METHODS OF TREATING DISEASES CHARACTERISED BY VASOCONSTRICTION

Abstract

There is herein provided a compound that is an mPGES-1 inhibitor, or a prodrug thereof, for use in the treatment or prophylaxis of a disease or disorder characterised by vasoconstriction.

Claims

1. A method of treating pulmonary arterial hypertension (PAH) in a subject, comprising administering to a subject an effective amount of a compound represented by the following structural formula: ##STR00203## or a pharmaceutically acceptable salt thereof.

2-17. (canceled)

18. A method of reducing the likelihood of developing pulmonary arterial hypertension (PAH) in a subject, comprising administering to a subject who is at risk of developing pulmonary arterial hypertension (PAH) an effective amount of a compound represented by the following structural formula: ##STR00204## or a pharmaceutically acceptable salt thereof, thereby reducing the likelihood of developing pulmonary arterial hypertension (PAH) in the subject.

Description

FIGURES

[0504] FIGS. 1 to 6 show results obtained from the experiments described in Examples 1 and 2 herein below.

[0505] FIG. 1 shows the effects of COX-2 inhibition using parecoxib and deletion of mPGES-1 on plasma levels of ADMA in mice (* shows p<0.05 by one-way ANOVA with Dunnett's post-hoc test).

[0506] FIG. 2 shows the effects of COX-2 inhibition using parecoxib and deletion of mPGES-1 on the expression of genes responsible for synthesis of ADMA (Prmt1) in mice (* shows p<0.05 by one sample t-test).

[0507] FIG. 3 shows the effects of COX-2 inhibition using parecoxib and deletion of mPGES-1 on the expression of genes responsible for degradation of ADMA (Agxt2) in mice (* shows p<0.05 by one sample t-test).

[0508] FIG. 4 shows that, in mPGES-1 deficient mice, deletion of mPGES-1 significantly improved the eNOS driven dilator response to acetylcholine in aorta (* shows p<0.05 by two-way ANOVA).

[0509] FIG. 5 shows that the effect observed in FIG. 4 was not mediated by an increased sensitivity of the vessels to NO, as responses to the exogenous NO donor, sodium nitroprusside, were not altered (* shows p<0.05 by two-way ANOVA).

[0510] FIG. 6 shows that the effect observed in FIG. 4 was not mediated by changes in contractility since responses to U46619 (a thromboxane mimetic) were not different between wild-type and mPGES-1 deficient mice (* shows p<0.05 by two-way ANOVA).

EXAMPLES

[0511] The present invention will now be illustrated by way of the following examples.

[0512] General Experimental Methods

[0513] Animals

[0514] Experiments were performed on male and female mPGES-1.sup.−/− and their wild-type mPGES-1.sup.+/+ littermate mice at 6-8 weeks of age. Mice were generated on a DBA/1J background and had a deletion in the Ptges gene by breeding heterozygous littermates and experimental animals and controls identified by genomic PCR as previously described (see Trebino, C. E. et al., Proc Natl Acad Sci USA., 100, (2003) 9044-9049). All mice were housed with a 12 h light/dark cycle in a climate-controlled environment, and were fed with standard rodent chow with water ad libitum. All mice experiments were conducted in line with EU directive 2010/63/EU and according to guidelines from the Swedish Veterinary board the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Studies were sanctioned by the Karolinska Institute ethics committee (dnr. N86_13 and N364_11), the Shantou University Institutional Animal Research and Use Committee and/or the Imperial College London Ethical Review Panel (PPL 70/7013 and 70/8422). Where indicated, wild-type (mPGES-1.sup.+/+) mice were treated with the selective COX-2 inhibitor, parecoxib (100 mg/kg; Pfizer, USA) added to their drinking water for 5 days prior to tissue collection as we have done before (see Ahmetaj-Shala, B. et al., Circulation, 131, 633-642 (2015)).

[0515] Circulating Mediators

[0516] Mice were killed by CO.sub.2 narcosis, and blood collected from the inferior vena cava into heparin (10 U/ml final; Leo Laboratories, UK). Plasma was separated by centrifugation for the following circulating mediators measured by immunoassay: ADMA (DLD Diagnostika, Germany), PGE.sub.2 (Cisbio Bioassays, France).

[0517] PG Release

[0518] PG release ex vivo was measured as previously described (see Kirkby, N. S. et al., Proc Natl Acad Sci USA., 109:17597-602 (2012); and Kirkby, N. S. Et al., PLoS One, 8, e69524 (2013)). Briefly, segments of aorta (2 mm length; cleaned of peri-adventitial material), renal medulla (2×2×2 mm) or renal cortex (2×2×2 mm) were removed from experimental animals and placed in wells of microtitre plates containing DMEM media (Sigma, UK) and Ca.sup.2+ ionophore A23187 (30 uM; Sigma, UK) or acetylcholine (10 uM; Sigma, UK).

[0519] Gene Expression

[0520] RNA was isolated from renal medulla homogenates using a magnetic silica-bead isolation kit (Life Technologies, UK) and converted to cDNA using reverse transcriptase (Thermo Fisher Scientific, UK) with oligo(dT) primers (Life Technologies, UK). Gene expression levels were determined using TaqMan hydrolysis probes (Life Technologies, UK) recognising Prmt1 (probe ID: Mm00480133_m1 or Agxt2 (probe ID: Mm01304088_m1). Data were normalised to expression of the housekeeping genes 18S (probe ID: Mm03928990_g1) and Gapdh (probe ID: Mm99999915_g1) and relative expression compared using the comparative Ct method.

[0521] Vascular Function

[0522] Mouse aorta was isolated, cleaned of peri-advential material and divided into 2 mm rings. These were loaded into organ baths of a Malveny-Halpern wire myography (DMT, Denmark) containing Krebs buffer (composition: 120 mM NaCl; 4.7 mM KCl; 1.2 mM MgSO.sub.4; 1.2 mM KH.sub.2PO.sub.4; 25 mM NaHCO.sub.3; 0.03 mM EDTA; 5.5 mM D-glucose) at 37° C. Vessel responses were recording via a force transducer connected to a digital signal acquisition system (AD Instruments, UK). Resting tension was gradually applied to model a transmural pressure of 13.3 kPa. Vessel responsiveness was refreshed and assessed by application of three consecutive challenges with 125 mM KCl with washing with Krebs buffer in between. Concentration-response curves where then recorded in response to the thromboxane mimetic, U46619 (1-300 nM; Cayman Chemical, USA), acetylcholine (1 nM-30 μM; Sigma, UK) and sodium nitroprusside (1 nM-30 μM; Sigma UK). Responses to dilator agents (acetylcholine and sodium nitroprusside) were determined after pre-contraction of vessels with an EC.sub.50 concentration of U46619 and changes in force normalised to the pre-existing tension present immediately before addition the first concentration of dilator agent.

[0523] Statistics and Data Analysis

[0524] Data were analysed using Prism 6.0 software (Graphpad software, USA) and are presented as mean±standard error for ‘n’ number of animals. Where duplicate measurements were made on tissue from the same animals, values were averaged and considered as n=1. Data were compared using Student's unpaired t-test, one sample t-test, one-way or two-away ANOVA with Dunnett's post-hoc test as indicated in individual figure legends. Differences were considered significant if p<0.05.

Example 1

[0525] The effect on vasoconstriction of deletion of mPGES-1 was compared to that resulting from inhibition of COX-2 through investigation of their effects on levels of asymmetric dimethylarginine (ADMA), which is a naturally occurring inhibitor of eNOS.

[0526] In accordance with the general experimental methods described above, it was found that in mice:

[0527] COX-2 inhibition using parecoxib increased levels of ADMA whereas deletion of mPGES-1 had no significant effect (as shown in FIG. 1);

[0528] COX-2 inhibition using parecoxib increased expression of genes responsible for synthesis of ADMA (Prmt1) whereas deletion of mPGES-1 had no significant effect on expression of these genes (see FIG. 2); and

[0529] COX-2 inhibition using parecoxib increased expression of genes responsible for degradation of ADMA (Agxt2) whereas deletion of mPGES-1 again had no significant effect on expression of these genes (see FIG. 3).

Example 2

[0530] The effect of mPGES-1 deletion on eNOS-dependent vasodilator responses induced by acetylcholine was examined.

[0531] It was found that, in contrast to the effect previously seen in COX-2 deficient mice (Ahmetaj-Shala, B. et al., Circulation, 131(7), 633-42 (2015)), deletion of mPGES-1 significantly improved the eNOS driven vasodilator response to acetylcholine in aorta (as shown in FIG. 4).

[0532] Further, it was shown that this effect was not mediated by either:

[0533] an increased sensitivity of the vessels to NO, as vasodilator responses to the exogenous NO donor, sodium nitroprusside, were not altered between wild-type and mPGES-1 deficient mice (see FIG. 5); or

[0534] changes in contractility, since contraction force responses to U46619 were not different between wild-type and mPGES-1 deficient mice (see FIG. 6).