WO2001068126A2

WO2001068126A2 - Methods and compositions for treating premature rupture of fetal membranes

Info

Publication number: WO2001068126A2
Application number: PCT/US2001/008038
Authority: WO
Inventors: Stephen Joseph Fortunato; Ramkumar Menon
Original assignee: Stephen Joseph Fortunato; Ramkumar Menon
Priority date: 2000-03-13
Filing date: 2001-03-13
Publication date: 2001-09-20
Also published as: AU2001243625A1; WO2001068126A3

Abstract

Methods and pharmaceutical compositions useful in the treatment and prevention of preterm premature rupture of the fetal membranes (PROM) in pregnant human patients are provided.

Description

METHODS AND COMPOSITIONS FOR TREATING PREMATURE RUPTURE OF FETAL MEMBRANES

FIELD OF THE INVENTION

The invention described herein relates to therapeutic methods and compositions useful in the management of preterm premature rupture of fetal membranes.

BACKGROUND OF THE INVENTION The placental fetal membranes protects the fetus from mechanical and physical factors during gestation. Rupture of the membranes occurs during labor and sometimes before the onset of labor (preterm premature rupture of the membranes). The incidence of preterm premature rupture of the fetal membranes (PROM) is between about 4% and 8% of all pregnancies. It is a major cause of preterm labor and increased neonatal morbidity and mortality (representing about 30% of all Prematurity). Prior history of PROM substantially increases one's risk of subsequent PROMs. There are currently no drugs indicated for the prevention or treatment of PROM.

Fetal membranes, comprised of amnion and chorion cell layers connected by an extra cellular matrix region (ECM), are made up of various types of collagens. Pathological complications like IAI during pregnancy were thought to contribute to the destruction of these collagens by substrate specific enzymes leading to membrane degradation.

Matrix metalloproteinases (MMPs), a family of zinc dependent enzymes have been studied extensively as possible agents of membrane degradation due to their collagen specificity. A balanced activity between MMPs and their tissue specific inhibitors (TIMPs) is involved in membrane remodeling throughout pregnancy allowing for accommodation to the increased pressure and volume of the growing pregnancy. However, during infection and other complications of pregnancy there is an imbalance between MMPs and TIMPs which favors MMP activity over inhibition leading to destruction of the ECM components rather than remodeling. Increased degradation of the ECM weakens the membrane and leads to rupture.

Fetal membrane rupture during labor at or before term (PROM) has been associated with increased matrix metalloproteinases (MMPs) activity (Draper D, McGregor J, Hall J, Jones W, Beutz M, Heine RP, Porreco R Elevated protease activities in human amnion and chorion correlate with preterm premature rupture of membranes. Am J Obstet Gynecol 1995;173:1506-12). An increased presence of MMPs (gelatinases and stromelysinl ) in human fetal membranes and amniotic fluid has been documented during PROM (Draper et al., 1995; Bryant-Greenwood GD; Yamamoto SY. Control of peripartai collagenolysis in the human chorion-decidua. Am J Obstet Gynecol 1995;172:63-70; Athayde N; Edwin SS; 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; Fortunato SJ, Menon R, Lombardi SJ. Stromelysins in placental membranes and its elevation in amniotic fluid during premature rupture of membrane. Obstet Gynecol 1999; 94:435-440; Fortunato SJ, Menon R, Lombardi SJ. Collagenolytic enzymes (gelatinases) and their inhibitors in human amniochorionic membranes). Am J Obstet Gynecol 1997; 177:731-741 ; Athayde N; Romero R; Gomez R; Maymon E; Pacora P; Mazor M; Yoon BH; Fortunato S; Menon R; Ghezzi F; Edwin SS. Matrix metalloproteinases-9 in preterm and term human parturition. J Matern Fetal Med 1999;8:213-9; Vadillo-Ortega F, Hernandez A, Gonzalez-Avila G, Bermejo L, Iwata K, Strauss JF 3^rd. Increased matrix metalloproteinase activity and reduced tissue inhibitor of metaIloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes. Am J Obstet Gynecol 1996; 174(4): 1371 -6).

Although the causes of PROM are multifactorial, several exogenous and endogenous factors have been linked. The main exogenous factor is infection, with consequent host inflammatory response, present in over 60% of cases of PROM. Lipopolysaccharide increases the expression and release of gelatinases and decreases tissue inhibitors of matrix metalloproteinase 2, which shifts the balance in favor of gelatinase activity leading to membrane degradation that predisposes to premature rupture of membranes (Fortunato et al., 2000, Obstet Gynecol 95: 240-244). See also, Fortunato et al. 1999, J Perinatal Med 27: 362- 368; Fortunato et al., 1999, Obstet Gynecol 94: 435-440).

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for the prevention and treatment of preterm premature rupture of fetal membranes (PROM). Specifically, the methods of the invention comprise inhibiting the expression of the matrix metalloproteinase MMP9 in fetal membranes. In a particular embodiment described herein, IL-10 is used to achieve the inhibition of MMP9 gene and protein expression in PROM. Preferred embodiments of the invention utilize IL-10 or IL-10 activating substances capable of inhibiting infection-induced uterine contractility and preterm labor. Other inhibitors of MMP9 gene expression, such as antisense, ribozymes, intracellular antibodies, may also be useful for treating PROM. In addition, antibodies and related molecules may be useful to block MMP9 activity, thereby preventing matrix degradation and PROM. BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1. Fetal membranes after LPS (Lipopolysaccharide and PGPS (peptidoglycan polysaccharide - cell wall material from Gram positive bacteria) stimulation and from women with Infection associated PROM shows induction of MMP9 mRNA in basic PCR, whereas no visible change in mRNA expression for MMP2 was seen.

FIG. 2. LPS induced the expression of MMP9 mRNA in LPS stimulated tissues (49 transcripts/0.5μg of total RNA; median 60; range 19.2- 79.18) whereas detectable levels of expression was not seen in control tissues. Co-stimulation with IL-10 significantly reduced the expression of MMP9 mRNA to 10 transcripts/0.5 μg of total RNA (median 6; range 0 - 26; p 0.03).

FIG. 3. LPS increases mRNA expression of MMP2 compared to control. LPS simulation resulted in 3.6 10⁶ transcripts/0.5 μg of total RNA (median 3.3 x 10⁶ , range 5.5 x 10⁵ - 7.6 x 10⁶) compared to 5.9 x 10⁴ transcripts/0.5 μg of total RNA (median 6 x 104; range 5.7 x 10⁴ - 6.0 x 10⁴; p = 0.009) in controls for MMP2. Co-stimulation of IL-10 did not result in a change in the expression pattern of MMP2 mRNA (6 x 10⁶ transcripts/0.5 μg of total RNA; p = ns).

FIG. 4. ELISA on culture media documented 29 ng/ml (median 39; range 11 - 44) of immunoreactive MMP9 after LPS stimulation (control 8.08 ng/ml [median 6; range 1.4 - 16; p <0.05). This level was reduced to 6.2 ng/ml (median 4, range 0 - 15.5; p <0.05) after co- stimulation with IL-10.

FIG. 5. ELISA on culture media samples documented a significant increase in MMP2 protein release from human fetal membranes after LPS stimulation (205.8 [median 193; range 174 - 285] vs. 114.8 ng/ml (median 85; range 44 - 219; p =0.009). However, these levels were not significantly reduced after IL-10 treatment (126 ng/ml [median 190; range 69 - 205]; p < 0.05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for treating, preventing and inhibiting preterm premature rupture of fetal membranes (PROM). In particular, the methods of the invention comprise inhibiting the expression of matrix metalloproteinase Gelatinase B (MMP9). The invention is based, in part, upon discovering that IL-10 is capable of inhibiting amniochorionic membrane MMP9 RNA and protein expression. Therefore, in one embodiment, IL-10 is used to prevent PROM, wherein the method comprises contacting the amniochorionic membrane with an amount of IL-10 sufficient for inhibiting the expression of fetal membrane MMP9 in PROM. In another, related embodiment, a composition which is capable of inducing the expression of IL-10 in situ may be used (an "IL-10 activator"). Such compositions comprise, for example, alpha-melanotropin (α-MSH) or its analogues (e.g., N, O- diacetyl-Ser1-alpha-MSH) (Buckley et al., 1981 , Int J Pept Protein Res 17: 508-13; U.S. Patent No. 5, 420,109). In yet another embodiment, the IL-10 gene or other nucleic acid molecules encoding IL-10, preferably under appropriate regulatory control, may be used to transduce cells of the amniochorionic membrane such that the cells express and secrete biologically active IL-10 using techniques well known in the art.

The Gelatinase A (MMP2) and gelatinase B (MMP9) proteases degrade the Type IV collagen that cements the amnion and chorion layers to the extracellular matrix (ECM) . Gelatinase A

(MMP2) is a constitutively expressed protein in pregnancy and it is likely more involved in membrane remodeling, whereas, gelatinase B (MMP9) is seen only during PROM or at the time of active term labor. We have documented an increase in the active and inhibitor free forms of both of these enzymes in fetal membranes, and in the amniotic fluid of women with infection and/or PROM. Active MMP9 is seen only in the amniotic fluid of women with PROM and not in any other conditions. Our studies and those of several other investigators document that gelatinases play a crucial role in PROM and are also active during labor causing cervical ripening and membrane rupture. These MMPs can initiate a vicious pathway of other MMP activation. Regulation of their expression and production might significantly reduce ECM degradation and thereby the risk of PROM.

The methods of treating PROM provided herein involve the inhibition of MMP9 expression or biological activity. In a particular embodiment described in the Examples herein, the cytokine IL-10 is used to inhibit the elevated expression of MMP9 induced by LPS stimulation. Inhibition of MMP9 expression may be achieved using several approaches, including inhibition at the transcriptional level, inhibition of processing, and inhibition of biological activity.

Several strategies may be used to inhibit MMP9 expression at the transcriptional or translational level. In one approach, a method of inhibiting the transcription of the MMP9 gene comprises contacting the MMP9 gene with a MMP9-specific antisense polynucleotide. In another approach, a method of inhibiting MP9 mRNA translation comprises contacting the MMP9 mRNA with a MMP9-specific antisense polynucleotide. In another approach, a MMP9 specific ribozyme may be used to cleave the MMP9 message, thereby inhibiting translation. Such antisense and ribozyme based methods may also be directed to the regulatory regions of the MMP9 gene, such as the MMP9 promoter, NFkB response element, or enhancer elements. Similarly, proteins capable of inhibiting a MMP9 gene transcription factor may be used to inhibit MMP9 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors which inhibit the transcription of MMP9 through interfering with MMP9 transcriptional activation may also be useful for the treatment of cancers expressing MMP9. Similarly, factors which are capable of interfering with MMP9 processing may be useful for the treatment of PROM. Cancer treatment methods utilizing such factors are also within the scope of the invention. Additionally, the biological activity of MMP9 may be inhibited (e.g., using MMP9-specific antibodies or other molecules that inhibit MMP9 function).

As mentioned above, the preferred embodiment of the method of the invention involves the use of the cytokine IL-10. The experimental data presented in the Example section below indicate that:

1. Simulated infection in vitro increases the expression and release of MMP2 and induces MMP9 mRNA from human fetal membranes;

2. During infection, IL-10 transcriptionally regulates the expression of MMP9 and therefore the release of MMP9 protein from fetal membranes; and

3. IL-10 does not regulate MMP2 gene expression or protein release from human fetal membranes, suggesting that the remodeling role of MMP2 is unaffected by cytokine regulation.

Thus, administration of IL-10 during PROM is expected to regulate the production of MMP9 and thereby reduce ECM degradation causing PROM. Since the MMP9 gene promoter contains an Nf-kB (transcriptional activator protein) responsive element, the mechanism of IL- 10 action is possibly exerted by preventing Nf-kB activation and binding.

As used herein, "IL-10" means human interleukin-10 protein as well as allelic variants, conservative substitution mutants, or mammalian homologues which retain IL-10 biological activity. The extent to which (and the dose at which) a particular IL-10 is effective at preventing PROM may be evaluated using the ex vivo amniochorionic membrane culture system described in the Examples presented herein.

In the practice of the method of the invention, any route of administration of an IL-10 or an IL- 10 activator which results in the inhibition of MMP9 expression may be employed, including but not limited to intraamniotic, intrauteral, intravenous, subcutaneous, and intramuscular administration. Recombinant human IL-10 administered as single doses by intravenous injection is well tolerated in human subjects, and the pharmacokinetics and pharmacodynamics of recombinant human IL-10 have been studied (see, for example, Huhn et al., 1997, Clin Pharmacol Therap 62: 171-180 and the publications cited therein). The dose of IL-10 or IL-10 activator used in the practice of the method of the invention will depend on various factors, including but not limited to the route of administration and clinical considerations. Doses for intravenous administration may be in the range of 25 to 50 μg/kg or higher. Dosages for intraamniotic administration may be significantly lower due to decreased volume of distribution.

In a preferred embodiment, IL-10 is initially administered intraamniotically and intravenously as infusions in appropriately preserved solutions of, for example, phosphate buffered saline (PBS) or another appropriate buffer, at doses of between approximately 1 and 10 mg. Treatment will generally involve the multiple administrations of the IL-10 preparation. It may be preferable for the initial dose to be administered as an intraamniotic infusion with or without intravenous co- administration. Subsequent doses are preferably administered intravenously, although intraamniotic infusions may also be acceptable depending upon the patient's response to the initial dose. However, as one of skill in the art will understand, various factors will influence the ideal dose regimen.

PROM may be treated with IL-10 alone or in combination with other agents, including for example, tocolytic agents and antibiotics. The treatment methods of the invention may be applied prophylactically in pregnancies carrying a significant risk of PROM or after the onset of PROM. Risk of PROM increases substantially with prior history of PROM. For example, a patient with one prior PROM has a risk of about 30-40%, compared with about 4-8% in the general population. Patients with multiple prior histories carry significantly greater risk. For example, a patient with a history of two prior PROMs has a 50-60% risk of a subsequent PROM, and a patient with three or more prior PROMs has an 80% or greater risk. Therefore, the methods and compositions of the invention may be applied prophylactically to patients with a prior history of PROM, at a time based upon when the prior PROM event(s) occurred during term. For example, in a patient who experienced a prior PROM, administration of the methods and compositions of the invention may be most useful when initiated two to four weeks or more earlier in the treated pregnancy.

The invention also provides a pharmaceutical composition comprising IL-10 and, optionally, a suitable pharmaceutical carrier, for use in the treatment of PROM. The invention additionally provides a pharmaceutical composition comprising an IL-10 activator and, optionally, a suitable pharmaceutical carrier, for use in the treatment of PROM. Suitable carriers for pharmaceutical compositions include any material which when combined with the IL-10 or IL- 10 activator retains the molecule's activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, various standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, etc. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Excipients which have utilized in the administration of IL-10 in other indications include sodium citrate dihydrate, sucrose, and glycine mixtures. Compositions comprising such carriers are formulated by well known conventional methods.

The invention is further described and illustrated by way of the following example and the experimental details therein. This section is set forth as an aid to understanding the invention, but is not intended to, nor should it be construed as, limiting the scope of the invention.

EXAMPLE 1 : 1NTERLEUKIN-10-MEDIATED INHIBITION OF MMP9 mRNA AND PROTEIN EXPRESSION IN AMNIOCHORIONIC MEMBRANES EX VIVO

MATERIALS AND METHODS

Ex Vivo Organ Culture of Amniochorionic Membranes: Placentas (n=4) were obtained from uncomplicated gestations at term undergoing elective repeat cesarean section (C-section) prior to the onset of labor. Membranes were harvested under strictly sterile conditions. Amniochorionic membranes were dissected free of the placenta and were placed in PBS with heparin 100U/ml, penicillin 100U/ml, streptomycin 100 mg/ml, and amphotericin B 0.25 mg/ml. All adherent blood clots were removed using sterile cotton gauze. Portions of cleared membrane without decidual contamination, adherent clots, or blood vessels were cultured in an organ explant system as previously described (Fortunato et al., 1994, Am J Reprod Immunol 32: 188-195). Briefly, membranes were washed in HBSS with antibiotics, and pieces of tissue were cut into circles using a 6 mm skin biopsy punch (Euro-Med Cooper Surgical, Richmond, VA). Tissues were then washed in 3 changes of HBSS supplemented with antibiotics. The membrane discs were placed in Falcon 9 mm cell culture inserts which were then placed in a Falcon 24 well tissue culture plate. The culture medium consisted of Dulbecco's modified Eagle's medium with Ham's F12 nutrient mixture (1 :1), antibiotics as above, 2 mM glutamine, and 15% heat inactivated fetal bovine serum (all from Sigma, St. Louis Mo.). Media and fetal bovine serum used in this culture system contained the lowest endotoxin levels commercially available (<0.05 ng/ml). The cell culture insert contained 0.2 ml and the culture well 0.6 ml of medium. Cultures were incubated at 37°C in an atmosphere of 5%C0₂:room air and media were changed on a daily basis.

Bacterial Lipopolysaccharide and IL-10 Treatments: Amniochorion tissues were incubated for a period of 48 hrs before stimulation. This was done to achieve a base line expression of cytokines as defined in earlier studies (Menon et al., 1995, Am J Obstet Gynecol 172: 493- 500). LPS, (E.coli 055:B5, Sigma chemicals, St. Louis, MO) and recombinant IL-10 (R&D Systems, Minneapolis, MN) were added to the culture medium as follows. After a pre- incubation for 48 hours, membranes were stimulated with 50 ng/ml of LPS. Some membranes were simultaneously treated with 500 ng/ml of recombinant IL-10. Tissue and media samples were collected after a 24 hr treatment and frozen for mRNA and protein analysis.

Quantitative competitive PCR for MMP2 and MMP9 (QPCR): In this competitive PCR assay one set of primers was used to amplify both the target gene cDNA and another neutral DNA fragment (MIMIC). The MIMIC fragment competes with the target cDNA fragment for the same primers and acts as an internal standard. This protocol uses a non-homologous DNA fragment engineered to contain the desired gene template primers and thus is recognized by a pair of gene specific primers. Serial dilutions of the MIMIC (known concentrations) were added to PCR amplification reactions containing constant amounts of experimental cDNA samples. A constant amount of the cDNA was then co-amplified with known concentrations of the MIMIC for 30 cycles. This MIMIC utilizes the same primer as the target cDNA but yields a PCR product of different size. Following PCR, the products were resolved by 1.6% agarose gel electrophoresis and visualized on an ethidium bromide stained gel. A visual comparison was made of the intensities of the bands produced by the target gene and the neutral DNA. Quantitation of PCR products was achieved by gel band spot densitometry using the Alpha Ease software program (Alphalnnotech Corporation, San Leandro, CA). Integrated density values for two matching bands (neutral DNA and target genes) were obtained and the densitometric ratio was calculated. This value was multiplied by the known concentration of the neutral DNA to get the approximate (~) number of molecules. Since the molar concentrations of the neutral DNA were known, the relative levels of expression of target mRNA was estimated (# fold increase/decrease).

ELISA for MMP9 and MMP2: The release of MMPs into the culture medium was quantitated using ELISA. The assay procedure involved a multiple-site two step sandwich immuno assay using oligoclonal antibodies (several monoclonal antibodies directed against different epitopes of each specific protein of interest). These assays were performed in our laboratory with commercially available kits (Amersham- Pharmacia, Piscataway, NJ). Manufacturers instructions were followed for each kit to perform ELISA. Standard curves were developed using duplicate samples of known quantities of recombinant proteins. Sample concentrations were determined by relating the absorbance obtained to the standard curve by linear regression analysis. Colorimetric absorption was read at 450 nm using an Inc Star Automatic microplate reader. Controls consisted of assay buffer, plain culture media and LPS and IL-10 containing media incubated in tissue culture wells without tissues.

Statistical Analysis: Statistical significance of QPCR and ELISA was analyzed by unpaired nonparametric, two-sample Mann-Whitney U test and Tukey-Kramer Multiple Comparison test. (p 0.05 was significant)/

RESULTS

LPS stimulation of amniochorionic membranes induced 49 transcripts of MMP9 compared to no MMP9 transcripts in control tissues (See FIGS. 1 and 2). Co-stimulation of IL-10 with LPS resulted in a significant drop in MMP9 mRNA levels (10 transcripts; p=0.03) (See FIG. 2). MMP2 mRNA levels were higher in LPS stimulated tissues (3.6 x 10⁶ transcripts) compared to control (5.9 x 10⁴ transcripts; p=0.009) (See FIG. 3). No significant change was seen in MMP2 mRNA levels in co-stimulated tissues (6 x 10⁶ transcripts) (FIG. 3).

ELISA on culture media documented 29 ng/ml of immunoreactive MMP9 after LPS stimulation (control - 8.08 ng/ml; p<0.05)(FIG. 4). This level was reduced to 6.2 ng/ml (p<0.05) after co- stimulation with IL-10 (FIG. 4). MMP2 levels were increased after LPS stimulation compared to control (205.8 vs. 114.8 ng/ml; p=0.009); however, these levels were not significantly reduced after IL-10 treatment (126 ng/ml; p>0.05) (FIG. 5).

The data set used to generate the above results was expanded to n=8 (compared to n=5 for the above results). Similar results were calculated from the larger set of data, as follows. Lipopolysaccharide stimulation induced 58 transcripts of matrix metalloproteinase 9, compared with none in control tissues. Co-stimulation with interleukin-10 and lipopolysaccharide significantly reduced matrix metalloproteinase 9 messenger RNA levels to ten transcripts (P < 0.05). Lipopolysaccharide stimulation produced 29 ng/ml of immunoreactive matrix metalloproteinase 9 which was reduced to 6.3 ng/ml (P<0.05) after co-stimulation with interleukin-10. Matrix metalloproteinase 2 messenger RNA levels were higher in lipopolysaccharide stimulated tissues (4.3 x 10⁶ transcripts) compared to control (2.8 x 10⁵ transcripts; P < 0.01) with no change in matrix metalloproteinase 2 messenger RNA levels in interleukin-10 co-stimulated tissues (2.6 x 10⁶;P > 0.05). Interleukin-10 co-stimulation resulted in a less than significant decrease in matrix metalloproteinase 2.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any which are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

Throughout this application, various publications are referenced within parentheses. The disclosures of these publications are hereby incorporated by reference herein in their entireties.

Claims

CLAIMS:

1. A method of treating, inhibiting or preventing premature rupture of the fetal membranes in a pregnant human patient, which comprises inhibiting the expression of the matrix metalloproteinase MMP9 in the fetal membranes of said patient.

2. The method according to claim 1 , wherein MMP9 expression is inhibited by inhibiting MMP9 mRNA expression.

3. The method according to claim 2, wherein MMP9 mRNA expression is inhibited by contacting the fetal membranes within said patient with an amount of IL-10 sufficient for inhibiting the expression of MMP9 mRNA therefrom.

4. The method according to claim 2, wherein MMP9 mRNA expression is inhibited by contacting the MMP9 gene in cells of the amniochorion with an MMP9-specific antisense polynucleotide.

5. The method according to claim 1 , wherein MMP9 expression is inhibited by inhibiting the translation of MMP9 mRNA.

6. The method according to claim 5, wherein the translation of MMP9 mRNA is inhibited by contacting the MMP9 mRNA expressed by amniochorion cells with an antisense polynucleotide complementary to said MMP9 mRNA.

7. The method according to claim 5, wherein the translation of MMP9 mRNA is inhibited by contacting the MMP9 mRNA expressed by amniochorion cells with a ribozyme capable of cleaving said MMP9 mRNA.

8. A method of treating, inhibiting or preventing premature rupture of the fetal membranes in a pregnant human patient, which comprises contacting the amniochorionic membrane within said patient with an amount of IL-10 sufficient for transcriptionally inhibiting the expression of MMP9 therefrom.

9. The method according to claim 8, wherein the patient has a prior history of premature rupture of the fetal membranes.

10. The method according to claim 8, wherein the patient presents with symptoms of intraamniotic infection.

11. A pharmaceutical composition comprising IL-10 for use in the treatment, inhibition or prevention of premature rupture of the fetal membranes in a pregnant human patient.

12. A pharmaceutical composition comprising an IL-10 activator for use in the treatment, inhibition or prevention of premature rupture of the fetal membranes in a pregnant human patient.