PHARMACEUTICAL COMPOSITION COMPRISING ANCROD FOR THE TREATMENT OR PROPHYLAXIS OF ENDOCARDITIS

Abstract

The present invention concerns ancrod for endocarditis prophylaxis or the treatment of endocarditis, especially for the treatment of damage-induced endocarditis. According to the present invention ancrod is preferably administered by intravenous injection, intravenous infusion, intramuscular injection, subcutaneous injection, intraperitoneal injection or combinations thereof. The present invention also concerns pharmaceutical compositions, especially aqueous solutions or dispersions of ancrod for endocarditis prophylaxis or the treatment of endocarditis.

Claims

1. Ancrod for endocarditis prophylaxis or the treatment of endocarditis.

2. Ancrod according to claim 1 for endocarditis prophylaxis.

3. Ancrod according to claim 1 for the treatment of endocarditis.

4. Ancrod according to claim 1, wherein the endocarditis is a damage-induced endocarditis.

5. Ancrod according to claim 1, wherein ancrod is administered by a method selected from the group consisting of injection directly into the vascular system, infusion directly into the vascular system, injection into tissue, injection into the peritoneal cavity of the abdominal cavity and combinations thereof.

6. Ancrod according to claim 1, wherein ancrod is administered by a method selected from the group consisting of intravenous injection, intravenous infusion, intramuscular injection, subcutaneous injection, intraperitoneal injection and combinations thereof.

7. Ancrod according to claim 1, characterized in that ancrod is administered by a method selected from the group consisting of intravenous injection, intravenous infusion and combinations thereof.

8. Ancrod according to claim 1, characterized in that ancrod is administered by intravenous injection.

9. Ancrod according to claim 1, characterized in that ancrod is administered in an amount of from 0.1 to 10 iU per kg of body weight.

10. Ancrod according to claim 1, characterized in that ancrod is administered in an amount of from 0.5 to 5 iU per kg of body weight.

11. Ancrod according to claim 1, characterized in that ancrod is administered in an amount of from 1 to 2 iU per kg of body weight.

12. Ancrod according to claim 1, characterized in that the endocarditis is a damage-induced endocarditis, wherein the damage is induced by surgery.

13. Ancrod according to claim 1, characterized in that the endocarditis is a damage-induced endocarditis, wherein the damage is induced by cardiac catheter examination.

14. A pharmaceutical composition comprising ancrod according to claim 1.

15. A method for endocarditis prophylaxis or the treatment of endocarditis, comprising: administering to a patient in need thereof an effective amount of ancrod.

16. The method according to claim 15, wherein the method is for endocarditis prophylaxis.

17. The method according to claim 15, wherein the method is for the treatment of endocarditis.

18. The method according to claim 15, wherein the endocarditis is a damage-induced endocarditis.

19. The method according to claim 15, wherein the administering is performed by a method selected from the group consisting of injection directly into the vascular system, infusion directly into the vascular system, injection into tissue, injection into the peritoneal cavity of the abdominal cavity and combinations thereof.

20. The method according to claim 15, wherein the administering is performed by a method selected from the group consisting of intravenous injection, intravenous infusion, intramuscular injection, subcutaneous injection, intraperitoneal injection and combinations thereof.

21. The method according to claim 15, wherein the ancrod is administered in an amount of from 0.1 to 10 iU per kg of body weight.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0018] The figures show the following:

[0019] FIG. 1: Adhesion of wild type S. aureus on damaged and undamaged cardiac valves

[0020] FIG. 2: Adhesion of a clinical strain of S. aureus on damaged cardiac valves dependent on the amount of damage.

[0021] FIG. 3: Scanning electron microscopy image of an intact aortic valve (162×)

[0022] FIG. 4: Scanning electron microscopy image of a valve with damage-induced endocarditis (162×)

[0023] FIG. 5: Scanning electron microscopy image of a valve with damage-induced endocarditis (9140×)

[0024] FIG. 6: Effect of ancrod on the amount of vegetation produced by adhesion of S. aureus to the inner walls of cardiac valves at a dosage of 6 iU

EXPERIMENTAL PART

Methods

Bacterial Strains

[0025] The reference strains used in this study were S. aureus Newman, originally isolated from a case of osteomyelitis, and S. aureus USA300. Clinical strains of S. aureus were isolated from blood cultures of patients from the University Hospitals of Leuven. Before usage the bacteria were grown overnight in Tryptic Soy Broth (Sigma-Aldrich, Darmstadt, Germany) at 37° C., washed twice in phosphate-buffered saline (PBS) and quantified by recording optical densitometry at a wavelength of 600 nm (OD600). The inoculum size was verified by plating on blood agar.

Animals

[0026] Mice of the wild type (WT) C57BL/6 were used. Before surgery mice were anesthetized with ketamine (125 mg/kg body weight) and xylazine (12.5 mg/kg body weight) and anesthesia was checked by pedal reflex. Mice that were not immediately sacrificed after the procedure also received buprenorphine (0.1 mg/kg body weight) subcutaneously 20 to 30 min prior to the surgery and twice daily thereafter.

Spontaneous Endocarditis

[0027] To see whether mice develop endocarditis spontaneously, old mice (>18 months of age and 27 months of age) and young mice (10 to 15 weeks of age) were injected via tail vein with 2×10.sup.6 to 3×10.sup.7 CFU S. aureus Newman. Mice were sacrificed three days after injection and the hearts were excised, fixed overnight in paraformaldehyde 4% and embedded in paraffin. Sections of the aortic valve were stained with Brown-Hopps tissue Gram staining and imaged with light microscopy (Axiovert 200 m, Carl-Zeiss, Oberkochen, Germany). A researcher analyzed the sections for presence of endocarditis without knowledge of the treatment conditions.

Endocarditis Mouse Model to Study Early Bacterial Adhesion

[0028] Bacteria were fluorescence-labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimidylester (Sigma-Aldrich, Darmstadt, Germany; 30 μg/mL) or Texas Red®-X, succinimidyl ester (Thermo Fisher, Waltham, Mass., USA; 10 μM) for 45 minutes before usage. The mice were anesthetized as described above and injected with 3×10.sup.7 CFU fluorescent S. aureus bacteria via tail vein. Subsequently, the carotid artery was dissected and a 32-gauge polyurethane catheter was inserted (RecathCo, Allison Park, Penn., USA). The catheter was moved upstream beyond the aortic valve until pulsation of the catheter was detected, assuring its position in the left ventricle, beyond the aortic valve. The time between injection of the bacteria and placement of the catheter was approximately 15 minutes.

[0029] To generate endothelial damage for the examination of the effect of ancrod, the catheter was left in place during 15 or 30 minutes, damaging the valves with every heartbeat. Afterwards, the catheter was removed and the carotid artery ligated to prevent blood loss. The control group consisted of mice that underwent sham operation: the catheter was placed as described above, but immediately removed and the carotid artery was ligated.

[0030] After removal of the catheter, the mice were immediately sacrificed and transcardially perfused with saline and paraformaldehyde 4% both for two minutes. The hearts were then fixed in paraformaldehyde 4% overnight, transferred to sucrose 25%, embedded and frozen in Tissue-Tek o.c.t. compound (commercially available from Sakura Finetek Europe B.V., in Alphen aan den Rijn, The Netherlands). Afterwards, 4 to 7 cryosections, 200 μm in thick, covering the entire aortic valve were made. Of each 200 μm in section a Z-stack was made with confocal microscopy (LSM 700 or LSM880 Carl-Zeiss, Oberkochen, Germany) as to image the entire aortic valve. These images were then analyzed in 3D with Imaris (Bitplane, Zurich, Switzerland) or Image J (Image J, NIH, Bethesda, USA).

Studying Long-Term Endocarditis Development

[0031] In these experiments non-fluorescent bacteria (2×10.sup.6 to 2×10.sup.7 CFU/mouse) were injected and a catheter cardiac valve damage was induced as described above. Afterwards, the catheter was removed, the carotid artery was ligated and the skin closed. The mice recovered from surgery and were monitored for wellbeing four times per day, up to day three, after which they were sacrificed. Hearts were excised, fixed overnight in paraformaldehyde 4% and embedded in paraffin. Sections of the aortic valve were stained with Brown-Hopps tissue Gram staining and imaged with light microscopy (Axiovert 200 m, Carl-Zeiss, Oberkochen, Germany). A researcher analyzed the sections for presence of endocarditis without knowledge of the experimental conditions.

Electron Microscopy

[0032] Aortic valves were dissected and fixed with 2% paraformaldehyde, 2.5% glutaraldehyde and 0.02% sodium azide mixture in 0.05 M sodium cacodylate buffer. After double treatment with osmium tetroxide and thiocarbohydrazide with OTO protocol (Friedman P L, Ellisman M H. Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation. J Neurocytol; 1981; 10(1); 111-31) samples were dehydrated in ethanol followed by hexamethyldisilazane, dried, mounted on aluminium stubs, sputter coated with chromium and examined in a digital field emission scanning electron microscope (Carl Zeiss) at 5 kV accelerating voltage.

Statistical Analysis

[0033] All calculations were done with GraphPad Prism 5.0 d (GraphPad Software, La Jolla, Calif., USA). If normally distributed the two-tailed Student's t-test was used. The early adhesion measurements were found to be skewed and were log transformed, after which t-testing could be applied. All values are reported as mean±standard deviation (SD). To test proportion (endocarditis or not), the Fisher's exact test was used. A P-value of <0.05 was considered statistically significant.

EXAMPLES

Example 1: Spontaneous Occurrence of Infective Endocarditis in Mice

[0034] The validity of mice to study infective endocarditis and whether endocarditis spontaneously occurs in bacteremic mice was studied in a first experiment. 10 young mice (10 to 15 weeks old) were injected intravenously with 2×10.sup.6 CFU of S. aureus Newman. None of them developed endocarditis. The experiment was repeated with 3×10.sup.7 CFU with the same result. However, when 10 old mice (>1.5 years of age and 27 months of age) were injected with 2×10.sup.6 CFU S. aureus Newman, two out of ten developed endocarditis, without any preceding surgical procedure. These lesions shared remarkable similarity with human endocarditis lesions, consisting of large bacterial colonies growing unimpeded in a meshwork of platelets and fibrin, mostly devoid of leukocytes.

[0035] Mice are therefore in principle suitable as a model for infective endocarditis.

Example 2: Early Bacterial Adhesion to the Heart Valves

[0036] Mice were injected intravenously with fluorescence-labeled S. aureus and bacterial adhesion to the valve leaflets was quantified using 3D confocal microscopy on 200 μm thick cryosections of the aortic valves. A first group of mice only received an injection of S. aureus and underwent no manipulation of the valves, representing the bacteremic patient without risk factors. In a second group, cardiac valve damage was simulated by insertion of a 32-gauge polyurethane catheter in the carotid artery, which was advanced beyond the aortic valve. The catheter was left in place damaging the valve during 15 minutes, after which it was removed.

[0037] FIG. 1 shows the result. As can be seen by the common logarithm of the volume of the bacterial vegetation, in the bacteremia group without manipulation of the aortic valve, hardly any bacteria adhered to the valves (see entries for “No catheter” in FIG. 1). However, in mice where the cardiac valves were damaged, a significant increase in bacterial adhesion was seen (P<0.01; see entries for “Catheter” in FIG. 1). These results were confirmed with a clinical strain, which was a CC5-positive S. aureus from an endocarditis patient; the longer the cardiac valves were damaged, the stronger the clinical strain adhered (see FIG. 2, here the volume of the bacterial vegetation is shown, not its common logarithm). Similar results were obtained with the S. aureus USA300-strain (data not shown).

Example 3: From Early Bacterial Adhesion to a Mature Endocarditis

[0038] In a next step it was tested, whether this observed early bacterial adhesion was indeed the first step in the development of a mature endocarditis. To this end, again an intravenous injection with S. aureus was conducted and a 32-gauge polyurethane catheter was used to induce cardiac valve damage (for 30 minutes this time). Afterwards, the catheter was immediately removed and the mice were followed over a three-day period. Sections of the aortic valve were analyzed at various time points. Ten mice for each experiment were used.

[0039] None of the control mice (sham operation) developed endocarditis after injection of 2×10.sup.6 CFU S. aureus. In contrast, in some mice where the aortic valves were damaged during 30 minutes, endocarditis did occur: 0/5 (0%) for S. aureus Newman, 1/12 (8%) for S. aureus USA300 and 1/11 (9%) for the clinical strain (P>0.05 for all experiments vs. sham operation). However, when a higher bacterial load was used (2×10.sup.7 CFU) a larger proportion of mice developed endocarditis: 2/6 (33%) for S. aureus Newman, 1/12 (8%) for S. aureus USA300 and 8/10 (80%) for the clinical strain, which was a CC5-positive S. aureus from an endocarditis patient (P=0.45, P=0.9, P<0.01 vs. sham operation for Newman, USA300 and the clinical strain respectively).

[0040] Using light microscopy the different stages in the development of these endocarditis lesions could be captured, observing how small, early lesions would grow to large, destructive vegetation that ultimately destroyed the whole aortic valve. Importantly, these experimental vegetation looked very similar to those that spontaneously occurred in older mice as described in Example 1. Echocardiography confirmed that these vegetation originated from the aortic valve and could in some cases cause serious aortic regurgitation. In addition, electron microscopy was used to study these lesions in more detail (FIGS. 3 to 5), revealing individual Staphylococci in large destructive vegetation consisting of platelets and fibrin. FIG. 3 shows a scanning electron microscopy image of an intact aortic valve. However, FIGS. 4 and 5 show a valve damaged by endocarditis. FIG. 4 is the picture of a valve in the same size as that of FIG. 3 and shows in comparison to FIG. 3 that a large amount of vegetation is produced. In a larger magnification FIG. 5 shows individual Staphylococci in the vegetation of the cardiac valve shown in FIG. 4. It is therefore apparent, that the induced endocarditis develops upon time into a full-blown endocarditis.

Example 4: Effect of Ancrod on Bacterial Adhesion

[0041] Experiments as described in Example 2 were conducted. However, as a first step C57BL/6-mice were injected intravenously with 6 international units (iU) per mouse of ancrod (Nordmark Arzneimittel GmbH & Co. KG, Uetersen, Germany) before the experiments. The concentrations used in this example are much higher than the preferred dose range for a therapeutic use in humans. The reason is that the present examples serve as a proof of concept. In a control group ancrod was not used. Instead an equal volume of saline was administered as placebo. 5 to 7 hours after the administration of ancrod or placebo, respectively, 3×10.sup.7 CFU of fluorescence-labeled Staphylococcus aureus Newman were injected intravenously into the mice and a 32-gauge polyurethane catheter was inserted in the carotid artery and advanced beyond the aortic valve. To create cardiac valve damage, the catheter was left in place for 15 minutes. Thereafter the mice were immediately sacrificed and bacterial adhesion was measured with confocal microscopy.

[0042] FIG. 6 shows a comparison of the results for ancrod-treated mice vs. control mice, which were not treated with ancrod. The results in FIG. 6 represent vegetation volumes transformed by the common logarithm, every dot corresponds to a single mouse. Mean±standard deviation range is given, *p<0.05, two-tailed unpaired Student's t-test. As can clearly be seen, mice treated with ancrod showed significantly reduced bacterial adhesion (P<0.05).