COMPOSITIONS AND METHODS
20230167155 · 2023-06-01
Inventors
Cpc classification
C07K2319/033
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
International classification
A61P9/10
HUMAN NECESSITIES
Abstract
The invention relates to a compound comprising: CH.sub.2—CH.sub.2—F-Cha-Cha-RKPNDK-NH.sub.2 joined via a linking group to [Myr2]-KSSKSPSKKDDKKPGD. The invention also relates to its use in treatment of atheroma, use in treatment of atherosclerosis, use in inducing regression of atherosclerosis, and use in treatment of Acute Kidney Injury (AKI).
Claims
1. A compound comprising: CH2-CH2-F-Cha-Cha-RKPNDK-NH2 joined via a linking group to [Myr2]-KSSKSPSKKDDKKPGD.
2. A compound according to claim 1 which has the formula: ##STR00008## wherein A is CH2-CH2-F-Cha-Cha-RKPNDK-NH2 M is [Myr2]-KSSKSPSKKDDKKPGD X is S, O, or NR B is optional and is an optionally substituted C1 to C6 alkyl Y is S, O, or NR m is 1, 2, 3, 4, 5, or 6 wherein each R is independently selected from H, or optionally substituted C1-6 alkyl
3. A compound according to claim 1 or claim 2 wherein said linking group comprises a disulphide bridge.
4. A compound according to claim 1 or claim 2 wherein said linking group consists of a cysteine residue-disulphide bridge.
5. A compound according to claim 1 or claim 2 wherein said linking group comprises a thioether group.
6. A compound according to claim 1 or claim 2 wherein said linking group consists of a lysine residue-thioether group.
7. A compound according to any of claims 1 to 4 which has the formula: ##STR00009##
8. A compound according to any of claims 1, 2, 5 or 6 which has the formula: ##STR00010##
9. A compound according to any of claims 1 to 8 for use in medicine.
10. A compound according to any of claims 1 to 8 for use as a medicament.
11. Use of a compound according to any of claims 1 to 8 for the manufacture of a medicament for atheroma.
12. Use of a compound according to any of claims 1 to 8 for the manufacture of a medicament for atherosclerosis.
13. Use of a compound according to any of claims 1 to 8 for the manufacture of a medicament for inducing regression of atherosclerosis.
14. A compound according to any of claims 1 to 8 for use in treatment of atheroma.
15. A compound according to any of claims 1 to 8 for use in treatment of atherosclerosis.
16. A compound according to any of claims 1 to 8 for use in inducing regression of atherosclerosis.
17. A method of treatment comprising administering a therapeutic amount of a compound according to any of claims 1 to 8 to a subject in need of same.
18. A method of treating atheroma in a subject comprising administering a therapeutic amount of a compound according to any of claims 1 to 8 to said subject.
19. A method of treating atherosclerosis in a subject comprising administering a therapeutic amount of a compound according to any of claims 1 to 8 to said subject.
20. A method of inducing regression of atherosclerosis in a subject comprising administering a therapeutic amount of a compound according to any of claims 1 to 8 to said subject.
21. A method of treating or preventing Acute Kidney Injury (AKI) in a subject comprising administering a therapeutic amount of a compound according to any of claims 1 to 8 to said subject.
22. Use of a compound according to any of claims 1 to 8 for the manufacture of a medicament for treatment or prevention of Acute Kidney Injury (AKI).
23. A compound according to any of claims 1 to 8 for use in treatment or prevention of Acute Kidney Injury (AKI).
24. A method of treating or preventing delayed type hypersensitivity (DTH) in a subject comprising administering a therapeutic amount of a compound according to any of claims 1 to 8 to said subject.
25. Use of a compound according to any of claims 1 to 8 for the manufacture of a medicament for treatment or prevention of delayed type hypersensitivity (DTH).
26. A compound according to any of claims 1 to 8 for use in treatment or prevention of delayed type hypersensitivity (DTH).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0208] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:
[0209]
A-E. Three colour immunofluorescence images of sections through donor aortas, 6-12 weeks post-transplantation. Recipients were ApoE−/− mice, fed a high fat diet (HFD) for two weeks from age 6 weeks, prior to transplantation of aorta from CD31-TFPI-Tg (A,B), CD31-Hir-Tg (C-D) or C57BL/6 mice (E). Blue—nuclear stain 4′,6-diamidino-2-phenylindole (DAPI). Red—anti-hTFPI (A,B), anti-hirudin (Hir-C,D) or anti-CD31 (E). Green—MIF (A,C,E) or CD31 (B,D). Each panel of three images shows consecutive sections.
F-J—Analysis of atheroma development in whole aorta (F,G,H) and aortic root (H,I,J) after a HFD for 6 weeks post-transplantation. F&G: representative Oil Red O-stained en face preparations of aorta from ApoE−/− mice transplanted with aorta from CD31-TFPI-Tg (F) or BL/6 (G) mice. The transplanted section is highlighted by arrows. H: Quantitative assessments show the area occupied by atheroma, assessed at three different sites (as indicated) as a proportion of the total area (n=6 males each group) in ApoE−/− mice transplanted with aortas from CD31-TFPI-Tg (white bars) or BL/6 (grey bars) donors. Graphs show box plots with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. I&J Representative light photomicrographs of elastic/van Gieson stained sections from aortic root of mice transplanted with aortas from CD31-TFPI-Tg (I) or BL/6 (J) mice.
K-O—Analysis of atheroma development in the whole aorta (K, L,M) and aortic root (M, N, O) after a HFD for 12 weeks post-transplantation. K&L: representative Oil Red O-stained en face preparations of aorta from ApoE−/− mice transplanted with aorta from CD31-Hir-Tg (K) or BL/6 (L) mice. The transplanted section is highlighted by arrows. M: Quantitative assessments show the area occupied by atheroma, assessed at three different sites (as indicated) as a proportion of the total area (n=6 males each group) in ApoE−/− mice transplanted with aortas from CD31-Hir-Tg (white bars) or BL/6 (grey bars) donors. Graphs show box plots with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. N&O representative light photomicrographs of elastic/van Gieson stained sections from aortic root of mice transplanted with aortas from CD31-Hir-Tg (N) or BL/6 (O) mice. Quantitative analyses were performed by a member of the team blinded to the mouse strain.
[0210]
A&B: Two colour IF images of cross sections through aorta harvested at 6 hours post-IV injection of 10 mg/g PTL060 (A) or equimolar (5 mg/g) HLL (B) stained with isotype control or RICS2 antibody (which recognises HLL) as indicated. Blue-DAPI. (NB Sections examined at all other time points showed less evidence of binding by PTL060).
C-E: Flow cytometric assessment of binding to erythrocytes (C), CD11b+ leukocytes (D) and platelets (gated on CD41+) (E) obtained from mice given either saline control, HLL (2.5 mg/g), or PTL060 (5 mg/g). Graphs show percentage of population binding RICS antibody (left column) and the geometric mean of the fluorescence intensity of binding (right column). Samples were taken from mice at the time points post-injection as indicated. n=3 per group.
F&G: Thrombin clotting times (seconds f SEM) in plasma. Blood was collected into citrated tubes at the times specified under terminal anaesthesia before spinning at 15000 g for 10 minutes to separate out cellular components and plasma. Thrombin times performed by adding 25 U (F) or 50 U (G) thrombin to 100 ml of plasma and recording time for a fibrin clot to form. Mice (n=3 per group) injected with PTL060 (5 mg/g—filled squares) or equimolar dose of HLL (2.5 mg/g—circles). Plasma from mice treated with PTL060 was centrifuged for a further 20 minutes at 10000 g, to remove any membrane bound PTL060, before repeating assessments (open squares).
H: Graph depicting tail bleeding times in minutes f SEM at various times after IV injection of control phosphate buffered saline (open circles), PTL060 10 mg/g (squares) or equimolar (5 mg/g) HLL (closed circles). N=6 per group. Mouse euthanised at 20 minutes if tail still bleeding.
[0211]
A: Quantitative impact of PTL060 on MIF expression by endothelium (left axis), represented as the proportion of CD31+ cells staining for MIF, plotted against time or development of atheroma (right axis) 4 weeks post injection. Mice (n=6) given either PBS control (white) or PTL060 10 mg/g (grey) by IV injection, 2 weeks after starting a HFD and analysed at the time points indicated. Graphs show box plots with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’.
B&C: Representative light photomicrographs of elastic/van Gieson stained sections from aortic root of ApoE−/− mice treated with PBS (B) or 10 mg/g PTL060 (C).
D: Quantitative impact of PTL060 on MIF expression 1-week post injection (left axis), represented as the proportion of CD31+ cells staining for MIF, or development of atheroma (right axis) 4 weeks post injection. Mice (n=6) given either PTL060 2.5 mg/g (white bars), PTL060 5 mg/g (grey bars), PTL060 10 mg/g (striped bars) or HLL 5 mg/g (diamond bars) by IV injection, 2 weeks after starting a HFD and analysed at the time points indicated. HLL 5 mg/g is equimolar to PTL060 10 mg/g. Graphs show box plots with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’.
E-H: Representative light photomicrographs of elastic/van Gieson stained sections from aortic root of ApoE−/− mice treated with PTL060 2.5 mg/g (E), 5 mg/g (F), 10 mg/g (G), or HLL 5 mg/g (H).
[0212]
A-D: Representative Oil Red O-stained en face preparations of aorta from ApoE−/− mice fed a HFD from age of 6-22 weeks (baseline: A), or 6-28 weeks with weekly injections (weeks 23-28) of saline (B), control cytotopic ‘tail’ compound (C) or PTL060 10 mg/g (D).
E-H: Representative light photomicrographs of elastic/van Gieson stained sections from aortic root of ApoE−/− mice fed a HFD from age of 6-22 weeks (baseline: E), or 6-28 weeks with weekly injections (weeks 23-28) of saline (F), control cytotopic ‘tail’ compound (G) or PTL060 10 mg/g (E).
I: Qualitative comparison of impact of PTL060 on atheroma formation in mice on HFD aged 6-22 weeks (white bars) or 6-28 weeks with weekly injections (weeks 23-28) of saline (grey bars), control ‘tail’ compound (striped bars) or PTL060 10 mg/g (diamond bars).
J-M: Representative Oil Red O-stained en face preparations of aorta from ApoE−/− mice fed a HFD from age of 6-22 weeks (Baseline: J), or 6-28 weeks with weekly injections (weeks 23-28) of saline (K), control ‘untailed’ HLL (L) or PTL060 10 mg/g (M).
N-Q: Representative light photomicrographs of elastic/van Gieson stained sections from aortic root of ApoE−/− mice fed a HFD from age of 6-22 weeks (N), or 6-28 weeks with weekly injections (weeks 23-28) of saline (O), control untailed HLL (P) or PTL060 10 mg/g (Q).
R: Qualitative comparison of impact of PTL060 on atheroma formation in mice on HFD aged 6-22 weeks (white bars) followed by weekly injections, for 6 weeks of saline (grey bars), control untailed HLL (striped bars) or PTL060 10 mg/g (diamond bars).
S-T: Impact of PTL060 on foam cells in atherosclerosis. Representative light photomicrographs of elastic/van Gieson stained sections from aortic root (S) with consecutive sections analysed by two-colour immunofluorescence (T) stained with DAPI (blue) or anti-CD68 (green). ApoE−/− mice were fed a HFD from age of 6-22 weeks, followed by weekly injections, for 6 weeks of saline, control untailed HLL or PTL060 10 mg/g as indicated.
U: Graphical representations of the % of plaque area staining with Oil Red O (upper panel) and, in lower panel, the % of area occupied by CD68+ cells (white bars) with the proportion of those CD68+ cells co-localising with lipid (grey bars). Each graph is a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Data is derived from an assessment of each of the three aortic root plaques from 6 individual mice, from consecutive sections as illustrated in S&T.
V-W: Representative Oil Red O-stained en face preparations of aorta from ApoE−/− mice fed a normal chow diet to the age of 28 weeks, followed by weekly injections, for 6 weeks of saline (V) or PTL060 10 mg/g (W).
X-Y: Representative light photomicrographs of elastic/van Gieson stained sections from aortic root of ApoE−/− mice fed a chow diet age to the age of 28 weeks, followed by weekly injections, for 6 weeks of saline (X) or PTL060 10 mg/g (Y).
Z: Qualitative comparison of impact of PTL060 on atheroma formation in mice on chow diet to for 28 weeks followed by weekly injections, for 6 weeks of saline (white bars) or PTL060 10 mg/g (grey bars).
[0213]
[0214]
All panels: CD11b cells, harvested from either BL/6 or CD31-Hir-Tg mice were labelled in vitro with PKH26 (red) and adoptively transferred into ApoE−/− mice fed a HFD between ages of 6-22 weeks. Aortic roots were collected 48 hours post-injection, for confocal IF analysis of the phenotype of adoptively transferred cells. Graphs are a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Each is derived from an assessment of at least 3 aortic root plaques from 6-35 individual mice.
A: To illustrate the expression of MIF (green) at baseline age 22 weeks, throughout the plaque area in a mouse that received BL/6 CD11b+ cells.
B-D: Comparison of the recruitment of CD11b+ cells from BL/6 (B) and CD31-Hir-Tg (C) mice. Hirudin (green) only seen in cells from CD31-Hir-Tg mice. D illustrates quantitative assessment of the proportion of plaque area occupied by PKH26+ cells.
E-G: To illustrate expression of Ly6G (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (E) or CD31-HIr-Tg (F) mice. G illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing Ly6G.
I-J: To illustrate expression of CCR2 (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (H) or CD31-HIr-Tg (I) mice. J illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing CCR2.
K-M: To illustrate expression of ABCA1 (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (K) or CD31-HIr-Tg (L) mice. M illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing ABCA.
N-P: To illustrate expression of CCR7 (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (N) or CD31-HIr-Tg (O) mice. P illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing CCR7.
[0215]
Confocal microscopic analysis of three colour immunofluorescence images through consecutive sections of aortic roots of ApoE−/− mice, fed a high fat diet from 6 to 26 weeks, with mice administered weekly injections of saline or PTL060 as indicated below between weeks 22-25. 1 week after the last injection, mice were injected with PKH2-labelled CD11b cells (green) and aortic roots harvested 48 hours later. Graphs are a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Each is derived from a double assessment of each of the three aortic root plaques from 3 individual mice.
A-C: To illustrate the expression of MIF (red) after adoptive transfer of BL/6 CD11b+ cells in mice treated with saline (A) or PTL060 (B). C illustrates quantitative assessment of the proportion of plaque area occupied by PKH2+ cells. D-F: To illustrate the expression of CCR2 (red) after adoptive transfer of BL/6 CD11b+ cells in mice treated with saline (D) or PTL060 (E). F illustrates quantitative assessment of the proportion of PKH2+ cells co-expressing CCR2. G-I: To illustrate the expression of CCR7 (red) after adoptive transfer of BL/6 CD11b+ cells in mice treated with saline (G) or PTL060 (H). I illustrates quantitative assessment of the proportion of PKH2+ cells co-expressing CCR7. J-K: To illustrate the expression of ABCA1 (red) after adoptive transfer of BL/6 CD11b+ cells in mice treated with saline (J) or PTL060 (K). L illustrates quantitative assessment of the proportion of PKH2+ cells co-expressing ABCA1.
[0216]
Samples represented here are from ApoE−/− mice fed a HFD from age of 6-28 weeks with weekly (weeks 23-28) injections of CD11b+ cells from BL/6 mice pre-incubated with saline (A, E), control ‘tail’ molecule (B, F), PTL060 10 mg/g (C,G) or with CD11b cells from CD31-Hir-Tg mice (D, H)
A-D: Representative Oil Red O-stained en face preparations of aorta
E-H: Representative light photomicrographs of elastic/van Gieson stained sections from aortic root
I: Qualitative comparison of atheroma regression in the whole aorta (en face) or aortic root of mice fed a HFD from age of 6-28 weeks with weekly (weeks 23-28) injections of CD1b+ cells from BL/6 mice pre-incubated with saline (white bars), control ‘tail’ molecule (grey bars), PTL060 10 mg/g (striped bars) or with CD1b cells from CD31-Hir-Tg mice (diamond bars).
J: Graph is a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Each is derived from an assessment of at least 3 aortic root plaques from 6-24 individual mice. It illustrates the proportion of plaque area occupied by cells expressing the various markers (as indicated on abscissa) from mice receiving CD11b+ cells from BL/6 mice pre-incubated with saline (white bars) or CD31-Hir-Tg mice (grey bars).
[0217]
[0218]
[0219] A-C. Three colour immunofluorescence images of sections through donor aortas, 6 weeks post-transplantation. Recipients were ApoE−/− mice, fed a high fat diet (HFD) for two weeks from age 6 weeks, prior to transplantation of aorta from BL/6 (A) CD31-TFPI-Tg (B) or CD31-Hir-Tg (C). Blue—nuclear stain 4′,6-diamidino-2-phenylindole (DAPI). Red—anti-CD31 (A) anti-hTFPI (B) or anti-hirudin (Hir-C). Green—CCL2. Each panel of three images shows consecutive sections.
[0220] D&E: Three colour IF images of consecutive sections through aortic root, taken 1, 2 or 3 weeks post IV injection of 10 mg/g of PTL060 (D) or PBS (E). ApoE−/− mice were commenced on a high fat diet 2 weeks prior to the injections. Blue—DAPI. Red—anti-CD31. Green—MIF.
[0221]
In vitro analysis of MIF (A) or CCL2 (B) production by cultured mouse SMCs, following stimulation by thrombin, with addition of reagents to demonstrate that PTL060 predominantly inhibits PAR-1 mediated chemokine production.
[0222]
Graphs show box plots with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. P values by Mann Whitney U test.
Plasma TNFα (A), IFNg (B), MIF (C) and CCL2 (D) in different groups of ApoE−/− mice, as indicated on abscissa.
[0223]
Confocal microscopic analysis of three colour immunofluorescence images through consecutive sections of aortic roots of ApoE−/− mice, fed a high fat diet from 6 to 22 weeks (‘Baseline’, all panels) or 6-28 weeks, with mice administered weekly injections of saline, HLL, or PTL060 as indicated between weeks 22-28. Panels show the plaque expression of CD68 (red) with (green) either IL-10 (A) IFNγ (E) or TNFα (I). Yellow in overlay image indicates co-localisation. The plaque area is demarcated by the lumen (L) and the dotted white line. Le=aortic leaflet.
Each panel of images is accompanied by graphical representations of the % of plaque area staining for the molecule of interest (B-IL-10, F-IFNγ, J-TNF α) and the % of plaque area occupied by CD68+ (C, G, K) and the proportion of CD68+ cells (white bars) and CD68-negative cells (grey bars) co-staining for IL-10 (D), IFNγ (H), or TNF α (L). Each graph is a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Each is derived from an assessment of each of the three aortic root plaques from at least 6 individual mice.
[0224]
Confocal microscopic analysis of three colour immunofluorescence images through consecutive sections of aortic roots of ApoE−/− mice, fed a high fat diet from 6 to 22 weeks (‘Baseline’, all panels) or 6-28 weeks, with mice administered weekly injections of saline, HLL, or PTL060 as indicated between weeks 22-28. Panels show the plaque expression of CD68 (red) with (green) either iNOs (A) or CD206 (E). Yellow in overlay image indicates co-localisation. The plaque area is demarcated by the lumen (L) and the dotted white line. Le=aortic leaflet.
Each panel of images is accompanied by graphical representations of the % of plaque area staining for the molecule of interest (B-iNOS, F-CD206) and the % of plaque area occupied by CD68+ (C, G) and the proportion of CD68+ cells (white bars) and CD68-negative cells (grey bars) co-staining for iNOS (D) or CD206 (H). Each graph is a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Each is derived from an assessment of each of the three aortic root plaques from at least 6 individual mice.
[0225]
[0226]
[0227]
[0228]
[0229]
[0230]
[0231]
[0232]
[0233]
[0234]
Impact of adoptive transfer of CD11b+ cells expressing hirudin_2
All panels: CD11b cells, harvested from either BL/6 or CD31-Hir-Tg mice were labelled in vitro with PKH26 (red) and adoptively transferred into ApoE−/− mice fed a HFD between ages of 6-22 weeks. Aortic roots were collected 48 hours post-injection, for confocal IF analysis of the phenotype of adoptively transferred cells. Graphs are a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Each is derived from a double assessment of each of the six aortic root plaques from 6 individual mice.
A-C: To illustrate expression of IFNγ (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (A) or CD31-HIr-Tg (B) mice. (C) illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing IFNγ.
D-F: To illustrate expression of IL-10 (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (D) or CD31-HIr-Tg (E) mice. (F) illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing IL-10.
G-I: To illustrate expression of iNOS (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (G) or CD31-HIr-Tg (H) mice. (I) illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing iNOS.
J-L: To illustrate expression of CD206 (green) within the plaque after adoptive transfer of CD11b+ cells from BL/6 (J) or CD31-HIr-Tg (K) mice. (L) illustrates quantitative assessment of the proportion of PKH26+ cells co-expressing CD206.
[0235]
Monocyte recruitment and phenotype after systemic PTL060_2.
Confocal microscopic analysis of three colour immunofluorescence images through consecutive sections of aortic roots of ApoE−/− mice, fed a high fat diet from 6 to 26 weeks, with mice administered weekly injections of saline or PTL060 as indicated below between weeks 22-25. 1 week after the last injection, mice were injected with PKH2-labelled CD11b cells (green) and aortic roots harvested 48 hours later. Graphs are a box plot with median with interquartile range (IQR) with whiskers showing upper and lower limits and outliers indicated as single data points. Means are represented with ‘x’. Each is derived from a double assessment of each of the three aortic root plaques from 3 individual mice.
A-C: To illustrate the expression of IL-10 (red) after adoptive transfer of BL/6 CD11b+ cells in mice treated with saline (A) or PTL060 (B). (C) illustrates quantitative assessment of the proportion of PKH2+ cells co-expressing IL-10.
D-F: To illustrate the expression of TNFα (red) after adoptive transfer of BL/6 CD11b+ cells in mice treated with saline (D) or PTL060 (E). (F) illustrates quantitative assessment of the proportion of PKH2+ cells co-expressing TNFα.
G-I: To illustrate the expression of IFNγ (red) after adoptive transfer of BL/6 CD11b+ cells in mice treated with saline (G) or PTL060 (H). (I) illustrates quantitative assessment of the proportion of PKH2+ cells co-expressing IFNγ.
[0236]
[0237]
[0238]
[0239]
[0240]
[0241]
Interpretation:
[0242] MIF and CCL2 are associated with recruitment of CCR2+ (Ly6Chi) monocytes CX3CL1 and CCL5 associated with recruitment of CCR2− (Ly6Clo) monocytes
The two chemokines on the left of the panel (MIF, CCL2) are secreted after stimulation by a PAR-1 agonist, but less so by a PAR-2 agonist.
In contrast, those on the right of the panel (CX3CL1 and CCL5) are secreted after stimulation by either PAR-1 or -2 agonists.
3-MP in this assay behaves as a pure PAR-2 agonist.
[0243] SMC and EC respond equivalently
[0244]
Thrombin-induced secretion of MIF and CCL2 are reduced by 30-50% by both a PAR-1 antagonist and 3-MP
In contrast, thrombin-induced secretion of CX3CL1 and CCL5 are reduced by 50% by the PAR-1 antagonist, but by only 0-10% by 3-MP
*3-MP=3-mercaptopropionyl-F-Cha-Cha-RKPNDK
[0245]
The enhanced sensitivity to IFNγ induced by thrombin is inhibited by a PAR-1 antagonist—this is via expression of ABCA1 and membrane lipid rafts.
A pure PAR-2 agonist also reduces the sensitivity of macrophages to IFNγ induced by thrombin.
3-MP completely (bottom) or near completely (top) abolishes the effect of thrombin on sensitivity to IFNγ. It also inhibits the background response of control macrophages to IFNγ.
BMM purified cells cultured in MCSF for 5 days. Cells then removed for 24 hours and incubated with thrombin (50 units) (if an antagonist or 3-mercaptopropionyl-F-Cha-Cha-RKPNDK was used cells were incubated for 2 hours with the antagonist or 3MP prior to thrombin stimulation to allow time for antagonist to dock with PAR). Cells then had media replaced with thrombin+ PAR reagent and IFN gamma at the dose described. For the PAR2 experiments it was at this point that PAR2 was added. Cells were then incubated for 24 further hours and then analysed by flow cytometry for iNOS. Pure PAR-1 antagonist (αagonist)=FLLRN; Pure PAR-2 agonist=2-Furoyl-LIGRLO-amide; 3-MP=3-mercaptopropionyl-F-Cha-Cha-RKPNDK; All used at 100 microM. Thrombin used at 50 iu
[0246]
A pure PAR-2 agonist induces SOCS3 expression, as does 3-MP (middle graph). This is a well-described inhibitor of IFNγ signalling.
In the right graph, the thrombin-induced sensitivity to IFNγ, as assessed by change in iNOS expression, is shown. Incubation with a PAR-2 agonist dampens the enhanced sensitivity. A control siRNA does not inhibit the effect of the PAR-2 agonist. However, an siRNA against SOCS3, to inhibit SOCS3 expression, abolishes the effect of the PAR-2 agonist, suggesting that PAR-2 signalling works through SOCS3 upregulation.
[0247]
Extent of atherosclerosis was explored by en face examination of the surface area of aorta containing plaques using Oil-Red-O
Interpretation:
[0248] Treatment of mice with adoptively transferred monocytes only, after incubation with either PTL060 or PTL0GC-1, is sufficient to induce the same degree of regression associated with IV treatment of the same reagents, suggesting that a direct effect on monocytes may be the dominant mechanism of action.
EXAMPLES
Example 1—PTL060: A Cytotopic Direct Thrombin Inhibitor
[0249] In this example, PTL060, a cytotopic direct thrombin inhibitor, exemplifies the point that cytotopic modification uncouples the anti-inflammatory effect from systemic anticoagulation during regression of atheroma.
[0250] Our experience with the cytotopic compound PTL060, which is a tailed version of the direct thrombin inhibitor bivalirudin, is that the cytotopic tailing process endows bivalirudin with prolonged biological activity, such that weekly (or less frequent) dosing of the tailed moiety is sufficient for therapeutic activity (see
[0251]
[0252] Using ApoE−/− mice that were fed a high fat diet (HFD) for 16 weeks, IV injection of PTL060 (10 μg/g) weekly for six weeks induced regression in the burden of atheromatous lesions present at the aortic root. The lesional burden reduced by >40% compared to control mice receiving saline injection, but importantly, also reduced by >35% compared to baseline mice examined at week 16, prior to beginning any PTL060 injections. In stark contrast, the mice receiving equimolar doses of parental bivalirudin on the same dosing schedule developed increasing atheroma burden equivalent to that seen in saline control mice. Interestingly, these effects were seen without reductions in plasma total cholesterol levels.
[0253] Most importantly, mice treated with PTL060 were systemically anticoagulated for only 1/7.sup.th of the time between doses (see example below). Thus, the addition of the cytotopic tail to bivalirudin uncouples the pharmacodynamics of its impact on haemostasis from its effects on inflammation, at doses that both prevent plaque formation and induce plaque regression. To our knowledge, this is the first demonstration of such uncoupling, and represents a significant advance in understanding the true therapeutic potential of targeting coagulation proteases to influence inflammatory disease.
[0254] In this model, PTL060 works via two distinct mechanisms (see example below). First, it acts at the vascular wall to promote the recruitment of predominantly CCR2-Ly6Clo monocytes, which are known to be precursors of M2 polarised macrophages. These recruits express CD206, IL-10, ABCA1 and CCR7 and have a phenotype previously associated with regression.
[0255] Second, it acts directly on circulating monocytes, and protects them from the effects of thrombin signalling through PAR-1. This means that upon recruitment into the plaque, all monocytes adopt a ‘regression’ phenotype (see example below).
Example 2—Regression of Atherosclerosis in Apoe−/− Mice Via Modulation of Monocyte Recruitment and Phenotype, Induced by Weekly Dosing of a PTL060 ‘Cytotopic’ Anti-Thrombin without Prolonged Anticoagulation
[0256] Background: Coagulation proteases play an important role in atherogenesis. Accordingly, anticoagulants can induce regression in animal models of atherosclerosis, but exploiting this clinically has been limited by major bleeding events that occur after systemic anticoagulation. Here we test a novel thrombin inhibitor, PTL060, that comprises hirulog covalently linked to a synthetic myristoyl electrostatic switch to tether it to cell membranes.
[0257] Methods: ApoE−/− mice, fed either chow or high fat diets were used. Transplantation of congenic aortic segments was used to demonstrate the impact of expressing anticoagulants on endothelium. PTL060, parental hirulog or controls were tested to assess suppression of vessel wall chemokine gradients, impact on plaque development and regression of existing plaques. Adoptive transfer of labelled CD11 b positive cells was used to assess recruitment of monocytes and inform on how PTL060 influenced monocyte phenotype.
[0258] Results: Transgenic expression of anticoagulant fusion proteins based on TFPI or hirudin on EC led to complete suppression of MIF and CCL2 expression throughout the vessel wall and segments of aorta transplanted into ApoE−/− mice did not develop atherosclerosis. A single IV injection of PTL060, but not parental (unmanipulated) hirulog inhibited the same chemokines for >1 week and atheroma formation was reduced by >50% compared to controls when assessed 4 weeks later. Mice had prolonged bleeding times for only 1/7.sup.th of the time that PTL060 was biologically active. Repeated weekly injections of PTL060 but not parental hirulog caused regression of atheroma in ApoE−/− mice fed either chow or high fat diets. Mechanistically, 100% of circulating monocytes quickly became coated with PTL060 after the first dose, following which >70% of CCR2+ monocytes recruited into plaques expressed CCR7, ABCA1 and IL-10, a phenotype associated with regression, compared to <20% of CCR2+ recruits in control mice. Multiple doses caused a significant reduction in the number of monocytes recruited, and a switch to recruitment of CCR2-negative cells, the majority of which (>90%) had a similar regression-associated phenotype. The impact of PTL060 on circulating monocytes appeared dominant, as regression equivalent to that induced by IV PTL060 was induced by adoptive transfer of CD11b+ cells pre-coated with PTL060.
[0259] Conclusions: PTL060, a novel tethered direct thrombin inhibitor causes regression of atherosclerosis in ApoE−/− mice, via an effect at the endothelial surface but also through a direct effect on monocytes, causing differentiation into macrophages capable of plaque regression. Covalent linkage of a myristoyl electrostatic switch onto hirulog uncouples the pharmacodynamic effects on haemostasis and atherosclerosis, such that regression is accompanied by only transient anticoagulation.
INTRODUCTION
[0260] Atherosclerosis, is a chronic inflammatory disease that causes coronary artery, peripheral vascular and cerebrovascular disease. It is a major cause of death in the Western world. Important early steps in atherogenesis, in the context of a high lipid microenvironment include secretion of chemokines such as CCL-2 and macrophage migration inhibitory factor (MIF).sup.1, by activated endothelial cells (ECs) and smooth muscle cells (SMCs).sup.2,3. These promote infiltration of monocytes into the subendothelial space, where they become macrophages and take up very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) to become foam cells, initiating the process of atheroma formation.
[0261] Coagulation proteases, such as thrombin, signal though protease activated receptors (PAR) as well as catalysing fibrin formation and are known to play a role in this process. Increased activity of tissue factor (TF), the 47-Kd cell membrane-bound glycoprotein that initiates the serine protease cascade, is seen in the neointima and underlying media of atherosclerotic plaques.sup.4-6 and TF is expressed by EC.sup.7, monocytes/macrophages.sup.8 and SMC.sup.9.
[0262] In previous work, we crossed a strain of transgenic mice expressing a membrane-tethered human Tissue Factor Pathway Inhibitor (hTFPI) fusion protein on α-smooth muscle actin (SMA).sup.+ cells (called α-TFPI-Tg mice).sup.10 with Apolipoprotein E-deficient (ApoE.sup.−/−) mice to generate a new strain (called ApX4). These mice were resistant to atheroma formation.sup.11. In dissecting the mechanism of resistance, we showed that TF expression by SMC was necessary to generate MIF, via generation of thrombin and signalling primarily through PAR-1. Inhibition of either the TF, thrombin mediated PAR-1 signalling or MIF secretion prevented atherosclerosis in mice fed either a high fat diet (HFD) or a regular chow-based diet.
[0263] One of the observations from that study was that MIF continued to be secreted by EC in ApX4 mice fed a HFD, beneath which small atherosclerotic plaques developed.sup.11. This suggested that targeting SMC with hTFPI was not completely efficient at inhibiting atheroma. In this new study, we explored how transgenically-expressed tethered anticoagulants on EC impacted on atherosclerosis development, and assessed the translational potential of a novel thrombin inhibitor containing the potent peptide hirulog (a direct thrombin inhibitor), that has been chemically modified (HLL) to accept a lipid membrane-binding anchor or ‘cytotopic’ tail. This new compound is called PTL060 (thrombalexin-3). PTL060 has previously been localised within organs before transplantation to successfully inhibit thrombosis and rejection in several models.sup.12-14. In the process we describe an unpredicted impact of PTL060 on the phenotype of monocytes recruited into atherosclerotic plaques, by two interrelated pathways, one of which occurs by virtue of its ability to tether directly to monocytes. We also provide a mechanistic insight into the role that thrombin appears to play in driving plaque progression, as evidenced by the regression seen when PTL060 is administered systemically.
Methods
Mice and In Vivo Procedures
[0264] C57BL/6J (BL/6) mice were purchased from Harlan UK Ltd (Bicester, UK) and ApoE−/− mice were from the Jackson Laboratory (Bar Harbor, Me. 04609, USA). CD31-TFPI-Tg and CD31-Hir-Tg.sup.15 mice were bred in house. Mice were housed in a temperature-controlled Specific Pathogen-Free environment at 22-24° C. and all animal surgical protocols, animal experiments and care were approved by the UK Home Office. To assess the distribution of PTL060, male BL/6 mice weighing 25-28 g (n=6 per group) were injected IV through a tail vein with either PTL060 (10 μg/g in 100 μl saline), equimolar HLL (5 μg/g) or saline. At 5, 30 minutes and 2, 4, 6, 24 and 48 hours mice were sacrificed to collect citrated whole blood for separation into cells and plasma and to harvest aortas for immunofluorescence analysis.
[0265] Bleeding times were assessed as previously described.sup.15. Briefly, mice were anesthetized and placed in a restrainer (Becton Dickinson), before a distal 3-mm segment of tail was severed with a razor blade. The tail was immediately immersed in 0.9% saline at 37° C. Bleeding time was defined as the time required for bleeding to stop. Experiments were terminated at this time or at 20 minutes.
[0266] For all atherosclerosis work, male ApoE−/− mice, from the age of 6-weeks, were fed with a high fat diet (HFD) consisting 35 kcal % fat, 1.25% cholesterol, and 0.5% cholic acid (Special Diet Services, Essex UK). Aortas were transplanted 2 weeks after starting the HFD, using a sleeve anastomosis technique described previously.sup.11. Briefly, a 5 mm of the segment of the infrarenal donor aorta, flushed with 300 μl of saline containing 50 U of heparin, was transplanted into ApoE−/− recipient abdomen aortas (N=6 per group). Blood flow was confirmed by direct inspection after removal of the clamps. Mice were fed a HFD for 6-12 weeks post-transplantation before the experiment was terminated (Suppl
Cell Isolation and Labelling.
[0267] Leukocytes were isolated from the blood of mice aged 8-10 weeks, using anti-CD11b MicroBeads (Miltenyi Biotec Ltd, Surrey, UK) according to manufacturers' instructions. For cell labelling, 2×10.sup.7 CD11b+ cells were incubated with 4×10.sup.−6 M of either PKH26 or PKH2 fluorescent dyes (Sigma, UK) for 5 minutes at 25° C. according to manufacturers' protocols, with the reaction stopped using 1% BSA in PBS followed by three washes. Each recipient mouse received 0.5×10.sup.6 cells by IV injection; in some experiments, the cells were incubated with PTL060 (100 μM in 0.5 mls) or equimolar controls for 30 minutes at room temperature and washed three times before immediate injection. For viability assays, murine bone marrow cells were incubated for 5 days in 6 well plates, counted and re-seeded at 2×10.sup.5 cells/ml in 24 well plates with 25 ng/ml MCSF. After 1 day, media was replaced with new DMEM/FCS containing different concentrations of PTL060 (or a fixed volume of control PBS), and incubated for 30-120 minutes.
Histological Analysis
[0268] Atherosclerotic lesions were evaluated as previously described.sup.11. Simply, the entire length of the aorta was perfused with PBS, dissected using a dissecting microscope, longitudinally opened and stained with Oil Red O (ORO) solution (Sigma, UK) for 30 minutes, before being photographed with a digital camera (DSC-W320, Sony, Japan). The total aortic area and lesional area were measured by using Image J. Aortas from every animal were assessed. To assess lesions in the aortic sinus, hearts were embedded in paraffin, sectioned through the aortic root and incubated with elastin/van Gieson stain using the Accustain™ Elastin Stain kit (Sigma). Sections were examined on an Olympus U-ULH optical microscope (Olympus Optical Co. Ltd, Tokyo, Japan). Atheromatous lesional and total aortic root area was determined using Image-Pro Plus TM software version 4.0 (Media Cybernetics, Silver Spring, USA). At least three random sections were examined from each mouse in all groups.
[0269] Immunohistology of frozen cross sections were prepared and examined as previously described.sup.11. Briefly, isolated tissues were snap-frozen and embedded in OCT compound (VWR International, Dorset, UK), sectioned at 5 μm thickness and fixed in methanol at −20° C. Frozen sections were immersed in 1% bovine serum albumin-phosphate-buffered saline (BSA-PBS) for 30 minutes and then incubated overnight at 4° C. with one or more of the following antibodies: rabbit polyclonal antibody to CD68, iNOs, CD206, TNFα, MIF, CCR7 ABCA1 (all from Abcam, Cambridge, UK), or hirudin (Biobyt, Cambridge, UK) or CCL2 (Lifespan BioScience, Inc., WA 98121, USA); goat polyclonal antibody to CD31 (Santa Cruz Biotechnology, Texas 75220, USA); rat anti-mouse CD68, CD11b (Serotec, Oxford, United Kingdom), CD31, IFNγ (BD Bioscience Pharmingen, Oxford, United Kingdom), Ly-6G (BioLegend, London, UK), IL-10 (Abcam) or biotinylated anti-HLL (RICS-2).sup.14; mouse anti-CCR2 (Abcam). The following were used as isotype controls; goat anti-rat antibodies to IgG2a, IgG2b (BD Bioscience, Berkshire, UK) and polyclonal rabbit IgG (Abcam). The following anti-IgG FITC or TRITC-conjugated antibodies were used: sheep anti-mouse, rabbit anti-rat, goat anti-rabbit and rabbit anti-goat (all from Sigma). Fluorescein-conjugated streptavidin (Jackson Immunoresearch, Cambridge, UK) was used to detect RICS-2. Stained sections were mounted in Vectashield with DAPI (Vector Laboratories Inc, CA USA). Sections were directly captured and examined by a Leica DMIRBE confocal microscope (Leica, Wetzlar, Germany) equipped with Leica digital camera AG and a confocal laser scanning system with excitation lines at 405, 488, 543, and 560 nm at magnifications 10×/0.40 CS and 20×/0.70 IMM (Leica, Planapo, Wetzlar, Germany). Images were processed using Leica-TCS-NT software associated with the Leica confocal microscope. All immunohistochemistry was performed at 22° C. Quantification of staining was achieved by expressing the area of positive staining as a ratio of the total lesion area, calculated using Image-Pro Plus TM, version 4.0. All quantification was performed by members of the team blinded to the identity of the sections. For estimations of positive stained area, average measurements were derived from examination of at least six random sections from each tissue sample. To detect macrophage-derived foam cells, frozen sections of aortic sinus were analysed by a combination of ORO staining and CD68 immunostaining. Sections were incubated with rat anti-mouse CD68 antibody (overnight at 4° C.) and goat anti-rat antibody (1 hour at room temperature) before staining with filtered ORO solution (0.5% in propylene glycol, Sigma) for 15 minutes at room temperature.
Plasma Assays.
[0270] Anticoagulated whole blood (EDTA 30 mM pH8) was separated into plasma and cells by centrifugation (14,000 g for 10 mins). Plasma TNFα, IFN-γ, MIF and CCL2 were detected using separate specific ELISA kits (R&D Systems, Abingdon, UK) according to the manufacturers' instructions. Total cholesterol, high-density lipoprotein and low-density lipoprotein were determined using kits from Cell Biolabs, and Tryglycerides with a kit from Abcam, (both Cambridge, UK) according to the manufacturer's protocol. Thrombin clotting times were measured in 3.2% trisodium citrated plasma according to the protocol of Ignjatovic.sup.16. Briefly, 100 μl mouse plasma was incubated with 2.5 U of human thrombin in a total volume of 300 μl (Enzyme Research Laboratories (ERL), Swansea, UK) at 37° C., and the time for a clot to form was measured (n=6 per group). For some experiments plasma was further centrifuged (20,000 g for 10 mins) to minimise the presence of extracellular vesicles.
Flow Cytometry
[0271] The cells obtained from whole blood were washed twice in PBS with 2% FCS before staining with either anti-CD11b-FITC (Abcam) or anti-CD41-FITC (eBioscience) with biotinylated RICS2 followed by Streptavidin-PE (Bio-rad). Cells were then washed twice before analysis on a BD FACSCALIBUR with CellQuest Pro software.
[0272] Erythrocytes were identified by forward/side scatter profile.
[0273] For viability assays, cells were washed twice with PBS and then incubated with Fixable LIVE/DEAD Near-IR fluorescent reactive dye (Life Technologies) for 30 minutes at 4° C. Cells were washed, fixed for 15 minutes in 1% paraformaldehye, then washed with PBS-5% FCS and stored at 4° C. before acquisition and analysis within 24 hours on an LSRII/Fortessa flow cytometer at the BRC Flow Cytometry Laboratory, King's College London with Flowjo software (Treestar Inc). Macrophages identified by forward/side scatter profile.
SMC-MIF/CCL2 Release Assay In Vitro
[0274] SMCs, cultured as previously described.sup.11 and seeded at a density of 1×10.sup.6 cells/well of a 24-well plate were serum-starved for 24 hours before addition of PTL060 (100 μM) for 1 hour, followed by PAR agonists or antagonists (all from ERL) for 12 hours, followed by thrombin 10 nM or active site inhibited thrombin (ERL) for 48 hours, before collection of supernatants. Chemokines were measured by ELISA according to the manufacturers' instructions (R&D systems, Abingdon, UK)
Statistical Analysis
[0275] Statistical analysis was performed with GraphPad Prism software.
Results.
Anticoagulants Transgenically Localised to EC Completely Inhibit Vessel Wall Expression of Chemokines and Prevent Formation of Atheroma.
[0276] To assess the impact of expressing hTFPI fusion protein on EC alone, we used the congenic aortic transplant model previously described.sup.11, and compared the extent of atheroma development in transplanted aortas from CD31-TFPI-Tg mice (expressing hTFPI transgene on EC.sup.15) and BL/6 mice. The recipients were 8-week old ApoE−/− mice fed a HFD for 2 weeks prior to the transplant, and the experiment was terminated 6 weeks after the transplant (suppl
[0277] Next we transplanted aortas from a second transgenic strain (CD31-Hir-Tg).sup.15, expressing a tethered hirudin fusion protein on EC (suppl
IV Injection of PTL060, a Novel Tethered Therapeutic Anti-Thrombin.
[0278] After IV administration of PTL060 into BL/6 mice, linear deposition of the anticoagulant moiety could be found on the luminal surface of the aorta mice several hours later (
TABLE-US-00001 SUPPLEMENTARY TABLE 1 Incubation time (minutes) 30 60 120 Control PBS — — 99 25 μM PTL060 99 95 96 50 μM PTL060 96 98 92 100 μM PTL060 97 87 80 % viability within Macrophage Forward Scatter/Side scatter gate, as assessed by LIVE/Dead aqua fluorescent dye. See methods for details
[0279] Thrombin clotting times of citrated plasma were prolonged for >6 hours post injection of PTL060, indicating the presence of a thrombin inhibitor, to a similar extent as was seen after injection of an equimolar amount of HLL (
[0280] We assessed the differential impact of PTL060 and HLL on expression of MIF by the vasculature, as a biomarker of potential efficacy at suppressing atheroma formation. A single IV injection of PTL060 was accompanied by complete suppression of MIF expression throughout the vessel wall for almost 1-week (suppl
[0281] These data indicate that equimolar doses of PTL060 and HLL induce similar degrees of systemic thrombin inhibition lasting approximately 24 hours, but only PTL060 promotes prolonged suppression of MIF expression by vessel wall cells.
Impact of PTL060 on Atherosclerosis
[0282] A single injection of PTL060 caused significant inhibition of atheroma formation in ApoE−/− mice fed a HFD for two weeks prior to, and four weeks after the injection (
[0283] To assess the impact of PTL060 on established atheroma, 6-week old ApoE−/− mice were fed a HFD until the age of 22 weeks, before receiving IV injections of saline, HLL, control cytotopic tail compound or PTL060, weekly for a further 6 weeks. PTL060 caused a significant reduction in atheroma burden, when measured either by en face analysis or by cross sectional analysis of the aortic root (
The Phenotype of Regressing Plaques after PTL060 Treatment
[0284] Atheromatous plaques in ApoE−/− mice fed a HFD from 6-22 weeks of age (baseline) contained a significant number of CD68+ cells (monocytes/macrophages), occupying approximately 45% of plaque area (
[0285] Plaques developing in mice fed a HFD between 6-22 weeks were almost devoid of cells expressing the chemokine receptor CCR7 (
[0286] Thus, six weeks of weekly PTL060 injections promoted a significant reduction in plaque CD68+ cells and a significant shift in their phenotype, towards a phenotype that has previously been associated with plaque regression (CCR7+, ABCA1+, IFNγ-, IL-10+, iNOS-, CD206+).
Mechanism of Regression: Impact of Thrombin Inhibitor Tethered to the Surface of Circulating Monocytes.
[0287] As shown already, PTL060 rapidly adheres to the surface of circulating leukocytes. To investigate the impact of this leukocyte-tethered thrombin inhibitor, in isolation to that tethered by EC or platelets and erythrocytes, we adoptively transferred CD11b+ cells labelled with the fluorescent dye PKH26 into ApoE−/− mice fed a HFD from weeks 6-22, before assessing the phenotype of the labelled plaque cells by confocal immunofluorescence microscopy 48 hours later. To avoid the potential confounding influence of transfer of PTL060 from the adoptively transferred cells to vascular membranes, for these experiments we used CD11b+ cells from CD31-Hir-Tg mice, which express covalently tethered cell surface hirudin on all monocytes, and compared the impact of labelled BL/6 cells.
[0288] At the point of adoptive transfer, MIF was expressed throughout the plaques (
Monocyte Recruitment and Phenotype after Systemic PTL060.
[0289] To assess whether PTL060 reduced numbers of monocytes recruited, ApoE−/− mice were fed a HFD from 6-22 weeks, before administration of weekly PTL060 or saline for 3 weeks to the age of 25 weeks. PKH2-labelled CD11b cells from BL/6 mice were administered one week after the last injection of PTL060 (by which time all PTL060 should have left the circulation (see
[0290] As at baseline, the monocytes recruited into plaques of saline treated animals were predominantly CCR2+(
[0291] However, monocytes recruited to plaques in PTL060-treated mice were predominantly CCR2-neg, suggesting they were predominantly Ly6Clo monocytes (
[0292] Compared to cells recruited in saline treated mice, the majority of labelled cells recruited into the plaques of PTL060 mice expressed CCR7 (
A Thrombin Inhibitor on the Surface of CD11 b+ Cells is Sufficient to Induce Regression.
[0293] To assess whether tethering of PTL060 to leukocytes alone was sufficient to induce plaque regression, we fed ApoE−/− mice a HFD from 6-22 weeks, and then adoptively transferred, by weekly IV injection during weeks 23-28, CD11b+ cells, while continuing the HFD. Control mice received cells from BL/6 mice incubated, prior to transfer, with either saline or the cytotopic tail compound only. Experimental mice received BL/6 cells pre-incubated with PTL060 or, as a positive control, cells from CD31-Hir-Tg mice. All mice receiving control cells showed progression of atherosclerosis between 23-28 weeks (
[0294] Taken together with the data from the adoptive transfer of labelled cells, these data indicate that mechanistically, the impact of systemic PTL060 treatment can be reproduced entirely by isolating a thrombin inhibitor onto the surface of circulating monocytes, suggesting that inhibiting thrombin activity on only these cells is sufficient to promote regression.
DISCUSSION
[0295] The involvement of coagulation proteases in atherosclerosis and the impact of inhibiting them has been described by multiple groups in previous studies. For instance, ApoE.sup.−/− mice made deficient in HCII, a natural thrombin inhibitor.sup.17, or carrying a DNA variant resulting in defective thrombomodulin-mediated generation of activated protein C.sup.18 develop severe atheroma, indicating that in this model, endogenous regulators of thrombin act to limit disease severity. Conversely, factor (F)Xa inhibitors.sup.19,20 and direct thrombin inhibitors.sup.21-24 prevent atheroma progression and maintain plaque stability Systemic anticoagulants can also induce regression of atherosclerosis in ApoE−/− mice. Bea et al used megalatran in 30-week-old animals and showed reduced burden of advanced atheromatous lesions associated with plaque stability.sup.25. More recently, Posthuma et al.sup.26 reduced atheroma burden in 22 week old animals by 25% after daily treatment for 6 weeks with clinically relevant doses of the FXa inhibitor rivaroxaban.
[0296] These data from animal models have fed into clinical practice, and the benefit of systemic anticoagulation in patients with atherosclerosis has been most recently confirmed by the COMPASS trial, which showed that addition of rivaroxaban to aspirin in patients with stable atherosclerotic cardiovascular disease led to fewer deaths, strokes and myocardial infarction.sup.27. Moreover, the PAR-1 antagonist vorapaxar has also been shown to reduce the risk of myocardial infarction in patients with stable atherosclerosis.sup.28. However, these benefits were associated with a significant increase in the incidence of major bleeding events: this is the biggest drawback to using systemic anticoagulants or antiplatelet drugs for non-thrombotic diseases, as their impact on haemostasis cannot be separated from their clinical efficacy.
[0297] The development of thrombalexins built upon a foundation of tethering anti-complement compounds using a generic tail based on the myristoyl-electrostatic switch .sup.29,30 We have demonstrated that several versions of thrombalexin, including PTL060 effectively bind to cell membranes, maintain potent thrombin inhibitory activity, and prevent intravascular thrombosis when infused into rodent.sup.12 or primate kidneys.sup.14 prior to transplantation. Under these circumstances, PTL060 remains detectable in tissue for several days.
[0298] In this work, we have shown that after IV injection, PTL060 inhibits secretion of vessel wall chemokines for 1 week and prevents atheroma formation but increases the risk of bleeding for only 24 hours. Therefore, the addition of the cytotopic tail uncouples the pharmacodynamics of hirulog's effects on haemostasis from its effects on atheroma formation, so that an increased bleeding tendency is seen for only 1/7.sup.th of the period between doses that both prevent plaque formation and induce plaque regression. To our knowledge, this is the first demonstration of such uncoupling, and represents a significant advance in understanding the true therapeutic potential of targeting coagulation proteases to influence inflammatory disease.
[0299] Our interest in this area began with the idea that targeting anticoagulants to cell membranes would achieve high concentrations in localised environments, such as the endothelium of an organ transplant, to inhibit vascular thrombosis. We demonstrated proof of concept using transgenically expressed fusion proteins.sup.15, 31, and in the process showed that inhibiting thrombin-mediated signalling through protease activated receptors on vessels inhibited local chemokine gradients, which reduced monocyte recruitment to sites of inflammation, including after transplantation, and prolonged survival.sup.32. We then went on to show that thrombin was similarly involved in chemokine gradient generation in atherosclerosis, such that expression of a tethered anticoagulant on SMC significantly reduced the development of atheroma in ApoE−/− mice.sup.11. In this new work we have confirmed that expression of tethered anticoagulants on EC is equally efficacious at suppressing vessel wall chemokine expression by both EC and SMC in ApoE−/− mice and equally effective at preventing atherosclerosis as expression on SMC. Although there was some variation in the extent of atherosclerosis development by control ApoE−/− mice fed a HFD for 4-6 weeks across temporally distinct experiments, one consistent feature was that single doses of PTL060 caused significant inhibition (≥50%) of atheroma formation compared to controls. We have not investigated the mechanism by which targeted thrombin inhibition on EC influences the phenotype of underlying SMC, but the data are consistent with the known importance of EC/SMC interplay for atheroma development.sup.33, and one possibility is that it acts via regulation of angiopoietin-2 secretion, known to be important in atherosclerosis.sup.34, which we have shown to be thrombin-dependent in a separate model system.sup.35. Our most important finding was that in ApoE−/− mice fed a HFD for 16 weeks prior to weekly injections of PTL060 for six weeks, atheroma burden was significantly reduced, compared not only to control mice given either saline or an equimolar dose of parental HLL, but also in comparison to baseline, indicating that PTL060 caused regression of existing disease. This was achieved without impacting plasma lipid concentrations. A similar reduction in plaque burden was seen in mice fed a normal Chow diet. There were significantly fewer CD68+ macrophages and foam cells present after 6 weeks treatment in the regressing plaques.
[0300] In assessing the mechanisms of regression, we considered the importance of inhibiting vessel wall chemokine gradients. Continuous monocyte recruitment into the vessel wall is one of the major steps in the pathogenesis of atherosclerosis, as evidenced by studies showing that simultaneous inhibition of CCL2, CX3CR1 and CCR5 near abolishes development of atheroma in ApoE−/− mice.sup.36. In addition, deficiency of MIF also impairs atheroma development in LDL-R deficient mice.sup.37 an inhibitory anti-MIF antibody has been shown to prevent atherosclerosis in ApoE.sup.−/− mice.sup.38, and our previous work illustrated that MIF secretion was important. We confirmed that a single dose of PTL060 led to prolonged suppression of vessel wall MIF (and CCL-2), and that this associated with prevention of plaque development. In addition, recruitment of labelled CD11 b+Ly6G-monocytes, adoptively transferred 1 week after the last of three doses of PTL060, was reduced by 90%, compared to that seen in control, saline-treated mice. This is consistent with the idea that suppression of vessel wall chemokine expression, interrupting the continuous cycle of monocyte recruitment, foam cell development, cell death and vessel wall inflammation might be an important contributory mechanism of how PTL060 induces plaque regression.
[0301] However, PTL060 also modulated the phenotype of recruited monocytes/macrophages. Thus, plaque cells in the regressing plaques in PTL060-treated mice had a significantly different phenotype compared to those detected in the progressing plaques in control animals, with reduced expression of pro-inflammatory IFNγ, TNFα and iNOS, and significant increases in the proportions of cells expressing CD206, IL-10, ABCA1 and CCR7.
[0302] These phenotypic characteristics have all been associated with mechanisms of regression defined in other studies. For instance after transplantation of atheromatous aorta from ApoE−/− mice into BL/6 mice.sup.39-41, the chemokine receptor CCR7 was shown to be important for emigration of foam cells, as demonstrated by inhibiting the chemokine ligands for CCR7.sup.42. In another model, LDLR−/− mice treated with an antisense to miR-33 showed regression associated with upregulated ABCA1 expression in plaque macrophages and enhanced reverse cholesterol transport.sup.43, in association with increased levels of circulating HDL, consistent with the known importance of ABCA1 for cholesterol loading into HDL and with the phenotype of ABCA1-deficient mice.sup.44. Finally, the importance of polarising new monocyte recruits to the plaque towards an M2 phenotype has been recently demonstrated in the aortic transplant model, by confirming that regression is dependent on the expression of both appropriate chemokine receptors (CCR2/CX3CR1) and the transcription factor STAT6 by recipient monocytes.sup.45.
[0303] These phenotypic changes were evident in newly recruited monocytes, but our adoptive transfer experiments suggested that CCR2+ or CCR2− monocytes were recruited at different times following PTL060 treatment. In the first experimental setting, using CD11b+ cells from CD31-Hir-Tg mice, transferred into ApoE−/− mice fed a HFD for 16 weeks without PTL060 treatment, we showed that CCR2+ monocytes were predominantly recruited, and these displayed the phenotypic traits associated with regression. In a second experimental setting, we showed that the CD11b+ cells recruited after adoptive transfer into mice already treated with 3 doses of PTL060 were predominantly CCR2− cells but also displaying the same phenotypic traits associated with regression. In this situation, labelled cells were transferred one week after the last of three doses of PTL060, into mice in which PTL060 had been cleared from the circulation, but importantly, into mice in which significant changes in plaque phenotype had already been induced. We suggest that the differential recruitment of the CCR2-(Ly6Clo) subset, known to be precursors of M2 polarised macrophages.sup.46, is most likely due to the conditions within the plaque already established by the PTL060. We postulate that, after the first dose, the immediate uptake of PTL060 onto the surface of circulating CCR2+ monocytes, protects them from thrombin as they are recruited into established plaques, significantly skews their phenotype as they become macrophages, and this rapidly establishes the microenvironmental conditions inside the plaque that are required to initiate regression.
[0304] Although the focus of this work has been on the impact of thrombin inhibition on the vessel wall and circulating leukocytes, they also provide a potential explanation for the mechanisms through which FXa inhibitors induce regression, though these agents would also influence signalling through PAR-2. We also showed immediate uptake of PTL060 onto circulating platelets after IV injection. Since interactions between platelets and EC and between platelets and leukocytes, via CD40, have been shown to promote leukocyte recruitment and exacerbate plaque formation in this model.sup.47, we cannot exclude the possibility that PTL060 might be modulating these interactions.
[0305] However, the data generated showing that weekly adoptive transfer of CD11b+ cells pre-treated with PTL060, or expressing a transgenic hirudin fusion protein can induce the same degree of regression as systemic PTL060, suggests that protecting plaque-recruited monocytes from the direct effects of thrombin is the key factor required for regression. Since thrombin, via protease activated receptor-1 and cullin 3-mediated degradation is known to promote post-transcriptional downregulation of ABCA1 in macrophages.sup.48, and is also known to promote M1 polarization of microglia after intracerebral haemorrhage.sup.49, our data is most consistent with the hypothesis that thrombin plays a hitherto unrecognised but pivotal role in determining the inflammatory phenotype of plaque macrophages and promoting plaque progression.
References to Example 2
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Borissoff J I, Otten J J, Heeneman S, Leenders P, van Oerle R, Soehnlein O, Loubele S T, Hamulyak K, Hackeng T M, Daemen M J, Degen J L, Weiler H, Esmon C T, van Ryn J, Biessen E A, Spronk H M and ten Cate H. Genetic and pharmacological modifications of thrombin formation in apolipoprotein e-deficient mice determine atherosclerosis severity and atherothrombosis onset in a neutrophil-dependent manner. PLoS One. 2013; 8:e55784. [0325] 19. Zhou Q, Bea F, Preusch M, Wang H, Isermann B, Shahzad K, Katus H A and Blessing E. Evaluation of plaque stability of advanced atherosclerotic lesions in apo E-deficient mice after treatment with the oral factor Xa inhibitor rivaroxaban. Mediators Inflamm. 2011; 2011:432080. [0326] 20. Hara T, Fukuda D, Tanaka K, Higashikuni Y, Hirata Y, Nishimoto S, Yagi S, Yamada H, Soeki T, Wakatsuki T, Shimabukuro M and Sata M. Rivaroxaban, a novel oral anticoagulant, attenuates atherosclerotic plaque progression and destabilization in ApoE-deficient mice. Atherosclerosis. 2015; 242:639-46. [0327] 21. Lee I O, Kratz M T, Schirmer S H, Baumhakel M and Bohm M. The effects of direct thrombin inhibition with dabigatran on plaque formation and endothelial function in apolipoprotein E-deficient mice. The Journal of pharmacology and experimental therapeutics. 2012; 343:253-7. [0328] 22. Kadoglou N P, Moustardas P, Katsimpoulas M, Kapelouzou A, Kostomitsopoulos N, Schafer K, Kostakis A and Liapis C D. The beneficial effects of a direct thrombin inhibitor, dabigatran etexilate, on the development and stability of atherosclerotic lesions in apolipoprotein E-deficient mice: dabigatran etexilate and atherosclerosis. Cardiovasc Drugs Ther. 2012; 26:367-74. [0329] 23. Pingel S, Tiyerili V, Mueller J, Werner N, Nickenig G and Mueller C. Thrombin inhibition by dabigatran attenuates atherosclerosis in ApoE deficient mice. Arch Med Sci. 2014; 10:154-60. [0330] 24. Preusch M R, Ieronimakis N, Wijelath E S, Cabbage S, Ricks J, Bea F, Reyes M, van Ryn J and Rosenfeld M E. Dabigatran etexilate retards the initiation and progression of atherosclerotic lesions and inhibits the expression of oncostatin M in apolipoprotein E-deficient mice. Drug Des Devel Ther. 2015; 9:5203-11. [0331] 25. Bea F, Kreuzer J, Preusch M, Schaab S, Isermann B, Rosenfeld M E, Katus H and Blessing E. Melagatran reduces advanced atherosclerotic lesion size and may promote plaque stability in apolipoprotein E-deficient mice. Arterioscler Thromb Vase Biol. 2006; 26:2787-92. [0332] 26. Posthuma J J, Posma J J N, van Oerle R, Leenders P, van Gorp R H, Jaminon A M G, Mackman N, Heitmeier S, Schurgers L J, Ten Cate H and Spronk H M H. Targeting Coagulation Factor Xa Promotes Regression of Advanced Atherosclerosis in Apolipoprotein-E Deficient Mice. Sci Rep. 2019; 9:3909. [0333] 27. Eikelboom J W, Connolly S J, Bosch J, Dagenais G R, Hart R G, Shestakovska O, Diaz R, Alings M, Lonn E M, Anand S S, Widimsky P, Hori M, Avezum A, Piegas L S, Branch K R H, Probstfield J, Bhatt D L, Zhu J, Liang Y, Maggioni A P, Lopez-Jaramillo P, O'Donnell M, Kakkar A K, Fox K A A, Parkhomenko A N, Ertl G, Stork S, Keltai M, Ryden L, Pogosova N, Dans A L, Lanas F, Commerford P J, Torp-Pedersen C, Guzik T J, Verhamme P B, Vinereanu D, Kim J H, Tonkin A M, Lewis B S, Felix C, Yusoff K, Steg P G, Metsarinne K P, Cook Bruns N, Misselwitz F, Chen E, Leong D, Yusuf S and Investigators C. Rivaroxaban with or without Aspirin in Stable Cardiovascular Disease. N Engl J Med. 2017; 377:1319-1330. [0334] 28. Kidd S K, Bonaca M P, Braunwald E, De Ferrari G M, Lewis B S, Merlini P A, Murphy S A, Scirica B M, White H D and Morrow D A. Universal Classification System Type of Incident Myocardial Infarction in Patients With Stable Atherosclerosis: Observations From Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events (TRA 2 degrees P)-TIMI 50. J Am Heart Assoc. 2016; 5. [0335] 29. Thelen M, Rosen A, Nairn A C and Aderem A. Regulation by phosphorylation of reversible association of a myristoylated protein kinase C substrate with the plasma membrane. Nature. 1991; 351:320-2. [0336] 30. Smith R A. Targeting anticomplement agents. Biochem Soc Trans. 2002; 30:1037-41. [0337] 31. Chen D, Weber M, McVey J H, Kemball-Cook G, Tuddenham E G, Lechler R I and Dorling A. Complete inhibition of acute humoral rejection using regulated expression of membrane-tethered anticoagulants on xenograft endothelium. Am J Transplant. 2004; 4:1958-63. [0338] 32. Chen D, Carpenter A, Abrahams J, Chambers R C, Lechler R I, McVey J H and Dorling A. Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1-dependent leukocyte recruitment in vivo. J Exp Med. 2008; 205:1739-46. [0339] 33. Li M, Qian M, Kyler K and Xu J. Endothelial-Vascular Smooth Muscle Cells Interactions in Atherosclerosis. Front Cardiovasc Med. 2018; 5:151. [0340] 34. Yu H, Moran C S, Trollope A F, Woodward L, Kinobe R, Rush C M and Golledge J. Angiopoietin-2 attenuates angiotensin II-induced aortic aneurysm and atherosclerosis in apolipoprotein E-deficient mice. Sci Rep. 2016; 6:35190. [0341] 35. Chen D, Li K, Tham E L, Wei L L, Ma N, Dodd P C, Luo Y, Kirchhofer D, McVey J H and Dorling A. Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice. Frontiers in immunology. 2018; 9:1517. [0342] 36. Combadiere C, Potteaux S, Rodero M, Simon T, Pezard A, Esposito B, Merval R, Proudfoot A, Tedgui A and Mallat Z. Combined inhibition of CCL2, CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost abolishes atherosclerosis in hypercholesterolemic mice. Circulation. 2008; 117:1649-57. [0343] 37. Pan J H, Sukhova G K, Yang J T, Wang B, Xie T, Fu H, Zhang Y, Satoskar A R, David J R, Metz C N, Bucala R, Fang K, Simon D I, Chapman H A, Libby P and Shi G P.
[0344] Macrophage migration inhibitory factor deficiency impairs atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation. 2004; 109:3149-53. [0345] 38. Burger-Kentischer A, Gobel H, Kleemann R, Zernecke A, Bucala R, Leng L, Finkelmeier D, Geiger G, Schaefer H E, Schober A, Weber C, Brunner H, Rutten H, Ihling C and Bernhagen J. Reduction of the aortic inflammatory response in spontaneous atherosclerosis by blockade of macrophage migration inhibitory factor (MIF). Atherosclerosis. 2006; 184:28-38. [0346] 39. Feig J E. Regression of atherosclerosis: insights from animal and clinical studies. Ann Glob Health. 2014; 80:13-23. [0347] 40. Fisher E A. Regression of Atherosclerosis: The Journey From the Liver to the Plaque and Back. Arterioscler Thromb Vase Biol. 2016; 36:226-35. [0348] 41. Moore K J, Sheedy F J and Fisher E A. Macrophages in atherosclerosis: a dynamic balance. Nat Rev Immunol. 2013; 13:709-21. [0349] 42. Trogan E, Feig J E, Dogan S, Rothblat G H, Angeli V, Tacke F, Randolph G J and Fisher E A. Gene expression changes in foam cells and the role of chemokine receptor CCR7 during atherosclerosis regression in ApoE-deficient mice. Proc Natl Acad Sci USA. 2006; 103:3781-6. [0350] 43. Rayner K J, Sheedy F J, Esau C C, Hussain F N, Temel R E, Parathath S, van Gils J M, Rayner A J, Chang A N, Suarez Y, Fernandez-Hernando C, Fisher E A and Moore K J. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest. 2011; 121:2921-31. [0351] 44. Aiello R J, Brees D and Francone O L. ABCA1-deficient mice: insights into the role of monocyte lipid efflux in HDL formation and inflammation. Arterioscler Thromb Vase Biol. 2003; 23:972-80. [0352] 45. Rahman K, Vengrenyuk Y, Ramsey S A, Vila N R, Girgis N M, Liu J, Gusarova V, Gromada J, Weinstock A, Moore K J, Loke P and Fisher E A. Inflammatory Ly6Chi monocytes and their conversion to M2 macrophages drive atherosclerosis regression. J Clin Invest. 2017; 127:2904-2915. [0353] 46. Woollard K J and Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nat Rev Cardiol. 2010; 7:77-86. [0354] 47. Gerdes N, Seijkens T, Lievens D, Kuijpers M J, Winkels H, Projahn D, Hartwig H, Beckers L, Megens R T, Boon L, Noelle R J, Soehnlein O, Heemskerk J W, Weber C and Lutgens E. Platelet C D40 Exacerbates Atherosclerosis by Transcellular Activation of Endothelial Cells and Leukocytes. Arterioscler Thromb Vase Biol. 2016; 36:482-90. [0355] 48. Raghavan S, Singh N K, Mani A M and Rao G N. Protease-activated receptor 1 inhibits cholesterol efflux and promotes atherogenesis via cullin 3-mediated degradation of the ABCA1 transporter. J Biol Chem. 2018; 293:10574-10589. [0356] 49. Wan S, Cheng Y, Jin H, Guo D, Hua Y, Keep R F and Xi G. Microglia Activation and Polarization After Intracerebral Hemorrhage in Mice: the Role of Protease-Activated Receptor-1. Transl Stroke Res. 2016; 7:478-487.
TABLE-US-00002 TABLE 1 Effect of PTL060 on body mass and plasma lipids in ApoE-/- mice Aortic Tx recipients*-fed HFD 6-14 weeks Prevention CD31- CD31- P experiments BL/6 TFPI-Tg P value BL/6 Hlr-Tg value† Body Age 6 weeks 18.6 ± 0.27 18.6 ± 0.25 0.89 19.6 ± 0.42 19.6 ± 0.31 0.96 mass End of Exp. 25.2 ± 1.71 24.6 ± 1.36 0.70 26.8 ± 0.34 25.9 ± 0.77 0.32 Cholesterol (mmol/L) 53.1 ± 3.31 52.9 ± 3.67 0.97 50.1 ± 4.98 50.2 ± 5.77 0.99 Triglycerides (mmol/L) 2.1 ± 0.14 2.0 ± 0.20 0.72 2.0 ± 0.13 2.0 ± 0.14 0.70 HDL(mmol/L) 1.5 ± 0.11 1.5 ± 0.10 0.90 1.6 ± 0.07 1.5 ± 0.08 0.60 LDL (mmol/L) 57.2 ± 2.73 55.2 ± 3.49 0.46 63.9 ± 9.97 61.0+6.28 0.83 Effect of PTL060 on body mass and plasma lipids in ApoE-/- mice Single injection*-fed HFD 6-12 weeks Prevention HLL PTL060 PTL060 PTL060 P experiments PBS (5 μg/g) (2.5 μg/g) (5 μg/g) (10 μg/g) value† Body Age 6 weeks 19.0 ± 0.40 19.4 ± 0.22 19.4 ± 0.44 19.4 ± 0.38 19.2 ± 0.24 0.87 mass End of Exp. 32.0 ± 1.63 32.0 ± 0.99 31.4 ± 0.84 29.8 ± 0.35 29.4 ± 0.73 0.36 Cholesterol (mmol/L) 51.0 ± 5.56 54.1 ± 5.70 49.3 ± 4.56 50.3 ± 4.36 50.1 ± 5.44 0.97 Triglycerides (mmol/L) 2.1 ± 0.31 2.1 ± 0.36 2.2 ± 0.28 2.2 ± 0.27 2.1 ± 0.30 1.00 HDL(mmol/L) 1.4 ± 0.28 1.5 ± 0.08 1.6 ± 0.12 1.5 ± 0.09 1.5 ± 0.09 0.92 LDL (mmol/L) 55.6 ± 6.64 54.7 ± 4.48 54.1 ± 4.72 51.7 ± 5.89 52.8 ± 5.42 0.99 Series 1 HFD 6-28 weeks Regression 6-week PTL060 P experiments old Baseline.sup.Ø PBS.sup.¥ Tail only (10 μg/g) value† Body Age 6 weeks — 19.3 ± 0.28 19.6 ± 0.37 19.1 ± 0.15 19.3 ± 0.38 0.74 mass Age 22 weeks — 30.5 ± 0.58 32.8 ± 0.44 30.4 ± 0.90 32.0 ± 0.81 0.19 End of Exp. — — 33.2 ± 0.31 31.8 ± 0.73 28.7 ± 1.55 0.03 Cholesterol (mmol/L) 10.3 ± 4.6 56.7 ± 6.08 61.5 ± 6.51 61.9 ± 6.35 57.9 ± 3.61 0.88 Triglycerides (mmol/L) 0.36 ± 0.14 2.08 ± 0.15 2.25 ± 0.11 2.49 ± 0.13 2.19 ± 0.16 0.26 HDL(mmol/L) 4.46 ± 1.1 1.54 ± 0.007 1.49 ± 0.06 1.46 ± 0.06 1.56 ± 0.06 0.65 LDL (mmol/L) 14.4 ± 2.8 46.5 ± 3.39 56.1 ± 2.92 57.4 ± 2.57 50.8 ± 2.84 0.07 Series 2 HFD 6-28 weeks PTL060 PTL060 P Baseline.sup.Ø PBS HLL (5 μg/g) (5 μg/g) (10 μg/g) value† Body Age 6 weeks 20.2 ± 0.39 20.1 ± 0.38 20.6 ± 0.07 20.1 ± 0.30 20.3 ± 0.26 0.73 mass Age 22 weeks 31.5 ± 1.34 31.4 ± 0.84 31.1 ± 0.66 31.2 ± 1.00 31.9 ± 0.78 0.85 End of Exp. — 32.9 ± 1.19 31.4 ± 0.33 30.2 ± 0.93 29.8 ± 0.59 0.11 Cholesterol (mmol/L) 54.9 ± 6.25 56.9 ± 6.93 54.1 ± 5.39 53.9 ± 6.31 54.3 ± 13.1 0.41 Triglycerides (mmol/L) 2.27 ± 0.28 1.68 ± 0.31 2.21 ± 0.38 2.2 ± 0.33 2.16 ± 0.69 0.86 HDL(mmol/L) 1.61 ± 0.12 1.58 ± 0.11 1.78 ± 0.2 1.6 ± 0.16 1.68 ± 0.38 0.27 LDL (mmol/L) 53.8 ± 3.51 62.7 ± 4.43 63.5 ± 3.55 60.6 ± 6.79 63.3 ± 16.7 0.97 HFD; high fat diet. BL/6; C57BL/6J. HLL; hirulog modified to accept the myristoyl tail (NB: HLL 5 μg is equimolar to PTL060 10 μg). PBS; phosphate buffered saline. Exp.; experiment. HDL; high density lipoprotein. LDL; low density lipoprotein. *In prevention experiments, aortic transplants performed and single injections given to mice aged 8 weeks, 2 weeks after starting HFD. .sup.ØBaseline = week 22. Mice in the ‘baseline’ groups were harvested at this timepoint prior to any treatment .sup.¥Treatments given weekly by IV injection for 6 weeks (mice aged 22-28 weeks). †Kruskal Wallis Test for multiple groups (NB: values from 6-week old mice not included in comparisons)
Example 3—PTL032 and PTL0GC1
[0357] Inspired by their new understanding of how PTL060 works, the inventors devised the idea that generation of a tethered PAR-1 antagonist, which would inhibit the signalling of thrombin through PAR-1 (by virtue of the PAR-1 antagonist domain), and be endowed with the uncoupled pharmacodynamic impact on haemostasis and inflammation (by virtue of the cytotopic tail) would have the same impact on atheroma regression as PTL060.
[0358] Following on from this idea, the inventors worked to generate a new cytotopic PAR-1 antagonist, called PTL032, by conjugating a known PAR-1 antagonist, 3-mercaptopropionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH.sub.2 (3-mercaptoproprionyl-F-Cha-Cha-RKPNDK amide), to the cytotopic tail via a linker.
[0359] The inventors also generated another new cytotopic PAR-1 antagonist, called PTL0GC-1, containing the same active PAR-1 element joined to the cytotopic tail via a different linker, which lacks a disulphide bond.
[0360] The structures are shown in
[0361]
Manufacture
[0362] The component parts (peptides/peptidic components) are prepared by standard techniques, for example prepared in the appropriate activated form by solid phase synthesis, unless otherwise stated.
[0363] Particular embodiments of the invention may be finally prepared by conjugation, for example as described below:
PTLOGC-1
[0364] The final conjugation conditions comprised (Myr)2KS SKSPS KKDDK KPGDK(Bromoacetate)-NH2 (1 eq) and 3-Mercaptopropionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH2 (1.2 eq) at 2 mg/ml concentration, based on the (Myr)2KS SKSPS KKDDK KPGDK(Bromoacetate)-NH2 in sodium phosphate buffer (50 mM, pH 6.5). The reaction was carried out at room temperature with stirring for 18 hours. Upon completion, the conjugation reaction was filtered and purified using an AKTA HPLC system using a Phenomenex Luna C18(2) 15 μm 100 Å 250×20 mm column. For the method, mobile phase A was 0.1% v/v TFA in water and mobile phase B was 0.1% v/v TFA in acetonitrile. Flow rate was variable with UV monitoring at 220, 240 and 280 nm. The gradient was 10-68% B over 12 CV. Fractions containing the product at >95% purity were pooled where appropriate. Solutions were freeze dried in round bottomed flasks for 2 days to give a lyophilized TFA salt.
PTL032
[0365] (Myr)2KS SKSPS KKDDK KPGDC acid was prepared in the activated S-(2-pyridyl)thiocysteine form by solid phase synthesis. The conjugation reaction between (Myr)2KS SKSPS KKDDK KPGDC(S-2-thiopyridyl)-OH (5 mg, 0.932 micromoles thiol equivalent) in DMSO (0.1 ml) and 3-Mercaptopropionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH2 (1.22 mg, 0.94 micromoles) in PBS buffer (0.1 ml) was performed on ice for 3 hours, then 100 ul fractionated on a Superdex 10/300GL peptide column (GE Healthcare, Uppsala, Sweden) at 22° C. using an AKTA purifier pump system (GE Healthcare, Sweden) in 0.02 mM sodium phosphate buffer pH 7.0 run at 0.5 ml/min. The product eluted in a UV-detectable peak at 8.1 ml and a pool between 7.8 and 8.5 ml was collected and stored at −80° C.
Example 4—Comparative Data
[0366] In a direct comparison of PTL060 with PTL032, given at equimolar doses, the inventors were surprised to find that the latter demonstrated considerably better regression of atherosclerosis than PTL060 (
[0367]
[0368] As evidenced by ELISA testing of plasma, regression of the atheroma in the mice given PTL032 was associated with suppression of important chemokines and cytokines, almost back to levels seen in very young mice prior to the development of atherosclerosis (
[0369]
[0370] All these data provide evidence that PTL032 induces significant regression of established disease in this model of atherosclerosis, with an efficiency better than the tethered direct thrombin inhibitor PTL060 in these direct comparative experiments.
Example 5—Further Experimental Support
[0371] Experimental evidence to explain the remarkable unexpected potency of PTL032/PTL0GC-1 is provided.
[0372] Firstly we show how 3-mercaptopropionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH.sub.2 is different to other PAR-1 antagonists.
[0373] This example uses a model of contact hypersensitivity. Here, mice are challenged on the abdomen with the contact sensitizer oxazolone, before a re-challenge 4 days later on both sides of the ear with oxazolone (on the right) or vehicle alone (on the left).
[0374] Swelling of the ears, measured with a micrometer, reflects the influx of monocytes/macrophages and T cells in a typical delayed-type hypersensitivity (DTH) response. Dual inhibition of both PAR-1 and PAR-2 signalling in mice expressing the tissue factor inhibitor was associated with exacerbated responses to re-challenge with oxazolone (i.e. increased ear swelling), whereas in the mice expressing hirudin, in which PAR-2 signalling was allowed, ear swelling was significantly prevented, even compared to negative littermate controls.
[0375] We have followed up these early observations with further experiments using selective PAR agonists and antagonists administered to WT mice simultaneously with the second exposure to oxazolone. As illustrated in
[0376]
[0377] The swelling directly associates with the proportion of CD68+ cells (macrophages) that can be found infiltrating the ears.
[0378] However, the effects of PAR-2 specific reagents are exactly opposite: a PAR-2 antagonist exacerbates ear swelling whereas a PAR-2 agonist inhibits it (
[0379] Finally, in the same model, we have directly compared the impact of 3-mercaptoproprionyl-F-Cha-Cha-RKPNDK amide to the other PAR reagents. As can be seen in
[0380]
[0381] We have also studied the impact of 3-mercaptoproprionyl-F-Cha-Cha-RKPNDK amide on the chemokine expression by both SMC and EC, relevant as influencing chemokine expression at the vessel wall is one of the mechanisms by which PTL060 induces regression (see earlier example). In this model, cells are purified from a WT mouse and grown in vitro as previously described (Chen et al 2015 Circulation vol 131 pages 1350-1360. Once confluent, cells are incubated with PAR agonists/antagonists for 12 hours, followed by, where appropriate, a further 1 hour incubation with thrombin. The chemokines secreted over the following 48 hours are measured in the supernatant by ELISA. As shown in
[0382] In contrast, the chemokines CX3CL1 (and CCL5), both of which promote recruitment of Ly6Clo (CCR2-) monocytes, is stimulated better by a PAR-2 agonist and 3-mercaptoproprionyl-F-Cha-Cha-RKPNDK amide than by a PAR-1 agonist.
[0383] These differences in PAR-1-dependent and PAR-2 dependent chemokine secretion impact significantly when thrombin-mediated secretion is antagonised by either a pure PAR-1 antagonist or 3-mercaptoproprionyl-F-Cha-Cha-RKPNDK amide. As shown in
[0384]
[0385] In summary the inventors have performed the work above to illustrate the differences between the parental compound (3-mercaptoproprionyl-F-Cha-Cha-RKPNDK amide) and other pure PAR-1 antagonists.
[0386] The inventors show a comparative experiment with PTL032, indicating that in a formal ‘head to head’ test it is more potent than PTL060.
[0387] The inventors have performed multiple experiments in vivo with PTL060, which serve to exemplify how cytotopic proteins with the same membrane binding compound behave in vivo after IV injection.
[0388] It is being confirmed that PTL0GC-1 causes significant regression of atherosclerosis in ApoE−/− mice and is more effective than equimolar doses of either PTL060 or a pure PAR-1 antagonist.
[0389] It is being confirmed that PTL0GC-1 inhibits development of renal fibrosis in a model of aristolochic acid nephropathy (AAN) and is more effective than equimolar doses of either PTL060 or a pure PAR-1 antagonist.
[0390] Studies in a large animal model (pig), formal toxicology studies, and generation of GMP-grade material for clinical use follow.
Example 6: Further Experimental Support
[0391] Research published after the earliest priority date includes (Chen D, Li K, Festenstein S, et al. Regression of Atherosclerosis in ApoE−/− Mice Via Modulation of Monocyte Recruitment and Phenotype, Induced by Weekly Dosing of a Novel “Cytotopic” Anti-Thrombin Without Prolonged Anticoagulation. J Am Heart Assoc 2020; 9:e014811; and Wilkinson H, Leonard H, Chen D, et al. PAR-1 signalling on macrophages is required for effective in vivo delayed type hypersensitivity responses. iScience 2021; 21). These papers together set out:
a) The beneficial impact of PTL060 on both atherosclerosis and delayed type hypersensitivity (DTH);
b) PTL060 operates via two mechanisms in atherosclerosis.
i) A switch from recruitment of Ly6Chi (CCR2-pos) monocytes into atherosclerotic plaques, to recruitment of Ly6Clo (CCR2-neg) monocytes that express a ‘regression phenotype’ (CD206+, ABCA1+, IL-10+, CCR7+, iNOS−).
ii) A direct effect of PTL060 on Ly6Chi monocytes causing expression of a ‘regression phenotype’ (CD206+, ABCA1+, IL-10+, CCR7+, iNOS−);
c) Of these, the second mechanism appears dominant, as evidenced by adoptive transfer of PTL060 pre-treated monocytes, which induces the same degree of regression associated with IV treatment of PTL060;
d) The direct effect of thrombin on monocytes is explained by PAR-1 signals mediating downregulation of the reverse cholesterol transporter ABCA1, resulting in increased membrane expression of cholesterol rich microdomains, and movement of the receptor for interferon gamma (IFNg) into these areas. This enhances the sensitivity of thrombin-treated cells to IFNg by 1000×, such that monocytes are polarised away from the ‘regression phenotype’ by very small concentrations of IFNg. PTL060 inhibits these mechanisms.
[0392] In this example we further show: [0393] Compared to PTL060, equimolar PTL0GC-1 injected IV is better at inducing atheroma regression (
[0403] Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.
SEQUENCE LISTING
[0404]
TABLE-US-00003 SEQ ID NO: 1 CH2-CH2-F-Cha-Cha-RKPNDK-NH2 (PTLo32 and/or PTLoGC1) SEQ ID NO: 2 [Myr2]-KSSKSPSKKDDKKPGD (PTLo32-including Cysteine residue) SEQ ID NO: 3 [Myr2]-KSSKSPSKKDDKKPGDC (PTLoGC1-including Lysine residue) SEQ ID NO: 4 [Myr2]-KSSKSPSKKDDKKPGDK