WO2004041115A2 - Novel human elastases and stress urinary incontinence - Google Patents

Novel human elastases and stress urinary incontinence Download PDF

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Publication number
WO2004041115A2
WO2004041115A2 PCT/US2003/018696 US0318696W WO2004041115A2 WO 2004041115 A2 WO2004041115 A2 WO 2004041115A2 US 0318696 W US0318696 W US 0318696W WO 2004041115 A2 WO2004041115 A2 WO 2004041115A2
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WO
WIPO (PCT)
Prior art keywords
elastase
woman
women
sui
urinary incontinence
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PCT/US2003/018696
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French (fr)
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WO2004041115A3 (en
Inventor
Leslie Kushner
Mahesh Mathrubutham
Srinivasa K. Rao
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North Shore - Long Island Jewish Research Institute
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Priority to AU2003299526A priority Critical patent/AU2003299526A1/en
Publication of WO2004041115A2 publication Critical patent/WO2004041115A2/en
Publication of WO2004041115A3 publication Critical patent/WO2004041115A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6448—Elastases, e.g. pancreatic elastase (3.4.21.36); leukocyte elastase (3.4.31.37)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/966—Elastase

Abstract

Novel elastases are provided that are associated with stress urinary incontinence. Also provided are methods for determining whether a woman has stress urinary incontinence, and methods for treating or preventing stress urinary incontinence.

Description

AR&E Docket No.50425/175
NOVEL HUMAN ELASTASES AND STRESS URINARY INCONTINENCE
Background
(1) Field of the Invention The present invention generally relates to proteases and diseases affected by altered protease concentrations. More particularly, the invention relates to novel elastases and their relation to stress urinary incontinence.
(2) Description of the Related Art Relevant Published Abstracts Desautel, M.G., Burney, T.L., Moak, S.A., Greenwald, R.A., Kushner, L. and Badlani, G H. Elevated Collagenase Activity in Skin and Endopelvic Fascia of Women with Pelvic Floor Weakness. J. Urology, (1997). Mathrubhutham, M., Austria, A., Badlani, G., Kushner, L. and Rao, S. Capacity of Plasma to Inhibit Elastase Activity is Reduced Under Certain Conditions, The FASEB J., (1997). Aybek, Z., Mathrubutham, M., Rao, S., Kushner, L. and Badlani, G. Capacity of Plasma to Inhibit Elastase Activity is Reduced in Patients with Stress Urinary Incontinence. J. Urology, (1998). Mathrubutham, M., Aybek, Z., Fogarty, J., Lee, J., Rao, S.K., Badlani, G H. and Kushner, L. Plasma Elastase Regulation in Stress Urinary incontinence. Neurourology and Urodynamics 18:281 (1999).
Kushner, L., Chen, Y., Desautel, M., Moak, S., Greenwald, R. and Badlani, G. Collagenase
Activity is Elevated in Conditioned Media from Fibroblasts of Women with Pelvic Floor Weakening. Neurourology and Urodynamics 18:282 (1999). Mathrubutham, M., Kushner, L., and Rao, S.K. Kinetic Comparison of Elastase Substrates. The FASEB Journal (2000).
Mathrubutham, M., Maytal, A., Rao, S.K., Badlani, G.H. and Kushner, L. Elastolytic and Collagenolytic Activity is Elevated in Conditioned Media from Skin and Endopelvic Fascia Explants of Women with Pelvic Floor Weakening. J. Urology 163: 95 (2000). Mathrubutham, M., Rao, S.K., Badlani, G.H. and Kushner, L. Partial Characterization of the Elastolytic Activity Secreted by Skin and Endopelvic Fascia Explants from Women with
Pelvic Floor Weakening. The FASEB Journal (2001). Shah, D., Kushner, L., Rao, S.K., Mathrubutham, M., Mutyala, M., Badlani, G. Elastase
Activity in Plasma: Screening Tool for Stress Urinary Incontinence. J. Urology (2002).
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U.S. Patent No. 6,197,537. Urinary incontinence is a significant problem affecting over 10 million Americans of all ages. Nearly 85% of those affected are women (1). One of every six women over the age of 45 is affected. Urinary incontinence dramatically impairs social and economic functioning which results in increased isolation and deterioration of life-style. With the current methods of therapy, nearly half of the patients are dissatisfied and most would prefer another form of treatment (1). Stress urinary incontinence (SUI) is the involuntary leakage of urine due to an increase in abdominal pressure, as may occur during a cough. Most (85-90%) of SUI patients are categorized as having urcthral hypermobility. That is, the patient loses urine with sudden increases in abdominal pressure but never during sleep (grade I) or the patient loses urine during physical activities such as walking or standing up from a sitting position (grade II). In the remaining 10-15% of SUI patients, incontinence is the result of a defective urethral closure mechanism and is termed intrinsic sphincter deficiency. Urethral hypermobility, which is the cause of most SUI, is the result of weakened support from the connective tissues and ligaments of the pelvic floor. The consequence of this weakened support is an unequal transmission of intra-abdominal pressure to the bladder and bladder neck during episodes of stress thus leading to the loss of urine. Weakened support from the connective tissues and ligaments of the pelvic floor also contributes to pelvic organ prolapse (POP) which may be present alone or in addition to stress urinary incontinence. The etiology of this weakened support is unknown, but may result from repeated pregnancy (1, 2) obesity, chronic coughing or alterations in collagen metabolism (3). There is a three-fold prevalence of SUI among first-degree related female patients (4), suggesting the potential for genetic factors predisposing to weakened pelvic floor support.
Urge incontinence (UI) is characterized by the involuntary loss of urine concomitant with a sensation of the sudden need to urinate. This type of incontinence is associated with involuntary contractions of the detrusor muscle and is often referred to as overactive bladder. Although UI is often idiopathic, it is sometimes associated with degenerative nerve or muscle diseases. Thus, the etiology of UI is likely to be distinct from SUI and not involve processes that lead to weakening of the connective tissue supporting the bladder and urethra.
There is no preventative therapy for SUI. One option for treatment of SUI is a bladder neck suspension which is aimed at restoring support to the bladder neck and proximal urethra. A subset of these patients is destined to fail correction with this procedure and require later surgical placement of a pubovaginal sling. The cause of the failed surgery is probably multifactorial. However, for some of these patients, the cause may involve continued weakening of the endopelvic fascial support due to altered metabolism of extracellular matrix proteins. If these patients could be identified prior to surgery by simple diagnostic tests, more appropriate choices for treatment could be offered. For example, if patients can be identified early in the disease process to have an increased level of collagenolysis and/or elastolysis, a potential preventative therapy might include treatment with a protease inhibitor. Several inhibitors of matrix metalloproteinase (MMP) (5, 6) and elastase (7-9) are being developed for clinical use for inhibition of protease activity for a variety of pathologies. Similar approaches might prove effective in blocking progressive endopelvic fascia connective tissue weakening and serve as an adjunctive medical therapy for patients who undergo bladder neck suspension or serve as a medical therapy to slow progression of the disease in patients who are identified early.
Connective Tissue Weakening in Stress Urinary Incontinence (SUI). Urinary continence in women is dependent on the integrity of the pubourethral ligaments and the muscles and connective tissue of the pelvic floor (10). The ligaments and pubocervical fascia are fibrous connective tissue composed primarily of collagen I and III (11). Elastin is a major component of the extracellular matrix of connective tissue, as well. This elastic protein present in the submucosa and lamina propria of the urinary bladder is thought to contribute to bladder compliance (12, 13). The low density of elastin in the bladder wall is suggestive of only a minor role in bladder contraction during voiding (14). However, elastin present in the urethral wall may contribute to maintaining urethral closure during periods between voiding (15). Alterations in collagen metabolism has been suggested as an underlying etiology for stress urinary incontinence in women (4). The following experimental evidence suggests that the collagen matrices in SUI women are different from unaffected women.
Ulmsten et al. (16) demonstrated that the skin and ligamentum rotundum of women with stress urinary incontinence had 25-40% less hydroxyproline than that of unaffected women of similar age, indicating a lower total collagen content. The hydroxyproline content of the vesico- vaginal fascia was significantly lower (33%) in women with SUI and cystocoele compared to that of women with cystocoele only (4).
Versi et al. (17) demonstrated a correlation between skin hydroxyproline content and various urethral pressure measurements under resting and stress (cough) conditions. These data suggest that greater skin collagen content is associated with better urethral function and a lower probability of SUI.
Bergman et al. (18) demonstrated that women with both SUI and genital prolapse (GP) have lower amounts of collagen III (with no change in total collagen) in their uterosacral ligament, round ligament and perineum skin compared to women with GP only and women without GP or SUI.
Falconer et al. (19) demonstrated that fibroblast cultures established from skin biopsies from SUI women incorporated 30-40% less 3H-proline into collagen compared to fibroblasts from age-matched continent women. Moreover, total collagen secreted (as measured using Sircol Red) was 30% less in the conditioned medium of skin fibroblasts from SUI women compared to age-matched continent women. These data suggest a reduced capacity for collagen synthesis in fibroblasts from women with SUI.
Elastin content in the connective tissue structures and ligaments of the pelvic floor probably contribute to the resiliency of those tissues. A decrease or alteration of elastin content in the structures of the pelvic floor would be expected to result in weakened support of the bladder and urethra. However, elastin has not been studied in this context.
In women with SUI, the shear strength of the endopelvic fascia is significantly lower than in age- and parity-matched women without SUI or genital prolapse (20). Shear strength of the endopelvic fascia did not correlate with age, suggesting an age-independent factor was operating to reduce endopelvic fascia sheer strength in SUI women. Moreover, the lower shear strength was not limited to endopelvic fascia, but was evident in the rectus fascia, which has no role in bladder neck support. These data suggest that factors which contribute to connective tissue shear strength are altered in women with SUI, that these factors are independent of age, and that these factors affect connective tissue in structures that are not responsible for bladder support.
Taken together, these data suggest that alterations in the connective tissue composition of endopelvic fascia weakens the pelvic floor, leading to hypermobile SUI, and that biochemical processes responsible for altering endopelvic fascia connective tissue may be part of a systemic defect in connective tissue processing. However, the mechanism underlying changes in collagen composition and/or altered collagen metabolism has not been fully explored. Reduced collagen content may be a result of decreased synthesis, increased collagenolysis or both. Notably, the role of collagenolysis in pelvic floor weakening has not been addressed. Alterations in elastin content of the underlying endopelvic fascia have also not been examined. Reduced elastin is likely due to increased elastolysis, rather than decreased synthesis, because elastin synthesis is at minimal levels in adulthood.
Collagen. There are approximately twenty genetically distinct collagens (12). The ligaments and pubocervical fascia supporting the urinary bladder and urethra are fibrous connective tissue composed primarily of collagens of types I and III (12) (For a review of interstitial collagens, see Bienkowski, 1991 (21)). Collagen I is usually a heterotrimer composed of two αl(I) and one c 2(I) polypeptide chains. Transcription of the genes encoding pro l(I) and pro 2(I) is usually coordinately regulated to produce a 2: 1 ratio of messages. However, independent regulation of transcription of these genes has been described (22). Collagen III is a homotrimer of α 1 (III) polypeptide chains. The collagen molecule is a cleavage product of the procollagen precursor which consists of three proα chains in a triple helix, stabilized by posttranslational hydroxylation of approximately half the proline residues. Procollagen is normally secreted and cleaved to form collagen in the extracellular milieu. The extracellular collagen molecules associate to form fibrils.
Genetic Defects in Collagen I and III. A number of hereditary disorders have been described which are the result of alterations in collagen types I and III. Mutations in the genes that encode the chains of type I procollagen (COLA1 and COL1A2) and type III procollagen (COL3A1) usually result in osteogenesis imperfecta (OI) or Ehlers-Danlos syndrome (EDS) (23). A variety of mutations have been described which result in different phenotypic consequences. In some families the same mutation may result in great variability in phenotype, even producing a lethal phenotype in the child but a relatively mild phenotype in the parent (24- 26). Mutations can result in abnormal procollagen chains, such that the abnormal chains are not secreted but retained within the cell (27, 28). Mutations can result in synthesis of less than the normal amount of proα chains or failure to incorporate some proα chains into procollagen molecules (29-32). Mutations that affect transcription, splicing or transport of the hnRNA (33) would result in lower procollagen mRNA levels and corresponding lower synthesis of collagen. Mutations may cause the synthesis of an unstable pro-α chain that does not get incorporated into procollagen molecules, thus resulting in the secretion of less than normal amount of procollagen (34). Mutations which result in the secretion of abnormal procollagen would alter fibrillogenesis (35-36).
Collagenolysis. Enzymes that degrade collagen play an important role in the integrity of connective tissue matrix and, thus, its function in supportive tissues. The collagenases are matrix metalloproteinases (MMPs), metal-dependent enzymes which degrade connective tissue matrix. Although over 20 soluble MMPs have been identified (37), the term "collagenase" most often is used to refer to interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8) due to the ability of these enzymes to cleave triple helical collagen. MMP-1 is synthesized by fibroblasts and is equally active in degrading collagen I and III (38). MMP-8 is synthesized by neutrophils and is more active in degrading collagen I than collagen III (39). Constitutive synthesis and activity of collagenase is very low in most tissues, but can be stimulated by various cytokines and is often greatly increased in pathologic states characterized by connective tissue degradation (40). Collagenase has been identified in homogenates and cultures of rheumatoid synovium
(41-43) detected in inflammatory synovial fluids (44, 45) and localized immunologically and by in situ hybridization in proliferative pannus and synovium (46-49). In explants of ruptured anterior cruciate ligament of the rabbit, collagenase activity is elevated 82% compared to explants from uninjured tissue (50). This corresponds to a 34% loss of total collagen from the injured ligament, which suggests that response to ligament injury involves degradation of the collagenous matrix.
Systemic degradation of collagen often results in increased urinary excretion of collagen crosslinks (51) and short collagen-derived peptides (53, 52). Urinary excretion of collagen degradation products has been detected in diseases involving collagenolysis such as osteoporosis, rheumatoid arthritis (54), hyperparathyroidism, and Paget's disease (55).
Elastolysis. Elastic fibres, composed primarily of elastin, is the component of extracellular matrix responsible for elasticity and resiliency of connective tissues. Elastin deposition occurs primarily during development, with very little elastin synthesis and deposition in the adult under normal conditions (56). However, elastin degradation by increased elastase activity has been implicated in emphysema (58, 57) abdominal aortic aneurysm (59), atherosclerosis (60), pancreatitis (61) and inflammatory diseases (62). Elastases are released from inflammatory cells such as neutrophils and macrophages. Human neutrophil elastase (HNE) is a serine protease, while macrophage elastase (MMP-12) is a matrix metalloproteinase. Elastases are also secreted by the fibroblasts of connective tissue (63). Several matrix metalloproteinases (MMP-2 and MMP-9) have significant elastolytic activity.
In several pathologic conditions, levels of anti-protease are reduced resulting in increased elastolytic activity in the affected tissues. Previous investigators have hypothesized that one of the factors contributing to connective tissue destruction in certain pathological conditions is an imbalance in the ratio of protease to antiprotease (64). Three classes of endogenous elastase inhibitors, α-1 protease inhibitor, α-2 macroglobulin and secreted leukoproteinase inhibitor have been identified. The best-characterized inhibitor of elastase activity is αl-antitrypsin (65). A reduced level of α 1 -antitrypsin in the lungs of patients with hereditary emphysema results in increased elastase activity (66) which is thought to be responsible for the destruction of the alveolar walls characteristic of the emphysematous lung. A similar imbalance has been described as the cause of the loosening of hip prostheses (67).
Since resynthesis of elastic fibers in adult tissues is considered rare (69) and is not a demonstrated response to elastolysis in multiple pathologies, any factors which increase elastolytic activity may contribute to permanent reduction of elastin in those tissues. During normal aging, elastase activity increases in the aortic wall. However, patients who develop abdominal aortic aneurysm have significantly elevated elastolytic activity in the plasma (70), as well as in the aortic wall in the region of the aneurysm, compared to age-matched controls. This increased elastolytic activity may be involved in the reduction of elasticity and structural integrity of the aortic wall. Without stimulation of elastin expression and deposition, repair of the compromised aortic wall does not occur (71) and aneurysm development is progressive. Factors that increase plasma elastolytic activity, such as cigarette smoking, may contribute to aneurysm development.
Summary of the invention Accordingly, the inventors have discovered that elevated levels of novel elastases are produced by fibroblasts of the skin and pubocervical fascia of women with stress urinary incontinence (SUI). Elevated concentrations of these elastases are also present in the plasma of SUI patients. These novel elastases are likely major factors in the etiology of SUI. These discoveries enable new methods of studying, detecting and treating SUI. Thus, in some embodiments, the present invention is directed to isolated human elastases. These elastases have a pH optimum of about 8.5, and are inhibited by α-1-antitrypsin, 1 , 10-phenanthroline and EDTA, but not phenylmethyl-sulfonyl fluoride.
In other embodiments, the invention is directed to antibodies or antibody fragments that specifically bind to the above-described elastases. The invention is also directed to aptamers that specifically bind to the above-described human elastases.
In additional embodiments, the invention is directed to methods of determining whether a woman has, or is likely to develop, stress urinary incontinence. The methods comprise determining whether the woman has an elastase having a pH optimum of about 8.5, which is inhibited by α- 1 -antitrypsin, 1 , 10-phenanthroline and EDTA, but not phenylmethyl-sulfonyl fluoride. In these methods, an elastase level elevated above an average elastase level of a group of women that do not have stress urinary incontinence indicates that the woman has or is likely to develop, stress urinary incontinence.
Additionally, the invention is directed to methods of preventing or treating stress urinary incontinence in a woman. The methods comprise inhibiting the above-described elastases in pubocervical fascia of the woman. In related embodiments, the invention is directed to methods of preventing or treating, in a patient, a disease characterized by undesired elastin degradation in the patient. The methods comprise inhibiting an invention elastase in the patient.
Brief Description of the Drawings
Figure 1 shows representative micrographs from a histopathologic evaluation of endopelvic fascia and skin sections (10 μM), 400X magnification. Representative sections are shown. The sections in the left panel (A,B,C,D) are from endopelvic fascia. The sections in the right panel (E,F,G,H) are from skin. Sections A,B,E and F are hematoxylin and eosin stained sections. Sections C,D,G, and H are trichrome stained adjacent sections. The collagen bundles of the endopelvic fascia stain dense, heavy blue and are more abundant in sections from continent women (C) compared to those from women with SUI (D). Similarly, the collagen bundles of the skin stain dense, heavy blue and are more abundant in the sections from continent women (G) compared to those from women with SUI (H).
Figure 2 shows the results of experiments establishing that collagenase activity is elevated in the conditioned medium from explants obtained from women with SUI compared to continent women. Skin and pubocervical (PC) fascia biopsies were immediately transferred to sterile tissue culture media without serum. [A] Conditioned medium (4d) was assayed by soluble lysis assay. [B] A representative autoradiogram demonstrating 30% digestion of collagen by the 4 day conditioned medium from skin explants obtained from a woman with SUI (lanes 4-6) and 1% digestion in the 4 day conditioned medium for skin explants obtained from a woman of the same age without SUI is shown (lanes 1-3). Lanes 1 and 2 are duplicates of the same sample; Lane 3 is the same sample in the presence of 1 , 10-phenanthroline to block the reaction. Lanes 4 and 5 are duplicates of the same sample; lane 6 is the same sample in the presence of 1,10 phenanthroline. There was 0% digestion of collagen in lanes 3 and 6.
Figure 3 is a graph summarizing results from experiments measuring immunoreactive matrix metalloproteinase 1 (MMPl) in conditioned media (CM) from skin and endopelvic fascia (PC) explants taken from women with stress urinary incontinence (SUI) compared to women without pelvic floor weakening (Control). MMPl level was determined by ELISA (Biotrak) and normalized to protein. Data is reported as ng MMPl/mg protein and expressed as mean ± SEM. Statistical significance was determined by Students' T-test.
Figure 4 is a bar graph summarizing results of experiments measuring collagenolytic activity in the conditioned medium from explants obtained from women with SUI compared to continent women. Skin and pubocervical (PC) fascia biopsies were immediately transferred to sterile tissue culture media without serum. Conditioned medium (4d) was assayed by the method of Rao et al (75). Briefly, free amino ends generated from succinylated collagen substrate was quantified by measuring A450 after reaction with TNBSA. Figure 5 is a representative gel showing PCR products using primers that detect MMPl transcripts in fibroblasts. Lanes 3-5 are the PCR products from pubocervical fascia (PC) fibroblasts from women with SUI. Lanes 6-8 are the PCR products from skin fibroblasts from with SUI. Lane 9 is the PCR product obtained when RNA is replaced with sterile water (negative control). Lane 10 is the PCR product obtained when the RNA sample is used in the amplification (negative control). Lane 11 is the PCR product derived from MMPl -producing cultured fibroblasts (positive control).
Figure 6 is a bar graph summarizing experiments showing that collagen synthesis in not different in fibroblasts from women with SUI compared to women without SUI. Late log phase cells were incubated with 3H-proline and the cells and medium, combined, incubated with bacterial collagenase. The amount of 3H-proline released was determined by liquid scintillation counting. Data is expressed as % of total protein synthesis after correction for the high proline content of collagen.
Figure 7 shows results from experiments demonstrating that the ratio of collagen al(III)/al(I) synthesized is not different in fibroblasts from women with SUI compared to women without SUI. Late log phase cells were incubated with 3H-proline in medium containing ascorbic acid. Collagen α 1 (III) was separated from collagen al(I) by the technique of interrupted gel electrophoresis. [A] Representative autoradiogram; each lane is a digestion from fibroblasts cultured from a separate biopsy specimen. [B] Data is presented as mean ± SEM of the ratios of collagen I1I/I synthesis for each group. Figure 8 is a bar graph summarizing results from experiments showing that plasma proteolytic activity is elevated in women with SUI.
Figure 9 is a bar graph showing results from experiments establishing that elastolytic activity is higher in conditioned media from explants from women with SUI than from women without SUI, both for endopelvic fascia and skin.
Figure 10 shows results from experiments establishing that a serine protease-type elastase is not detected in endopelvic fascia fibroblasts. The figure is a representative gel showing RT-PCR products generated via methods described in Examples 2 and 6 using the specific primers set forth therein. PCR amplifications of the cDNA (5 μl) were performed in a 50 μl reaction mixture containing 10 mM Tris CI, pH 9.0, 500 mM KC1, 2.5 mM MgCl2, 1 M dNTPs, and 2.5 units Taq polymerase (Promega, Madison, WI), with 0.2 μM of each of the primers. PCR was carried out in a DNA thermal cycler (Perkin-Elmer Cetus) using the following conditions for 30 cycles: denaturation at 96°C for 1 min, annealing of primer at 55°C for 1 min, polymerization at 72°C for 1 min, in each cycle, with a final extension at 72°C for 7 min. Lanes labeled P are from fibroblasts derived from explants from women with SUI. Lanes labeled C are from fibroblasts derived from explants from women without SUI. Lanes labeled "Control" are from cultured U937 cells that had been stimulated with phorbol 12-myristate 13- acetate (PMA). Figure 11 shows duplicate zymographs using an elastin substrate. Electrophoresis was performed on an SDS (1%)- polyacrylamide (9%) gel containing lmg/ml elastin (Elastin Products Company, Owensville, Missouri) under non-reducing conditions and without boiling. Gels were run in 25 mM Tris CI, 0.2 M glycine, 0.1% SDS for 4-6 hr at 18 mA on ice. After elctrophoresis, gels were washed 2 X 15 min with 2.5% Triton X-100 and incubated overnight at 37°C in 0.04 M Tris CI, pH 7.5, 0.2 M NaCl, 0.01 M CaCl2 with [A] or without [B] 10 mM
EDTA. Gels were stained in 0.2% Coomassie blue in 50% methanol and 10% glacial acetic acid for 2 hr with shaking. Gels were destained with 20% methanol and 10% glacial acetic acid. Enzymes added to each well are indicated as follows: CM, 17X concentrated conditioned medium from explant culture (10 μl); PE, pancreatic elastase (10 ng); 12, MMPl 2 (20 μl); 9, MMP9 (100 ng of APMA-activated); 2, MMP2 (100 ng of APMA-activated). Standard enzymes were obtained from Calbiochem, (San Diego, CA).
Detailed Description of the Invention
The present invention is based on the discovery and isolation of two novel elastase enzymes. The concentration of these elastases are greatly elevated in the plasma of women with stress urinary incontinence (SUI), and fibroblasts from skin and pubocervical fascia of SUI patients also produce far greater amounts of these elastases than similar tissues from women without SUI. Based on these discoveries and other findings (see Examples), it is likely that these two enzymes are major factors contributing to he etiology of SUI.
Accordingly, in some embodiments, the present invention is directed to the isolated human elastases, described in the examples, that are associated with SUI. These elastases are novel, in that they have particular characteristics that distinguish them from other elastases known in the art. These characteristics include a pH optimum of about 8.5. Additionally, the elastase activity of these enzymes are inhibited by α-1-antitrypsin, 1,10-phenanthroline and EDTA, but not phenylmethyl-sulfonyl fluoride. The inventors believe that there are no other elastases described in the prior art that have the above characteristics. Other fibroblast elastases are described and partially characterized in Refs. 100-109. As used herein, the term "isolated", when referring to the elastases of the present invention, means separated from living cells and present in a preparation as a greater proportion of the proteins in the preparation than found in the tissue from where the elastase naturally occurs. Preferably, the isolated elastases comprise at least 5% of the protein in the preparation. More preferably, the elastases comprise at least 10%, even more preferably 20% of the protein in the preparation. In most preferred embodiments, the elastases comprise at least 50% of the protein in the preparation.
The elastases of the present invention can be prepared and isolated by any means known in the art. For example, the elastases can be produced by incubating fibroblasts, preferably from a woman with SUI, in a culture medium, where the elastases will be elaborated into the medium. In preferred embodiments, the fibroblasts are part of a skin or pubocervical fascia explant. The elastases can then be further isolated by any of a number of well-known methods for purifying proteins, for example salt precipitation (either to concentrate the elastase or remove other proteins), liquid chromatography, gel chromatography, affinity chromatography (for example by standard methods using an affinity column comprising beads to which an antibody that is specific for the elastase is bound), HPLC, etc.
The elastases of the invention can also be produced by standard molecular biology methods. In these methods, the elastase is cloned, e.g., by preparing an antibody to the elastase, and screening a fibroblast expression library with the antibody. The fibroblast expression library is preferably prepared using fibroblasts from the skin or pubocervical fascia of a woman with SUI, since the elastases of the invention are apparently more highly expressed in those fibroblasts. The cloned sequence from the fibroblast expression library that encodes a protein reactive to the elastase antibody is then sequenced and confirmed to encode an elastase, for example by cloning the sequence into an expression vector and expressing the protein, then performing an elastase assay (e.g., by using zymography to also confirm the apparent molecular weight) on the expressed protein. The expression vector can also be used to produce the protein, e.g., in yeast or E. coli.
The elastases of the invention are further characterized as having an apparent molecular weight of about 48 kDa or 58 kDa, as determined by zymography on a 1% SDS-9% polyacrylamide gel containing 1 mg/ml elastin under non-reducing conditions and without boiling. See Example 6 for details in preferred methods for determining the apparent molecular weight using zymography.
As previously discussed, the elastases of the invention are produced by fibroblasts, preferably fibroblasts from skin or endopelvic fascia. The elastases are also produced more abundantly in fibroblasts from women with SUI than from women without SUI. However, the elastases may also be expressed in other cells, particularly other fibroblasts. The circulating concentration of the elastases is also greater in women with SUI than in women without SUI.
In other embodiments, the invention is directed to agents that specifically bind to the invention elastases. Examples of such agents are antibodies or antibody fragments, and aptamers. These agents are useful for detecting and/or quantifying the elastases, e.g., in biological samples to diagnose SUI. In some embodiments, the agents are also used therapeutically, to inhibit the activity of the elastases. For those latter embodiments, it is preferred that the binding agents bind to the elastase in such a way as to prevent the elastases from catalyzing the cleavage of elastin. Such binding agents would usually, but not invariably, bind to the active site of the elastase.
As used herein, an agent that specifically binds to an elastase binds with greater affinity to the elastase than to unrelated proteins or other biological molecules. Where the agent is an antibody, the elastase binding occurs in the variable region of the antibody molecule, as is well known in the art.
The antibody or antibody fragments that specifically bind to the elastases can be produced by any known method, for example by inoculating a vertebrate with the elastases by well established methods, then obtaining the polyclonal antibodies from the serum of the vertebrate, or fusing B cells of the vertebrate with a myeloma cell to create hybridomas that produce monoclonal antibodies. Alternatively, genes for antibodies or antibody fragments that specifically bind to the elastases can be isolated and cloned by known phage display methods, then expressed and the antibodies isolated.
Another example of a useful binding agent is an aptamer. Aptamers are single stranded oligonucleotides or oligonucleotide analogs that bind to a particular target molecule, such as a protein or a small molecule (e.g., a steroid or a drug, etc.). Thus, aptamers are the oligonucleotide analogy to antibodies. However, aptamers are smaller than antibodies, generally in the range of 50-100 nt. Their binding is highly dependent on the secondary structure formed by the aptamer oligonucleotide. Both RNA and single stranded DNA (or analog), aptamers are known. See, e.g., Refs. 92-97 and U.S. Pats. No. 5,773,598; 5,496,938; 5,580,737; 5,654,151; 5,726,017; 5,786,462; 5,503,978; 6,028,186; 6,110,900; 6,124,449; 6,127,119; 6,140,490; 6,147,204; 6,168,778; and 6,171,795.
Aptamers that bind to virtually any particular target can be selected by using an iterative process called SELEX, which stands for Systematic Evolution of Ligands by Exponential enrichment. Several variations of SELEX have been developed which improve the process and allow its use under particular circumstances. See, e.g., Refs. 96-99 and U.S. Pats. No. 5,472,841; 5,503,978; 5,567,588; 5,582,981; 5,637,459; 5,683,867; 5,705,337; 5,712,375; and 6,083,696. Thus, aptamers to the invention elastases can be isolated using these known methods without undue experimentation.
The invention is also directed to methods of determining whether a compound is an elastase inhibitor. The methods comprise contacting the compound with an invention elastase, and determining whether the elastase is inhibited. These methods can be useful for evaluating any compound, for example a protein such as an antibody, a peptide, a small molecule, an oligonucleotide such as an aptamer, or a chelator.
In additional embodiments, the invention is directed to methods of determining whether a woman has, or is likely to develop, stress urinary incontinence. The methods comprise determining whether the woman has one of the invention elastases at a concentration that is elevated above the average elastase level of a group of women that do not have stress urinary incontinence. In these methods, an elevated concentration of one of the elastases indicates that the woman has stress urinary incontinence.
It is envisioned that these methods can be used to diagnose women that have had episodes of incontinence to determine whether their incontinence is due to SUI. The methods can also be used as a screen for women to determine the risk for having, or later developing, SUI. Since SUI is more common in post-menopausal women, it is contemplated that those women would achieve the most benefit from such a screening program.
In these methods, the elastase levels are preferably measured in blood or a blood fraction, such as plasma. In other embodiments, the elastase levels are measured in media conditioned with skin or pubocervical fascia explants.
These methods are not narrowly limited to any particular procedure for quantifying the invention elastases. Examples of such procedures are elastase assays or zymography as described in the examples; assays using labeled binding agents, such as assays using aptamers or assays using antibodies or antibody fragments such as ELISA or radioimmunoassay; and quantitation of mRNA of the elastases using, e.g., northern hybridization, RNA dot blots or RT- PCR. All of the above procedures are well known and the adoption of any of those procedures for the invention elastases could be accomplished without undue experimentation.
The elastase level in the woman that is indicative of SUI can be set without undue experimentation at a particular level in relation to the average elastase level of the group of women, in order to minimize false positive and/or false negative SUI diagnoses when performing these methods. For example, the level indicative of SUI can be set at twice, three times, four times, or more, the average elastase level of the group of women. Alternatively, the elastase level indicative of SUI can be set at two, three, four, or more standard deviations above the average elastase level of the group of women.
It is believed that the elastases of the present invention are a major contributing factor in the etiology of SUI. The Examples provide strong evidence for this assertion. In light of this, inhibiting the elastases in the pubocervical fascia of a woman with SUI would be expected to reduce the severity of SUI in that woman. Additionally, inhibiting the elastases in the pubocervical fascia of a woman at risk for SUI (i.e., not incontinent but having high concentrations of the invention elastases) would be expected to prevent, delay, or limit the severity of future episodes of incontinence for those women.
Accordingly, the invention is also directed to methods of treating stress urinary incontinence in a woman with SUI, or preventing, delaying, or limiting the severity of SUI in a woman at risk for SUI. In these embodiments, the methods comprise inhibiting the invention elastases in the pubocervical fascia of the woman.
For these methods, the elastases can be inhibited by any means known in the art. For example, in some embodiments, the elastase is inhibited by contacting the pubocervical fascia of the woman with a known chemical inhibitor of the enzyme, such as a peptide, small molecule or chelator that is known to inhibit the enzyme. Examples include α-1-antitrypsin, 1,10- phenanthroline and EDTA. In other embodiments, the elastase is inhibited by contacting the pubocervical fascia of the woman with a specific elastase binding agent, such as an antibody or an aptamer, provided that the binding agent also inhibits the activity of the elastase. Combinations of any of these inhibitors is also contemplated.
The agent used in these methods to inhibit the invention elastases can be administered to the woman by any means capable of providing contacting of the agent with the pubocervical fascia of the woman, including orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, bucally, intrabuccaly and transdermally to the patient. Accordingly, pharmaceutical compositions of the agents designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example with an inert diluent or with an edible carrier. The compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Some examples of disintegrating agents include alginic acid, corn starch and the like. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring and the like. Materials used in preparing these various compositions should be pharmaceutically pure and nontoxic in the amounts used.
Pharmaceutical compositions of the inhibiting agents can easily be administered parenterally such as for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the inhibitor compositions of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. Rectal administration includes administering the pharmaceutical compositions into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120° C, dissolving the cholinergic agonist in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.
Transdermal administration includes percutaneous absorption of the agents through the skin. Transdermal formulations include patches (such as the well-known nicotine patch), ointments, creams, gels, salves and the like. The present invention includes nasally administering to the mammal a therapeutically effective amount of the inhibitory agent. As used herein, nasally administering or nasal acirninistration includes administering the inhibitory agent to the mucous membranes of the nasal passage or nasal cavity of the patient. As used herein, pharmaceutical compositions for nasal administration of an inhibitory agent include therapeutically effective amounts of the agent prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder. Administration of the inhibitory agent may also take place using a nasal tampon or nasal sponge.
In preferred embodiments, the agent is administered topically, since it is believed that the elastases are secreted by the fibroblasts into the extracellular matrix of the pubocervical fascia, where their enzymatic action causes weakening of the fascia. An exogenously applied agent would thus not necessarily need to cross a cell membrane in order to contact and inhibit the elastases believed to contribute to SUI.
In other embodiments, the elastases can be inhibited by preventing translation of the elastases using ribozymes or antisense nucleic acids or analogs, as are known in the art. These ribozymes and antisense compounds can be developed against the invention elastases without undue experimentation.
More generally, the invention is directed to methods of preventing or treating, in a patient, a disease characterized by undesired elastin degradation in the patient. The methods comprise inhibiting an invention elastase in the patient. As with the previously described treatment methods, the elastases can be inhibited in these methods by any means known in the art, for example with a known chemical inhibitor of the enzyme, such as a peptide, small molecule or chelator that is known to inhibit the enzyme.
Examples include α-1-antitrypsin, 1,10-phenanthroline and EDTA. In other embodiments, the elastase is inhibited by contacting the elastase with a specific elastase binding agent, such as an antibody or an aptamer, provided that the binding agent also inhibits the activity of the elastase. Use of ribozymes, antisense compounds, or RNA interference oligonucleotides is also contemplated, as discussed previously in the context of SUI treatments.
Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.
Example 1. MMPl and Collagenolytic Activity is Elevated in Conditioned Media from Skin and Pubocervical Fascia Explants and Fibroblasts from Women with Stress Urinary Incontinence.
A major objective of this study was to demonstrate that weakened pelvic floor support of the lower genitourinary tract in women with stress urinary incontinence (SUI) is due to increased collagenolysis in the endopelvic fascia and that increased collagenase activity is a systemic phenomenon that could be measured in other tissues.
Skin and endopelvic fascia biopsies were obtained from postmenopausal women undergoing vaginal surgery. The experimental group included women with videourodynamic evidence of SUI undergoing bladder neck suspension surgery (n=33). The age range of these women was 52-80 years old (mean = 65). The control group consisted of continent women without evidence of pelvic floor weakening undergoing laparoscopic-assisted vaginal hysterectomy for symptomatic uterine fibroids, endometrial cancer, or benign ovarian pathology (n=23). The age range of these women was 50-78 years old (mean = 57). Biopsies of the lower abdominal skin and pubocervical fascia were taken at the time of surgery and either fixed in formalin or immediately transferred to tissue culture media using sterile technique. Fixed tissue specimens were embedded in paraffin and thin-sectioned (10 μm). Sections of each specimen were stained in trichrome for detection of collagen. Adjacent sections were hematoxylin and eosin stained. Figure 1 demonstrates qualitative histopathological differences in collagen content of endopelvic fascia and/or skin were detected between women with SUI and continent women without evidence of pelvic floor weakening. Specimens taken from women with SUI had a qualitatively lower collagen content in both skin and endopelvic fascia compared to specimens taken from women without SUI. This is consistent with the observations of Rechberger et al. (73), who found a highly significant decrease in collagen content in fascial tissue from incontinent women, and Keane et al. (74), who demonstrated that nulliparous women with SUI had significantly lower collagen in their periurethral skin than continent controls.
Specimens transferred to sterile media were washed twice in sterile phosphate-buffered saline (PBS) divided, and placed individually in 1 ml Earle's Modified Eagles Medium (MEM) (Gibco), without serum, containing nonessential amino acids, L-glutamine, 100 mM vitamin E, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Explants were incubated at 37°C in a humidified environment of 5% C02.
Mean 1,10-phenanthroline inhibitable collagenase activity, as determined by soluble lysis assay and normalized to protein, in the 4 day conditioned media from skin explants was significantly higher in the SUI group (10.79 ± 2.71 U/mg protein) than in the control group (2.88 ± 1.12 U/mg protein) (Figure 2A). Collagenase activity in the conditioned media from pubocervical fascia explants was also higher in the SUI group (1.71 ± 0.69 U/mg protein) compared to the control group (1.25 ± 0.39 U/mg protein). The specificity of the soluble lysis assay for collagenase was confirmed in representative samples by SDS-PAGE fluorography (Figure 2B).
Similarly, immunoreactive MMPl levels, as determined by ELISA (Biotrak) and normalized to protein, were significantly (p<0.05) higher in the conditioned media (CM) from skin and endopelvic fascia explants from SUI women compared to controls (Figure 3). Mean collagenolytic activity, as determined by generation of free amino groups from succinylated collagen (75) was more than 5 fold higher in the CM from explants from women with SUI than that from the controls for both the endopelvic fascia (0.57 ± 0.08 vs. 0.11 ± 0.04 U/mg protein, pO.OOl) and the skin (0.86 ± 0.18 vs. 0.16 ± 0.09 U/mg protein, p<0.05) (Figure 4). The difference in values obtained with the two assay methods could be attributed to the difference in sample number (n) in the groups, difference in the substrate, and the difference in the product measured. The soluble lysis assay is specific for collagenolytic activity as defined by the ability of the enzyme to cleave triple helical collagen at a single point. In addition, this data represents activity subsequent to aminophenyl mercuric acetate (APMA) activation. However, the degradation of succinylated collagen was performed with CM that had not been APMA-activated and probably represents gelatinase activity due to the nature of the substrate and reaction conditions.
Taken together, these data demonstrate, by several different methods, that women with SUI have significantly higher collagenase secreted from skin and endopelvic fascia, compared to women without pelvic floor weakening. The fact that the difference was apparent in the skin suggests a systemic change.
Fibroblasts were cultured from the explants by standard techniques. Collagenase activity was determined in the serum-free four-day conditioned media (CM) by soluble lysis assay and normalized to protein. Mean collagenase activity in the skin fibroblast CM, determined by soluble lysis assay, was more than 6 fold higher in the SUI (30.25 ± 13.08 U/mg protein) compared to the control group (4 65 ± 1.43 U/mg protein). Collagenase activity in the CM from endopelvic fascia fibroblasts was also higher in the SUI group (10.83 ± 3.01 U/mg protein) compared to the control (6.46 ± 1.49 U/mg protein). Immunoreactive MMPl was more than 10 fold higher in the CM from skin fibroblasts from SUI women (1197.0 ± 367.1 ng/mg protein) compared to controls (92.6 ± 32.5 ng/mg protein). Immunoreactive MMPl was more than 5 fold higher in the conditioned media from endopelvic fibroblasts from SUI women (159.0 ± 62.6 ng/mg protein) compared to controls (26.6 ± 3.13 ng/mg protein). These data suggest that fibroblasts from women with weakened pelvic support, such that they have SUI, synthesize and secrete significantly more matrix metalloproteinase (MMP) 1 resulting in higher collagenase activity in their skin and endopelvic fascia compared to women of similar age without pelvic floor weakening. In addition, these data indicate that the elevation in collagenase synthesis and secretion, in fibroblasts from women with SUI compared to those from continent women, is maintained with cell culturing.
Example 2. The Steady-state Level of Transcripts Encoding Mmpl Is Not Different in
Fibroblasts from Women with Stress Urinary Incontinence Compared to Continent Women. A major objective of this study was to determine whether the higher levels of MMPl detected in the conditioned media (CM) of skin and endopelvic fascia explants and fibroblasts from women with SUI is due to increased transcription of the gene encoding the enzyme in the fibroblasts of these tissues.
Total RNA isolated from fibroblasts cultured from PC and skin biopsies from women with and without SUI, as described in Methods, was reverse transcribed using the Omniscript
Kit (Qiagen, Valencia, CA) with oligo-dT primers. Resulting cDNA was amplified by polymerase chain reaction (PCR) using the MMP-1 specific oligonucleotide primers, forward 5'-
CATCCAAGCCATATATGGACGTCC-3', reverse 5'-
TCTGGAGACTCAAAATTCTCTTCGT-3' (Clontech Laboratories, Palo Alto CA) (76) and the β-actin specific oligonucleotide primers, forward 5'- ATCTGGC ACCACACCTTCTAC AATGAGCTGCG-3 ' , reverse 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'. PCR amplifications of the cDNA (5 μl) were performed in a 50 μl reaction mixture (10 mM Tris CI, pH 9.0, 500 mM KC1, 2.5 mM MgCl2, 1 mM dNTPs, 2.5 units Taq polymerase (Promega, Madison, WI)) with 0.2 μM of each of the primers. PCR was carried out in a DNA thermal cycler (Perkin-Elmer Cetus) using the following conditions for 40 cycles: denaturation at 94°C for 1 min, annealing of primer at 55°C for 2 min, polymerization at 72°C for 3 min, in each cycle, with a final extension at 72°C for 7 min. PCR products (5 μl) were electrophoresed in 2% pre-cast E-gels (Invitrogen, Carlsbad, CA) at 70V for 30 min. MMP-1 (61 lbp) and β-actin (838bp) PCR products were visualized (Figure 5) and quantified by a UN digital imaging system Alpha Innotech Corp, San Leandro, CA). The mean peak area ratios (MMP-1/β-actin, MMP-1 /β-actin) were compared by Students' t-test.
MMP-1 expression in pubocervical fascia fibroblasts from SUI patients (n=19, 0.220 ± 0.059 SEM) versus control patients (n=17, 0.151 ± 0.044 SEM) was not significantly different. MMP-1 expression in skin fibroblasts from SUI patients (n=l 1, 0.189 ± 0.129 SEM) versus control patients (n=13, 0.565 ± 0.165 SEM) was not significantly different. These data suggest higher collagenolytic activity in tissues from women with SUI is not due to increased transcription of MMPl . Thus, the elevated MMPl in conditioned media (CM) of explants and fibroblasts from women with SUI is regulated subsequent to transcription.
Example 3. Excretion of Collagen Degradation Products Is Increased in Women with Stress
Urinary Incontinence.
A major objective of this study was to demonstrate whether weakened pelvic floor support of the lower genitourinary tract in women with stress urinary incontinence (SUI) is due to increased collagenolysis which could be measured by excretion of collagen degradation products in the urine.
When mature crosslinked fibrillar collagen is destroyed by collagenase, a small fragment of the molecule, a pyridinoline crosslink (PYD), is released and excreted into the urine (52). The pyridinoline crosslink is not found in immature or newly synthesized collagen and its excretion is not affected by dietary gelatin, non-collagen proteins, or change in collagen synthesis. Collagen breakdown can therefore be assessed by measurement of pyridinoline (PYD) crosslinks in the urine. In addition, degradation of collagen results in peptide fragments of various lengths which are excreted in the urine (53, 54). Therefore, degradation of collagen which has not been crosslinked can be assessed independently, by measurement of collagen derived peptides in the urine.
A 24 hr urine collection was obtained from 23 women with stress urinary incontinence (mean age 57.6 years old) and 39 women without urinary incontinence (mean age 47.4 years old). Aliquots of the 24 hr urine collections were stored frozen at -20°C until analysis. PYD in a single aliquot from each subject was assayed in duplicate using ELISA plates from Metra Biosystems, Inc. and absolute amounts determined from a standard curve constructed from standard PYD assayed simultaneously. Helical peptide l(I) 620-622 was assayed in triplicate by competitive enzyme immunoassay (Quidel Corp, CA) and absolute amounts determined from a standard curve constructed from standard helical peptide assayed simultaneously. Values were normalized to urinary creatinine.
The mean urine PYD concentration for women with stress urinary incontinence (110.8 ± 19.7) was not significantly different than that for women without stress urinary incontinence (85.2 ± 13.7). The mean urine concentration of helical peptide αl(I) 620-633 for women with SUI (78.3 ± 15.8 μg/mg creatinine) was significantly (p<0.05) higher than that for women without SUI (48.4 ± 5.6 μg/mg creatinine). No correlation between age and concentration of collagen degradation products in urine was demonstrated in either group.
These data suggest that collagenolytic activity in women with SUI is elevated compared to continent controls, as measured by urinary helical peptide αl(I) 620-633 excretion. The lack of difference in urinary PYD excretion between the two populations suggests that the increased collagenolytic activity in women with SUI, compared to continent controls, is restricted to uncrosslinked collagen. That is, increased collagenolysis of mature fibrillar collagen does not significantly contribute to SUI. Degradation of nascent collagen is not detected by measuring PYD.
Example 4. Collagen Synthesis Is Similar in Women with and without SUI.
A major objective of this study was to determine whether weakened pelvic floor support of the lower genitourinary tract in women with stress urinary incotinence is due, in part, to decreased collagen synthesis and secretion and or an altered ratio of collagen III/I synthesis by the fibroblasts of the endopelvic fascia and skin compared to that of women without evidence of pelvic floor weakening.
Endopelvic fascia and skin biopsies were obtained from women with SUI (n=14) and women without evidence of SUI or genital prolapse (n=12). Fibroblast cultures were established from the biopsies by standard techniques. Cells were incubated with 3H-proline in medium containing ascorbic acid. The radiolabeled collagens were precipitated and digested with pepsin. The percent collagen synthesized (as a percent of total protein) was determined (77). Collagen synthesis, expressed as percent of total protein synthesis, was not significantly different between fibroblasts obtained from women with or without SUI (Figure 6). Collagen α 1 (III) was separated from collagen αl(I) and α2(I) by SDS-PAGE according to the technique of interrupted electrophoresis (78). Samples of highly purified collagen I and collagen III were run on each gel as internal controls. The gels were fixed and processed for autoradiography in order to determine the position of the bands (Figure 7A). Amount of 3H-proline in each band was determined by liquid scintillation counting. The mean of collagen III/I synthesized in fibroblasts was not significantly different between fibroblasts obtained from women with or without SUI (Figure7B).
These data suggest that the rate of collagen synthesis and the ratio of collagen III/I synthesized is not different between women with SUI and women without SUI. The observation that collagen content of connective tissue is lower in women with SUI compared to women without SUI is not likely to be attributable to differences in collagen synthesis.
Example 5. Plasma Proteolytic Activity Is Elevated in Patients with Stress Urinary Incontinence. A recently developed method for determining collagenolytic and elastolytic activity in plasma (75), was employed in this study. This assay is based upon the principal that free amino groups, generated during proteolytic digestion of substrates derivatized to block pre-existing amino groups, can react with trinitrobenzene sulfonic acid (TNBSA) to produce a chromophore which is detected spectrophotometrically. Plasma components which interfere with other methods of determination of elastolytic activity do not interfere with this assay. Therefore, this assay can be used to detect low levels of active elastolytic, as well as other proteolytic, activity in samples such as blood or plasma (See U.S. patent # 6, 197,537).
Plasma was prepared from blood taken from women with SUI (63.8 ± 2.5 years old; n=33) and age-matched women without evidence of SUI or pelvic organ prolapse (POP) (61.0 ± 2.7 years old; n=33). Diagnoses of SUI were made by physical and urodynamic evaluation. Plasma (10 μl) was added to an assay mixture containing succinylated collagen or elastin substrate to initiate the reaction, in order to measure plasma collagenolytic or elastolytic activity, respectively.
The collagenolytic activity in the plasma of women without SUI was 0.13 ± 0.03 U/ml, while that of age-matched women with SUI was 0.67 ± 0.08 U/ml (p<0.001) (Figure 8). The elastolytic activity in the plasma of women without SUI was 0.28 ± 0.06 U/ml, while that of age- matched women with SUI was 1.07 ± 0.07 U/ml (p<0.001) (Figure 8).
These data suggest that SUI patients have significantly elevated plasma proteolytic activity compared to age-matched women without SUI. Degradation of succinylated collagen probably represents gelatinolytic activity rather than collagenolytic activity. Further characterization of the enzymatic activities are necessary to identify the specific enzymes which have elevated activity in the plasma of women with SUI. In addition, these results represent net activity and may be the net result of both increased activated enzyme levels and/or decreased levels of inhibitors. Nonetheless, elevated proteolytic activity, in these women, may result in increased collagen and elastin degradation in connective tissues supporting the bladder and urethra and, thus, contribute to the development of SUI. Both advancing age and estrogen deficiency has been implicated in the development of
SUI in postmenopausal women (79). Estrogen replacement is thought to improve symptoms of SUI in this population. As estrogen is also involved in the regulation of expression of several collagenolytic enzymes, we hypothesized that a reduction in plasma estrogen would be associated with elevated proteolytic activity in women with SUI. Plasma was analyzed for 17-β-estradiol (the major estrogenic hormone) by ELISA (DRG Diagnostics). Plasma 17-β-estradiol in women with (52.43 ± 5.59 pg/ml) and without SUI (49.91 ± 6.38 pg/ml) was not significantly different, but demonstrated a similar significant (p<0.001) degree of negative correlation (as determined by Pearson Product Moment Correlation) with age in both groups. There was no correlation between plasma proteolytic activity and either age or 17-β-estradiol in either group. These data suggest that the age-associated decrease in circulating estrogens is similar in women with and without SUI. However, there is no correlation between circulating estrogen and elevated plasma proteolytic activity demonstrated in women with SUI.
Example 6. Elastolytic activity in the Conditioned Media from Skin and Pubocervical Fascia Explants From Women with Stress Urinary Incontinence (SUI). A major objective of this study was to determine whether weakened pelvic floor support of the lower genitourinary tract in women with stress urinary incontinence (SUI) is due to increased elastolysis in the endopelvic fascia and whether increased elastolytic activity is a systemic phenomenon that could be measured in other tissues.
Conditioned media (CM) from explant cultures was prepared as described in Example 1. Elastolytic activity in the conditioned medium was determined by the generation of free amino groups from succinylated elastin (75). Briefly, conditioned medium (5 μl) was added to an assay mixture (150 μl) containing 200 μg succinylated elastin in 50 mM phosphate buffered saline (PBS), pH 8.5, and incubated for 0-5 min at 37°C The reaction was stopped by the addition of trinitrobenzene sulfonic acid (TNBSA; 50 μl of a 0.03% soution in sodium borate, pH 8.5) and incubated at room temperature for 20 min. A450nm was determined. All reactions were carried out in triplicate in a 96 well plate. One unit (U) of activity is defined as the amount of activity to generate 1 μmol of digest product per minute at 37°C
Mean elastolytic activity was significantly higher in the CM from explants from women with SUI than that from the controls for both the endopelvic fascia (0.69 ± 0.10 vs. 0.05 ± 0.02 U/mg protein, p<0.0001) and the skin (0.89 ± 0.14 vs. 0.08 ± 0.03 U/mg protein, p<0.005) (Figure 9). This elastolytic activity was distinguishable from the collagenolytic activity by inhibition with α-1-antitrypsin. α-1-antitrypsin (1.45 μM) inhibited the elastolytic activity by 92 ± 4% and collagenolytic activity by 3 ± 2%. This suggests that elastolytic activity and collagenolytic activity are due to at least two different enzymes, distinguishable by α-1- antitrypsin inhibition.
As SUI is not associated with an inflammatory infiltrate, a potential source of the elastolytic activity is resident fibroblasts. In order to characterize the elastolytic activity secreted by skin and endopelvic fascia biopsies, the steady state level of human serine protease type elastase transcripts in cultured skin and endopelvic fascia fibroblasts was determined using reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR, using primers for the conserved exon 4 and 5 regions for the serine proteases human neutrophil elastase, pancreatic elastase, proteinase 3, azurucidin and adipsin (forward 5'-CTCAACGGGTCGGCCACC-3'; reverse 5'GTGGGTCCTGCTGGCCGG-3') (80), failed to detect transcripts in fibroblasts from women with or without SUI, although they were detected in other cell lines (Figure 10).
The elastolytic activity activity secreted into the CM by skin and pubocervical fascia fibroblasts was optimum at pH 8.5, inhibited 92 ± 4% by α-1-antitrypsin (1.45 μM), 97 ± 3% 1,10-phenanthroline ( 1 mM) and
75 ± 12% EDTA (10 mM), but was inhibited by only 15 ±15% by phenylmethyl-sulfonyl fluoride (PMSF; ImM), 18 ± 8% by elastinal (1.45 μM), 0-5% by TIMPs (1,2,3 and 4 at 50nM (Oncogene Research Products, Cambridge, MA)). Matrix metalloproteinase- 12 (MMPl 2), a macrophage elastase, could not be detected by Western analysis of the CM using a specific polyclonal antibody directed against the amino terminal end of human MMP-12 (Oncogene Research Products, Cambridge, MA)(data not shown). Transcripts encoding MMP12 were not detected by RT-PCR, using two different primer sets: set 1 (sense, 5'-TTCCCCTGAACAGCTCTACAAGCCTGGAAA-3'; antisense, 5'- GATCCAGGTCCAAAAGCATGGGCTAGGATT-3'); set 2 (forward 5'- TCACGAGATTGGCCATTCCTT-3', reverse 5'-TCTGGCTTCAATTTCATAAGC-3'), under the conditions described in Example 2; although transcripts were detected in PMA- stimulated U937 cells (data not shown). Matrix metalloproteinase-9 (MMP-9), an MMP with known elastolytic activity, could not be detected by Western analysis of the CM using a specific monoclonal antibody (Oncogene Research Products, Cambridge, MA); although standard MMP- 9 was detected (data not shown).
Zymography using elastin substrate revealed two bands, at approximately 48 and 58 kDa, with elastolytic activity (Figure 11A). This elastolytic activity is inhibited by EDTA (10 mM) (Figure 1 IB). This is characteristic of matrix metalloproteinases, but not serine elastases, as demonstrated. MMP-12, -9, and -2 have elastolytic activity under the conditions of zymography, as does the serine elastase, pancreatic elastase (Figure 11A). However, EDTA inhibits the elastolytic activity of the MMPs, but not the serine elastase, as expected (Figure 11B). The elastolytic enzymes in the CM of pubocervical explants from women with SUI appear to have molecular weights of 48 and 58 kDa (Figure 11A), indicating that at least two elastolytic enzymes are produced. The elastolytic activity at both 48 and 58 kDa, in the CM, is inhibited by EDTA (Figure 1 IB).
Taken together, the above data indicates that the elastolytic activity produced by skin and endopelvic fibroblasts from women with SUI is due to at least two proteins. The biochemical characteristics of this enzymatic activity suggest that it is similar to matrix metalloproteinases in that it is inhibited by the zinc chelators, EDTA and 1,10-phenanthroline. However, this activity is distinguished from known MMPs in that it is not inhibited by the TIMPs. The enzymatic activity is also similar to serine protease type of elastases in that it is inhibited by α-1-antitrypsin. However, sizes of the elastolytic enzymes from the CM are generally larger than serine proteases with elastolytic activity. In addition the elastolytic activity in CM is not very sensitive to elastinal inhibition. Example 7. Screening for Stress Urinary Incontinence by Elastase Activity
Introduction and Objective: Development of SUI has been associated with parity, obesity, aging and hormonal loss. Increased proteolytic activity manifested by elevated plasma collegenolytic and elastolytic activity has been demonstrated in patients undergoing SUI surgery when compared to controls. That elastolytic activity is elevated in the plasma of women with SUI was tested in a random population-based screening study.
Material and Methods: Plasma was assayed for elastase activity, by the method of Rao, et al, in 252 females over the age of 21 years being screened for presence of human papilloma virus (HPV) and carcinoma of cervix in a village in southern India. Detailed questionnaires included questions related to presence or absence of SUI. Physical examination and PAP smear was performed in 214 of 252 attendees. (Post hysterectomy and patients menstruating were excluded.)
Results: Analysis of the questionnaires and physical examination revealed 52/252 women with SUI. 124 patients were excluded from analysis due to conditions known to affect proteolytic activity (AIDS, tuberculosis, chronic liver diseases, etc). 76 subjects served as controls. Elastolytic activity in the plasma of women with SUI (5.56 ± 0.20 U/ml) was significantly (p<0.05) greater than women without SUI (4.01 ± 0.15 U/ml).
Conclusion: Elastolytic activity is significantly higher in subjects identified as having SUI when compared to controls in a random population-based screening study. A larger study is needed to confirm whether plasma elastolytic activity can be used to detect SUI in women.
In view of the above, it will be seen that the several advantages of the invention are achieved and other advantages attained.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

Claims

What is claimed is:
1. An isolated human elastase having a pH optimum of about 8.5, which is inhibited by α-1-antitrypsin, 1,10-phenanthroline and EDTA, but not phenylmethyl-sulfonyl fluoride.
2. The isolated human elastase of claim 1, having an apparent molecular weight of about 48 kDa as determined by zymography on a 1% SDS-9% polyacrylamide gel containing 1 mg/ml elastin under non-reducing conditions and without boiling.
3. The isolated human elastase of claim 1, having an apparent molecular weight of about 58 kDa as determined by zymography on a 1% SDS-9% polyacrylamide gel containing 1 mg/ml elastin under non-reducing conditions and without boiling.
4. The isolated human elastase of claim 1, wherein the elastase is produced by a fibroblast.
5. The isolated human elastase of claim 4, wherein the fibroblast is from skin or endopelvic fascia.
6. The isolated human elastase of claim 4, wherein the fibroblast is from a woman having stress urinary incontinence.
7. The isolated human elastase of claim 6, wherein the woman is post-menopausal.
8. The isolated human elastase of claim 6, wherein the woman has an elevated concentration of the elastase.
9. The isolated human elastase of claim 8, wherein the elastase concentration is elevated in blood of the woman.
10. The isolated human elastase of claim 8, wherein the elastase concentration is elevated in skin or pubocervical fascia of the woman, or in media conditioned with skin or pubocervical fascia explants of the patient. -sel l . An antibody or antibody fragment that specifically binds to the isolated human elastase of claim 1.
12. The antibody or antibody fragment of claim 11, which is a monoclonal antibody.
13. The antibody or antibody fragment of claim 11, which is a polyclonal antibody.
14. An aptamer that specifically binds to the isolated human elastase of claim 1.
15. The aptamer of claim 14, wherein the aptamer is an RNA aptamer.
16. A method of determining whether a woman has, or is likely to develop, stress urinary incontinence, the method comprising determining for the woman a level of an elastase having a pH optimum of about 8.5, which is inhibited by α-1-antitrypsin, 1,10-phenanthroline and EDTA, but not phenylmethyl-sulfonyl fluoride, wherein an elastase level elevated above an average elastase level of a group of women that do not have stress urinary incontinence indicates that the woman has stress urinary incontinence.
17. The method of claim 16, wherein the elastase has an apparent molecular weight of about 48 kDa, as determined by zymography on a 1% SDS-9% polyacrylamide gel containing 1 mg/ml elastin under non-reducing conditions and without boiling.
18. The method of claim 16, wherein the elastase has an apparent molecular weight of about 58 kDa, as determined by zymography on a 1% SDS-9% polyacrylamide gel containing 1 mg/ml elastin under non-reducing conditions and without boiling.
19. The method of claim 16, wherein the woman is postmenopausal.
20. The method of claim 16, wherein the elastase levels are measured in blood or a blood fraction.
21. The method of claim 16, wherein the elastase levels are measured in media conditioned with skin or pubocervical fascia explants.
22. The method of claim 16, wherein an elastase level in the woman that is at least twice the average elastase level of the group of women indicates that the woman has stress urinary incontinence.
23. The method of claim 16, wherein an elastase level in the woman that is at least three times the average elastase level of the group of women indicates that the woman has stress urinary incontinence.
24. The method of claim 16, wherein an elastase level in the woman that is at least four times the average elastase level of the group of women indicates that the woman has stress urinary incontinence.
25. The method of claim 16, wherein an elastase level in the woman that is at least two standard deviations above the average elastase level of the group of women indicates that the woman has stress urinary incontinence.
26. The method of claim 16, wherein an elastase level in the woman that is at least three standard deviations above the average elastase level of the group of women indicates that the woman has stress urinary incontinence.
27. The method of claim 16, wherein an elastase level in the woman that is at least four standard deviations above the average elastase level of the group of women indicates that the woman has stress urinary incontinence.
28. A method of treating stress urinary incontinence in a woman, the method comprising inhibiting an elastase in a pubocervical fascia of the woman, wherein the elastase has a pH optimum of about 8.5 and an apparent molecular weight of about 48 kDa or about 58 kDa as determined by zymography on a 1% SDS-9% polyacrylamide gel containing 1 mg/ml elastin under non-reducing conditions and without boiling.
29. The method of claim 28, wherein the elastase is inhibited by contacting the pubocervical fascia of the woman with at least one inhibitor selected from the group consisting of α-1-antitrypsin, 1,10-phenanthroline and EDTA.
30. The method of claim 28, wherein the elastase is inhibited by contacting the pubocervical fascia of the woman with at least one inhibitor selected from the group consisting of an antibody and an aptamer.
31. A method of preventing or treating, in a patient, a disease characterized by undesired elastin degradation in the patient, the method comprising inhibiting an elastase in the patient, wherein the elastase has a pH optimum of about 8.5 and an apparent molecular weight of about 48 kDa or about 58 kDa as determined by zymography on a 1% SDS-9% polyacrylamide gel containing 1 mg/ml elastin under non-reducing conditions and without boiling.
32. The method of claim 31, wherein the elastase is inhibited by contacting the elastase with at least one inhibitor selected from the group consisting of α-1-antitrypsin, 1,10- phenanthroline and EDTA.
33. The method of claim 31, wherein the elastase is inhibited by contacting the elastase with at least one inhibitor selected from the group consisting of an antibody and an aptamer.
34. The method of claim 31, wherein the disease is SUI, the patient is a woman, and the elastase is in the pubocervical fascia of the woman.
35. The method claim 31 , wherein the disease is selected from the group consisting of pelvic organ prolapse, emphysema, abdominal aortic aneurysm, atherosclerosis, pancreatitis, and an inflammatory disease.
36. A method of determining whether a compound is an elastase inhibitor, the method comprising contacting the compound with the elastase of claim 1, and determining whether the elastase is inhibited.
37. The method of claim 36, wherein the compound is selected from the group consisting of a protein, a peptide, a small molecule, an oligonucleotide, and a chelator.
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WO2006016353A3 (en) * 2004-08-10 2006-07-13 Yeda Res & Dev Elastase inhibitor in leukemia
US7740576B2 (en) 2005-04-05 2010-06-22 Ams Research Corporation Articles, devices, and methods for pelvic surgery
US9060839B2 (en) 2005-07-26 2015-06-23 Ams Research Corporation Methods and systems for treatment of prolapse
US9084664B2 (en) 2006-05-19 2015-07-21 Ams Research Corporation Method and articles for treatment of stress urinary incontinence
US10271936B2 (en) 2006-06-16 2019-04-30 Boston Scientific Scimed, Inc. Surgical implants, tools, and methods for treating pelvic conditions

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* Cited by examiner, † Cited by third party
Title
CHEN ET AL. NEUROLOGY AND URODYNAMICS vol. 26, no. 2, 2007, pages 274 - 279 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006016353A3 (en) * 2004-08-10 2006-07-13 Yeda Res & Dev Elastase inhibitor in leukemia
US7740576B2 (en) 2005-04-05 2010-06-22 Ams Research Corporation Articles, devices, and methods for pelvic surgery
US7811223B2 (en) 2005-04-05 2010-10-12 Ams Research Corporation Articles, devices, and methods for pelvic surgery
US8801595B2 (en) 2005-04-05 2014-08-12 Ams Research Corporation Articles, devices, and methods for pelvic surgery
US9060839B2 (en) 2005-07-26 2015-06-23 Ams Research Corporation Methods and systems for treatment of prolapse
US9084664B2 (en) 2006-05-19 2015-07-21 Ams Research Corporation Method and articles for treatment of stress urinary incontinence
US10271936B2 (en) 2006-06-16 2019-04-30 Boston Scientific Scimed, Inc. Surgical implants, tools, and methods for treating pelvic conditions

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