Patent Application: US-68929100-A

Abstract:
methods are disclosed for diagnosing urinary incontinence or a predisposition to urinary incontinence in a subject by determining the ratio of expression levels of mmps / timps in pelvic supporting tissue of the subject and comparing the ratio to a predetermined indicator . a diagnostic kit is also disclosed . methods are disclosed for treating urinary incontinence by reducing proteolysis of collagen in pelvic supporting tissue of the subject and for identifying therapeutic agents in in vitro screens .

Description:
it should be appreciated by those skilled in the art that while the experimental data is directed towards stress urinary incontinence (“ sui ”) in female subjects , the invention is intended to encompass urinary incontinence and accompanying conditions in both males and females which involves abnormal collagen metabolism causing increased collagen degradation in pelvic supporting tissues . the embodiments of the invention have utility in veterinary medicine as well as human medicine . the examples below are intended to describe the experimental support for the invention , but not to limit the scope of the invention in any way . the methods of the present invention are based on the inventors &# 39 ; discovery that urinary incontinence in women is related to increased extracellular matrix proteolysis by matrix metalloproteinases . experimental support for this discovery , and the methods disclosed herein , are explained in the experiments described below . the experiments were designed to identify the presence of mmp - 1 mrna in vaginal tissue from incontinent and control , continent women , and to measure quantitative mrna expression of mmp - 1 , mmp - 2 and mmp - 9 and their inhibitors , timp - 1 , timp - 2 and timp - 3 ( 41 , appendix a ). the gelatinases were examined because mmp - 2 and mmp - 9 in combination with mmp - 1 degrade the major pelvic collagens completely to amino acids and are primarily inhibited by timp - 2 and timp - 3 , respectively . the results from these experiments document the presence of mmp - 1 mrna in vaginal tissue from continent and incontinent women ( fig1 ) and show that the ratio of mmp - 2 / timp - 2 mrna expression is significantly ( p & lt ; 0 . 04 ) higher in women with stress urinary incontinence ( sui ) compared to control women with no urinary dysfunction ( fig2 ). other studies comparing the ratio of mmp - 1 / timp - 1 and mmp - 9 / timp - 3 mrna expression also show higher ratios in women with sui compared to control women ( fig3 fig4 ). the mmp - 1 / timp - 1 ratio has been found to be significantly higher ( p = 0 . 04 ) in the stress incontinent group compared to controls . in addition , the total collagen content of vaginal cuff tissue ( fig5 ) from women with sui is lower when compared to normal women , as are pyridinium crosslinks ( fig6 ), consistent with an elevated mmp / timp ratio . these data suggest that women with sui may have increased or abnormal collagen proteolysis resulting from increased expression of matrix metalloproteinases or decreased expression of timps in pelvic tissue , and thus provide a a molecular pathophysiologic explanation for sui . vaginal cuff tissue was obtained from women undergoing hysterectomy and surgery for sui . tissue was obtained from the anterior vaginal cuff of three premenopausal women ( ages 36 - 52 ) with normal estrogen levels and one postmenopausal woman ( age 75 ) on a standard dose of hormone replacement therapy , so that all patients were estrogen - replete . cuff tissue was taken from the anterior vaginal surface at the time of hysterectomy and placed immediately in ice cold pbs solution in the operating room to remove contaminating blood . after surgical removal of vaginal epithelium in the laboratory , tissue was processed immediately for rna extraction . all women underwent urodynamic studies including a cystometrogram to confirm adequate bladder compliance and size as well as the absence of detrusor instability . all women had sui secondary to hypermobility of the bladder neck . all women were evaluated on physical examination and found to have moderate to severe sui of greater than one year duration requiring the daily wearing of pads with a positive cough stress test , leakage of urine , and moderate prolapse with vault desensus and a cystocele . all women were parous with 1 to 7 vaginal births . women were treated with a variety of procedures including hysterectomy with anterior - posterior repair and pubovaginal sling , depending on the degree of stress incontinence . control samples of anterior vaginal cuff tissue were taken from premenopausal women ( ages 37 - 46 ) with normal estrogen levels undergoing hysterectomy for either dysfunctional bleeding or fibroids . women with pelvic inflammatory disease , endometriosis , or malignant lesions were excluded from the study . the extraction of rna from the tissue samples was carried out with the rna - stat - 60 reagent ( tel - test inc ., friendswood , tex .) ( 28 ). briefly , tissue samples were washed three times in pbs ( gibco brl , grand island , n . y .) to remove blood contamination . one hundred milligrams of tissue were homogenized in 1 ml of rna - stat - 60 reagent . total rna was separated from dna and proteins by adding chloroform and was precipitated using isopropanol . the precipitate was washed two times in 75 % ethanol , air - dried , and rediluted in diethylpycocarbonate ( depc )- treated dh 2 o . amount and purity of extracted rna was quantitated by spectrophotometry in a genquant rna / dna calculator ( pharmacia biotech ltd ., cambridge , uk ). in order to measure the quantitative expression of mmp - 1 , mmp - 2 , mmp - 9 , timp - 1 , timp - 2 , and timp - 3 mrna , the technique of quantitative competitive reverse transcription polymerase chain reaction ( qc - rt - pcr ) was chosen . rt - pcr can be used to analyze low abundance mrnas derived from cells or tissue and is a well established technique whose sensitivity provides a major advantage . quantitative analysis of these messages can be achieved by a modification known as qc - pcr ( 40 , 42 ), in which an internal control molecule possessing a small deletion in the amplified portion of the specific molecule is amplified simultaneously with the target sample instead of another control molecule such as β - actin or rrna . because the efficiency of the amplification of the internal control molecule is identical to that of the target template , quantitative pcr avoids discrepancies associated with tube - to - tube or sample - to - sample variations in the kinetics of the rt reaction ( 42 , 43 ). as quantitative pcr is based on the competitive status between the target molecules and the internal standard molecules within the same pcr reaction , the relative amount of each product expressed is determined as a ratio of target to internal standard molecules . differences as small as 20 % between two samples can be determined with an accuracy of 95 % ( 44 ). 3 . primers for reverse transcription ( rt ) and polymerase chain reaction ( pcr ) specific sequences of oligonucleotide primers for mmp - 9 , timp - 1 , and timp - 3 were obtained from gene bank database of the national center for biotechnology information ( ncbi ) of the national institutes of health ( nih , internet address : http :// www . ncbi . nlm . nih . gov ). one corresponding set of primers for mmp - 9 , timp - 1 and timp - 3 was found with the help of the program oligo 5 . 0 primer analysis software ( national bioscience , plymouth , minn .) and synthesized by the “ protein , aminoacid and nucleicacid ( pan )- facility ,” beckman center , stanford university , stanford , calif . sequences for oligonucleotide primers for mmp - 2 and timp - 2 were obtained from the breast cancer literature ( 24 ). primers for the interstitial collagenase mmp - 1 were identified ( 45 ) and synthesized in the beckman center pan - facility . the human β - actin primers that were used to amplify an external standard were obtained from clontech laboratories , inc ., palo alto , calif . β - actin mrna expression was employed as an external positive control and was detected in all the samples studied , thus confirming the integrity of the rna and the rt - pcr process . for rt - pcr , the gen amp rna pcr kit ( perkin - elmer , foster city , calif .) was used . nineteen microliters of rt - mastermix for each sample were prepared containing 5 mmol / l mgcl 2 , 1 × pcr buffer ii , 1 mmol / l of each deoxy - ntp , 2 . 5 μl / l oligo ( deoxythymidine ) 16 , 20 iu ribonuclease inhibitor ( all perkin - elmer ), and 100 iu moloney murine leukemia virus reverse transcriptase ( gibco brl ), and 1 μg total rna diluted in 1 μl depc - treated h 2 o and filled into 0 . 5 ml thin wall pcr tube ( applied scientific , south san francisco , calif .). rt - mastermix in pcr tubes was covered with 50 μl of light white mineral oil and kept on ice until the rt . rt was carried out in the dna thermal cycler 480 ( perkin - elmer ) using a program with the following parameters : 42 ° c ., 15 min . ; 99 ° c ., 5 min . ; then quenched at 4 ° c . after the reaction completed , samples were stored at − 20 ° c . until the pcr . as negative control , 1 μl depc - treated h 2 o without rna sample was subjected to the same rt reaction . a 440 base pair ( bp ), 473 bp , 590 bp , 969 bp fragment of native mmp - 2 , mmp - 9 , timp - 2 , and timp - 3 cdna ( the target ) ( table 1 ) was obtained by pcr amplification of reverse - transcribed total rna from vaginal cuff samples with the regular 3 ′ and 5 ′ primers . the pcr product was visualized by agarose gel electrophoresis stained with ethidium bromide ( etb ), and the cdna was extracted from the gel , purified with an agarose gel extraction kit ( amersham pharmacia biotech ltd ., arlington heights , ill . ), and quantitated by spectrophotometry ( pharmacia biotech ltd ., cambridge , uk ). to construct a competitive cdna fragment : a floating primer with a sequence complementary to the cdna between the 3 ′ and 5 ′ primer binding sites was designed by attaching the complementary sequence of the binding site of the original 3 ′- end mmp - 2 , mmp - 9 , timp - 2 , and timp - 3 primer ( table 1 ). after pcr with the regular 5 ′- end primer and the 3 ′- end floating primer , the pcr product was visualized by agarose gel electrophoresis stained with ethidium bromide , cdna extraction , purification and determination of the concentration performed as described above . this step resulted in cdna fragments of mmp - 2 ( 174 bp ), mmp - 9 ( 196 bp ), timp - 2 ( 227 bp ), and timp - 3 ( 464 bp ). primers for mmp - 1 ( table 1 ) were identified to yield a 180 bp fragment on rt - pcr analysis . the standard curves for mmp - 1 , mmp - 2 , mmp - 9 , timp - 2 , and timp - 3 were constructed by a coamplification of a constant amount of competitive cdna with declining amounts of target cdna obtained by serial dilution ( 28 ). sixty microliters of the cdna mix were added to 40 μl pcr - mastermixture containing 1 . 9 mm mgcl 2 solution , 10 × pcr buffer ii , 0 . 2 mm each dntp , 2 . 5 u taq - polymerase ( all perkin - elmer ), corresponding paired primers in a concentration of 0 . 2 μmol / l of each primer to a total volume of 100 μl and 14 . 5 μl depc - treated h 2 o . the reaction was covered with 50 μl light white mineral oil and put in the perkin - elmer dna thermal cycler 480 . pcr cycles were composed of 1 cycle at 95 ° c . for 5 min . to denature all proteins , 30 cycles at 94 ° c . for 45 sec ., at 55 ° c . for 45 sec ., and at 72 ° c . for 60 sec . for mmp - 9 and timp - 3 . pcr was performed in 35 cycles including 2 min . denaturation at 94 ° c ., 1 min . primer annealing at 56 ° c ., and 2 min . extension at 72 ° c . for mmp - 2 and timp - 2 . the reaction was terminated at 72 ° c . for 6 min . and was quenched at 4 ° c . pcr conditions for mmp - 1 amplification were 30 cycles at 94 ° c . for 1 minute , 50 ° c . for 1 minute , and 72 ° c . for 3 minutes , followed by reaction termination at 72 ° c . for 6 minutes and quenching at 4 ° c . two percent agarose gel ( life technologies , grand island , n . y .) electrophoresis was carried out in a h5 electrophoresis chamber . gels were stained with ethidium bromide . aliquots ( 25 μl ) of each pcr product and dye buffer were analyzed in parallel with a 100 - bp dna ladder ( life technologies , grand island , n . y .) as a standard . after completion of electrophoresis , the gel blot was analyzed , and photocopies of the blot were printed by uv densitometry ( gel - doc 1000 system , bio - rad laboratories , hercules , calif .). the logarithmically transformed ratios of target cdna to competitive cdna were plotted against the log amount of initially added target cdna in each pcr to obtain the standard curve . fig7 and fig8 show the standard curves for mmp - 9 and timp - 3 and are representative of the standard curves obtained for mmp - 1 , mmp - 2 and timp - 2 . these standard curves were highly reproducible and linear . the values obtained from the regression line of the standard curves ( y = b + mx ) allowed us to calculate the amount of cdna transcripts in an unknown sample . therefore , competitive cdna was added to each unknown sample before pcr . the ratio of the densities of sample target cdna band to competitive cdna were logarithmically transformed and compared to the values obtained from standard curve to quantitative mrna expression in the experimental samples . fibroblast cultures are started from biopsy specimens of vaginal tissue using an explant method ( 46 ). ten small tissue samples of approximately 1 mm 3 are placed into 25 cm 2 of untreated plastic tissue culture flasks for primary explantation . after allowing tissue fragments to attach to the plastic surface for 15 minutes , 5 ml of culture medium consisting of 90 % dmem / 10 % fetal bovine serum , is added to the flasks . cultures are incubated at 37 ° c . in an atmosphere of 95 % air and 5 % co 2 and experiments performed on confluent cultures between passages 3 and 7 as described ( 46 ). 8 . tests of putative modulators ( e . g ., gonadal steroids , cytokines and growth factors ) time course studies of both mrna and protein expression in fibroblast cell cultures will determine an appropriate timeframe for these experiments which is expected to be 24 hours . dose response studies are then performed as follows . increasing concentrations of estrogen ( 0 - 10 − 5 m ), progesterone ( 0 - 10 − 5 m ), dihydro - testosterone ( 0 - 10 − 5 m ), interleukin - 1β ( 0 - 1 , 000 iu / ml ), and tgf - β ( 0 - 40 ng / ml ) are incubated with confluent human fibroblast cultures for 24 hours at 37 ° c . in an atmosphere of 95 % air / 5 % co 2 . after 24 hours of incubation , supernatants are isolated and cells lysed by rna - stat 60 . both supernatants and cell lysates are examined for mmp - 1 , mmp - 2 , mmp - 9 , timp - 1 , timp - 2 , and timp - 3 protein levels by elisa ( oncogene research , cambridge , mass .). cells are harvested and rna extracted as previously described ( 30 ). quantitative mrna expression of mmp - 1 , mmp - 2 , mmp - 9 , timp - 1 , timp - 2 , and timp - 3 is measured using qc - rt - pcr . in experiments examining the ability of il - β and tgf - β to modulate mrna and protein expression of mmps and timps , appropriate anti - il - 1β and anti - tgf - β antibodies are employed to reverse the effect and thus confirm specificity of the response . the antiprogestin , ru - 486 , is used to reverse the effect of progesterone on mmp and timp expression , thus confirming specificity of the response . vaginal cuff tissue is analyzed for collagen protein using a protease / acid methodology to determine hydroxyproline content for total collagen ( 47 ). conditioned medium from fibroblast cultures and the cell monolayers themselves are both analyzed for hydroxyproline to determine total collagen content ( 47 ). the specific antibodies for both type i and type iii collagens are available ( accurate chemical and scientific corp ., westbury , n . y .) and are used to determine protein concentrations in vaginal cuff tissue and fibroblast cultures from control and incontinent patients using western analysis ( 26 , 48 ). through a combination of qc - pcr and western analysis , the ratio of type i / type iii collagen in in vivo isolated vaginal cuff tissue and in vitro fibroblast cultures from control and incontinent patients is analyzed . vaginal cuff tissue is homogenized and treated with 6n hcl to degrade collagen and release pyridinoline crosslinks ( 49 ). total pyd and dpd crosslinks is assayed by elisa ( metra biosystems , mountain view , calif ., appendix e ) to determine the level of collagen crosslinking in tissue isolated from women with stress incontinence and control women . supernatants from fibroblast cultures are collected and concentrated . monolayer cultures are collected , treated with triton x - 100 , and both supernatants and cell homogenates are assayed for pyridinoline crosslinks by elisa ( metra biosystems , mountain view , calif .). both the three - quarter ( tc a ) and one - quarter ( tc b ) length collagen fragments are a primary measure of initial collagen cleavage ( 18 ). initial collagen cleavage of the triple helix generates a new carboxy - terminal end , col2 - 3 / 4c short to which antibodies have been developed by dr . robin poole . the level of the carboxy - terminal neoepitope is a direct measure of collagenase activity in osteoarthritic articular cartilage ( 18 ) and atheromatous plaques ( 23 . in vaginal cuff tissue harvested from incontinent and control women , immunohistochemistry is used to assess the location and intensity of staining for the carboxy - terminal neoepitope as a measure of collagenolysis . in addition , total collagen is extracted and the level of the carboxy - terminal neoepitope is quantitated by elisa . using cultured fibroblasts from vaginal wall tissue of continent and incontinent women , the level of carboxy - terminal neoepitope secreted into conditioned medium is determined using an elisa ( 18 ). in addition , collagen is isolated from the cell fibroblast layer to determine intracellular neoepitope content . fibroblast cultures from vaginal cuff tissue of premenopausal women with and without stress incontinence are established in untreated plastic dishes ( 46 ). between passage 3 and 7 , confluent cultures from each group of women are treated for an appropriate length of time with increasing concentrations of a putative therapeutic agent . ( e . g ., the roche mmp inhibitor rs 113 , 456 ). a suitable dosage range for this inhibitor is 1 μg / ml - 100 μg / ml in dmem . initial experiments are performed to determine the time course for inhibition of mmp activity by rs 113 , 456 . after addition of rs 113 , 456 to fibroblast cultures , the earliest period at which steady state mmp inhibition is reached is identified . that time is subsequently used as a standard time of incubation for dose response curves . cell supernatants are isolated and concentrated and fibroblast monolayers are homogenized using triton x - 100 . to measure the specific proteolytic inhibition produced by rs 113 , 456 , the mmp activities in supernatants and cell homogenates are examined by zymography with electrophoresis on premade gelatin gels ( biorad , hercules , calif .) ( 50 , 51 ). using antibody which recognizes the col2 - 3 / 4c short epitope ( 18 ), the level of collagenase activity is determined directly . while the present invention has been described with reference to the specific embodiments thereof , it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention . in addition , many modifications may be made to adapt a particular situation , material , composition of matter , process , process step or steps , to the objective spirit and scope of the present invention . all such modifications are intended to be within the scope of the claims appended hereto . all of the publications , patent applications and patents cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication , patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety . 1 . wall l l . birth trauma and the pelvic floor : lessons from the developing world . j women &# 39 ; 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