrunken, along with the putamen and globus pallidus. The pathologic signature of Huntington's Disease is the loss of virtually all medium spiny neurons in the caudate. The brainstem and cerebellum are normal. Microscopically, there is extensive loss of neurons in the caudate and putamen, with evidence for apoptosis and necrosis (Portera-Caillau et al, 1995).
Huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. Huntingtin is a cytosolic protein primarily found in somatodendritic regions (Sharp et al. 1995; Strong et al, 1993). Recently, immunocytochemrical studies, using antibodies generated against peptides corresponding to the huntingtin N-terminus, suggest that inclusions containing huntingtin are present in the nucleus of striatal neurons of Huntington's Disease patients, but not in their cerebellar or brainstem neurons (DeFiglia et al, 1997). In the adult form of Huntington's Disease, axonal inclusions in dystrophic neurites are far more common than nuclear inclusions (DiFiglia et al, 1997). These inclusions are never found in normal individuals. These inclusions contain aggregates of huntingtin. These inclusions do not have the appearance of amyloid: “searches for amyloid deposits in brains of Huntington's Disease patients have been negative.” (Lunkes et al, 1997).
These aggregates stain with antibodies directed to the N-terminus of huntingtin but not to the C-terminus. The huntingtin N-terminal fragment, containing the polyglutamine domain, is most likely bound to ubiquitin via a lysine ubiquitin bond (Ciechanover, 1994). Somehow, in the pathogenesis of Huntington's Disease, the mutant huntingtin translocates to the nucleus and forms inclusions composed of aggregated N-terminal fragments of huntingtin. This is a pathological feature of the disease (Davies et al, 1997; Scherzinger et al, 1997; DiFiglia et al, 1997). Recently ubiquitinated intranuclear inclusions containing expanded polyglutamine domains were also seen in neurons in dentatorubralpallidoluysian atrophy (Igarashi et al, 1998), spinocerebellar ataxia type 3 (Paulson et al, 1997) and in spinocerebellar ataxia type 1 (Skinner et al, 1997).
Two mechanisms have been postulated to explain the cross-linking of huntingtin; these mechanisms may not be mutually exclusive. Molecular modeling had shown that &bgr;-strands made of polyL-glutamine can be assembled into sheets or barrels by hydrogen bonds between their main-chain and side-chain amides (Perutz et al, 1994). Perutz and colleagues (Stott et al, 1995; Perutz, 1996) tested this model experimentally. They showed that synthetic polyL-glutamine (Asp2-Q15-Lys2) (SEQ ID NO:1) forms &bgr;-strands, which are held together by hydrogen bonds between their amide groups. These aggregates maintain their secondary structure at pH 7 and pH 3. Interestingly, at pH 7 the peptide gradually precipitated. They postulated that these polymers comprised of polar zippers may be responsible for the neurodegeneration seen in Huntington's Disease. Recently, Scherzinger and colleagues showed that a glutathione S-transferase (GST) fusion protein encoding part of exon 1 of huntingtin, containing a polyglutamine domain of 51Q, spontaneously aggregates into amyloid-like fibrils, after enzymatic cleavage of the GST protein together with a few amino acids of exon 1 of huntingtin (Scherzinger et al, 1997) The GST-huntingtin Q51 construct was soluble; aggregates were formed only upon total enzymatic cleavage of the GST tag from GST-httQ51. Somehow, covalent fusion of the peptide with the polyglutamine domain to an unrelated protein, GST, prevented aggregation.
The GST-htt intermediate may serve as a nucleation factor for ordered protein aggregation in this system (Scherzinger et al, 1997). Indeed, this model is supported by the experimental finding of intermediate structures, termed “clots”, on one or both ends of the growing fibrils. Scherzinger and colleagues stated, “These clots w
Browdy and Neimark PLLC
Padmanabhan Sreeni
Yeda Research and Development Co. Ltd.
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