NON-INVASIVE BIOMARKER TO IDENTIFY SUBJECT AT RISK OF PRETERM DELIVERY

Abstract

Methods for diagnosis to allow prediction of the likelihood of preterm birth based upon the concentration of lipocalin-type prostaglandin D2 synthase (L-PGDS) in cervical vaginal secretions. In addition, specific prostaglandin D2 receptor antagonists may represent novel tocolytic therapeutics.

Claims

1-21. (canceled)

22. A pharmaceutical formulation comprising a therapeutic amount of a composition selected from the group consisting of at least one of a prostaglandin DP1 receptor antagonist, or a prostaglandin DP2 receptor antagonist, and a selective L-PGDS inhibitor, in an effective and non-teratogenic amount to delay preterm delivery in a pregnant human when administered in repeated doses without harming the fetus.

23. The pharmaceutical formulation according to claim 22, wherein the composition is selected from the group consisting of AM156 ({2′-[(cyclopropanecarbonyl-ethyl-amino)-methyl]-6-methoxy-4′-trifluoro-methyl-biphenyl-3-yl}acetic acid, sodium salt), and AM206 (5-{2-[(benzoyloxycarbonyl-ethyl-amino)-methyl]-4-trifluoromethyl-phenyl}-pyridin-3-yl)-acetic acid, sodium salt), MK-0524 ([(3R)-4-(4-Chloro-benzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic Acid), AM-853 (2-(4-(4-(tert-butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenyl sulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid), BW868C (3-benzyl-5-(6-carbohexyl)-I-(2-cyclohexyl-2-hydroxyethylamino)-hydantoin), S-5751 ((Z)-7-[(IR,2R,3S,5S)-2-(5-hydroxy benzo[b]thiophen-3-ylcarbonylamino)-10-norpinan-3-yl]hept-5-enoic acid), BAY-u3405 (Ramatroban, 3(R)-[[(4-fluorophenyl) sulphonyl]amino]-1,2,3,4-tetrahydro-9H-carbazole-9-propanoic acid), and AT-56 (4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine).

24. A method for predicting and delaying preterm delivery of a fetus, comprising: collecting a cervical vaginal secretion sample from a pregnant woman having an intact amniotic membrane, with a sponge treated with at least a protease inhibitor and a bacteriocide; measuring lipocalin-type prostaglandin D2 synthase concentration and ratios of isoforms of lipocalin-type prostaglandin D2 synthase in the collected cervical vaginal secretion sample; determining a likelihood of preterm delivery based on a comparison of the measured lipocalin-type prostaglandin D2 synthase concentration and ratios of the isoforms of lipocalin-type prostaglandin D2 synthase with population statistics with respect to the gestational age of the fetus; and selectively applying a tocolytic therapy in dependence on the determined likelihood.

25. The method according to claim 24, wherein the tocolytic therapy comprises oral administration of a prostaglandin D2 receptor antagonist

26. A method for delaying preterm delivery of a human fetus, comprising: collecting a cervical vaginal secretion sample from a pregnant woman having an intact amniotic membrane; employing at least one antibody to measure lipocalin-type prostaglandin D2 synthase concentration and lipocalin-type prostaglandin D2 synthase isoform ratios in the collected cervical vaginal secretion sample; comparing the measured lipocalin-type prostaglandin D2 synthase concentration and lipocalin-type prostaglandin D2 synthase isoform ratios in the collected cervical vaginal secretion sample with a relevant standard population, to determine a preterm delivery risk; and selectively administering a therapeutic amount of at least one composition selected from the group consisting of a prostaglandin DP1 receptor antagonist, a prostaglandin DP2 receptor antagonist, and a selective L-PGDS inhibitor to the pregnant woman dependent on the determined preterm delivery risk.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS. 1A and 1B present data from a preliminary study of samples of pregnant women correlating L-PGDS levels in cervical vaginal secretions with term or preterm delivery.

[0027] FIG. 2 present data from a study of samples of pregnant women correlating L-PGDS levels in cervical vaginal secretions with term or preterm delivery.

[0028] FIG. 3 presents data from (murine) animal studies to demonstrate the relationship between lower L-PGDS levels and likelihood of viability and successful term birth.

[0029] FIG. 4 shows 2D PAGE of Human CVS L-PGDS Isoforms.

DETAILED DESCRIPTION

[0030] Methods and Procedures for Risk Assessment for Preterm Delivery.

[0031] Cervical vaginal secretions (CVS) are readily collected and constitute a minimally invasive procedure. During a sterile speculum examination of a pregnant woman, a Week-gel sponge is applied into the cervical os for approximately one minute. The sponge is then removed and placed in a buffer solution containing physiological saline, Trizma buffer, protease inhibitors (EDTA, phenylmethylsulfonyl fluoride, pepstatin) and antibacterial agents (such as sodium azide). This sample collection process is very similar to that used for other routine clinical assays (e.g., fetal fibronectin) that use cervical secretions for the starting sample material. Preferably, the buffer solution has a low protein concentration, e.g., is produced with a small amount or no bovine serum albumin (BSA). This BSA is not required to stabilize the L-PGDS, and may interfere with its measurement.

[0032] L-PGDS concentration levels from the cervical vaginal secretion samples are then determined by an antibody sandwich ELISA. As an initial step, antibody against L-PGDS raised in rabbits is purified by hydroxyapatite chromatography, followed by an immunoaffinity purification on an Affi-Gel 15 Gel column.

[0033] The purified antibody is then diluted to 10 μg/ml in 0.2M sodium carbonate buffer, pH 9.0, and 50 μl added to each well of an Nunc-Immuno plate with Maxi Sorp surface overnight at 4° C. The wells are then washed with 0.1% BSA/0.05% Tween 20 in PBS 4 times and blocked with 1% BSA in PBS for 1 hour. Diluted plasmas or L-PGDS standards (1.2 to 75 ng/ml) are incubated in Block for 90 minutes, then washed as above. A secondary anti-L-PGDS antibody, raised in chickens, at 4 ng/ml in wash buffer, is incubated in the wells for 1 hour and then washed, as above. Horseradish peroxidase-tagged goat anti-chicken antibody at 1:10,000 in wash buffer is added for 1 hour and then the plate is washed, as above, developed in a 0.01 3,3′, 5,5′ tetramethylbenzidine solution, stopped with 50 μl of 1N sulfuric acid, and read at 450 nm in a microplate reader. L-PGDS concentration is determined by the standard curve obtained by plotting the absorbance versus the corresponding concentration of an L-PGDS standard.

[0034] This assay is also adapted to a BioPlex suspension array for similar quantitation. The assay is also amenable for development to run the samples on a 2-D PAGE and determine the particular L-PGDS isoforms present/absent from the CVS samples that may be indicative of an increased propensity toward preterm birth. It is understood and believed that an antibody to the specific isoform may be created to fine-tune the detection method.

[0035] In assessing test samples, L-PGDS concentrations determined by ELISA are normalized to protein levels in the CVS. Protein content of the samples is determined by BCA method, which has been commonly used to quantitate protein levels in cell lysates.

[0036] The new markers of the present invention are highly sensitive and specific (e.g., 90% or higher). In an preliminary study, at a level of 90% specificity, the sample size yields a 95% confidence level, indicating high precision, with a margin of error of about 5%. The collection of test data from pregnant women is ongoing and studies producing significant results have been established as demonstrated in the following Table 1.

TABLE-US-00001 TABLE 1 CVS L-PGDS (ng/ml) Term Preterm Patient CVS Term Days to Patient CVS Preterm Days to ID # (weeks) Delivery Delivery ID # (weeks) Delivery Delivery 17 26 607 84 20 30 7653 11 19 26 725 86 21 30 4839 24 24 26 818 93 23 32 2589 2 26 31 1163 58 28 28 1033 2 27 33 282 32 30 32 4926 1 29 25 782 85 MEAN 30 4208 8 31 25 872 SEM 1 761 3 33 30 1798 57 34 29 2717 66 36 25 1056 37 27 1308 MEAN 28 1103 70 SEM 1 201 6 Notes: Current samples collected at between 28-30 weeks of gestation Level of L-PGDS ~4-fold higher than in pregnancies that go to term Average time until delivery based on elevated L-PGDS 8 days vs. 70 days with lower levels Currently a cutoff of <1.8 μg/ml L-PGDS would appear to distinguish ~91% of term deliveries

[0037] Statistical analysis of this data indicates that high L-PGDS levels (i.e., L-PGDS levels above 1.8 μg/ml) predict an increased likelihood of preterm delivery in human subjects. Graphical illustration of this analysis is depicted in FIGS. 1A and 1B. Findings from the studies indicate that L-PGDS levels in pregnant women susceptible to preterm delivery average a 4-fold increase over levels in women whose pregnancies go to term, and that the average time until delivery for women with elevated L-PGDS levels is 8 days, compared to 70 days for those women that do not demonstrate such elevated levels.

[0038] The pattern of elevated L-PGDS in preterm is generally consistent with the predicted rise. Because the L-PGDS level has a normal change over gestation, CVS samples are preferably taken and L-PGDS assays performed every two weeks.

[0039] A key advantage of this assay over the current fetal fibronectin (fFN) test is that the present assay would predict preterm birth over the entire gestational period up to parturition. A negative fFN, although very accurate, is only valid for a 2-week window. Interestingly, in the study reported below, there were several samples that were fFN negative, but ultimately went preterm. The present assay showed elevated L-PGDS, and was predictive for preterm birth.

[0040] FIG. 2 shows results from a larger study. The data shows that for 163 patients, 36 were preterm (birth less than 37 weeks gestation), with a median of L-PGDS of 8,566 ng/ml/ng protein and 127 went to term with an median of 28,247 ng/ml/ng protein. Results showed that difference in the median values between the two groups is greater than would be expected by chance; there is a statistically significant difference (P=<0.001). See Table 2.

TABLE-US-00002 TABLE 2 Mann-Whitney Rank Sum Test Group N Missing Median 25% 75% Preterm 36 0 28247.587 17051.503 47944.682 Term 127 0 8566.720 4663.511 12857.063 Mann-Whitney U Statistic = 694.000 T = 4544.000 n(small) = 36 n(big) = 127 (P = <0.001)

[0041] The difference in the median values between the two groups is greater than would be expected by chance; there is a statistically significant difference (P=<0.001)

[0042] Animal studies conducted to determine the correlations between L-PGDS concentrations and birth viability and/or risk of preterm birth were conducted with L-PGDS knockout mice and transgenic L-PGDS overexpressor mice. Results are demonstrated in FIG. 3. The studies strongly validate the present findings that lower concentration levels of L-PGDS tend to increase the chances of successful term birth, as per the present murine model. The findings from these animal studies cross-validate the findings from human studies (e.g., as presented above in Table 1).

[0043] Table 3 shows data demonstrating that in LPS-induced preterm birth, there is a three-fold increase in viable mouse pups/pregnancy in the L-PGDS knockouts and a 15-fold decrease in viable pups/pregnancy in the L-PGDS transgenic overexpressors when compared to the C57BL/6 controls. This implies that L-PGDS is casually related to preterm birth, and not merely correlated with it.

TABLE-US-00003 TABLE 3 C57BL/6 L-PGDS KO L-PGDS KI Experiment # 1 2 3 4 5 Total SEM 1 2 3 4 5 Total SEM 1 2 3 4 5 Total SEM Pregnant 2 4 3 5 3 17 0.5 0 3 2 3 2 10 0.5 0 1 4 4 6 15 1.1 Females Viable Pups 1 5 0 5 7 18 1.3 0 8 9 16 0 33 3.0 0 0 0 1 0 1 0.2 Adverse 15 19 21 12 10 77 2.1 0 2 1 11 2 16 2.0 0 4 23 25 56 108 9.9 Outcome Viable 1.06 3.30 0.07 Pups/Pregnancy

[0044] In a study of pregnant C57BL/6 mice implanted with Alzet osmotic pumps containing both DP1 and DP2 antagonists (BWA868C and 11-deoxy-11-methylene prostaglandin D2 at 1.0 μg/μl or PBS vehicle) were injected with lipopolysaccharide (LPS) (20 μg) at day 14 of pregnancy to induce preterm birth. In the LPS-only control group, 80% suffered adverse outcomes (premature birth or fetal death), and 20% had normal outcomes. In the experimental group, LPS plus DP1 and DP2 antagonist, 50% suffered adverse outcomes and 50% had normal outcomes. This demonstrates that administration of prostaglandin D2 receptor antagonists may be administered to pregnant mammals to interrupt preterm labor, and more particularly, to avoid preterm labor induced by infectious agents, especially those which produce lipopolysaccharides. Because the preterm labor pathway has common elements, it is likely that the prostaglandin D2 receptor antagonists will also act to block preterm labor from other causes.

[0045] Chemical compounds which influence the levels of L-PGDS in pregnant women might be administered as tocolytic therapeutic agents. Preferred embodiments of such effective tocolytic agents include prostaglandin D2 (DP1 or DP2) receptor antagonists, such as BWA868C, see, e.g., U.S. Pat. Nos. 6,395,499; 6,878,522; 6,884,593; 7,144,913; 7,217,725; 7,517,889; 7,642,249; 8,067,445; 8,071,807; 8,193,183; 8,242,145; 8,338,484; 8,362,044; 8,378,107; 8,383,654; 8,426,449; 8,497,381; 8,501,959; 8,524,748; U.S. Pat Pub. Nos. 20020022218; 20030027854; 20040162323; 20040180934; 20040185509; 20040197834; 20050215609; 20070054951; 20070244131; 20070249686; 20070265278; 20070265291; 20080194600; 20080261922; 20090036469; 20090176804; 20090197959; 20100004331; 20100075990; 20100081673; 20100113503; 20100130574; 20100298368; 20110021573; 20110039852; 20110098302; 20110098352; 20110112134; 20110130453; 20110144160; 20110152338; 20110190227; 20110245303; 20110301168; 20110312974; 20110318308; 20110319445; 20120004233; 20120016029; 20120022119; 20120058123; 20120059055; 20130005728; 20130005741; 20130065902; 20130079375; 20130109685; 20130158036; each of which is expressly incorporated herein by reference. Methods of treatment and preventing preterm delivery include the administration of a therapeutic amount of prostaglandin D2 receptor antagonists to a pregnant woman in need thereof. Proper routes of administration, dosages and frequencies of administration of these tocolytic agent prostaglandin D2 receptor antagonists may be readily determined by one of skill in the art, e.g., medical practitioners.

[0046] More particularly, the present invention provides for methods of treating preterm labor via the administration of pharmaceutical compositions comprising active tocolytic therapeutic agents with a pharmaceutically acceptable carrier. Pharmaceutical composition of the present invention are intended to encompass a product comprising the active ingredient(s), e.g., prostaglandin D2 receptor antagonists, and the inert ingredient(s) (pharmaceutically acceptable excipients) that constitute the carrier, as well as any product which may result, directly or indirectly, from combination, complex formation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or form other types of reactions or interactions of one or more of the ingredients.

[0047] For example, available prostaglandin D2 (DP1/DPGTR, DP2/CRTH2) receptor antagonists include AM156 ({2′-[(cyclopropanecarbonyl-ethyl-amino)-methyl]-6-methoxy-4′-trifluoro-methyl-biphenyl-3-yl}-acetic acid, sodium salt), and AM206 (5-{2-[(benzoyloxycarbonyl-ethyl-amino)-methyl]-4-trifluoromethyl-phenyl}-pyridin-3-yl)-acetic acid, sodium salt), MK-0524 ([(3R)-4-(4-Chloro-benzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]acetic Acid), AM-853 (2-(4-(4-(tert-butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenyl sulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid), BW868C (3-benzyl-5-(6-carbohexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)-hydantoin), S-5751 ((Z)-7-[(1R,2R,3S,5S)-2-(5-hydroxy benzo[b]thiophen-3-ylcarbonylamino)-10-norpinan-3-yl]hept-5-enoic acid), BAY-u3405 (Ramatroban, 3(R)-[[(4-fluorophenyl) sulphonyl]amino]-1,2,3,4-tetrahydro-9H-carbazole-9-propanoic acid). Such agents, or others that are known or become known, may be used alone, in combination or subcombination. In some cases, the effects may be enhanced by selectively acting on DP1 or DP2, and therefore appropriate agents may be selected. Likewise, as may be appropriate, a mixed agonist/antagonist comprising a single or multiple agents, may be administered or concurrently administered.

[0048] For the treatment of preterm labor, the tocolytic therapeutic agents may be administered orally, by inhalation spray, topically, transdermally, parenterally or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Tocolytic therapeutic agents, e.g., prostaglandin D2 receptor antagonists, may be co-administered with other therapeutic agents and are suitable for simultaneous and/or sequential combination therapies. Methods of the present invention further encompass co-administration to a pregnant woman of a non-toxic therapeutically effective amount of a tocolytic therapeutic agent, such as a prostaglandin D2 receptor antagonist, optionally with other active therapeutic agents, e.g., other prostaglandin D2 receptor antagonists, either simultaneously or sequentially as part of a combination therapy treatment regimen. Similarly, a selective L-PGDS inhibitor, such as AT-56 (4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine) may be used alone or in combination with a DP1 and/or a DP2 antagonist. The therapeutic amounts of active therapeutic agents as administered in combination therapy may be those as commonly used for each active therapeutic agent when administered alone, or the amounts may result in lower dosage(s) for one or more of the active therapeutic agents.

[0049] As shown in FIG. 4, different isoforms of L-PGDS appear in preterm birth CVS samples than for at term births. Therefore, a selective immunoassay may be implemented based on the ability of certain antibodies to distinguish between the isoforms. Immunoassays may include radio immunoassays, enzyme-linked immunoassays, fluorescent immunoassays, and the like.

[0050] On the other hand in a ratiometric assay which seeks to determine a ratio between isoforms, or between a single isoform and the total, during purification, it may be preferred to avoid selective enrichment, and thus immunopurification may employ a non-specific antibody or a balanced mix of antibodies to ensure that the CVS sample concentrations are not altered during processing. Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. It is preferred, therefore, that the present invention be limited not by the specific disclosure herein, but only by the appended claims.