TREATMENTS FOR HEART FAILURE AND CARDIAC ISCHAEMIC REPERFUSION INJURY

Abstract

The present invention relates to the use of extracorporeal cardiac Shockwave therapy in the treatment or prophylaxis of heart disease, in particular the invention provides a dipeptidyl peptidase-4 (DPP-4) inhibitor, or a pharmaceutical composition containing said inhibitor, for use in: (a) treating or preventing heart failure; or (b) treating cardiac ischaemic reperfusion injury, in a subject, wherein said subject has been administered extracorporeal cardiac Shockwave therapy and wherein said inhibitor is for administration prior to, simultaneously with, and/or after administration of said Shockwave therapy.

Claims

1.-20. (canceled)

21. A method of treating or preventing heart failure or treating cardiac ischaemic reperfusion injury comprising administering extracorporeal cardiac shockwave therapy and a DPP-4 inhibitor, or a pharmaceutical composition comprising said inhibitor, to a subject in need thereof, wherein said inhibitor or composition is administered prior to, simultaneously with, and/or after administration of said shockwave therapy.

22. The method of claim 21 further comprising administering a pharmacological agent suitable for mobilizing stem cells to said subject, wherein said agent is administered separately, simultaneously or sequentially to said inhibitor or composition.

23. The method of claim 22, wherein said pharmacological agent suitable for mobilizing stem cells is parathyroid hormone or a fragment thereof.

24. The method of claim 23, wherein said parathyroid hormone fragment comprises teriparatide.

25. The method of claim 21, wherein said inhibitor or composition is administered prior to said shockwave therapy and continuously throughout the course of the shockwave therapy.

26. The method of claim 21, wherein said pharmacological agent suitable for mobilizing stem cells is administered at least 8 hours before the shockwave therapy.

27. The method of claim 21 further comprising administering stem cells to said subject, wherein said stem cells are administered separately, simultaneously or sequentially to said inhibitor or composition.

28. The method of claim 27, wherein said stem cells are administered at least 8 hours before the shockwave therapy.

29. The method of claim 27, wherein said progenitor cells are derived from or comprise bone marrowderived mononuclear cells.

30. The method of claims 21, wherein the DPP-4 inhibitor is Sitagliptin, Linagliptin, Vildagliptin, Gemigliptin, Anagliptin, Teneligliptin, Trelagliptin, Dutogliptin, Omarigliptin, Lupeol or a combination thereof.

31. The method of claim 21, wherein said heart failure is chronic heart failure or post-infarction heart failure.

32. The method of claim 21, wherein said shockwave therapy comprises administering shockwave pulses with energy of about 0.05-0.25 mJ/mm.sup.2.

33. The method of claim 21, wherein said shockwave therapy comprises administering at least about 500 shockwave pulses, preferably at least about 1000 shockwave pulses.

34. The method of claim 21, wherein said shockwave therapy comprises administering a dose of about 500-2000 shockwave pulses.

35. The method of claim 21, wherein said shockwave therapy comprises administering shockwave pulses during the isovolumic contraction and/or isovolumic relaxation periods of the cardiac cycle.

36. The method of claims 21 comprising targeting the focal point of the shockwaves to a specific region of the heart.

37. The method of claim 36 further comprising determining the region of the heart to be targeted by echocardiography (echo) visualisation and/or electrocardiogram (ECG) pattern recognition.

38. The method of claim 36 further comprising determining the region of the heart to be targeted by magnetic resonance imaging.

39. The method of claims 36, wherein specific region of the heart to be targeted comprises scar tissue.

40. The method of claims 21, comprising administering the dose of shockwave pulses over more than one session.

41.-50. (canceled)

Description

[0148] The invention will be further described with reference to the following non-limiting Examples with reference to the following drawings in which:

[0149] FIG. 1 shows a bar chart showing the percentage of viable rat cardiomyocytes under various treatment conditions (error bars represent the standard deviation, ****p0.001, ns no significance).

[0150] FIG. 2 shows bar charts of the relative amount (fold change) of (A) SDF-1 gene expression and (B) MCP-1 gene expression in human ventricular tissue four hours after various shockwave treatments (*p>0.05, **p0.05, ***p0.001 and ****p0.0001).

[0151] FIG. 3 shows bar charts of the relative amount (fold change) of (A) ANGP-1 (Angiopoietin) gene expression and (B) VEGFA (Vascular endothelial growth factor A) gene expression in human ventricular tissue four hours after various shockwave treatments (*p>0.05, **p0.05, ***p0.001 and ****p0.0001).

[0152] FIG. 4 shows bar charts of the relative amount (fold change) of (A) NOS-3 (Nitric Oxide Synthase 3) gene expression and (B) TAC-1 (Tachykinin Precursor 1) gene expression in human ventricular tissue four hours after various shockwave treatments (*p>0.05, **p0.05, ***p0.001 and ****p0.0001).

[0153] FIG. 5 shows graphs of the relative amount (fold change) of SDF-1 gene expression over time in (A) Human umbilical vein endothelial cells (HUVECs) and (B) human cardiac fibroblasts after various shockwave treatments (*p>0.05, **p0.05, ***p0.001 and ****p0.0001).

[0154] FIG. 6 shows graphs of the relative amount (fold change) of (A) VEGFA gene expression and (B) MCP-1 (Monocyte chemotactic protein 1) gene expression over time in human cardiac fibroblasts after various shockwave treatments (*p>0.05, **p0.05, ***p0.001 and ****p0.0001).

[0155] FIG. 7 shows bar charts of the relative amount (fold change) in AKT phosphorylation in rat cardiomyocytes under various conditions, wherein (A) shows the relative change of normalised p-AKT308 and (B) show the relative change of the ratio of p-AKT308 and unphosphorylated AKT.

[0156] FIG. 8 shows bar charts of the relative amount (fold change) of (A) SDF-1 gene expression and (B) VEGFA gene expression in rat cardiomyocytes four hours after various shockwave treatments.

[0157] FIG. 9 shows photomicrographs of frozen tissue sections from rat hearts subjected to the following treatments: (A) no treatment; (B) extracorporeal cardiac shockwave treatment and daily water gavages; (C) extracorporeal cardiac shockwave treatment and daily DPP4i gavages; and (D) daily DPP4i gavages, as described in Example 7. The circles show areas of brown deposits that are evidence of the presence of SDF-1.

[0158] FIG. 10 shows photographs of fixed rat hearts subjected to the following treatments: (A) no treatment; (B) extracorporeal cardiac shockwave treatment and daily water gavages; (C) extracorporeal cardiac shockwave treatment and daily DPP4i gavages; and (D) daily DPP4i gavages, as described in Example 7.

[0159] The error bars in FIGS. 2-6 and 8 represent the 95% confidence interval (Cl).

EXAMPLES

Example 1: Effect of Shockwave Treatment on Hypoxia Induced Apoptosis in Rat Cardiomyocytes

[0160] Primary rat cardiomyocytes were isolated using the Langendorff method, where the explanted hearts were retrogradely perfused through the aorta with oxygenated low calcium solutions and collagenase solutions. The hearts were then diced and agitated in collagenase solutions to release the cells. The cells were washed and the cardiomyocytes were then positively selected using low-speed centrifugation and by adherence to laminin-coated culture plates (Petri dishes).

[0161] The rat cardiomyocytes were exposed to severe hypoxia for 30 minutes (0% O.sub.2, 100% N.sub.2). The groups receiving shockwave treatments (1000 pulses of 1 bar or 2 bar) were treated immediately upon return to normoxia. Shockwaves were delivered from the underneath the Petri dishes via direct contact between the shockwave applicator, on the handpiece of Swiss Dolarclast (EMS) shockwave system, coupled with ultrasound gel. Notably the group receiving exogenous SDF received no shockwave treatment upon returned to normoxia. The viability of the cells was assessed by counting the number of rod-shaped and round-shaped cells (wherein rod-shaped cells are viable) in triplicate 24 hours after shockwave or SDF treatment. The results are shown in FIG. 1 and demonstrate that post-conditioning using shockwave therapy immediately after hypoxia increases cardiomyocyte viability relative to untreated controls, which suggests that shockwave therapy may attenuate ischemic reperfusion injury.

Example 2: Effect of Shockwave Treatment on Gene Expression in Human Ventricular Tissue

[0162] Explanted human cardiac ventricular tissue was cut into small pieces and individually cultured in 24-well plates in M199 media. Exposure to various shockwave conditions was performed by temporarily placing the tissue pieces in 1.5 ml Eppendorf tubes in M199 media. The shockwave applicator described in Example 1 was directly coupled to the tubes using ultrasound gel. The tissue pieces were then returned to their respective culture media in the incubator (37 C. and 5% CO.sub.2) for the 4-hour time points and then stored in RNA-Later at 80 C. In batches, the samples were completely homogenised in Trizol using a power homogenizer. The RNA was purified using chloroform and commercial spin columns. The quality of RNA was inspected using a Nanodrop spectrophotometer and reverse transcription reactions were performed. The gene expression of SDF1 (Stromal derived factor 1), VEGFA (Vascular endothelial growth factor A), MCP1 (Monocyte chemotactic protein 1), ANGP1 (Angiopoietin), TAC1 (Tachykinin Precursor 1) and NOS3 (Nitric Oxide Synthase 3) was assessed using Taqman probes in TaqMan Gene Expression Master Mix normalised using GAPDH, on the Applied Biosystems 7900HT Fast Real-Time PCR System. The fold changes were calculated using the CT method. The results are shown in FIGS. 2-4 and demonstrate that there is a statistically significance difference between the untreated controls and the 4-hour time points for all of the genes measured.

Example 3: Effect of Shockwave Treatment on SDF-1 Gene Expression in Endothelial and Human Cardiac Fibroblast Cells

[0163] Human cardiac ventricular tissues were dissociated using collagenase and the single-cell suspensions were washed, plated and cultured in DMEM supplemented with 10% FCS. HUVECs were harvested from the human umbilical cords and cultured in EGM2. Cells passages 3-5 were used in the experiments.

[0164] The human cardiac fibroblasts and HUVECs were subjected to shockwave treatment as described in Example 1 and upon reaching the time points, the media was removed and the cells lysed using Trizol. SDF1 gene expression was measured at various time points after shockwave treatment in accordance with the method described in Example 2. The results are shown in FIG. 5.

[0165] In human cardiac fibroblasts SDF1 gene expression continued to increase at the 24-hour time point whereas SDF1 gene expression reached maximum expression between 2-6 hours in HUVECs and returned to baseline levels within 24 hours of shockwave treatment. These results demonstrate that shockwave treatment induced sustained SDF1 gene expression specifically in cardiac fibroblasts.

Example 4: Effect of Shockwave Treatment on Gene Expression in Human Cardiac Fibroblasts

[0166] Human cardiac fibroblasts were obtained according to the method described in Example 3 and subjected to shockwave treatment as described in Example 1. VEGFA and MCP1 gene expression was measured at various time points after shockwave treatment in accordance with the method described in Example 2. The results are shown in FIG. 6 and show that, in contrast with SDF1, VEGFA and MCP1 gene expression rapidly increased by the 3 hour time point and returned to baseline levels by the 24 hour time point. This data demonstrates that SDF1 expression in fibroblasts lags behind the SDF1 expression of the endothelial cells by 24 hr, creating a temporal and spatial gradient from intravascular to cardiac tissue.

Example 5: Effect of Shockwave Treatment on AKT Phosphorylation in Rat Cardiomyocytes

[0167] Rat cardiomyocytes were cultured in standard conditions and subjected to treatment with: shockwaves (1000 pulses at 1 bar); exogenous SDF; or a PI3 kinase inhibitor (LY294002). AKT phosphorylation was measured at various time points using Western Blot normalised using pan-AKT and COX IV as a loading control.

[0168] The results are show in in FIG. 7 and show that AKT phosphorylation is increased in shockwave treated cells. However, the effect was not blocked by PI3 kinase inhibitor (LY294002) indicating that the phosphorylation of AKT by shockwave is independent of Phosphatidylinositol 3-kinase, an activator of the AKT pathway.

Example 6: Effect of Shockwave Treatment on SDF-1 Gene Expression in Rat Cardiomyocytes

[0169] Rat cardiomyocytes were cultured according to the method in Example 1 and subjected to treatment with shockwaves (1000 pulses at 1 bar or 2 bar). SDF1 and VEGFA gene expression was measured 4 hours after treatment as described in Example 2. The results are shown in FIG. 8 and demonstrate that there is no statistically significance difference between the untreated controls and the shockwave conditions for both SDF1 and VEGFA gene expression. This indicates that the anti-apoptotic effect of shockwaves on cardiomyocytes is independent of anti-apoptotic factors such as SDF1 and VEGFA.

Example 7: Effects of Shockwave and DPP-4 Inhibitor (DPP4i) Treatments on Rat Hearts

[0170] Male Lewis rats (250-275 g) were subjected to the following treatments for four days: (1) no treatment; (2) extracorporeal cardiac shockwave treatment on day 2 of 4 and daily water oral gavages; (3) extracorporeal cardiac shockwave treatment on day 2 of 4 and daily DPP4i oral gavages; and (4) daily DPP4i oral gavages.

[0171] Shockwave treatments (0.25 mJ/mm.sup.21000 pulses at 4 Hz using a Storz Medical DUOLITH SD1 device) were administered under general anaesthesia (1.5-2% isoflurane in 100% O.sub.2). The shockwaves were administered continuously and targeted to the heart, which was located using finger palpation and echocardiography coupled with ultrasound gel.

[0172] DPP4 inhibitor (Linagliptin, 3 mg per rat delivered as a 1 mg/ml solution) or water was administered directly into the stomach via a gavage needle.

[0173] The animals were culled and the hearts were explanted 4 days after the onset of the experiment. Blood was washed off the hearts with phosphate buffered saline, fixed in 4% paraformaldehyde overnight, cryoprotected in 30% sucrose overnight and then embedded in OCT for cryosectioning using a Cryostat.

[0174] Immunohistochemistry was performed on sections blocked with 1% BSA in Tris-buffered saline+Tween using rabbit anti-rat SDF1 primary antibodies and goat anti-rabbit HRP-conjugated (horse radish peroxidase-conjugated) secondary antibodies. The tissue sections were counterstained using hematoxylin and 3,3-diaminobenzidine (DAB) substrate was used for chromogenic detection of HRP. DAB results in a brown insoluble product in the presence of HRP. Sections were dehydrated using ethanol series, mounted using DPX-new and Xylene replacement as the solvent. Bright-field light microscopy was used to visualise the sections.

[0175] The results are shown in FIG. 9, wherein treatments 1-4 are shown in FIGS. 9A-D, respectively. For ease of reference, deposits of the brown insoluble reaction product, which are evidence of the presence of SDF-1, are circled. It was observed that the SDF-1 was induced by extracorporeal shockwave therapy (see FIGS. 9B and 9C); no brown deposits were observed in heart tissue from untreated rats (FIG. 9A) or rats treated only with DPP4i (FIG. 9D). Moreover, the rats treated with DPP4i showed a significant increase in SDF-1 relative to rats treated only with shockwaves or DPP4i. These results indicate that the combination of shockwave and DPP4i treatment results in a more than additive effect on the presence of SDF-1 in cardiac tissue.

[0176] FIG. 10 shows appearance of rat hearts after overnight incubation with 4% paraformaldehyde: A) untreated normal control, B) shockwave only; C) shockwave and DPP4i, and D) DPP4i only. It was observed that hearts from rats treated with shockwave and DPP4i have very prominent blood vessels compared to hearts from rats treated only with shockwaves. Hearts from untreated rats and rats treated only with DPP4i are very similar. It was concluded that DPP4i enhances the angiogenic process induced by shockwave and DPP4i on its own has a neutral effect.