Patent Application: US-76547601-A

Abstract:
the usage of compounds that improve fetal and neonatal outcome of preterm birth is described . these compounds are scavengers of ros , their precursors , and agents that induce production of the scavengers . examples of these compounds are glutathione , nac , antioxidants , and spin trapping compounds . these compounds improve fetal outcome by inhibiting a fetal inflammatory process that may affect the fetus independently of prematurity . this fetal inflammatory response is characterized by increased cytokine and matrix metalloproteases levels both in the mother and fetus and may be modulated by ros at different levels . targeting ros formation with compounds such as specific antioxidants , glutathione or spin trapping compounds , their precursors , and / or agents which induce their production will suppress both the direct effects of ros and its indirect effects through cytokines and mmps already circulating in the system . this therapeutical intervention would limit the pathophysiologoical chain of events that ultimately leads to pprom , preterm birth and / or adverse fetal and neonatal outcome .

Description:
patients likely to benefit from the present invention are identified as either high risk or having an acute problem . high risk patients include but are not limited to : cocaine users , preeclamptic patients , patients with preterm labor , a history of preterm labor , or pprom . fetuses with intrauterine growth restriction are likely to benefit . acute problems include intrapartum hypoxia . possible routes of administration for free radical scavengers and other agents of the present invention are : to a mother — include oral , iv , sublingual , inhaled , rectal , vaginal , transmucosal , transcutaneous , intraamniotic , and to the fetus include intraamniotic or via cordocentesis . preferred dosages for effective amounts of the agent — oral regimen : in women at risk : 600 mg twice daily ( dose used by tepel et al ., new engl j med 2000 for prevention of renal damage ). in acute situations the patient will be given a loading dose of 140 mg / kg followed by 70 mg / kg every 3 - 4 hours ( the fda approved protocol for acetaminophen intoxication ). the drug can also be administered as a 5 % solution in cola or fruit juice . a ) effect of ros modulation in vitro on amniochorionic mmp activity iv dosage : 10 % n - acetyl cysteine in 5 % dextrose or other drugs such as 3 % procysteine ( l - 2 - oxothiazolidine - 4 - carboxylate ) administrated at 0 . 4 mmoles / kg body weight . drugs are administrated over 30 minutes , every 8 hours ( from bernard et al ., chest 1997 — for protective effect against adult respiratory distress syndrome ). intraamniotic via amniocententesis or amnioinfusion catheter : infused liquid contains 10 % buffered n - acetyl cysteine . ( no referencejust guess . this is an important route of administration to the fetus because the fetus swallows the amniotic fluid so it is equivalent to an oral administration to the fetus . the drug dosage and bioavailability to the mother and the fetus will indicate therapeutic effectiveness . kits or assays are envisioned that may measure , for example , nac ( or thiols — not only nac ) in maternal uterine , amniotic fluid , maternal or neonatal blood at delivery or postpartum may have commercial value . antioxidant compounds of the present invention include cysteine , glutathione , n - acetylcysteine , l - alpha - acetamido - beta mercaptopropionic acid , s - nitroso - gutathione , n - acetyl - 3 - mercapto - alanine , butylated hydroxyanisole , butylated hydroxytoluene , l - 2 - oxothiazolidine - 4 - carboxylate , vitamin c ( ascorbate ) and vitamin e ( tocopherol ). these may be used alone and in combination with each other and / or with desferrioxamine , allopurinol , superoxide dismutase and superoxide dismutase mimetics such as salen - manganese complexes ( see u . s . pat . no . 6 , 046 , 188 ). spin trapping compounds that can be used as ros scavengers include nitrones ( e . g ., phenyl - butyl nitrone , trimethoxyphenyl - butyl nitrone ), nitroxides and salicylates . spin trapping compounds combined with mri canbe used as a noninvasive method to detect free radical damage to the fetus or maternal reproductive tissues during pregnancy . tissues : full thickness amniochorionic membranes were collected from seven patients without complications of pregnancy undergoing elective c - section at term . the fragment collected was selected from a region at least 10 cm away from the placenta . immediately after collection , membranes were placed in minimun essential medium ( mem ) ( gibco , grand island , n . y .) on ice . tissue samples were then cut under sterile conditions with a scalpel blade into smaller pieces of similar size . over the next 30 min ., the pieces of membranes were rinsed thoroughly with several changes of mem and then sterile phosphate buffered saline ( pbs ) ph = 7 . 8 . preweighed amounts of tissue ( approximately 500 mg ) consisting of several pieces selected at random were placed in a co 2 incubator with a humidified chamber at 37 ° c . for 24 hours . incubations were carried out in a total volume of 2 ml buffer . both wet weight and dry weight of tissue pieces were determined after incubation and after drying overnight at 60 ° c ., respectively . after incubation the culture - conditioned medium was collected , briefly spun at 1000 g to remove cellular debris and loaded directly on gelatin gels . the concentration of protein in the aliquots of medium was measured with a bca kit ( pierce , rockford , ill .). drugs and incubations : incubations were performed in the presence of xanthine ( x : 2 mm ), xanthine oxidase ( xo : 20 mu / ml ), the combination xanthine + xanthine oxidase ( x / xo ), superoxide dismutase ( sod : 500 u / ml ), x / xo / sod , nitro - l - arginine ( a nonspecific nitric oxide synthase inhibitor , lna : 1 mm ), x / xo / lna , s - nitroso - n - acetyl penicillamine ( a nitric oxide donor , snap 10 mm ), x / xo / snap , n - acetyl cysteine ( nac : 0 . 1 , 1 , 10 mm ), lipopolysaccharide ( lps 100 ng / ml ). all chemicals were purchased from sigma ( st . louis , mo .) unless otherwise specified . extracellular superoxide anion generation : o 2 − was generated enzymatically from the reaction between 2 mm xanthine ( x ) and 20 mu / ml xanthine oxidase ( xo ), as described by mccord and fridovich ( 1969 ). though these amounts are larger than those routinely used for in vitro generation of o 2 − in cell - free experiments , we have based our decision on the presence of both xo and sod in fetal membranes ( telfer et al ., 1997 ) and on our assumption that the expression of these enzymes will change the efficiency of the x / xo reaction . as a result we monitored the efficiency of the x / xo reaction in our experimental setting by a cytochrome c assay , both in tissue - free conditions as well as in the presence of the tissue ( babior et al ., 1973 ). the reduction of ferri - to ferro - cytochrome c is a process highly selective for extracellular o 2 − and associated with a change in absorbance at 550 nm within 15 min . as proof for the specificity of the reaction , sod ( 500 u / ml ) completely inhibited the change in absorbance at 550 nm . intracellular superoxide anion generation : pieces of fetal membranes were immersed in krebs bicarbonate buffer ( 118 mm nacl , 4 . 7 mm kcl , 1 . 18 mm mgso 4 , 1 . 18 mm kh 2 po 4 , 11 . 1 mm d - glucose , 0 . 016 mm edta , 2 . 2 mm cacl 2 , 15 . 8 mm nahco 3 , ph = 7 . 35 - 7 . 4 ) containing 1 mg / ml nitroblue tetrazolium ( nbt ), for 180 min . in the co 2 incubator with humidified chamber at 37 ° c . the soluble yellow form of nbt is reducedby o 2 . − generated within the tissue to form an insoluble precipitate of blue - purple formazan . after incubation , the fetal membranes were washed in saline , photographed , placed in 4 % formaldehyde and kept overnight in a refrigerator . specimens were then embedded in oct tissue tek embedding medium ( fisher scientific , pittsburgh , pa .) and frozen in liquid nitrogen . the frozen specimens were cut by cryostat into 20 μm sections , mounted onto electrostatically treated - glass slides ( fisherbrand superfrost plus , fisher scientific ) and counterstained with neutral red . they were immediately investigated by light microscopy using a nikon eclipse e1000 m microscope connected to an mti dc330 3ccd color camera ( dage mti inc ., michigan city , mich .). sds - substrate gel electrophoresis ( zymography ): for detection of mmps , equal volumes ( 15 μl ) of conditioned medium ( containing the activated mmps ) were mixed in a 1 : 3 ratio with substrate gel buffer ( 10 % sds , 4 % sucrose , 0 . 25 m tris - hcl , ph = 6 . 8 and 0 . 1 % bromphenol blue ) and loaded directly onto gels without boiling ( raj agopalan et al ., 1996 with modifications ). briefly , type i gelatin was added to a standard laemmli 10 % acrylamide polymerization mixture at a final concentration of 1 mg / ml . following electrophoresis , the proteins in the gel were renatured by exchanging sds with triton x - 100 and the gels were incubated overnight at 37 ° c . in 50 mm tris - hcl , ph = 7 . 4 containing 10 mm caci 2 and 0 . 05 % brij 35 . at the end of the incubation , the gels were stained with 0 . 5 % coomassie blue r - 250 ( bio - rad , hercules , calif .) in 40 % methanol / 10 % acetic acid , then destained in 40 % methanol / 10 % acetic acid for 1 h . proteins having gelatinolytic activity appeared as discrete translucent areas of lytic activity on an otherwise blue gel . when the gels are incubated in parallel with 0 . 01 m edta , disappearance of lytic bands confirms the metal dependence of mmp activity . migration of proteins was compared with that of prestained broad molecular weight markers ( bio - rad ). further , 10 ng / lane of zymography standards ( recombinant mmp - 2 / mmp - 9 , calbiochem , la jolla , calif .) were run along with the samples . in order to quantify the gelatinolytic activity of mmps , the wet gel was scanned on a gs - 670 inage densitometer ( bio - rad ), and analyzed using multianalyst v1 . 1 volume quantification system software ( bio - rad ). after image inversion and background subtraction , the amount of lytic activity ( which now appears as a black band on a white background ) is estimated as optical density over the area of the band ( od × mm 2 ). these values were normalized against the dry weight of the tissue incubated , and further normalized against the value from the lane where the tissue was incubated in pbs alone on the same gel . to evaluate the reliability of the image analysis system , differing amounts of zymography standard ( 5 , 10 , 20 ng / lane ) were loaded onto one gel and mnp - 2 ( 72 kda ) and mmp - 9 ( 92 kda ) activities quantified separately . the correlation coefficient between mmp concentration as quantified by substrate gel analysis and the amount of protein loaded approached 1 . to evaluate the reliability of the normalization , differing amounts of amnio - chorionic tissue ( 100 , 250 , 500 , 750 mg ) were incubated and mmp - 2 and mmp - 9 activities plotted against the dry weight of the tissue . the correlation coefficient was 0 . 98 . statistical analysis : the results are presented as mean + 1 sem of the percent activity from the lane incubated in pbs ( crl ) from the same patient and run in the same substrate gel electrophoresis . multiple comparisons between groups were performed using one - way analysis of variance ( anova ) followed by multiple post - hoc tukey tests . a p value & lt ; 0 . 05 was considered as the limit for statistical significance . mmp levels in human term amnio - chorionic membranes in the absence and presence of redox balance modulators : mmp - 9 and mmp - 2 corresponding to the 92 and 72 kda lytic bands , respectively , were detected in all tissues , at least after culture conditions ( fig1 a ). in some of the patients , an additional band corresponding to prommp - 9 was observed . there was a large variability in the intensity of the lytic bands among the seven patients justifying the need for the normalization described in the methods section . o 2 − generated by the x / xo reaction induced a nonsignificant increase in the total amount of protein released in the conditioned medium also justifying the need for a normalization against the dry weight of the tissue rather than against the protein concentration in the medium . the efficiency of the x / xo reaction was confirmed for each experiment by cytochrome c reduction assay . as illustrated in fig1 a incubation with x / xo significantly increased mmp - 9 concentration . this increase was reversed by simultaneous addition of sod . a small increase in mmp - 9 was observed in some of the patients with xanthine alone probably reflecting endogenous xo activity . incubation with 10 mmnac dramatically reduced both mmp - 2 and mmp - 9 activities to 20 % of the levels from the crl tissue nac also prevented the increase in mmp - 9 induced by x / xo ( not shown ). neither no synthase inhibition , nor the no donor snap had any significant effect on fetal membrane mmp activity ( nor shown ). fig2 a illustrates the gelatinolytic activity in fetal membranes in the presence or absence of lps . zymographically , we did not observe a significant increase in mmp concentration after lps alone . however , nac ( 10 mm ) dramatically inhibited mmp - 2 and mmp - 9 both in the presence and absence of lps . intracellular generation of o 2 − in fetal membranes : when exposed to nbt over the 3 hour incubation period , there was spontaneous formation of formazan as shown macroscopically in fig3 a . exposure to the x / xo reaction produced both intracellular ( intense purple discoloration of the tissue which is as a darkened area in fig3 a ) and extracellular formazan formation ( discoloration of the incubation medium ). the addition of sod inhibited completely the extracellular and only partially the intracellular formazan formation . nac ( 10 mm ) completely abolished formazan formation in the incubated tissue . when examined by light microscopy , the deposits of formazan induced by x / xo were localized mainly within chorion laeve , decidua and amniotic epithelium . scattered deposits were also seen in the connective tissue layer between amnion and chorion . in crl tissues , discrete deposits were localized in the chorion , decidua as well as in large areas of the amniotic epithelium ( fig3 b ). in tissues exposed to nac there were no formazan deposits indicating a complete inhibition of intracellular o 2 − formation . these experiments demonstrate that ( i ) mmp - 9 levels in human fetal membranes are directly increased by superoxide anion , ( ii ) the glutathione precursor n - acetyl - cysteine ( nac ) dramatically inhibits amnio - chorionic matrix metalloprotease activity in addition to inhibiting intrinsic superoxide generation within the tissue . it is , however , possible that the effect of nac on mmp activity to extend beyond inhibition of ros - induced effects since treatment with nac inactivated parallel mmp - 2 along with mmp - 9 . moreover , the ability of nac to inhibit mmp activity becomes more valuable as it involves total gelatinolytic activity examined by zymography ( the resultant of mmp release , mmp activation and mmp / timp interaction ). together , these findings suggest that the overall reduction / oxidation status of the local environment may be an important modulator of mmp levels . an increase in the oxidative state could enhance extracellular matrix degradation . conversely , any thiol - reducing agent ( nac being just one example ) could act as inhibitors of mmp activation within fetal membranes and thus may prevent premature rupture of membranes secondary to inflammation or other conditions associated with high ros production . animals and treatments : c57b16 inbred mice were housed together with cd57 / bl6 males under regular light and dark cycles , with stable ambient temperature conditions . the next morning the females were examined for the presence of a vaginal plug . the day of detection of the vaginal plug was designated as day 0 . on day 16 of pregnancy the pregnant mice were injected i . p . with either 10 μg lipopolysaccharide ( lps : from e coli 0111 . : b4 from calbiochem ) or saline ( controls ). n - acetylcysteine 1 g / kg ( nac : which is a membrane permeable gsh precursor , sigma , st . louis mo . and direct antioxidant ) was given p . o . by gavage 30 min before and 2 h after the lps injection . nac was diluted in phosphate buffered saline ( pbs ). after 12 hours the animals were inspected hourly for signs of parturition ( posture , bleeding ). the time between lps injection and delivery of the first pup was defined as latency . fetal viability at birth was recorded after tactile stimulation of the pup . to separate between the effect of prematurity on the fetus and the in utero consequence of endotoxin administration on the fetus , animals were sacrificed at 3 , 6 and 16 hours after endotoxin injection in a separate set of experiments . placental extracellular superoxide production : placental extracellular superoxide production was determined by the sod - inhibitable reduction of nitroblue tetrazolium . briefly , after sacrifice duplicate placentas where incubated each in 1 ml 1 mg / ml nitroblue tetrazolium ( nbt ) diluted in pbs , for 1 h . in a co 2 incubator with humidified chamber at 37 ° c . the soluble yellow form of nbt is reduced by o 2 . − generated within the tissue to form an insoluble precipitate of blue - purple formazan that discolorates the tissue . extracellular o 2 . − produces a discoloration of the incubation medium . the incubation was performed in the presence and absence of sod . the difference in absorbance at 655 nm normalized by the dry weight of placenta between the average of the duplicates in the presence and absence of sod represents the sod - inhibitable o 2 . − production and was expressed in normalized arbitrary units . measurement of total glutathione : glutathione content was measured in maternal and fetal livers of animals treated with and without nac and sacrificed at 3 , 6 and 16 hours after lps injection . fetal livers were pooled from all fetuses of each pregnant animal , snap frozen in liquid nitrogen and stored at − 80 ° c . until assayed . briefly , the frozen tissue was rapidly homogenized in 7 . 5 % trichloroacetic acid in a ratio of 1 : 20 ( w / v ). the homogenate was centrifuged at 3000 g for 10 min at 4 ° c . total glutathione content was assayed in the supernatant by a colorimetric reaction that involves the formation of a chromophoric thione ( bioxytech gsh - 420 , oxis health products , portland , oreg .) and was expressed in μg / ml by using oxidized glutathione standards and further normalized by the total protein content of the pellet . the protein pellet was solubilized in 0 . 1 n naoh and its protein content was estimated using a bca protein assay kit ( pierce , rockford , ill .). effects of n - acetylcysteine on the timing of birth : animals given two doses of 1 mg / kg nac per os , 1 h before and 2 h after lps , had a latency period of 35 . 13 ± 6 . 41 h ( n = 12 ). animals given nac alone delivered at 67 . 71 ± 5 . 8 h ( n = 7 ) compared to the saline injection ( fig4 a ). however , in our experiments there were also crl animals that delivered earlier . this is consistent with observations made by other authors when using this inbred stain of mice ( swaisgood et al ., 1997 ). though the kaplan - maier survival analysis of cumulative birth rates did not reveal a significant nac effect , the number of animals in each group was too small to exclude a type i error . however , the addition of nac shifted latency from a statistically significant decrease with lps alone to a value no longer different from either crl or lps animals ( fig4 b ). effects of n - acetylcysteine onfetal outcome : the mother cannibalizes the stillborn pups and covers the live born with her body . some of the animals given nac with lps delivered live and stillborn pups . there were no statistically significant differences in litter sizes ( number of pups / dam ) among the four groups . the percent of pups born alive versus stillborn is presented in fig5 a and 5b . it illustrates the statistically significant increase in the proportion of pups born alive in mice given nac before and after lps . to discriminate between a direct effect of lps on the fetus and the indirect effects of prematurity , and to identify the time frame when fetal death occurred , we repeated the experiment sacrificing the animals at 3 , 6 and 16 hours after lps . no mouse delivered prematurely , or had signs of labor ( vaginal bleeding ) at these times . all fetuses were alive in utero at 3 and 6 h . by 16 h after lps , 63 % of the fetuses were dead in the lps group compared to 37 % in the group that received lps plus nac ( p = 0 . 006 ). these results strongly suggest that death occurs in utero and is the result of inflammation , not prematurity . labor itself may further kill some compromised fetuses as in the lps group since the vaginal stillbirth rate was 100 %. this agrees with sastre et al ., ( 1994 ) who suggested that birth is an oxidative stress for the fetus , increasing by 11 fold hepatic gssg in neonatal rats and that this redox alteration induced by birth per se was prevented by the maternal administration of nac . these results suggest that nac improves fetal outcome in a murine model of preterm birth and fetal demise induced by inflammation . effect of nac on placental superoxide production : there was a 2 . 7 fold increase in extracellular o 2 − production in placentas from lps - injected animals at 3 hours . this increase was not seen in animals that had also received nac . at 16 hours after lps , placentas from all three groups produced a similar amount of formazan in the medium . these results suggest that nac acts as a free radical scavenger at the level of the placenta in a murine model of preterm birth and fetal demise induced by inflammation . effect of lps and nac supplementation on total glutathione content : hepatic glutathione was measured to test the efficacy of p . o . nac . there were no statistically significant differences in maternal or fetal liver gsh among groups 3 h after lps . at 16 h ( fig6 a ), livers from pregnant mice that received lps had significantly lower gsh compared to saline - crl . nac prevented the decrease in gsh of lps - treated animals . there was , however , a high degree of variability in the nac + lps group . of great potential importance , gsh content was lower in the livers of fetuses in mice injected with lps ( fig6 b ). nac elevated gsh content in fetal livers to values not different from either crl or lps . this strongly suggests that maternal treatment with nac may have a beneficial effect on the fetal oxidative state . co - administration of nac prevented maternal liver glutathione depletion , though the bioavailability of nac administered per os seems variable . we have further observed that fetal death was more likely if the maternal hepatic gsh was low ( fig6 c ). these results suggest that maternal administration of endotoxin , which results in an occult inflammatory process within the mother , induces increased glutathione consumption in the fetus . co - administration of nac was able to prevent maternal liver glutathione depletion during systemic inflammation . nac , a membrane permeable glutathione precursor and direct antioxidant significantly increases fetal survival in an animal model of preterm birth and fetal demise induced by inflammation . although nac likely delays the onset of preterm birth in this model and thus alters the duration of gestation . the protective effects ofnac on the fetus are independent of prematurity . these beneficial effects may be related to the decrease in free radical production in the placenta in response to endotoxin . in addition , adverse fetal outcome is strongly associated with an inflammatory state of the mother even in the absence of any general signs of maternal infection and inflammation . furthermore , the inflammatory state of the mother may result in a shift in the redox balance with glutathione depletion in the fetus . to date there are no therapeutical agents that target free radical production in the fetus either directly or transplacentally through the pregnant mother because the pathophysiological connection between free radicals , preterm birth and perinatal outcome is only now being elucidated . the findings of the present invention are unique . glutathione or other compounds that scavenge reactive oxygen species significantly improve fetal outcome when administered in amurine model ofpreterm birth with intrauterine fetal demise . although the timing of gestation with nac was not significantly altered , nac inhibits matrix metalloprotease activity in human fetal membranes . activation of mmp - 9 is followed by crucial catabolic processes that occur in human amniochorionic membranes during pprom . free radical trapping compounds , ( thiols and other chemical classes ), anti - oxidants , glutathiones , and similar compounds appear to have beneficial effects for the treatment and prevention of fetal complications leading to neonatal handicaps secondary to inflanunation that also causes preterm parturition . precursors to free radical trapping compounds also have this beneficial effect . agents that stimulate the production of endogenous free radical trapping compounds also have this beneficial effect . it has been suggested by others that one of the mechanisms by which ros act are as messenger molecules in mammalian cells through the activation of transcription factors such as the nuclear factor kappabeta ( nfκb ) or activator protein 1 ( ap - 1 ). nfκb consists of two subunits p65 and p50 and usually exists as a molecular complex with the inhibitory protein ikb in the cytosol ( baeuerle and baltimore , 1988 ). upon stimulation with proinflammatory cytokines , iκb is dissociated and nfkb is translocated to the nucleus were it activates expression of target genes . h 2 o 2 , for example , can induce early gene expression of cytokines ( los et al ., 1995 ). pretreatment of cells with antioxidants such as nac efficiently inhibited both cytokine - induced nfκb activation cascade ( khachigian et al ., 1997 ) and induction of ap - 1 ( bergelson et al ., 1994 ). in addition , both tyrosine kinase - mediated nfκb and c - jun / ap - 1 activation were proven to be essential to the induction of mmp - 9 by cytokines ( yokoo and kitamura , 1997 ). thus , compounds which inhibit nfkb may have the same therapeutic effect as ros inhibitors and are included under our claims . in addition , some of the compounds described herein for detecting and scavenging fr ( spin traps ) have been evaluated recently in conjunction with a new non - invasive imaging technique — 31 p nmr spin trapping by mri ( fuji et al ., 1999 ; khramtsov et al ., 1999 ). the present application describes evidence that in utero generation of fr is a cause of fetal damage and contribute to the development ofpreterm labor . we may expect to provide compounds ( spin traps ) that not only limit but also detect detrimental processes affecting the mother and child . a prerequisite for such a compound is the ability to trap fr in the fetus . we have experimentally investigated the transplacental passage of spin trapping compounds . methodology : c57b16 mice were injected ip with 4 mmols / kg pobn [ a -( 4 - pyridyl - 1 - oxide - n - t - butylnitrone )] on day 16 of pregnancy . animals were sacrificed at different times after injection , 2 - 5 fetuses collected and lipids extracted as described by brackett et al ., 1989 . electron paramagnetic spectra were obtained in a varian epr spectrometer equipped with an x - band microwave bridge with center field set at 3376 gauss and modulation frequency at 100 hz . the extracts were examined before and after in vitro addition of a hydroxyl radical generating fenton mixture ( fe 2 + + h 2 o 2 ). [ 0111 ] fig7 a represents epr spectra of the pobn - hydroxyl radical spin adducts trapped in the maternal liver at 1 h after intraperitoneal administration of pobn . fig7 b and 7c illustrated the pobn - hydroxyl radical spin adducts trapped by the amount of pobn that accumulated in the fetus within 1 h or 6 h aftermaternal i . p . administration of pobn . there is an additional 10 - fold difference in the magnitude of the signal between fig5 c and a or b ( receiver gain for fig5 a and b is 2 × 10 5 and for fig5 c is 2 × 10 4 ). these results indicate that fr may be trapped and detected in the fetus after maternal administration of spin trapping compounds . transplacental passage of such compounds has not been explored before . planned improvements include the further synthesis of specific spin traps that can be detected noninvasively by mri imaging . such compounds will have application not only to prevent fr damage to the fetus but also to detect in utero formation of specific fr which can induce damage to the fetus or lead to preterm labor . these findings have great clinical potential . mri is often performed for the imaging of fetal abnormalities and is believed safe . we believe that the application of spin traps which can be administered to the mother and cross the placenta will permit in vivo quantification of fr production in both the healthy and the at risk fetus . the technology that will enable this examination already exists . thus , treatment could be accurately initiated prior to the development of irreversible complications . changes may be made in the construction and the operation of various components , elements and assemblies described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the invention as defined in the following claims . the following citations are incorporated by reference herein for details supplementing this application : 1 . athayde n , edwin s s , romero r , gomez r , maymon e , pacora p , menon r . a role for matrix metalloproteinase - 9 in spontaneous rupture of the fetal membranes . am j obstet gynecol 1998 ; 179 : 1248 - 53 . 2 . babior b m , kipnes r s , cumutte j t . biological defense mechanisms . the production by leukocytes of superoxide , a potential bactericidal agent . j clin invest 1973 ; 52 : 741 - 4 . 3 . baeuerle p a , baltimore d . i kappa b : a specific inhibitor of the nf - kappa b transcription factor . science 1988 ; 242 : 540 - 6 . 4 . baylis c , mitruka b , deng a . chronic blockade of nitric oxde synthesis in the rat produces systemic hypertention and glomerular damage . j clin invest 1992 ; 20 : 278 - 81 . 5 . beckman j s , crow j p . pathological implications of nitric oxide , superoxide and peroxynitrite formation . biochem soc trans 1993 ; 21 : 330 - 34 . 6 . bergelson s , pinkus r , daniel v . intracellular glutathione levels regulate fos / jun induction and activation of glutathione s - transferase gene expression . cancer res 1994 ; 54 : 36 - 40 . 7 . berkovitz g s , papiemik e . epidemiology ofpreterm birth . epidemiol rev 1993 ; 15 : 414 - 3 . 8 . bernstein s , heimler r , sasidharan p . approaching the management of the neonatal intensive care unit graduate through history and physical assessment . 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