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The effect of phosphoramidon on inflammation-mediated preterm delivery in a mouse model
American Journal of Obstetrics and Gynecology (2004) 190, 528e31
www.elsevier.com/locate/ajog
The effect of phosphoramidon on inflammation-mediated preterm delivery in a mouse model Karen L. Koscica, DO,a Georges Sylvestre, MD,a Sandra E. Reznik, MD, PhDa,b Departments of Obstetrics and Gynecology and Women’s Healtha and Pathology,b Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY Received for publication May 2, 2003; revised July 28, 2003; accepted August 18, 2003
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Preterm delivery Phosphoramidon Metallopeptidase Cytokine
Objective: Several metallopeptidases have been implicated in both term and preterm parturition. We hypothesize that endotoxin-induced preterm delivery can be prevented by the administration of a metallopeptidase inhibitor. Study design: We used an animal model of endotoxin-induced preterm delivery in timed pregnancy C57Bl/6 mice. Test animals received lipopolysaccharide followed by phosphoramidon, either every 1.5 or every 3 hours. Control mice received lipopolysaccharide followed by buffer injections at the same intervals. The primary outcome was a preterm delivery rate. Results: The rate of preterm delivery for the control animals was 88.0% compared with the treatment groups of 45.5% for the mice that received phosphoramidon every 3 hours and 30.8% for the group that received it every 1.5 hours (P!.01). Conclusion: The administration of a metallopeptidase inhibitor resulted in a decreased rate of preterm delivery in this animal model. Ó 2004 Elsevier Inc. All rights reserved.
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Preterm delivery, defined as delivery at !37 weeks of gestation, occurs in 7% to 12% of pregnancies and is associated with 85% of perinatal morbidity and deaths.1,2 Unfortunately, the existing therapeutic approaches for preterm delivery have not decreased the incidence of prematurity and can be associated with risks to both the mother and fetus. Previous in vitro investigations have shown that endothelin-1 increases myometrial smooth muscle tone.3-6 Infection and inflammatory cytokines, which have been associated with preterm delivery,7,8 can stimulate endo-
Supported by National Institute for Child Health and Human Development grant No. 1K08HD01209. Reprints not available from the authors. 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2003.08.021
thelin-1 production.9 Endothelin-1, in turn, stimulates prostaglandin synthesis.10 Inflammatory cytokines may therefore trigger prostaglandin synthesis and, ultimately, contractions and delivery indirectly by increasing endothelin-1 synthesis. Recent data show that placental neutral endopeptidase (NEP) degrades locally produced gonadotropinreleasing hormone (GnRH).11 GnRH delays parturition by inhibiting the release of placental prostaglandins E and F and thromboxane B2 in a dose-dependent fashion.12 The inhibition of NEP would increase levels of GnRH in the myometrium and therefore decrease uterine activity. Because inflammatory cytokines are well known to decrease hypothalamic GnRH release,13 endotoxins in our mouse model may have a similar effect on GnRH that originated from placental
529
Koscica, Sylvestre, and Reznik Table
Results for mice that were treated with phosphoramidon versus control mice
Group
Treatment
No.
Preterm delivery (No.)
Preterm delivery (%)
I II III IV V VI
Buffer only mice Lipopolysaccharide + buffer every 3 h Lipopolysaccharide + phosphoramidon every 3 h Lipopolysaccharide + buffer every 1.5 h Lipopolysaccharide + phosphoramidon every 1.5 h Phosphoramidon every 1.5 h
5 8 11 9 13 4
0 7 5 8 4 0
0 88.0 45.5* 88.8 30.8* 0
*P!.01.
trophoblast. Inhibition of NEP would protect the reduced pool of paracrine GnRH. In the current study, we tested whether administering phosphoramidon, a compound that inhibits both endothelin-converting enzymes and NEP, affects endotoxin-induced preterm delivery in an animal model.
Material and methods All mice that were used for these experiments (February 2001 through August 2002) were purchased from either Jackson Laboratory (Bar Harbor, Me) or Charles River Laboratory (Wilmington, Mass). A total of 50 timed pregnant C57Bl/6 mice were used that were all at day 15.5 to 16 (of a 19-day gestation) and between 10 and 20 weeks of age. The Albert Einstein College of Medicine and Montefiore Medical Center Institutional Animal Care and Use Committees approved all animal experimental procedures. For mouse models of endotoxin-induced preterm delivery, 41 mice at day 15.5 to 16 were injected with 3.3 mg/kg lipopolysaccharide, serotype 026:B6 (Sigma Chemical Co, St Louis, Mo) intraperitoneally. Each injection contained 100 mg of lipopolysaccharide and 500 mL of phosphate-buffered saline solution (PBS). This followed the established method of the induction of inflammation-mediated preterm delivery in mice of Fidel et al14 and Kaga et al.15 Mice to receive the phosphoramidon (N-[a-rhamnopyranosyloxyhydroxyphosphinyl]-Leu-Trp; Sigma Chemical Co) were injected intraperitoneally at 6.7 mg/kg at scheduled interval dosings. Each injection contained 200 mg of phosphoramidon and 100 mL of PBS. Fifty mice were assigned to six groups. Group I served as control animals for the lipopolysaccharide injection and contained 5 mice that were injected with 500 mg of PBS intraperitoneally. Group II served as a control group that consisted of 8 mice that were treated with lipopolysaccharide and then received PBS intraperitoneally at 12, 15, 18, and 21 hours after the lipopolysaccharide injection. Group III consisted of 11 mice that were treated with lipopolysaccharide and were injected intraperitoneally with 6.7 mg/kg phosphoramidon at 12, 15, 18, and 21
hours after the lipopolysaccharide injection. Group IV served as a control group that consisted of 9 mice that were treated with lipopolysaccharide and received PBS intraperitoneally at 12, 13.5, 15, 16.5, and 18 hours after the lipopolysaccharide injection. Group V had 13 of the mice that were treated with lipopolysaccharide and were injected with 6.7 mg/kg phosphoramidon intraperitoneally at 12, 13.5, 15, 16.5, and 18 hours after the lipopolysaccharide injection. Group VI served as control animals for the phosphoramidon injections and contained 4 mice that were injected with 6.7 mg/kg phosphoramidon only at 12, 13.5, 15, 16.5, and 18 hours. All animals were observed for preterm delivery. After 24 hours, the mice were killed to confirm pregnancy and to collect placental and uterine tissue for future studies. The effect of phosphoramidon on lipopolysaccharide-induced labor was assessed with the log-rank test, with the time to labor as the outcome of interest. The primary outcome was the preterm delivery rate.
Results Of the 5 mice in group I (intraperitoneal PBS alone), none of the mice were delivered preterm. For mice that were assigned to control groups II and IV, 15 of the 17 mice that were treated with lipopolysaccharide followed by buffer were delivered only within 19 hours. (One mouse in each group failed to respond to lipopolysaccharide therapy). In group III that received lipopolysaccharide followed by phosphoramidon every 3 hours, 5 of 11 mice were delivered by 24 hours. Finally, in group V that received lipopolysaccharide followed by phosphoramidon every 1.5 hours, only 4 of 13 mice were delivered (Table). These results showed a significant decrease in the preterm delivery rate from 88.0% in the control groups to 45.5% in the 3-hour treated group to 30.8% in the 1.5-hour treated group. Statistical analysis, with the log-rank test, showed a significant difference (P!.01) in the effect of lipopolysaccharide in inducing preterm labor between mice that were treated with lipopolysaccharide followed by buffer only and mice that were treated with lipopolysaccharide followed by either dosing schedule of phosphoramidon every 3 hours
530
Figure 1 Effect of phosphoramidon on lipopolysaccharideinduced preterm delivery. Eight mice at day 15.5 to 16 were treated with lipopolysaccharide followed by buffer (closed squares). Eleven mice were treated with lipopolysaccharide, followed by phosphoramidon (closed triangles) at 12, 15, 18, and 21 hours. Statistical analysis, with the use of the log-rank test, shows a significant difference in the effect of lipopolysaccharide in the induction of preterm delivery between mice that were treated with lipopolysaccharide followed by buffer only and mice that were treated with lipopolysaccharide followed by either dosing schedule of phosphoramidon (P!.01).
(Figure 1) or every 1.5 hours (Figure 2). There was no significant difference between the two treatment groups. None of the 4 mice in group VI was delivered, as expected. These mice were killed at 24 hours, and an autopsy was performed to test for potential the harmful affects of the phosphoramidon injections. No abnormalities were found in either the mothers or the pups. The number of viable pups per pregnancy did not differ from the control animals in group I, and no intrauterine deaths occurred.
Comment Three closely related metallopeptidases of the M13 family of zinc peptidases are inhibited by phosphoramidon. Endothelin-converting enzyme-1 converts Big endothelin-1 to fully processed endothelin-1 by cleavage of a Trp21-Val22 peptide bond. Endothelin-converting enzyme-1 has not been found to synthesize any peptides other than endothelin-1 in vivo. Endothelin-converting enzyme-2 has been implicated recently in the nonclassic processing of regulatory peptides and also efficiently converts Big endothelin-1 to fully processed endothelin-1.16 Finally, phosphoramidon inhibits NEP, an integral plasma membrane ectopeptidase that functions to turn off peptide signaling events at the cell surface. NEP degrades enkephalins, tachykinins, natriuretic and chemotactic peptides, and GnRH.11,17
Koscica, Sylvestre, and Reznik
Figure 2 Effect of phosphoramidon on lipopolysaccharideinduced preterm delivery. Nine mice at day 15.5 to 16 were treated with lipopolysaccharide followed by buffer (closed squares). Thirteen mice were treated with lipopolysaccharide followed by phosphoramidon (closed triangles) at 12, 13.5, 15, 16.5, and 18 hours. Statistical analysis, with the use of the log-rank test, shows a significant difference in the effect of lipopolysaccharide in the induction of preterm delivery between mice that were treated with lipopolysaccharide followed by buffer only and mice that were treated with lipopolysaccharide followed by either dosing schedule of phosphoramidon (P!.01).
One possible mechanism for the effect of phosphoramidon on the number of preterm births in our mouse model is through the inhibition of the endothelin-converting enzymes and the decrease in fully processed endothelin-1. Endothelin-1, a 21 amino acid peptide originally isolated from porcine aortic endothelial cell culture supernatant,18 has emerged as an important mediator in the normal function of gestational tissues.19 Although the mechanism of endothelin-1 stimulation of myometrial contraction is not understood completely, it is likely that endothelin-1 works upstream of prostaglandins and, in fact, stimulates prostaglandin synthesis.10 Endothelin-1 acutely activates phospholipase A2 through phosphorylation and acute increases of intracellular calcium. Endothelin-1 also chronically enhances phospholipase A2 activity by transcriptional induction of this enzyme. In addition, endothelin-1 induces the transcription of prostaglandin endoperoxide synthase, the enzyme responsible for conversion of arachidonic acid to the endoperoxides prostaglandin G2 and prostaglandin H2. These endoperoxides are converted eventually to the prostaglandins by prostaglandin synthases. Previous investigators had found up-regulation of human placental endothelin-1 in labor at term and at preterm.20 We first showed that the distribution of human placental endothelin-converting enzyme-1 overlaps with that of human placental endothelin-1.21
531
Koscica, Sylvestre, and Reznik Another possible mechanism for the effect of phosphoramidon on our animal models is through the inhibition of NEP and decreased degradation of GnRH. NEP is highly expressed on the cell surface of human chorionic villous trophoblasts where it has access to the extrahypothalamic GnRH found in the placenta.11 The inhibition of NEP would lead to increased levels of GnRH, which could alter the quiescent state of the uterus. Because the substrate specificity of NEP is broad, an increase in any one of the many regulatory peptides that are normally degraded by NEP could be involved in the decrease in the number of premature deliveries that we observed in our study. Furthermore, other metalloenzymes for which phosphoramidon may have a mild inhibitory effect, such as the matrix metalloendopetidases, may also play a role. We report here a very dramatic effect of phosphoramidon in an animal model of preterm labor that was triggered by lipopolysaccharide, which is relevant to the preterm labor in the human that is observed with patients who experience pyelonephritis, overwhelming pneumonia, or sepsis and may be relevant in cases of idiopathic preterm labor in which clinical signs of infection are absent, but inflammatory cytokines are still present. Further studies are needed to elucidate the mechanism of the effect of phosphoramidon on preterm delivery.
7. 8.
9.
10.
11.
12. 13.
14.
15.
16.
References 1. Rush RW, Keirse MJNC, Howat P, Baum JD, Anderson AB, Turnbull AC. Contribution of preterm delivery to perinatal mortality. BMJ 1976;2:965-8. 2. Villar J, Ezcurra EJ, de la Fuente VG, Canpodonico L. Preterm delivery syndrome: the unmet need. Res Clin Forum 1994;16:9-33. 3. Kaya T, Cetin A, Cetin M, Sarioglu Y. Effects of endothelin-1 and calcium channel blockers on contractions in human myometrium: a study on myometrial strips from normal and diabetic pregnant women. J Reprod Med 1999;44:115-21. 4. Wolff K, Nisell H, Modin A, Lundberg JM, Lunell NO, Lindblom B. Contractile effects of endothelin 1 and endothelin 3 on myometrium and small intra-myometrial arteries of pregnant women at term. Gynecol Obstet Invest 1993;36:166-71. 5. Svane D, Larsson B, Alm P, Andersson KE, Forman A. Endothelin-1: imunocytochemistry, localization of binding sites, and contractile effects in human uteroplacental smooth muscle. Am J Obstet Gynecol 1993;168:233-41. 6. Kozuka M, Ito T, Hirose S, Takahashi K, Hagiwara H. Endothelin induces two types of contractions of rat uterus: phasic
17.
18.
19.
20.
21.
contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channel. Biochem Biophys Res Commun 1989;159:317-23. Goldenberg RL, Hauth MC, Andrews WW. Intrauterine infection and preterm delivery. N Engl J Med 2000;342:1500-7. Kniss DA, Iams JD. Regulation of parturition update: endocrine and paracrine effectors of term and preterm labor. Clin Perinatol 1998;25:819-36. Woods M, Mitchell JA, Wood EG, Barker S, Walcont NR, Rees GM, et al. Endothelin-1 is induced by cytokines in human vascular smooth muscle cells: evidence for intracellular endothelin-converting enzyme. Mol Pharmacol 1999;55:902-9. Shramek H, Coroneos E, Dunn MF. Interactions of the vasoconstrictor peptides, angiotensin II and endothelin-1, with vasodilatory prostaglandins. Semin Nephrol 1994;15:195-204. Kikkawa F, Kajiyama H, Ino K, Watanabe Y, Ito M, Nomura S, et al. Possible involvement of placental peptidases that degrade gonadotropin-releasing hormone(GnRH) in the dynamic pattern of placental hCG secretion via GnRH degradation. Placenta 2002; 23:483-9. Gohar J, Mazor M, Leiberman JR. GnRH in pregnancy. Arch Gynecol Obstet 1996;259:1-6. Kalra PS, Edwards TG, Xu B, Jain M, Kalra SP. The antigonadotropic effects of cytokines: the role of neuropeptides. Domest Anim Endocrinol 1998;15:321-32. Fidel PL, Romero R, Wolf N, Cutright J, Ramirez M, Araneda H, et al. Systemic and local cytokine profiles in endotoxin-induced preterm parturition in mice. Am J Obstet Gynecol 1995;170: 1467-75. Kaga N, Katsuki Y, Obata M, Shibutani Y. Repeated administration of low-dose lipopolysaccharide induces preterm delivery in mice: a model for human preterm parturition and/or assessment of the therapeutic ability of drugs against preterm delivery. Am J Obstet Gynecol 1996;174:754-9. Mzhavia N, Pan H, Che FY, Fricker LD, Devi LA. Characterization of endothelin-converting enzyme-2: implication for a role in the non-classical processing of regulatory peptides. J Biol Chem. In press. Turner AJ, Isaac RE, Coates D. The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function. Bioessays 2001;23:261-9. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayishi Y, Mitsui Y, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-5. Bouregeois C, Robert B, Rebourcet R, Mondon F, Mignot TM, Duc-Goiran P, et al. Endothelin-1 and ETA receptor expression in vascular smooth muscle cells from human placenta: a new ETA receptor messenger ribonucleic acid is generated by alternative splicing of exon 3. J Clin Endocrinol Metab 1997;82: 3116-23. Marinoni E, Picca A, Scucchi L, Cosmi EV, Dilorio R. Immunohistochemical localization of endothelin-1 in placenta and fetal membranes in term and preterm human pregnancy. Am J Reprod Immunol 1995;34:213-8. Ahmad Z, Reznik SE. Immunohistochemical localization of ECE1 in the human placenta. Placenta 2000;21:226-33.
www.elsevier.com/locate/ajog
The effect of phosphoramidon on inflammation-mediated preterm delivery in a mouse model Karen L. Koscica, DO,a Georges Sylvestre, MD,a Sandra E. Reznik, MD, PhDa,b Departments of Obstetrics and Gynecology and Women’s Healtha and Pathology,b Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY Received for publication May 2, 2003; revised July 28, 2003; accepted August 18, 2003
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Preterm delivery Phosphoramidon Metallopeptidase Cytokine
Objective: Several metallopeptidases have been implicated in both term and preterm parturition. We hypothesize that endotoxin-induced preterm delivery can be prevented by the administration of a metallopeptidase inhibitor. Study design: We used an animal model of endotoxin-induced preterm delivery in timed pregnancy C57Bl/6 mice. Test animals received lipopolysaccharide followed by phosphoramidon, either every 1.5 or every 3 hours. Control mice received lipopolysaccharide followed by buffer injections at the same intervals. The primary outcome was a preterm delivery rate. Results: The rate of preterm delivery for the control animals was 88.0% compared with the treatment groups of 45.5% for the mice that received phosphoramidon every 3 hours and 30.8% for the group that received it every 1.5 hours (P!.01). Conclusion: The administration of a metallopeptidase inhibitor resulted in a decreased rate of preterm delivery in this animal model. Ó 2004 Elsevier Inc. All rights reserved.
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Preterm delivery, defined as delivery at !37 weeks of gestation, occurs in 7% to 12% of pregnancies and is associated with 85% of perinatal morbidity and deaths.1,2 Unfortunately, the existing therapeutic approaches for preterm delivery have not decreased the incidence of prematurity and can be associated with risks to both the mother and fetus. Previous in vitro investigations have shown that endothelin-1 increases myometrial smooth muscle tone.3-6 Infection and inflammatory cytokines, which have been associated with preterm delivery,7,8 can stimulate endo-
Supported by National Institute for Child Health and Human Development grant No. 1K08HD01209. Reprints not available from the authors. 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2003.08.021
thelin-1 production.9 Endothelin-1, in turn, stimulates prostaglandin synthesis.10 Inflammatory cytokines may therefore trigger prostaglandin synthesis and, ultimately, contractions and delivery indirectly by increasing endothelin-1 synthesis. Recent data show that placental neutral endopeptidase (NEP) degrades locally produced gonadotropinreleasing hormone (GnRH).11 GnRH delays parturition by inhibiting the release of placental prostaglandins E and F and thromboxane B2 in a dose-dependent fashion.12 The inhibition of NEP would increase levels of GnRH in the myometrium and therefore decrease uterine activity. Because inflammatory cytokines are well known to decrease hypothalamic GnRH release,13 endotoxins in our mouse model may have a similar effect on GnRH that originated from placental
529
Koscica, Sylvestre, and Reznik Table
Results for mice that were treated with phosphoramidon versus control mice
Group
Treatment
No.
Preterm delivery (No.)
Preterm delivery (%)
I II III IV V VI
Buffer only mice Lipopolysaccharide + buffer every 3 h Lipopolysaccharide + phosphoramidon every 3 h Lipopolysaccharide + buffer every 1.5 h Lipopolysaccharide + phosphoramidon every 1.5 h Phosphoramidon every 1.5 h
5 8 11 9 13 4
0 7 5 8 4 0
0 88.0 45.5* 88.8 30.8* 0
*P!.01.
trophoblast. Inhibition of NEP would protect the reduced pool of paracrine GnRH. In the current study, we tested whether administering phosphoramidon, a compound that inhibits both endothelin-converting enzymes and NEP, affects endotoxin-induced preterm delivery in an animal model.
Material and methods All mice that were used for these experiments (February 2001 through August 2002) were purchased from either Jackson Laboratory (Bar Harbor, Me) or Charles River Laboratory (Wilmington, Mass). A total of 50 timed pregnant C57Bl/6 mice were used that were all at day 15.5 to 16 (of a 19-day gestation) and between 10 and 20 weeks of age. The Albert Einstein College of Medicine and Montefiore Medical Center Institutional Animal Care and Use Committees approved all animal experimental procedures. For mouse models of endotoxin-induced preterm delivery, 41 mice at day 15.5 to 16 were injected with 3.3 mg/kg lipopolysaccharide, serotype 026:B6 (Sigma Chemical Co, St Louis, Mo) intraperitoneally. Each injection contained 100 mg of lipopolysaccharide and 500 mL of phosphate-buffered saline solution (PBS). This followed the established method of the induction of inflammation-mediated preterm delivery in mice of Fidel et al14 and Kaga et al.15 Mice to receive the phosphoramidon (N-[a-rhamnopyranosyloxyhydroxyphosphinyl]-Leu-Trp; Sigma Chemical Co) were injected intraperitoneally at 6.7 mg/kg at scheduled interval dosings. Each injection contained 200 mg of phosphoramidon and 100 mL of PBS. Fifty mice were assigned to six groups. Group I served as control animals for the lipopolysaccharide injection and contained 5 mice that were injected with 500 mg of PBS intraperitoneally. Group II served as a control group that consisted of 8 mice that were treated with lipopolysaccharide and then received PBS intraperitoneally at 12, 15, 18, and 21 hours after the lipopolysaccharide injection. Group III consisted of 11 mice that were treated with lipopolysaccharide and were injected intraperitoneally with 6.7 mg/kg phosphoramidon at 12, 15, 18, and 21
hours after the lipopolysaccharide injection. Group IV served as a control group that consisted of 9 mice that were treated with lipopolysaccharide and received PBS intraperitoneally at 12, 13.5, 15, 16.5, and 18 hours after the lipopolysaccharide injection. Group V had 13 of the mice that were treated with lipopolysaccharide and were injected with 6.7 mg/kg phosphoramidon intraperitoneally at 12, 13.5, 15, 16.5, and 18 hours after the lipopolysaccharide injection. Group VI served as control animals for the phosphoramidon injections and contained 4 mice that were injected with 6.7 mg/kg phosphoramidon only at 12, 13.5, 15, 16.5, and 18 hours. All animals were observed for preterm delivery. After 24 hours, the mice were killed to confirm pregnancy and to collect placental and uterine tissue for future studies. The effect of phosphoramidon on lipopolysaccharide-induced labor was assessed with the log-rank test, with the time to labor as the outcome of interest. The primary outcome was the preterm delivery rate.
Results Of the 5 mice in group I (intraperitoneal PBS alone), none of the mice were delivered preterm. For mice that were assigned to control groups II and IV, 15 of the 17 mice that were treated with lipopolysaccharide followed by buffer were delivered only within 19 hours. (One mouse in each group failed to respond to lipopolysaccharide therapy). In group III that received lipopolysaccharide followed by phosphoramidon every 3 hours, 5 of 11 mice were delivered by 24 hours. Finally, in group V that received lipopolysaccharide followed by phosphoramidon every 1.5 hours, only 4 of 13 mice were delivered (Table). These results showed a significant decrease in the preterm delivery rate from 88.0% in the control groups to 45.5% in the 3-hour treated group to 30.8% in the 1.5-hour treated group. Statistical analysis, with the log-rank test, showed a significant difference (P!.01) in the effect of lipopolysaccharide in inducing preterm labor between mice that were treated with lipopolysaccharide followed by buffer only and mice that were treated with lipopolysaccharide followed by either dosing schedule of phosphoramidon every 3 hours
530
Figure 1 Effect of phosphoramidon on lipopolysaccharideinduced preterm delivery. Eight mice at day 15.5 to 16 were treated with lipopolysaccharide followed by buffer (closed squares). Eleven mice were treated with lipopolysaccharide, followed by phosphoramidon (closed triangles) at 12, 15, 18, and 21 hours. Statistical analysis, with the use of the log-rank test, shows a significant difference in the effect of lipopolysaccharide in the induction of preterm delivery between mice that were treated with lipopolysaccharide followed by buffer only and mice that were treated with lipopolysaccharide followed by either dosing schedule of phosphoramidon (P!.01).
(Figure 1) or every 1.5 hours (Figure 2). There was no significant difference between the two treatment groups. None of the 4 mice in group VI was delivered, as expected. These mice were killed at 24 hours, and an autopsy was performed to test for potential the harmful affects of the phosphoramidon injections. No abnormalities were found in either the mothers or the pups. The number of viable pups per pregnancy did not differ from the control animals in group I, and no intrauterine deaths occurred.
Comment Three closely related metallopeptidases of the M13 family of zinc peptidases are inhibited by phosphoramidon. Endothelin-converting enzyme-1 converts Big endothelin-1 to fully processed endothelin-1 by cleavage of a Trp21-Val22 peptide bond. Endothelin-converting enzyme-1 has not been found to synthesize any peptides other than endothelin-1 in vivo. Endothelin-converting enzyme-2 has been implicated recently in the nonclassic processing of regulatory peptides and also efficiently converts Big endothelin-1 to fully processed endothelin-1.16 Finally, phosphoramidon inhibits NEP, an integral plasma membrane ectopeptidase that functions to turn off peptide signaling events at the cell surface. NEP degrades enkephalins, tachykinins, natriuretic and chemotactic peptides, and GnRH.11,17
Koscica, Sylvestre, and Reznik
Figure 2 Effect of phosphoramidon on lipopolysaccharideinduced preterm delivery. Nine mice at day 15.5 to 16 were treated with lipopolysaccharide followed by buffer (closed squares). Thirteen mice were treated with lipopolysaccharide followed by phosphoramidon (closed triangles) at 12, 13.5, 15, 16.5, and 18 hours. Statistical analysis, with the use of the log-rank test, shows a significant difference in the effect of lipopolysaccharide in the induction of preterm delivery between mice that were treated with lipopolysaccharide followed by buffer only and mice that were treated with lipopolysaccharide followed by either dosing schedule of phosphoramidon (P!.01).
One possible mechanism for the effect of phosphoramidon on the number of preterm births in our mouse model is through the inhibition of the endothelin-converting enzymes and the decrease in fully processed endothelin-1. Endothelin-1, a 21 amino acid peptide originally isolated from porcine aortic endothelial cell culture supernatant,18 has emerged as an important mediator in the normal function of gestational tissues.19 Although the mechanism of endothelin-1 stimulation of myometrial contraction is not understood completely, it is likely that endothelin-1 works upstream of prostaglandins and, in fact, stimulates prostaglandin synthesis.10 Endothelin-1 acutely activates phospholipase A2 through phosphorylation and acute increases of intracellular calcium. Endothelin-1 also chronically enhances phospholipase A2 activity by transcriptional induction of this enzyme. In addition, endothelin-1 induces the transcription of prostaglandin endoperoxide synthase, the enzyme responsible for conversion of arachidonic acid to the endoperoxides prostaglandin G2 and prostaglandin H2. These endoperoxides are converted eventually to the prostaglandins by prostaglandin synthases. Previous investigators had found up-regulation of human placental endothelin-1 in labor at term and at preterm.20 We first showed that the distribution of human placental endothelin-converting enzyme-1 overlaps with that of human placental endothelin-1.21
531
Koscica, Sylvestre, and Reznik Another possible mechanism for the effect of phosphoramidon on our animal models is through the inhibition of NEP and decreased degradation of GnRH. NEP is highly expressed on the cell surface of human chorionic villous trophoblasts where it has access to the extrahypothalamic GnRH found in the placenta.11 The inhibition of NEP would lead to increased levels of GnRH, which could alter the quiescent state of the uterus. Because the substrate specificity of NEP is broad, an increase in any one of the many regulatory peptides that are normally degraded by NEP could be involved in the decrease in the number of premature deliveries that we observed in our study. Furthermore, other metalloenzymes for which phosphoramidon may have a mild inhibitory effect, such as the matrix metalloendopetidases, may also play a role. We report here a very dramatic effect of phosphoramidon in an animal model of preterm labor that was triggered by lipopolysaccharide, which is relevant to the preterm labor in the human that is observed with patients who experience pyelonephritis, overwhelming pneumonia, or sepsis and may be relevant in cases of idiopathic preterm labor in which clinical signs of infection are absent, but inflammatory cytokines are still present. Further studies are needed to elucidate the mechanism of the effect of phosphoramidon on preterm delivery.
7. 8.
9.
10.
11.
12. 13.
14.
15.
16.
References 1. Rush RW, Keirse MJNC, Howat P, Baum JD, Anderson AB, Turnbull AC. Contribution of preterm delivery to perinatal mortality. BMJ 1976;2:965-8. 2. Villar J, Ezcurra EJ, de la Fuente VG, Canpodonico L. Preterm delivery syndrome: the unmet need. Res Clin Forum 1994;16:9-33. 3. Kaya T, Cetin A, Cetin M, Sarioglu Y. Effects of endothelin-1 and calcium channel blockers on contractions in human myometrium: a study on myometrial strips from normal and diabetic pregnant women. J Reprod Med 1999;44:115-21. 4. Wolff K, Nisell H, Modin A, Lundberg JM, Lunell NO, Lindblom B. Contractile effects of endothelin 1 and endothelin 3 on myometrium and small intra-myometrial arteries of pregnant women at term. Gynecol Obstet Invest 1993;36:166-71. 5. Svane D, Larsson B, Alm P, Andersson KE, Forman A. Endothelin-1: imunocytochemistry, localization of binding sites, and contractile effects in human uteroplacental smooth muscle. Am J Obstet Gynecol 1993;168:233-41. 6. Kozuka M, Ito T, Hirose S, Takahashi K, Hagiwara H. Endothelin induces two types of contractions of rat uterus: phasic
17.
18.
19.
20.
21.
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