Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-05-28
2001-07-03
Romeu, David (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S300000, C530S836000, C530S848000, C530S850000, C930S250000
Reexamination Certificate
active
06255281
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the treatment of inflammatory and fibrotic conditions. More particularly, the invention provides a method for the treatment of inflammatory and fibrotic disease using recombinant human uteroglobin (rhUG). The invention identifies novel physiological roles for UG and their applications. Even more specifically, the invention relates to the treatment of inflammatory and fibrotic conditions by administering rhUG to inhibit PLA
2
s in vivo and to prevent fibronectin deposition in vivo. The invention further provides a method for the treatment of neonatal RDS/BPD, a critical clinical condition of the lung, and glomerular nephropathy, a disease of the kidney, both characterized by the involvement of inflammatory and fibrotic components.
BACKGROUND OF THE INVENTION
The search for improved therapeutic agents for the treatment of inflammatory, as well as fibrotic diseases, has received much attention in recent years. Neonatal Respiratory Distress Syndrome (RDS), a lung surfactant deficiency disease, is a condition of particular interest in that it is one of the major causes of mortality in premature neonates. While introduction of surfactant therapy dramatically improves survival of RDS patients, the development of chronic inflammatory and fibrotic disease in a significant percentage of this patient population is a major problem. Likewise, hereditary fibronectin-deposit glomerular nephropathy leads to end stage renal failure when patients' kidneys become blocked and no longer filter the blood. Hereditary glomerular nephropathy is characterized by fibronectin deposits and fibrosis of the kidneys. In both diseases, fibronectin deposition and fibrosis render the organ non-functional, and eventually, unable to support life. Thus, these patients require chronic hemodialysis or kidney transplantation.
PLA
2
is one of the enzymes responsible for hydrolysis of the surfactant phospholipids. Human uteroglobin, also known as “CC10”, inhibits the activity of phospholipase A
2
(PLA
2
) in vitro [Levin, S. W., et al. Uteroglobin inhibits phospholipase A
2
activity. Life Sci. 38:1813-1819 (1986); Singh, G. et al. Clara Cell 10 kda protein (CC10): Comparison of Structure and Function to Uteroglobin. Biochem. Biophys. Acta. 1039:348-355 (1990); Mantile, G. et al Human Clara Cell 10 kDa Protein Is The Counterpart of Rabbit Uteroglobin. J. Biol. Chem. 268:20343-20351 (1993)]. Human uteroglobin was first isolated in 1988 as a secretion product from the Clara cells of the lungs (Singh, G., et al. Identification, Cellular Localization, Isolation and Characterization of Human Clara Cell-Specific 10 kD Protein. J. Histochem. Cytochem., 36:73-80 (1987). (The designation “CC10” was derived from “Clara cell 10 kDa”.). It is a small globular homodimeric protein, and migrates in electrophoretic gels at a size corresponding to 10 kDa (Singh, G., et al. Identification, Cellular Localization, Isolation and Characterization of Human Clara Cell-Specific 10 kD Protein. J. Histochem.Cytochem., 36:73-80 (1987). (The designation “CC10” was derived from “Clara cell 10 kDa”). Human uteroglobin (hUG) is abundant in the adult human lung, and comprises up to about 7% of the total soluble protein (Bernard, A. et al, Protein 1 is a Secretory Protein of the Respiratory and Urogenital tracts identical to the Clara Cell Proteins. Clin. Chem. 38:434-435 (1992)). However, its expression is not fully activated in developing human fetus until the last few weeks of gestation. Consequently, the extracellular lung fluids of pre-term infants contain less human UG than those of adults, and may be deficient in these neonates (Dhanireddy, R., El-Ali, M., Murty, L., and A. B. Mukheijee, Pediatric Research 23:463A (1988) and Dhanireddy, R., Lim, M., and A. B. Mukheijee, Pediatric Research 33:323A (1993)).
PLA
2
s are a class of endogenous enzymes that hydrolyze the sn2 position ester bond of glycerophospholipids. They play critical roles in the inflammatory response because they release arachidonic acid (AA) from cellular phospholipid reservoirs. AA is metabolized to a number of potent inflammatory mediators in a process referred to as the arachidonic acid cascade (Piomelli, D. Arachidonic Acid in Cell Signaling. Op. In Cell Biol. 5:274-280 (1993)). Several acute and chronic clinical conditions have been characterized with elevated serum or local PLA
2
activity (see Table below). Native rabbit uteroglobin (UG), discovered in 1967 and purified from uteri of pregnant rabbits (Krishnan, R. S. and Daniel, J. C., Jr. “Blastokinin: Inducer and Regulator of Blastocyst Development in the Rabbit Uterus.” Science 158:490-492 (1967); Beier H. M. Verhandl Deut. Zool. Ges., Heidelberg (1968)), inhibits PLA
2
activity in vitro (Levin, 1988; supra). Clinical conditions in which elevated PLA
2
activity is documented are set forth in the following Table.
TABLE
Diseases
Sites
Rheumatoid arthritis
Serum, synovial fluid, WBC
Collagen vascular diseases
Serum
Pancreatitis
Serum
Peritonitis
Peritoneal fluid and cells
Septic shock
Serum
ARDS
a
Serum and alveolar fluid
Acute renal failure
Serum
Autoimmune uveitis
Serum, aqueous humor
Bronchial asthma
Bronchial fluid
a
Adult respiratory distress syndrome
(Modified from Mukherjee et al, 1992)
Amino acid analysis of purified human UG reveals that it is structurally similar to rabbit UG but not identical. 37 of 70 amino acids are identical between human and rabbit UG (see FIG.
1
). The “UG-like” proteins, including human UG/CC10, rat CC10, mouse CC10 and rabbit UG, exhibit species-specific and tissue-specific antigenic differences, as well as differences in their tissue distribution and biochemical activities in vitro. UG-like proteins have been described in many different contexts with regard to tissue and species of origin, and have also been identified using numerous different names, including uteroglobin, blastokinin, CC10 kDa protein (from rat lung), urine-protein 1 or “P1” (human), progesterone-binding protein (rabbit uterus), PCB-binding protein (rat lung), and CC16 (human lung), which has created confusion in the literature. At present there are no known physiological roles for these proteins (Umland, T. C., et al. Structure of a Human Clara Cell phospholipid-binding protein-ligand, complex at 1.9 Aresolution, Nature Struct. Biol. 1:538-545 (1994); Hard, T. et al, Solution Structure of Mammalian PCB-binding Protein in Complex with a PCB. Nat. Struct. Biol. 2:983-989 (1995); Umland, T. C. and M. Sax. Twixt form and function. Nat. Struct. Biol. 2:919-922 (1995); Stripp, B. R., et al. Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L656-L664 (1996).
The absence of structural identity among UG-like proteins makes it impossible to predict whether a protein will possess in vivo function based on in vitro or other activity exhibited by a structurally related protein in the context of an human therapeutic. For example, human uteroglobin binds less than 5% of the amount of progesterone than rabbit UG binds in the same assay (Singh, G., et al. Clara Cell 10 kDa Protein (CC10): Comparison of Structure and Function to Uteroglobin. Biochem Biophys Acta 1039:348-355 (1990). Human UG has a lower isoelectric point (4.6) than rabbit UG (5.4). Moreover, a UG knockout mouse recently generated to eliminate expression of UG has Clara cells which exhibit odd intracellular structures in place of UG secretion granules, but there is no other phenotype. This observation is highly significant since the anticipated phenotype was inefficient pulmonary finction accompanied by pulmonary inflammation and fibrosis. The knockout mouse shows no evidence of pulmonary impairment or abnormality, indicating that the UG protein has no significant role in surfactant homeostasis as had been previously suggested (Lesur, O., et al. Clara Cell Protein (CC16) Induces a Phospholipase A
2
-mediated Inhibition of Fibroblast Migration in Vitro. Am. J. Respir. Crit. Care Med. 152:290-297 (1995);
Mukherjee Anil B.
Pilon Aprile L.
Zhang Zhongjian
Claragen, Inc. and U.S. Government
Kramer Levin Naftalis & Frankel LLP
Romeu David
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