Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-08-21
2004-09-14
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S018000, C435S024000, C435S440000, C435S455000, C435S195000, C435S325000, C435S320100, C435S069100, C536S023200, C530S350000
Reexamination Certificate
active
06790649
ABSTRACT:
BACKGROUND OF THE INVENTION
The PHEX gene (formerly PEX; Phosphate regulating gene with homologies to Endopeptidases on the X chromosome) was identified by a positional cloning approach as the candidate gene for X-linked hypophosphatemia (XLH) (Francis et al., 1995). XLH is a Mendelian disorder of phosphate homeostasis characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal defects in phosphate re-absorption and vitamin D metabolism (Rasmussen and Tenenhouse, 1995). Using the information made available by the publication of the sequence of the PHEX gene, and standard techniques obvious to those skilled in the art, several groups have cloned and sequenced the human and mouse PHEX/Phex cDNAs (Du et al., 1996; Lipman et al., 1998; Grieff et al., 1997; Beck et al., 1997; Guo and Quarles, 1997; Strom et al., 1997) (PHEX/Phex refers to the human and mouse genes, respectively). Amino acid sequence comparisons have demonstrated homologies between PHEX/Phex protein and members of the neutral endopeptidase family as previously observed in the partial sequence of the candidate gene (Francis et al., 1995). The peptidases of the neutral endopeptidase family are zinc-containing type II integral membrane glycoproteins with a relatively short cytoplasmic N-terminal region, a single transmembrane domain, and a long extracytoplasmic domain, which contains the active site of the enzyme (Devault et al., 1987).
The mechanism by which loss of PHEX function elicits the bone and renal abnormalities observed in XLH patients is not clear. There are no data suggesting the presence of PHEX/Phex mRNA in the kidney (Du et al., 1996; Beck et al., 1997; Grieff et al., 1997). The increased renal phosphate excretion in Hyp mice is due to a down-regulation of the phosphate transporter, which is necessary for the re-absorption of the phosphate from the nephron (Tenenhouse 1998). The serum concentration of 1,25(OH)
2
D3 (calcitriol) was found to be the same in Hyp mice as in normal littermates (Meyer 1980). However, the Hyp kidney showed an accelerated degradation of the vitamin D metabolite to 1,24,25(OH)
3
D3, a metabolite with reduced activities (Tenenhouse 1988). In the presence of a phosphate rich diet, Hyp mice experienced an increase in serum 1,25(OH)
2
D3 and a fall in the C-24 oxidation products, while normal mice experienced no such changes (Tenenhouse 1990). To summarize, the renal disorder in vitamin D metabolism in Hyp mice appears to be secondary to the phosphate disorder.
PHEX/Phex mRNA was detected in bones by Northern blot hybridization and in other adult and fetal tissues such as lungs, liver, muscles, and ovaries by RT-PCR and RNase protection assays (Du et al., 1996; Beck et al., 1997). In situ hybridization performed on sections of embryos and newborn mice showed the presence of Phex mRNA in osteoblasts and odontoblasts (Ruchon et al., 1998). Phex gene expression was detectable on day 15 of embryonic development, which coincides with the beginning of intracellular matrix deposition in bones. Moreover, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the Phex transcript is decreased in adult bones and in non growing teeth. This result was confirmed when the presence of the Phex protein in newborn adult bones was investigated by Western blotting using a monoclonal antibody raised against the human PHEX. Immunohistochemical studies on a 2 month-old mouse showed exclusive labelling of mature osteoblasts and osteocytes in bones and of odontoblasts in teeth (Ruchon et al., 2000: J Bone Miner. Res. In press). Taken together these results suggest that PHEX/Phex plays an important role in the development and maintenance of mineralization in these tissues.
Further insights into the role of PHEX in bone metabolism were provided by experimental studies on cases of oncogenic osteomalacia (OOM), a tumor-associated sporadic condition with very similar clinical indications. There is strong evidence that a tumor-produced humoral factor inhibits renal phosphate re-absorption and vitamin D synthesis resulting in osteomalacia (Nelson et al., 1997). Experimental studies on Hyp and Gy mice, the murine model of human XL, also suggest the involvement of a humoral factor. In both mouse models, mutations have been identified in the Phex gene, which also appear to result in loss of function of the gene product (Strom et al., 1997; Beck et al., 1997).
Considering the similarities between PHEX protein and the other members of the metallopeptidase family of enzymes, it has been speculated that PHEX metabolizes a peptide hormone that modulates renal tubular phosphate re-absorption. Such an activity could involve either the processing of a phosphate reabsorbing hormone precursor to its active form or the inactivation of a circulating phosphaturic factor. There is evidence of intrinsic abnormalities in osteoblasts from Hyp mice (Ecarot et al., 1992). A defective phosphate transport was also observed in osteoblasts from Hyp mice (Rifas et al., 1994). PHEX might thus be involved in the control of bone metabolism both indirectly at the level of the kidney by controlling renal phosphate reabsorption and directly at the level of bones by inactivating a trophic peptide factor controlling either osteoblast or osteoclast functions or both.
Since absence of a functioning PHEX gene leads to hypophosphatemia, it should be possible to control human diseases involving hyperphosphatemia through the inhibition of this enzyme. Thus, inhibiting PHEX will cause a reduction in blood phosphate concentration, allowing for the prevention and reduction of hyperphosphatemiarelated disorders in humans and animals. Reduced renal excretion of phosphorus due to impaired kidney functions is the most common cause of hyperphosphatemia. In the specific case of secondary hyperparathyroidism (renal osteodystrophy), proper phosphate concentration would also benefit the patient by leading to an increase in endogenous calcitriol production and/or a lowering of PTH level. Therefore the early and adequate inhibition of PHEX activity could mitigate the serious consequences of renal osteodystrophy, giving patients an opportunity for an improved quality of life without the pain and mobility problems of advanced renal osteodystrophy. Hyperphosphatemia is defined in adults as an elevation of serum phosphorus above 1.67 mmol/L (5 mg/dL). Hyperphosphatemia is a common finding with many causes (Harrison's 14
Th
Ed CD-ROM, McGraw Hill Health Professions Division, New York N.Y., chapter 356).
Hyperparathyroidism or renal osteodystrophy results from the progressive nature of chronic renal failure. The leading causes of chronic renal failure are diabetes (43%), hypertension (35%) and glomerulonephritis (14%) among US Medicare patients (patients over 65 years of age). (Harrison's 14
Th
Ed CD-ROM, McGraw Hill Health Professions Division, New York N.Y., chapter 271, FIG. 271.1).
Hyperphosphatemia is potentially dangerous because of metastatic calcification. Although only an approximate guide, a calcium-phosphorus product [serum Ca (mg/dL) X serum P (mg/dL)] greater than 70 indicates a potential threat of calcification. Patients with this disease suffer from bone and joint pain, osteopenia, deformities, fractures, muscle weakness and extra-skeletal calcification.
Irrespective of the underlying cause, the disease is characterized by a progressive loss of the kidney ability to eliminate waste, to produce calcitriol (1,25(OH)
2
D3) and to excrete phosphate. Increased phosphate excretion is achieved with elevated PTH.
The direct effect of phosphate on PTH levels is well documented. In the presence of increasing phosphate concentration, intact fresh parathyroid gland showed increased PTH secretion (Almaden, 1996). A high phosphate diet causes elevated PTH while maintaining normal serum phosphate; in contrast, parathyroidectomized rats fed the same diet showed elevated phosphate levels (Borle, 1981 and Demeter 1991). Results in patients with mild to m
Boileau Guy
Crine Philippe
Nixon & Vanderhye
Prouty Rebecca E.
Ramirez Delia
Universite De Montreal
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