Hydrogen sulphide compounds

Abstract

The application describes Hydrogen Sulphide (H.sub.2S), or a (H.sub.2S) generating compound or compound capable of stimulating H.sub.2S production in a pregnant subject, for use in the treatment of pre-eclampsia (PE) or fetal growth restriction.

Claims

1. A method for treating pre-eclampsia or fetal growth restriction in a host animal, the method comprising (a) measuring an amount of H.sub.2S in a sample from the host animal; and (b) administering a therapeutically effective amount of a compound capable of generating hydrogen sulfide to the host animal.

2. The method of claim 1 wherein the compound is ACS14, ACS583, ACS84, ACS85, ACS86, DATS (diallyl trisulfide), S-diclofenac, sulfane, sulfur, thiocysteine, GSH hydropersulfide, GYY4137, SG1002, a H.sub.2S-donating derivative of sildenafil, ADT-OH, TBZ, 4-hydroxyphenyl isothiocyanate, thioglycine, 1-thiolysine, 1-thiovaline, H.sub.2S-Sartans, or H.sub.2S-L-DOPA or a salt of any of the foregoing, or a combination of any of the foregoing.

3. The method of claim 1 comprising administering GYY4137.

4. The method of claim 3 for treating pre-eclampsia.

5. The method of claim 3 for treating fetal growth restriction.

6. The method of claim 1 wherein the compound is ACS14, ACS583, ACS84, ACS85, or ACS86, or a salt of any of the foregoing, or a combination of any of the foregoing.

7. The method of claim 6 for treating pre-eclampsia.

8. The method of claim 6 for treating fetal growth restriction.

9. The method of claim 1 comprising measuring the amount of H.sub.2S in a sample of blood, serum, or plasma from the host.

10. The method of claim 1 comprising administering ACS14 or ADT-OH or a salt thereof.

11. The method of claim 10 for treating pre-eclampsia.

12. The method of claim 10 for treating fetal growth restriction.

13. The method of claim 11 comprising administering ACS14.

14. The method of claim 12 comprising administering ACS14.

15. The method of claim 1 wherein the amount of H.sub.2S is measured from CSE expression.

16. The method of claim 1 wherein the amount of H.sub.2S is measured from sFlt-1 expression.

17. The method of claim 1 wherein the amount of H.sub.2S is measured from PlGF production.

18. The method of claim 1 wherein the amount of H.sub.2S is measured from sEng production.

19. The method of claim 1 wherein the amount of H.sub.2S is measured from inhibition level of VEGF.

20. The method of claim 1 wherein the amount of H.sub.2S is measured from inhibition level of PlGF.

Description

(1) The invention will now be described by way of example only with reference to the following Figures:

(2) FIG. 1. CSE expression and H.sub.2S levels in preeclampsia.

(3) FIG. 2. Effects of CSE inhibition on angiogenesis factor release from human 1.sup.st trimester placenta.

(4) FIG. 3. CSE modulates sFlt-1 and sEng release in endothelial cells.

(5) FIG. 4. H.sub.2S rescues preeclamptic sera-induced inhibition of in vitro tube formation.

(6) FIG. 5. Inhibition of CSE reduces endogenous circulating H.sub.2S and promotes hypertension and abnormal placental vascularisation in pregnant mice.

(7) FIG. 6. H.sub.2S-generating compound, GYY4137 restored fetal growth and inhibits sFlt-1 and sEng induced by CSE inhibition in pregnant mice.

(8) FIG. 7. Effect of inhibition of CSE on trophoblast cell invasion.

(9) FIG. 8. H.sub.2S donor restores placental vascularisation in pregnant mice.

MATERIALS AND METHODS

(10) Placental Tissue Collection and Preparation

(11) Institutional Ethics Committee approved the blood and tissue collection and written informed consent was obtained. We analysed blood samples from women with singleton pregnancies recruited in the Low- and High-Risk Clinics and Labour and Delivery Unit. All women were followed prospectively from enrolment until delivery. Human placental tissues were collected from pregnancies complicated by preeclampsia and uncomplicated pregnancies delivered by elective Caesarean. Samples of placental tissue were processed for RNA extraction and maternal plasma from the same patients (n=14 PE and n=14 control) were used for analysis. From another set of patients placenta (n=5 PE and n=5 control) was collected for the immunohistochemical study. Preeclampsia was defined as blood pressure>140/90 mm Hg on at least 2 consecutive measurements and maternal proteinuria of at least 300 mg/24 h. First trimester placental tissues (6-9 weeks gestational age) were retrieved from normal pregnancies that had undergone elective termination. Villus explants were prepared as described previously..sup.29 Briefly, human placental villus explants were incubated with or without PAG for 24 hours, and conditioned media collected and assayed for sFlt-1 or sEng and PlGF.

(12) Animal Experimental Protocol

(13) Eight to ten week old C57/black6 mice were mated. The first day of pregnancy (E0.5) was defined by the presence of a vaginal plug the following morning. Pregnant mice were randomly assigned into four groups: (i) saline (vehicle control), (ii) 25 mg/kg DL-propargylglycine (PAG; Sigma, Poole, U.K.), (iii) 50 mg/kg group PAG and (iv) 50 mg/kg

(14) PAG with 0.25 mg/kg of slow-releasing H.sub.2S donor, GYY4137(Sigma). Mice were injected intraperitoneally (i.p.) with saline or PAG from E8.5. Blood pressure was measured by tail cuff-plethysmography. Mice were trained for measurement on alternate days from E4.5. Alternatively, mice were anesthetized by Ketamine/Xylazine cocktail. The carotid artery was isolated and cannulated with a 3-Fr high-fidelity microtip catheter connected to a pressure transducer (Millar Instruments, Houston, Tex., USA). Blood pressure was recorded and averaged over a 10-minute period. On E17.5, after blood pressure measurement and blood sample collection the animals were sacrificed and kidney, liver, and placenta were collected. The un-absorbed fetuses and placentas were counted and weighed.

(15) All experimentation was conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986 using procedures approved by the University of Edinburgh Ethical Review Committee.

(16) Histopathology

(17) Kidney, liver, and placenta were immersion fixed in 4% paraformaldehyde for 24 hours and processed to paraffin. A series of 5-m sections were cut and processed for hematoxylin & eosin (H&E) staining.

(18) Cell Culture

(19) Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as previously described..sup.30 Experiments were performed on third or fourth passage HUVEC.

(20) RNA Interference

(21) To silence human CSE expression, we performed transfection of small-interfering RNA (siRNA) duplex using electroporation (Nucleofector, Amaxa). Control and CSE siRNAs were synthesized by Eurogentec (Cologne, Germany). Knockdown of CSE in HUVEC was confirmed using Western blotting.

(22) Adenoviral Gene Transfer

(23) The recombinant, replication-deficient adenovirus encoding human CSE (AdCSE) and empty vector (AdEV) were purified on CsCl gradients, titered, and stored at 80 C. in viral storage buffer prior to use as described previously..sup.31 Optimal multiplicity of infection for AdCSE was determined to be 20 IFU/cell by Western blotting using a rabbit anti-CSE antibody (Abcam). AdEV infected HUVEC were used as a negative control.

(24) Enzyme-Linked Immunosorbent Assay

(25) Enzyme-linked immunosorbent assay (ELISA) kits for human and murine soluble Flt-1, soluble endoglin and PlGF were obtained from R&D Systems and performed according to the manufacturer's specifications.

(26) Immunohistochemistry

(27) Serial 3-5-m sections of formalin-fixed, paraffin-embedded human and murine placental tissue were prepared for immunohistochemistry as previously described..sup.29 Biotin-labelled isolectin B4, anti-CSE (5 mg/ml) and isotype control were used. The staining was analyzed using a Nikon inverted microscope and an Image Pro Plus image analysis software (Media Cybernetics).

(28) Real-Time Polymerase Chain Reaction (PCR)

(29) Sample preparation and real-time quantitative PCR was performed as described previously..sup.30 Briefly, mRNA from placental tissue was extracted using TRIzol and DNase-1 digestion/purification on RNAeasy columns (Qiagen), and reverse transcribed with the cDNA Synthesis Kit (Promega). Triplicate cDNA samples and standards were amplified in SensiMix containing SYBR green (Quantace) with primers specific for CSE (GCC-CAG-TTC-CGT-GAA-TCT-AA (SEQ ID NO: 1); CAT-GCT-GAA-GAG-TGC-CCT-TA) (SEQ ID NO: 2)) or -actin. The mean threshold cycle (CT) for CSE was normalized to -actin and expressed relative to control.

(30) In Vitro Angiogenesis Assay

(31) The spontaneous formation of capillary-like structures by HUVECs on growth factor-reduced Matrigel (Becton Dickinson, Bedford, Mass.), was used to assess angiogenic potential. HUVECs were treated with plasma samples collected from pregnancies complicated by preeclampsia and uncomplicated pregnancies with or without H.sub.2S donor (NaHS) and incubated at 37 C. for 24 hours. Ninety-six well plates were coated with Matrigel (10 mg/ml) according to the manufacturer's instructions. HUVECs (110.sup.4 cells/well) were then seeded on Matrigel-coated plates. After incubation for 6 hours, cells were observed with a Nikon inverted microscope and experimental results recorded using the Image Pro-Plus image analysis software (Media Cybernetics).

(32) Measurement of H.sub.2S in Plasma

(33) Citrated blood was obtained from women with uncomplicated pregnancies (n=14) and preeclampsia (n=14) and also from pregnant mice before termination of pregnancy. H.sub.2S levels were measured as described previously with modification..sup.32 Briefly, 75 l plasma was mixed with 250 l of 1% (w/v) zinc acetate and 425 l water, followed by 250 ml 50% trichloroacetic acid to remove protein. Then 133 l 20 mM N-dimethyl-p-phenylenediamine sulphate in 7.2 mM HCl and 133 l 30 M FeCl.sub.3 in 1.2 mM HCl were added to the mixture. After 10 min incubation at room temperature, reaction mixtures were pelleted by centrifugation at 10,000 g (2 minutes). The absorbance of the resulting solution was measured at 670 nm with a spectrophotometer in a 96-well plate. The concentration of H.sub.2S in the solution was calculated against a calibration curve of sodium hydrogen sulfide.

(34) Statistical Analysis

(35) Data are expressed as meanSEM. The significance of the difference between means was tested by non-parametric Man Whitney t-test. For statistical analysis of changes in clinical samples, one-way ANOVA was used, followed by the Student-Newman-Keuls test as appropriate. An observer blinded to treatment performed the analyses. Statistical significance was set at p<0.05.

(36) Results

(37) Placenta CSE Expression is Reduced in Preeclampsia

(38) To investigate whether CSE/H.sub.2S activity is altered in preeclampsia, H.sub.2S was measured in plasma obtained from gestational age-matched control pregnancies and those complicated by preeclampsia. Maternal plasma H.sub.2S levels were significantly reduced in preeclampsia compared with controls group (FIG. 1A). Quantitative real-time PCR revealed that the CSE mRNA expression was significantly reduced in preeclamptic placenta (FIG. 1B) and immunohistochemical staining confirmed that CSE immunoreactiovity was dramatically reduced in these samples (FIG. 1C iv) suggesting that the changes in placental CSE levels affect maternal circulating H.sub.2S levels. Expressed of CSE was located in the trophoblast, the endothelium and the mesenchymal cells within the core of the chorionic villus. The latter are possibly the Hofbauer cells, which are of mesenchymal origin (FIG. 1C iii). Clinical characteristics of the study patients are described in Table 1.

(39) Inhibition of CSE Activity Reduces PlGF Release in Placental Explants

(40) Angiogenic factors produced by placenta are important in regulating placental vascular development..sup.33 Imbalance of pro- and anti-angiogenic factors generated by the placenta.sup.29 may account for the widespread maternal endothelial dysfunction in preeclampsia..sup.34 To investigate whether reduced levels of CSE has any effect on the production of placental angiogenic factor production, sFlt-1, sEng, and PlGF levels were measured in conditioned medium from first trimester human placental explants in the presence of increasing concentration of CSE inhibitor PAG over 24 hours. While the levels of sFlt-1 and sEng remained unchanged by the inhibition of CSE activity, PlGF production was significantly reduced (FIG. 2). This suggests that reduction in endogenous H.sub.2S may alter the placental sFlt-1/PlGF ratio, which has been implicated in the pathogenesis of preeclampsia..sup.18, 29 In addition, a significant reduction (p<0.01) in cell invasion was observed when first trimester trophoblast cells (HTR-8/SVneo) were incubated with the CSE specific inhibitor PAG (50 M) compared with the vehicle control (Supplemental FIGS. S1A and S1B), suggesting that lack of CSE activity may affect placental perfusion which is essential for establishing normal pregnancy.

(41) CSE Modulates sFlt-1 and sEng Release in Endothelial Cells

(42) Although placenta has been considered to be the main source of sFlt-1 and sEng release in preeclampsia patients, some studies have shown that levels of sFlt-1 remained higher in women with a history of preeclampsia compared with those without preeclampsia an average of 18 months postpartum.sup.35, 36 suggesting that other antiangiogenic milieu are involved in the process. To investigate whether CSE affects sFlt-1 and sEng release in endothelial cells, CSE expression was modulated by siRNA or adenovirus in HUVECs. Down-regulation of CSE increased both sFlt-1 and sEng release (FIG. 3A, 3B) while over-expression of CSE inhibited sFlt-1 and sEng release by HUVECs (FIG. 3C, 3D). These data further support the concept that loss of CSE activity may contribute to the pathogenesis of preeclampsia.

(43) H.sub.2S Partially Rescues Preeclamptic Plasma-Induced Inhibition of In Vitro Tube Formation

(44) It has been demonstrated that excess sFlt-1 generated by preeclamptic placenta inhibits in vitro endothelial tube formation and removal of sFlt-1 from preeclampsia samples restores angiogenesis..sup.29 To assess whether H.sub.2S can reverse the anti-angiogenic effects of preeclampsia, plasma from normotensive or preeclamptic women was added to HUVEC grown on growth factor-reduced Matrigel in the presence of 100 mM NaHS, a H.sub.2S donor, and in vitro tube formation assay performed. Consistence with earlier findings, preeclamptic plasma inhibited capillary tube network formation compare with normal control sera (FIG. 4). More importantly, NaHS, a H.sub.2S donor, partially restored the ability of HUVECs to form tube-like structure (FIG. 4A, 4B).

(45) Blocking Endogenous H.sub.2S Causes Hypertension and Abnormal Placental Vascularisation in Pregnant Mice

(46) We predicted that inhibition of CSE in vivo would cause a preeclampsia-like syndrome in pregnant mice. Three groups (5-8/group) of pregnant C57Bl6/J mice were treated daily with vehicle or 25 mg/kg PAG or 50 mg/kg PAG from E8.5 to E16.5. After 8 days of treatment, plasma was pooled from all animals in each treatment group, and pooled H.sub.2S levels were measured. PAG caused a dose-dependent decrease in circulating H.sub.2S levels. The higher dose reduced plasma H.sub.2S levels by approximately 50% (FIG. 5A). In consistence of these data, we found that the mean blood pressure in high dose PAG treated group was significant higher compared with vehicle control (74.404.61 and 64.742.04) (Table 2). Although renal pathological changes such as proteinuria was not noted in our PAG treated animals (Table 2), liver damage represented by increased level of circulating liver enzyme aspartate transaminas (AST) was found in PAG-treated animals (Table 2). These date suggest that lack of CSE activity may cause non-proteinuric preeclampsia. Interestingly, all the changes of PAG-treated pregnant mice were abrogated by a slow releasing, H.sub.2S-generating compound GYY4137 (0.25 mg/kg) treatment (Table 2). Blinded histological analysis of placental sections showed that the maternal blood space in the labyrinth zone appeared larger in 50 mg/kg PAG-treated animals than in vehicle controls (FIG. 5B). The labyrinth zone consists of cells of trophoblast and mesodermal origin that together undergo branching morphogenesis, resulting in a large surface area for nutrient and gas exchange between the mother and fetus.

(47) The maternal blood space is lined by trophoblast. During placental development, this space becomes progressively more finely divided..sup.37, 38 Using isolectin B4 to highlight the fetal endothelial cell,.sup.39 we compared the anatomical features of the labyrinth zone in vehicle and 50 mg/kg PAG-treated mice. In control mice, the labyrinth appeared as organised fetal vessels with well-developed branching morphogenesis. In contrast, the fetal vasculature of the placenta in PAG-treated animals was observed as irregular branching (FIG. 5C) and maternal vasculature appear to be dilated. The morphology of placenta from PAG-treated animals suggests a placental vascular defect due to inhibition of CSE activity. This defect was also restored by H.sub.2S donor GYY4137 treatment (FIG. S2).

(48) The Effects of Inhibition of CSE Activity on Fetal Outcomes and sFlt-1 and sEng Production

(49) Fetal weight was significantly decreased in mice that received the higher dose of PAG (FIG. 6A). This could be explained by placental vascular defect induced by inhibition of CSE activity. GYY4137, at 0.25 mg/kg restored fetal growth compromised by the CSE inhibitor (FIG. 6A). Furthermore, GYY4137 inhibited the plasma levels of sFlt-1 and sEng induced by CSE inhibition (FIGS. 6B and 6C) in mice treated with 50 mg/kg PAG (FIGS. 5B and 5C). Plasma PlGF was below the detection limit of the assay. These data suggest that inhibition of CSE activity alters maternal angiogenic balance and H.sub.2S can help to restore normal angiogenic status.

(50) Discussion

(51) Chronic administration of a CSE inhibitor leads to reduced H.sub.2S and increased blood pressure in rats..sup.7 Thus it is plausible that a reduction in the circulating H.sub.2S level may contribute to hypertension in preeclampsia. In this study we provide evidence that preeclampsia is associated with reduced circulating H.sub.2S, which is accompanied by down-regulation of placental CSE, the key enzyme responsible for the generation of endogenous H.sub.2S. Furthermore, the inhibition of CSE in pregnant mice induces hypertension, increases sFlt-1 and sEng levels and causes placental abnormalities. This is due to inhibition of H.sub.2S production as a slow releasing, H.sub.2S-generating compound, GYY4137, inhibited circulating sFlt-1 and sEng levels and restored fetal growth compromised by CSE inhibition. These findings indicate that a dysfunctional CSE/H.sub.2S pathway may contribute to the pathogenesis of preeclampsia.

(52) H.sub.2S is a vasorelaxant factor that acts through K.sub.ATP channels causing smooth muscle relaxation.sup.1, 40 Studies using mice genetically deficient in CSE demonstrated that this enzyme is the major source of H.sub.2S in both the vasculature and the peripheral tissues..sup.9 Recently, CSE expression was found in the placenta and pregnant myometrium and it was shown to play a role in uterine contractility..sup.27, 28 In this study, placental CSE levels were dramatically reduced in preeclamptic patients compared with normotensive controls. A recent study also showed similar pattern in CSE in preeclamptic placenta..sup.41 These findings suggest that lack of the CSE leads to the reduction in circulating H.sub.2S.

(53) Angiogenic imbalance has been highlighted as the prime culprit in preeclampsia over systemic inflammation..sup.42, 43 In this study, CSE was found to be a negative regulator of anti-angiogenic factors, sFlt-1 and sEng, in endothelial cells, suggesting that dysregulation of CSE may contribute to the lasting endothelial dysfunction and an elevated risk of cardiovascular disease in women with a history of preeclampsia. In addition, the decrease in VEGF and PlGF activity in preeclampsia is believed to be the result of excess sFlt-1..sup.16, 18 As sFlt-1 levels are comparable to healthy controls during the first trimester of pregnancy, this theory does not explain why the circulating levels of PlGF are low in early pregnancy in women who subsequently develop preeclampsia..sup.44 Our findings that inhibition of endogenous placental H.sub.2S generation by CSE inhibitor attenuates the production of PlGF in first trimester placental explants provides a possible explanation and a new hypothesis for testing: namely, the decrease in PlGF expression in early pregnancy is due to loss or reduction in the enzymes producing H.sub.2S. Furthermore, inhibition of CSE activity abolished the invasion of first-trimester extravillus trophoblast cells suggesting that dysregulation of CSE/H.sub.2S pathway may not only change the balance of placental pro- and anti-angiogenesis factors, but also dysregulate maternal spiral artery remodeling and placental development.

(54) In pregnant mice, CSE inhibition reduced endogenous H.sub.2S and this was accompanied by an increase in blood pressure, and liver damage without renal pathological changes such as proteinuria and glomerular endotheliosis, a syndrome similar to non-proteinuric preeclampsia. However, it also suggests that other factors are also involved in the full spectrum of preeclampsia. Preeclampsia is also strongly associated with placental abnormalities including compromised villus volume and surface area, as well as reduced placental vascularisation..sup.15, 45 In the PAG-treated mice, the fetal labyrinth showed impaired branching morphogenesis, indicating endogenous H.sub.2S is required for placental development.

(55) Impaired placental perfusion and suboptimal oxygen and nutrient diffusion has been reported to occur as a result of inappropriate labyrinth vascularisation with altered patterning, branching and dilation..sup.46 Blood pressure, liver function and fetal weight compromised by PAG-treatment were rescued by the slow releasing, H.sub.2S-generating compound, GYY4137, demonstrating that the effects of CSE inhibitor were due to inhibition of H.sub.2S production. These results imply that endogenous H.sub.2S is required for healthy placental vasculature to support fetal wellbeing.

(56) Clinical Perspective

(57) The present study shows that dysregulation of CSE/H.sub.2S pathway is associated with preeclampsia and inhibition of CSE activity in pregnant mice produces some of the features of preeclampsia, including hypertension and impaired fetal outcomes. These findings support the concept that H.sub.2S is an important regulator of the placental vasculature development, a deficiency of which appears to be associated with preeclampsia and fetal growth restriction.

(58) Supplemental Methods

(59) Trophablast Cell Invasion Assay

(60) The human extravillus trophoblast (EVT) cell line HTR-8/SVneo was a kind gift from Professor Charles H. Graham, Queen's University, Kingston, Ontario, Canada. The invasion assay was performed as described previously, with modification.30 Briefly, HTR-8/SVneo (50,000) cells treated with or without PAG were placed in the upper chamber of Matrigel-coated (1 mg/ml) transwell inserts (8 m pore, Falcon, BD, UK) and housed in a 24-well plate. The cells were allowed to invade through the reconstituted extracellular matrix for 24 h in the presence or absence of 50 M PAG (n=3). Trophoblast cells located on the under-surface of the transwell membrane were fixed with ice-cold methanol and stained with hematoxylin, and brightfield images were obtained with Nikon inverted microscope and Image Pro Plus image analysis software (Media Cybernetics).

(61) Immunohistochemistry

(62) Serial 3-5-m sections of formalin-fixed, paraffin-embedded murine placental tissue were prepared for immunohistochemistry as previously described.sup.48. Biotin-labelled isolectin B4, were used. The staining was analyzed using a Nikon inverted microscope and an Image Pro Plus image analysis software (Media Cybernetics).

(63) FIG. 7. Effect of Inhibition of CSE on Trophoblast Cell Invasion.

(64) Transwell migration assays of HTR-8/SVneo cells in the presence of 50 M of PAG were performed as described in Methods. (A) Migrated HTR-8/SVneo were stained with hematoxylin, and brightfield images were captured. (B) Cell numbers were counted, and results are expressed as a percentage of the control (n=3).

(65) FIG. 8. H.sub.2S Donor Restores Placental Vascularisation in Pregnant Mice.

(66) Placental tissue from mice received either (A) PAG 50 mg/kg or (B) PAG 50 mg/kg plus GYY4137 injection were sectioned and stained with Isolectin B4 to visualize haemotrichorial labyrinth zone.

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