# Patent

## Title
Therapeutic use of agonists or antagonists of bradykinin receptor 1 or 2, for modulation collateral blood vessel growth

## Abstract

The present invention relates to bradykinin receptor modulators and pharmaceutical compositions thereof for use as a medicament for modulating collateral blood vessel growth of collateral arteries and/or other blood vessels of pre-existing arterial networks. The bradykinin receptor modulators of arteriogenesis are applicable in the treatment and/or prevention of disorders associated with defective blood flow or blood vessel malformation. A preferred aspect of the invention relates to bradykinin receptor agonists for use as a medicament for the prevention of cardiovascular ischemic disease in a patient at risk thereof. Further, the invention relates to a bradykinin receptor agonist for use in a method for treating a cardiovascular ischemic disease in a patient in need thereof, wherein said cardiovascular ischemic disease is a peripheral limb disease.

## Background
This is the U.S. national stage of International application PCT/EP2011/002521, filed May 16, 2011 designating the United States and claiming priority to EP 10075199.9, filed May 14, 2010 and EP10075353.2, filed Aug. 18, 2010.
The present invention relates to bradykinin receptor modulators for use as a medicament for modulating collateral blood vessel growth of collateral arteries and/or other blood vessels of pre-existing arterial networks. The bradykinin receptor modulators are applicable in the treatment and/or prevention of disorders associated with defective blood flow. The bradykinin receptor agonist of the present invention is intended for use as a medicament for the prophylactic treatment (prevention) of cardiovascular ischemic disease in a patient at risk thereof. The invention further relates to a bradykinin receptor agonist for use in a method for treating a cardiovascular ischemic disease in a patient in need thereof, wherein said cardiovascular ischemic disease is a peripheral limb disease. The present invention further relates to a bradykinin receptor antagonist for use in a method for treating or preventing the formation of vessel malformation in a patient. Finally, the present invention relates to a pharmaceutical composition comprising one or more bradykinin receptor agonists(s) and/or one or more bradykinin receptor antagonists(s) and a pharmaceutical acceptable carrier.
BACKGROUND OF THE INVENTION
Arteriogenesis (collateral blood vessel growth) is the most important endogenous mechanism of compensation for an arterial stenosis or occlusion. The driving force is a pressure gradient, for example through a stenosis or an AV-shunt, which initiates an increased blood flow through the collateral arteries in the areas of hypoperfusion. Arteriogenesis is induced by flow, namely by an increased shear stress which act on endothelial cells. Diseases related to ischemic arterial blockage represent the most common cause of death in the western world (Lloyd-Jones, Adams et al. 2009). The most common causes of stenosis or occlusive diseases of the main arteries of the brain, the heart or the periphery are progressive atherosclerotic modifications of the blood vessels. The possibilities for treating such diseases are at the present time very limited, whereby the therapies on offer are often unsatisfactory (Schiele, Niehues et al. 2000). Angioplasty (balloon dilatations) and bypass operations are the only methods of treatment available in clinics at the present time. An angioplasty can however only be applied in a case of an arterial obstructive or occlusive disease (for example ischemic heart diseases) and as a consequence leads to an increased risk of a restenosis. Bypass surgeries are in any case only applicable for certain arterial obstructive or occlusive diseases and are associated with the disadvantage that an invasive intervention is necessary. New therapeutic strategies are therefore particularly relevant when considering how to treat and to avoid vascular obstructive or occlusive diseases. A pharmaceutical stimulation of arteriogenesis (the concept of growing a biological bypass) represents enormous therapeutic potential and a very promising method in treating or preventing arterial obstructive or occlusive diseases (Love 2003). The present invention therefore relates to the modulation of arteriogenesis and flow-induced collateral blood vessel growth (FIG. 1).
The vascular system is organized in a highly complex manner. In order to ensure the essential functions of distributing oxygen and nutrients, the vascular system is constantly undergoing reconstruction. The reconstruction of blood vessels differs between arteriogenesis on the one hand and angiogenesis on the other hand.
In the context of the present invention it is important to distinguish between arteriogenesis and angiogenesis. Angiogenesis relates to the outgrowth of new capillaries from previously existing vessels. This relates specifically to the growth of new vessels (capillary sprouting). The present invention does not relate to modulation of neo-vascularisation (such as angiogenesis) but rather to modulation of arteriogenesis.
The diameter of an artery is actively increased in arteriogenesis. This mechanism does not deal with a passive dilatation, rather an active reconstruction of the vessel including the degradation of the extracellular matrix and a proliferation of artery cells (endothelial cells and smooth muscle cells), in order to achieve the required structure for a larger artery. The end result of arteriogenesis is that a pre-existent small artery grows into a large functional conductance artery. The initial stimulus for arteriogenesis is a change in the shear stress of the endothelial layer (not ischemia), which occurs through an increase in the blood flow through an artery.
It is therefore important to note that the present invention relates to arteriogenesis, the mechanism of which is fundamentally different from the mechanism of angiogenesis (Schaper 2009).
Previous studies have focused on the investigation of angiogenesis, whereby VEGF has been identified as a major regulator. Previous studies on rabbits, in which the femoral artery has been removed, show that under these hypoxic conditions VEGF is strongly up-regulated (Takeshita, Zheng et al. 1994). Additional application of the specific endothelial mitogen VEGF can stimulate artery growth. Such studies however do not distinguish between the outgrowth of capillaries (angiogenesis) and the mechanism related to the collateral growth of arteries (arteriogenesis). Angiogenesis occurs directly in the ischemic area and hypoxic conditions lead to the local production of VEGF. VEGF functions primarily as a mitogen for endothelial cells. However, arteriogenesis requires the reconstruction of endothelial cells, smooth muscles cells and a highly regulated end complex reconstruction of the extracellular matrix. In the case of an arterial obstructive or occlusive condition angiogenesis alone does not lead to a significant improvement in perfusion (Zadeh and Guha 2003, Storkebaum and Carmeliet 2004, Wang, Killic et al. 2005).
B1-receptor agonists are also known for modulating angiogenesis (Emaueli, Circulation, 2002), although only in relation to the outgrowth of capillaries (angiogenesis) and not in relation to modulation of the growth of collateral arteries (arteriogenesis).
In addition to flow-induced reconstruction, the migration of leucocytes plays a decisive role in arteriogenesis. Leucocytes, especially monocytes, produce inflammatory cytokines and growth factors, which are of significant importance for the remodeling of an artery. Such a remodeling leads to the enlargement of an arteriole into an artery with an outward growth of the artery lumen (positive outward remodeling). Arteriogenesis is therefore the most important endogenous compensatory mechanism for the maintenance of a stable blood flow in the case of a stenosis (mechanism of a biological bypass, FIG. 1). Arteriogenesis however generally does not occur in the ischemic region but rather in regions proximal to the occlusion and therefore effects the bypass circulation which provides blood supply to the distal hypoperfusion area (FIG. 1) (Pipp, Heil et al. 2003).
A schematic curve demonstrates the biological importance of arteriogenesis (FIG. 2a). Angiogenesis produces many new capillaries with a small diameter where as arteriogenesis enlarges the diameter of already existing arteries (FIG. 2b). The Hagen-Poiseuille rule demonstrates the relationship between blood flow and artery diameter and shows that the blood flow is enhanced by an exponential factor of 4 in relationship to the radius (FIG. 2b). According to this rule a minimal change in artery radius leads to significant improvements in perfusion. Comparable improvements in perfusion can not be achieved through the growth of many new small capillaries, as is the case in angiogenesis.
Previous studies have shown that collateral artery growth can be stimulated by an increase in blood flow (specifically an increase in the shear stress of the endothelial cells) or through the application of cytokines such as a GM-CSF (Buschmann, Busch et al. 2003). In a stroke or apoplexy model in the rat is has been shown that stimulation of arteriogenesis via the application of GM-CSF leads to an increase of the haemodynamic reserves of the brain (Schneeloch, Mies et al. 2004). Through the application of GM-CSF the ischemic region in an experimentally induced stroke could be significantly reduced. The stimulation of arteriogenesis is therefore an effective strategy for the treatment and/or prevention of ischemic obstructive or occlusive conditions, in addition to heart attacks and strokes.
Factors such as GM-CSF have been tested for their therapeutic stimulation of arteriogenesis (van Royen, Schirmer et al. 2005). The bradykinin signaling pathway has however not been examined in the prior art for its role in arteriogenesis, although its function as a vasodilator (antagonist of the Renin-angiotensin system) and its stimulatory effect in neo-vascularisation (Emanueli, Bonaria Salis et al. 2002) is known.
Bradykinin receptor 1 (B1R) and bradykinin receptor 2 (B2R) belong to the kallikrein-kinin system (KKS), which also contains the proteins kallikrein and kininogen. Kininogen is a substrate of the KKS and is enzymatically cleaved by kallikrein, through which a number of kinins are released.
Bradykinin, T-kinin and kallidin all belong to the kinin group. These proteins can be digested at their C terminus by a carboxypeptidase and can therefore be transformed to Des-Arg9-bradykinin and Des-Arg10-kallidin. Bradykinin, T-kinin and kallidin impart their signaling effect via the B2-receptor and Des-Arg9-bradykinin and Des-Arg10-kallidin via the B1-receptor (FIG. 3). All components of the kallikrein-kinin system can be produced in the artery wall. The bradykinin receptors in the artery walls can be expressed in either the endothelial cells, the smooth muscle cells or in leucocytes. A signaling effect via the bradykinin-receptors leads to vasodilatation, cell proliferation, inflammation and the production of various cytokines.
The effect of B1R and B2R agonists and antagonists on angiogenesis, which is the formation of new blood vessels, has been disclosed in the prior art. The relationship between angiogenesis and the bradykinin signaling pathway has also been investigated (Stone et al., 2009). It is known that treatment with agonistic substances of B1R and B2R leads to the promotion of angiogenesis and the formation of new vessels (WO 02/17958 A1). B1R and B2R antagonists have also been used for treating cerebral ischemic injury, arteriosclerosis, pain and inflammation (WO 98/06417, EP 0623350 A1, WO 2006/071775 A2).
Agonists of the B1R (such as increased B1R expression, peptides and various chemical derivatives of heterocyclic compounds) have been disclosed that may be useful in treatment of stroke patients, hypertension or heart failure (DE 10115668 A1, U.S. Pat. No. 6,015,818, WO 2004/069857).
Despite the extensive prior art surrounding the Bradykinin receptors and their role in angiogenesis, until the present time there has been no knowledge that connects Bradykinin receptor 1 (B1R) or bradykinin receptor 2 (B2R) with arteriogenesis.
Patients in need of stimulated or enhanced arteriogenesis, or patients who are at risk of suffering from disorders associated with an increased need for enhanced arteriogenesis, have long suffered through the lack of effective medicaments that target arteriogenesis. Similarly, patients with blood vessel malformation have been without effective treatment options, considering that until now no medicaments were known that could effectively reduce arteriogenesis in those patients where such treatment would be beneficial.
The pharmaceutical stimulation of arteriogenesis has previously been identified as exhibiting therapeutic potential and is a promising option for treating or preventing arterial obstructive or occlusive diseases or other conditions or disorders associated with defective blood flow (Love 2003). Despite this need, until now effective products and methods for modulating arteriogenesis were unknown.
SUMMARY OF THE INVENTION
In light of the prior art the technical problem underlying the present invention is to provide effective substances, methods and related medicaments for modulating collateral vessel growth (arteriogenesis), so that collateral artery growth can be either activated or inhibited depending on the patients needs.
This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.
The invention therefore relates to bradykinin receptor modulators for use as a medicament for modulating collateral blood vessel growth of collateral arteries and/or other blood vessels of pre-existing arterial networks.
The invention also relates to one or more bradykinin receptor modulators for use as a medicament for modulating collateral blood vessel growth of collateral arteries and/or other blood vessels of pre-existing arterial networks in the treatment and/or prevention of disorders associated with defective blood flow.
The bradykinin receptor modulator according to any one of the preceding claims, wherein said bradykinin receptor modulator is a bradykinin receptor 1 (B1R) and/or a bradykinin receptor 2 (B2R) modulator.
In a preferred embodiment the bradykinin receptor modulator of the present invention is a bradykinin receptor agonist.
In a preferred embodiment the bradykinin receptor agonist of the present invention is for use as a medicament for the prophylactic treatment (prevention) of cardiovascular ischemic disease in a patient at risk thereof.
In relation to the prevention of cardiovascular ischemic disease in a patient at risk thereof, the cardiovascular ischemic disease is

    
    
        (a) cerebral ischemia, in particular stroke, vascular dementia and/or infarct dementia;
        (b) myocardial ischemia, in particular a coronary heart disease and/or myocardial infarction; and/or
        (c) peripheral limb disease, in particular periphery arterial occlusive disease.
    
    


In relation to the prevention of cardiovascular ischemic disease in a patient at risk thereof, the patient

    
    
        (a) shows symptoms of being at risk of developing a cardiovascular ischemic disease;
        (b) shows any risk markers in ex vivo tests, in particular in blood samples;
        (c) has previously had a cerebral or myocardial ischemia; and/or
        (d) has a predisposition of developing a cardiovascular ischemic disease, in particular a genetic predisposition.
    
    


In another embodiment the bradykinin receptor agonist of the present invention for prevention of cardiovascular ischemic disease is defined in that said symptoms of (a) are selected from the group comprising of neurological malfunctions, transitory ischemic attack, congestive heart failure, angina pectoris, valvular heart disease, cardiomyopathy, pericardial disease, congenital heart disease, coarctation, atrial and/or ventricular septal defects and/or preferably combinations of two and more thereof.
The bradykinin receptor agonist of the present invention for prevention of cardiovascular ischemic disease can be further defined in that said patient at risk further exhibits one or more of the following conditions:
sclerosis, in particular atherosclerosis and/or transplantation-induced sclerosis; a cerebral occlusive disease, renal occlusive disease, a mesenterial artery insufficiency or an ophthalmic or retinal occlusion, post-operative or post-traumatic condition; thrombosis; embolism; restenosis, in particular primary restenosis, secondary restenosis and/or in-stent restenosis; trisomy 21; hypoglycemia; tachycardia; aneurysm; vasculitis; preeclampsia; placental hypoxia; sleep apnea; sexual dysfunction, in particular erectile dysfunction or female sexual dysfunction; post-operative hypoxia; Raynaud's disease; endothelial dysfunction; cancer; renal failure; varicose veins; edema, in particular venous edema and/or lymphedema; hypotension; decubitus; carbon monoxide poisoning; heavy metal poisoning; ulcers; sudden infant death syndrome; erythroblastosis; asthma; chronic obstructive pulmonary disease; sickle cell disease; induced g-forces which restrict the blood flow and force the blood to the extremities of the body; localized extreme cold, in particular frost bite; tourniquet application; an increased level of glutamate receptor stimulation or for any disease where atherosclerosic plaques in the vascular wall lead to an obstruction of the vessel diameter.
The bradykinin receptor agonist of the present invention for prevention of cardiovascular ischemic disease can be further defined in that said patient previously had been exposed to or is going to be exposed to

    
    
        (a) a pharmaceutical or medical treatment damaging one or more arteries;
        (b) a radiation treatment damaging one or more arteries; or
        (c) a surgical treatment damaging one or more arteries.
    
    


The bradykinin receptor agonist of the present invention can also be further defined in that said patient is tested for arteriogenesis.
The bradykinin receptor agonist of the present invention can be further defined in that said bradykinin receptor agonist is administered in a long-term application.
In a preferred embodiment the bradykinin receptor agonist is administered to the patient for at least one week, at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least two years, at least three years or enduringly.
In a preferred embodiment the bradykinin receptor agonist of the present invention is selected from the group comprising of

    
    
        Sar-[D-Phe8]-des-Arg9-bradykinin (R838);
        Lys-(Des-Arg9)-bradykinin;
        T-kinin;
        kallidin;
        des-Arg9-bradykinin;
        des-Arg10-kallidin;
        4-(2-pyridylmethoxy)-substituted quinoline (FR190997);
        3-(2-pyridylmethyl)-substituted benzimidazole (FR191413);
        Bradykinin;
        bradykinin/H1970;
        kallidin/Lys-bradykinin/H2180;
        SarLys[Hyp3,Cha5,D-Phe8]desArg9-bradykinin;
        SarLys[Hyp3,Igl5,D-Phe8]desArg9-bradykinin;
        SarLys[Hyp3,Cpg5,D-Phe8]desArg9-bradykinin;
        RMP-7 iobradamil/cereport;
        JMV1116 and analogues;
        Ile-Ser-bradykinin (B1643);
        [Phe8-(ψ-CH2NH)-Arg9]-bradykinin;
        [Hyp3,Tyr(Me)8]-bradykinin;
        [Hyp3]-bradykinin (B7775);
        a nucleic acid molecule coding for a bradykinin receptor or a bradykinin receptor agonist;
        an agent increasing the concentration or the half-life of a bradykinin receptor agonist;
        an agonistic bradykinin antibody; and
        a combination of two or more thereof.
    
    


In another embodiment the bradykinin receptor agonist of the present invention is selected from the group comprising of R838, T-kinin, kallidin, des-Arg9-bradykinin, des-Arg10-kallidin, FR190997, FR191413, a nucleic acid molecule coding for a bradykinin receptor and a bradykinin receptor agonist, together with an agent increasing the concentration or the half-life of the bradykinin receptor agonist, in particular wherein said bradykinin receptor agonist is R838, T-kinin and/or kallidin.
In another embodiment the bradykinin receptor agonist of the present invention is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a nucleic acid analogue.
In another embodiment the bradykinin receptor agonist of the present invention the deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a nucleic acid analogue is comprised in a vector, preferably, a viral vector or a plasmid.
In a preferred embodiment the agent increasing the concentration or the half-life of a bradykinin receptor agonist is an enzyme inhibitor, in particular a peptidase inhibitor.
In another embodiment the enzyme inhibitor is an angiotensin-converting enzyme (ACE) inhibitor, an amino peptidase P (APP) inhibitor and/or a carboxypeptidase N(CPN) inhibitor.
In another embodiment the ACE inhibitor is selected from the group consisting of ramipril, quinapril, cilazapril, spirapril-HCl 1H2O, catopril, enalaprilmaleat, lisinopril, perindopril-erbumin, trandolapril, fosinopril-sodium and/or a combination of two or more thereof.
In another embodiment of the present invention the bradykinin receptor agonist further induces migration of leukocyte cells and/or leucocyte precursor cells.
A further aspect of the present invention relates to a bradykinin receptor agonist used for treatment of cardiovascular ischemic disease in a patient in need thereof, wherein said cardiovascular ischemic disease is a peripheral limb disease, in particular periphery arterial occlusive disease.
A further aspect of the bradykinin receptor modulator of the present invention is a bradykinin receptor antagonist for treatment and/or prevention of blood vessel malformation in a patient.
In another embodiment the patient previously had been exposed to or is going to be exposed to

    
    
        (a) a pharmaceutical or medical treatment damaging one or more arteries;
        (b) a radiation treatment damaging one or more arteries; or
        (c) a surgical treatment damaging one or more arteries.
    
    


In another embodiment the bradykinin receptor antagonist is used in a method for treating a patient suffering from the Moja Moja Syndrome.
The bradykinin receptor antagonists of the present invention may also be administered in a long-term application.
An example of such long term application is that the bradykinin receptor antagonist is administrated to the patient for at least one week, at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least two years, at least three years or enduringly.
The bradykinin receptor antagonist of the present invention is in a preferred embodiment selected from the group consisting of

    
    
        icatibant (HOE140, H157);
        CP0597;
        D-Arg[Hyp3,Thi5,HypE(trans-propyl)7,Oic8]-bradykinin (NPC17731);
        (des-Arg9,Leu8)-bradykinin;
        AC-Lys-Arg-Pro-Pro-Gly-Phe-Ser-D□Nal-lie (R715);
        AC-Lys-Arg-Pro-Pro-Gly-(□Me)Phe-Ser-D□Nal-lie (R892);
        [Leu8,des-Arg9]-bradykinin;
        [Leu9,des-Arg10]-kallidin (CP0298);
        [des-Arg10]-HOE140;
        Lys-[Leu8,des-Arg9]-bradykinin;
        H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH (B9858);
        H-D-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-Arg-OH (B9430);
        FR167344;
        FR173657;
        WIN6433;
        bradyzide;
        a nucleic acid molecule inhibiting the formation of an intrinsic bradykinin receptor agonist or a bradykinin receptor;
        an inhibitory bradykinin antibody; and
        a combination of two or more thereof.
    
    


In another embodiment of the present invention the bradykinin receptor antagonist is selected from the group consisting of HOE140, CP0597, NPC17731, FR167344; FR173657; a nucleic acid molecule inhibiting the formation of an intrinsic bradykinin receptor agonist or a bradykinin receptor and/or a combination of two or more thereof.
In another embodiment the nucleic acid molecule is an siRNA, microRNA or a nucleic acid analogue.
In another embodiment the bradykinin receptor antagonist is comprised in a vector, preferably, a viral vector or a plasmid.
The invention also relates to bradykinin receptor antagonists that are intrinsic bradykinin receptor antagonists or functional analogues thereof.
A further aspect of the invention relates to a pharmaceutical composition comprising one or more bradykinin receptor agonists as described herein and a pharmaceutically acceptable carrier for treatment and/or prevention of cardiovascular ischemic disease in a patient exhibiting the disease or in a patient at risk thereof.
A further aspect of the invention relates to a pharmaceutical composition comprising one or more bradykinin receptor antagonists as described herein and a pharmaceutical acceptable carrier for treatment and/or prevention of blood vessel malformation in a patient exhibiting the disease or in a patient at risk thereof.
In another embodiment the pharmaceutical composition further comprises of one or more other compounds selected from the group consisting of

    
    
        (a) one or more colony stimulating factor(s) (CSF(s));
        (b) one or more eicosanoid(s), in particular one or more prostaglandin(s).
    
    


The invention further relates to a method for modulating collateral vessel growth (arteriogenesis) by the inhibition or stimulation of the bradykinin receptor 1 (B1R) and/or the bradykinin receptor 2 (B2R). In one embodiment the method of the present invention is characterized in that the modulation of the collateral vessel growth occurs via the modulation of collateral arteries from existing arterial networks, preferably by modulation of arterial radius.
In one embodiment the method of the present invention is characterized in that the modulation, such as an induction or a reduction, of the collateral vessel growth occurs through the stimulation (using an agonist) or the inhibition (using an antagonist) of the bradykinin receptor 1 and/or bradykinin receptor 2, preferably in the vessel wall by modulation of the bradykinin receptors of the endothelial cells, the smooth muscle cells and/or leucocytes that have migrated into the vessel wall. In one embodiment the method of the present invention is characterized in that the induction of the collateral vessel growth (arteriogenesis) occurs by stimulation of the bradykinin receptor.
The invention also relates to a method for enhancing collateral artery growth with kinins or other bradykinin receptor agonists. A further aspect of the present invention is characterized in that the inhibition of collateral vessel growth (arteriogenesis) occurs by inhibition of the bradykinin receptor.
The invention further relates to a method as described above, characterized in that the modulation of the collateral vessel growth occurs by means of nucleic acid molecules, preferably through nucleic acids (such as siRNA or microRNA) with complementarity to mRNA of components of the kallikrein-kinin system (for example kininogen, BR1 and/or BR2).
The invention further relates to a method as described herein, characterized in that the bradykinin receptor pathway stimulation is enhanced by bradykinin augmentation as a consequence of ACE inhibitor activity.
A further aspect of the present invention relates to a bradykinin receptor 1 (B1R) and/or bradykinin receptor 2 (B2R) inhibiting substance for use as a medicament to inhibit collateral vessel growth. A further embodiment of the present invention relates to a bradykinin receptor 1 (B1R) and/or bradykinin receptor 2 (B2R) stimulating substance for use as a medicament to stimulate collateral vessel growth.
The bradykinin receptor 1 (B1R) and/or bradykinin receptor 2 (B2R) stimulating substance of the present invention can be used as a medicament for the prevention and/or treatment of circulatory disorders, arterial conditions and/or ischemic obstructive or occlusive conditions or stroke, preferably coronary heart disease or peripheral obstructive disease, preferably via stimulation of the bradykinin signaling pathways. A further aspect of the invention relates to a pharmaceutical composition comprising at least one bradykinin receptor 1 and/or bradykinin receptor 2 modulating substance with a pharmaceutically acceptable carrier.
The invention also relates to the use of a bradykinin receptor 1 (B1R) and/or bradykinin receptor 2 (B2R) stimulating substance for the manufacture of a medicament to prevent and/or treat circulatory disorders, arterial diseases and/or ischemic obstructive or occlusive conditions or strokes, preferably coronary heart diseases or peripheral arterial obstructive diseases, preferably by a stimulation of the bradykinin signaling pathways. A further aspect of the invention relates to a method for the therapeutic or prophylactic treatment of circulatory disorders, arterial diseases and/or ischemic obstructive or occlusive conditions or strokes, preferably coronary heart diseases or peripheral arterial obstructive diseases, characterized in that a therapeutically effective amount of a bradykinin receptor 1 (B1R) and/or bradykinin receptor 2 (B2R) stimulating substance is administered to a mammalian patient.
DETAILED DESCRIPTION OF THE INVENTION
It is important to note that a stimulation of angiogenesis (neovascularisation) or a vasodilatation does not lead to a preventative treatment for patients with progressive constriction of blood vessels or arteries (for example as it is the case in atherosclerosis). Furthermore, a stimulation of angiogenesis can not sufficiently compensate for an endogenous stenosis in a major artery (see above prior art comparing angiogenesis and arteriogenesis). Similarly dilatation to its maximum extent cannot compensate for an endogenous stenosis in a major artery, as the hemodynamic reserves become very quickly exhausted when collateral vessels or arteries do not sufficiently grow in their diameter through arteriogenesis.
However, the activation or stimulation of arteriogenesis enables the preventative treatment of cardiovascular disease. This represents one surprising and beneficial aspect of the invention. Without being able to modulate the arteriogenesis, there has been until now no way of preventing blood flow restriction in patients at risk. The modulation of arteriogenesis, in contrast to angiogenesis, enables the treatment (or prevention) of restricted blood flow in new groups of patients, in comparison to what was previously possible in the prior art. The targeted modulation of arteriogenesis allows patients to be treated who were previously without appropriate or sufficient preventative or therapeutic options. The modulation of angiogenesis is simply not sufficient in providing effective relief or prevention of restricted blood flow. Although angiogenesis can be initiated quickly, the physiological effect of modulated, for example enhanced, angiogenesis is simply insufficient to provide beneficial therapeutic or preventative effects in patients (see FIG. 2b). For example diabetes patients with problems related to blood flow in their feet and other extremities could not be treated using the substances of the prior art that targeted enhanced angiogenesis. However, by targeting arteriogenesis a more effective therapeutic and preventative change is observed physiologically, allowing sufficient increases in blood flow via increased vessel diameter that prove significantly valuable to the patient.
The enablement of treating or prophylactically treating new groups of patients represents a significant and unexpected advance over the prior art. The use of bradykinin receptor modulators, particularly agonists, relates to a new use of known substances that provides new perspectives and therapeutic options for doctors when assessing patients with blood flow disorders. The substances of the present invention can also be administered in a preventative manner, something which was previously unknown and impracticable in the context of angiogenesis.
Basically cardiovascular ischemic diseases which occur due to the occlusion of a conductance artery can be prevented or treated by stimulation of arteriogenesis, but not due to stimulation of angiogenesis.
In most cases obstruction of conductance vessels occurs slowly, which results in the recruitment of the collateral artery network outside the ischemic region in oxygen-rich tissue that bypass the site of occlusion. Subsequent collateral arteries grow due to the driving force of blood flow induced shear stress, which leads to the expression of the components of the kinin-bradykinin receptor signaling pathway locally in the collateral network. Stimulation of arteriogenesis increases vessel diameter of the bypassing arteries, which produces a new functional conductance vessel to supply the endangered territory with blood. Increasing the number of capillaries within the ischemic region cannot significantly increase blood flow when its limiting structure lies upstream (FIG. 2b).
Furthermore, according to the law of Hagen-Poiseuille, perfusion v is mainly determined by the vessel radius r to the power of 4. Thus, only minimal changes in vessel diameter of a pre-existent arteriol lead to a big difference of the perfusion level (arteriogenesis). All other factors like viscosity η, pressure differences ΔP, and distance differences (vessel length) Δl as well as flow volume V are still proportional according to the equation. In conclusion, newly formed small vessels cannot compensate for the loss of a main supplying arterial vessel (angiogenesis). This basic physiological principle has been shown in a number of clinical trials, where therapeutically induced angiogenesis has led to a significant increase in capillary density, but without significant beneficial effects on tissue perfusion (Zadeh and Guha 2003; Storkebaum and Carmeliet 2004; Wang, Kilic et al. 2005). In contrast, adaptive proliferation of collateral vessels (arteriogenesis) represents the most important physiological protection mechanism against arterial stenosis (Carmeliet 2000).
It was until now unknown that bradykinin receptor modulators, especially agonists of the B1R, were capable of modulating arteriogenesis in a manner suitable for prophylactic or therapeutic application. Due to the fundamental differences between arteriogenesis and angiogenesis, new groups of patients with different sets of existing illnesses or risk factors may be treated, either therapeutically or prophylactically, with the bradykinin receptor modulators of the present invention.
As used throughout the invention, the term “bradykinin receptor” may be understood as any receptor that is selectively triggered by bradykinin. Presently, there are two bradykinin receptors known in the art, i.e., bradykinin receptor 1 (B1 receptor) and/or bradykinin receptor 2 (B2 receptor). The bradykinin receptor 2 is consecutively and permanently expressed, whereas the B1 receptor is only expressed when the cells are subjected any stress, such as, e.g., inflammation, a physical injury or trauma. The bradykinin receptor 1 may also play a role in the context of chronic pain.
It will be understood by a person skilled in the art that the terms “bradykinin receptor 1” and “bradykinin receptor 2” may refer to the naturally occurring receptors but may also refer to functional mutated forms thereof.
The term “bradykinin receptor 1” refers to a protein having the sequence of SEQ ID NO:1 or a sequence which has a sequence homology of at least 70%, preferably of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, even more preferably of at least 95%, and most preferably of 100% of the wildtype protein of SEQ ID NO:1.
The term “bradykinin receptor 2” refers to a protein having the sequence of SEQ ID NO:2 or a sequence which has a sequence homology of at least 70%, preferably of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, even more preferably of at least 95%, and most preferably of 100% of the wildtype protein of SEQ ID NO:2.
The term “functional” as used in this context refers to a protein that has at least 25%, preferably at least 50%, more preferable at least 75%, even more preferable at least 80%, even more preferable at least 85%, even more preferable at least 90%, even more preferable at least 95% and most preferably 100% or even more of the activity compared to the wildtype protein.
It will further be understood that that the receptors may be subjected to one or more posttranslational modifications. Therefore, the receptors may be, e.g., lipidated, phosphorylated, sulfated, cyclized, oxidated, reduced, decarboxylated, acetylated, acylated, amidated, deamidated, biotinylated or bound to one or more small molecule(s) and/or terpene(s). Further, the bradykinin receptor agonist may or may not form one or more intramolecular disulfide bond(s) and/or one or more intermolecular disulfide bond(s).
Further, the protein may be optionally truncated or elongated by one, two, three, four, five, up to ten, up to twenty, up to thirty, up to fifty or even more than fifty amino acid residues.
As used in the context of the present invention the term “bradykinin receptor modulator” may be understood in the broadest sense as any compound that can directly or indirectly modulate, for example stimulate or inhibit, the function of the bradykinin receptor and/or the signal transduction cascade related to a bradykinin receptor. Modulators therefore encompass both agonists and antagonists of the bradykinin receptor(s).
As used in the context of the present invention, the term “bradykinin receptor agonist” may be understood in the broadest sense as any compound that can directly or indirectly stimulate the signal transduction cascade related to a bradykinin receptor. The bradykinin receptor agonist may be, e.g., a peptide, a kinin precursor, a non-peptide agonist, a nucleic acid molecule encoding a kinin, a kinin precursor, or another bradykinin receptor agonist, or a compound inhibiting the degradation of any other bradykinin receptor agonist. Bradykinin receptor molecules themselves also fall under this definition, considering that over-expression or application of the bradykinin receptors themselves may lead to enhanced bradykinin receptor activity due to increased presence, e.g. cellular concentration of the receptors, so that the effect on Bradykinin signaling is enhanced by application or admission of additional molecules of the receptors themselves.
Most preferably, the bradykinin receptor agonist is a peptide or a peptidomimetic, in particular a nonapeptide or a decapeptide. Exemplarily, the peptidic bradykinin receptor agonist is bradykinin or a functional derivative thereof, a kinin precursor or a functional derivative thereof, kallidin or a functional derivative thereof.
As used in the context of the present invention, the term “bradykinin” refers to a peptide of the sequence H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH (SEQ ID NO:3). The term “kallidin” refers to a peptide of the sequence H-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH (SEQ ID NO:4). Bradykinin and kallidin are also designated as kinins.
In the context of the peptides bradykinin and kallidin, a functional derivative thereof may exemplarily be any protein or peptide that comprises the sequence of bradykinin. Therefore, one, two, three, four, five, six, seven, eight, nine, ten, up to 15, up to 20, up to 30, up to 40, up to 50, up to 100 or even more amino acids may be added to the amino or the carboxy terminus of said peptide.
Exemplarily, a functional peptide derivative may be Met-Lys-bradykinin bearing the sequence H-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH (SEQ ID NO:5) or
Ile-Ser-bradykinin bearing the sequence H-Ile-Ser-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH (SEQ ID NO:6). Ile-Ser-bradykinin is also designated as T-kinin.
Alternatively or additionally, the peptides bradykinin and kallidin, respectively, may be truncated by one or two amino acids at their amino or their carboxy terminus. Exemplarily, truncated bradykinin may be des-Arg9-bradykinin bearing the amino acid sequence H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-OH (SEQ ID NO:7), des-Arg10-kallidin bearing the amino acid sequence H-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-OH (SEQ ID NO:8), or Sar-[D-phe8]-des-Arg9-bradykinin (wherein Sar refers to a N-methylglycyl residue (aka sarcosine) and D-Phe refers to the D-amino acid phenylalanine).
Further, alternatively or additionally, one, two or even three amino acid residues may be replaced by other amino acid residues. These amino acid residues may be natural amino acid residues or non-natural amino acids. Moreover, alternatively or additionally, one, two or even three amino acid residues may be inserted into the sequence of said peptides, wherein these amino acid residues may be natural amino acid residues or non-natural amino acids.
Optionally, the bradykinin receptor agonist, in particular when it is a peptide, may be lipidated, phosphorylated, sulfated, cyclized, oxidated, reduced, decarboxylated, acetylated, acylated, amidated, deamidated, biotinylated or bound to one or more other small molecule(s) and/or terpene(s). Further, the bradykinin receptor agonist may or may not form one or more intramolecular disulfide bond(s) and/or one or more intermolecular disulfide bond(s). The bradykinin receptor agonist, in particular when it is a peptide, may also be conjugated or non-covalently associated to molecular structures that may facilitate uptake such as, e.g., positively charged polymers (e.g., polyethylene imine (PEI)), neutral lipids, positively charged lipids, viral vectors, liposomes, micro- and/or nanobeads, cell-penetrating peptides (=protein-transduction domains, e.g., polyarginines (e.g., R8, R9, R10), HIV Tat peptide, antennapedia/penetratin peptide, a Lactoferrin-derived peptide), antimicrobial peptides and/or a Chariot peptide.
Preferably, the peptide bears a free amino terminus and a free carboxy terminus or may have a blocked amino terminus and/or carboxy terminus. Exemplarily, the amino terminus may be acylated or acetylated and/or the carboxy terminus may be amidated.
Preferably, the peptide consists of natural L-amino acids. However, the peptide may also comprise one or more non-natural amino acid(s) such as, e.g., D-amino acid(s), beta amino acid(s), methylated amino acid(s) (e.g., N-methylated amino acid(s)). The peptide may even only consist of non-natural amino acids. The peptide may also be a retro-inverso peptide, thus, a peptide mainly comprising D-amino acids and a reverse amino acid sequence compared to the corresponding peptide mainly comprising L-amino acids. Many bradykinin receptor agonists, such as, e.g., the kinins bradykinin and kallidin, are inactivated by proteolytic cleavage, in particular proteolytic cleavage of the one, two or three C-terminal amino acid(s). Therefore, preferably, one or more amino acid(s) near the carboxy terminus, such as, e.g., the phenylalanine at position 8 (Phe8), may be replaced by one or more non-natural amino acid residue(s). Exemplarily, the peptide is the D-amino acid-containing peptide Sar-[D-phe8]-des-Arg9-bradykinin.
The bradykinin receptor agonist may also be a kinin precursor. As used herein, the term “precursor” may be understood in the broadest sense as any molecular structure that can be converted into a functional kinin peptide by metabolism. Typically, a kinin precursor is a longer polypeptide chain that is cleaved into the respective functional kinin peptide by proteolytic cleavage. Herein, the terms “proteolytic cleavage” and “peptidolytic cleavage” may be understood interchangeably. A bradykinin precursor may be, e.g., a kininogen cleavable by the protease kallikrein or kallikrein-like enzymes. Exemplarily, a kinin precursor is KNG1 kininogen 1 (also designated as BDK; KNG; KNG1).
The bradykinin receptor agonist may also be a non-peptide agonist such as, e.g., 4-(2-pyridylmethoxy)-substituted quinoline (FR190997); 3-(2-pyridylmethyl)-substituted benzimidazole (FR191413).
The bradykinin receptor agonist may also be a nucleic acid molecule encoding a kinin or a kinin precursor, a bradykinin receptor or another bradykinin receptor agonist.
Exemplarily, a nucleic acid may be a nucleic acid molecule comprising at least 30 consecutive nucleotides, preferably at least 50 consecutive nucleotides, more preferably at least 100 consecutive nucleotides, even more preferably at least 200 consecutive nucleotides, and most preferably all nucleotides of a gene encoding a kinin precursor (kininogen), such as, e.g., KNG1 kininogen 1 (SEQ ID NO:9). SEQ ID NO:9 relates to KNG1 kininogen 1 [Homo sapiens] according to NCBI:Pubmed Gene ID: 3827, Ensembl:ENSG00000113889, and is also known as BDK; KNG; KNG1, located on Chromosome 3: 186,435,065-186,461,743, forward strand, with protein lengths (transcript variants) of: 427 aa, 644 aa, 391 aa, 415 aa.
It will further be understood by a person skilled in the art that the KNG1 kininogen 1 gene may also be a nucleic acid sequence that has a sequence homology of at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of SEQ ID NO:9 and encodes a functional KNG1 kininogen 1 protein or functional mutant thereof. Naturally, said gene is expressible into several protein variants (transcript variants), such as protein variants of a protein length of approximately 427 amino acids, of approximately 644 amino acids, of approximately 391 amino acids, or of approximately 415 amino acids in length.
In an alternative example, a nucleic acid may be a nucleic acid molecule comprising at least 200 consecutive nucleotides, preferably at least 300 consecutive nucleotides, more preferably at least 400 consecutive nucleotides, even more preferably at least 500 consecutive nucleotides, and most preferably all nucleotides of a gene encoding a bradykinin receptor, such as, e.g., BDKRB1 bradykinin receptor B1 (SEQ ID NO:10) or BDKRB2 bradykinin receptor B2 (SEQ ID NO:11). SEQ ID NO:10 relates to the BDKRB1 bradykinin receptor B1 [Homo sapiens] according to the NCBI:Pubmed Gene ID: 623, Ensembl:ENSG00000100739, and is also known as B1R; BKR1; B1BKR; BKB1R; BRADYB1; BDKRB1, located on Chromosome 14: 96,722,559-96,731,095, forward strand, with a protein length of 353 aa. SEQ ID NO:11 relates to the BDKRB2 bradykinin receptor B2 [Homo sapiens] according to NCBI:Pubmed Gene ID: 624, Ensembl:ENSG00000168398, and is also known as B2R; BK2; BK-2; BKR2; BRB2; DKFZp6860088; BDKRB2, located on Chromosome 14: 96,671,016-96,710,666, forward strand, with possible protein lengths of 391 aa, 364 aa.
It will further be understood by a person skilled in the art that a bradykinin receptor gene may also be a nucleic acid sequence that has a sequence homology of at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of SEQ ID NO:10 or SEQ ID NO:11 and encodes a functional KNG1 kininogen 1 protein or functional mutant thereof. The full length protein of BDKRB1 bradykinin receptor B1 is typically approximately 353 amino acids in length. The full length protein of BDKRB2 bradykinin receptor B2 varies typically between approximately 391 and approximately 364 amino acids in length.
In an alternative example, a nucleic acid may be a nucleic acid molecule comprising at least 100 consecutive nucleotides, preferably at least 200 consecutive nucleotides, more preferably at least 300 consecutive nucleotides, even more preferably at least 400 consecutive nucleotides, and most preferably all nucleotides of a gene encoding a protease processing a kinin precursor into a kinin such as, e.g., bradykinin or kallidin. Said protease may be, e.g., a kallikrein, such as, e.g., KLK1 kallikrein 1 (SEQ ID NO:12). SEQ ID NO:12 relates to KLK1 kallikrein 1 [Homo sapiens] according to NCBI:Pubmed Gene ID: 3816, Ensembl:ENSG00000167748, also known as hK1; KLKR; Klk6; KLK1, located at Chromosome 19: 51,322,404-51,327,043, reverse strand, with a protein length of 262 aa or 160 aa.
It will further be understood by a person skilled in the art that a kallikrein gene may also be a nucleic acid sequence that has a sequence homology of at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of SEQ ID NO:12 and encodes a functional KNG1 kininogen 1 protein or functional mutant thereof. The full length protein variants of KLK1 kallikrein are typically approximately 262 amino acids or 160 amino acids in length.
A nucleotide as used herein may be any nucleotide known in the art. The nucleotide may be linear or circular. Preferably, the nucleotide is double-stranded deoxyribonucleic acid (DNA). The double-stranded DNA may be linear or circular. The double-stranded DNA may be a plasmid. Alternatively, the nucleotide may also be single-stranded DNA, ribonucleic acid (RNA) or a nucleotide composed of nucleic acid analogues, such as, e.g., a peptide nucleic acid (PNA) nucleotide, a Morpholino nucleotide, glycol nucleic acid (GNA) nucleotide, a threose nucleic acid (TNA) nucleotide or a methylated DNA nucleotide. It will be understood by a person skilled in the art that different types of nucleotides may also be combined with another.
The nucleotide may be inserted in the body of the patient by any means known in the art such as, e.g., electroporation, transfecting agents (e.g., Chariot), positively charged polymers (e.g., polyethylene imine (PEI)), neutral lipids, positively charged lipids, viral vectors, liposomes, micro- and/or nanobeads, cell-penetrating peptides (=protein-transduction domains, e.g., polyarginines (e.g., R8, R9, R10), HIV Tat peptide, antennapedia/penetratin peptide, a Lactoferrin-derived peptide) and/or antimicrobial peptides.
Optionally, the bradykinin receptor agonist of the present invention may be labeled radioactively (e.g., by 3H, 32P, 35S, 14C, 99mTc or lanthonoids (e.g., 64Gd)) or may be labeled with a spin label, such as one or more heavy isotopes, e.g., 13C, detectable by nuclear magnetic resonance (NMR) and magnetic resonance tomography (MRT).
Optionally, the bradykinin receptor agonist of the present invention may be labeled by one or more small molecule dye(s) (e.g., Cy dye(s) (e.g., Cy3, Cy5, Cy5.5, Cy7), Alexa dye/s (e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750), VisEn dye(s) (e.g. VivoTag680, VivoTag750), S dye(s) (e.g., S0387), DyLight fluorophore(s) (e.g., DyLight 750, DyLight 800), IRDye(s) (e.g., IRDye 680, IRDye 800), fluorescein dye(s) (e.g., fluorescein, carboxyfluorescein, fluorescein isothiocyanate (FITC)), rhodamine dye(s) (e.g., rhodamine, tetramethylrhodamine (TAMRA)) or HOECHST dye(s)) or one or more quantum dot(s).
The bradykinin receptor agonist of the present invention may be provided by any means known in the art. As used throughout the present invention, the term “providing a bradykinin receptor agonist” may be understood in the broadest sense as obtaining a bradykinin receptor agonist from any source. The bradykinin receptor agonist may be provided by chemical synthesis, obtained from a biotechnological method and/or extracted from a natural source. Preferably, the bradykinin receptor agonist is provided by chemical synthesis. In the context of peptides, the term “chemical synthesis” may refer to SPPS, liquid phase peptide synthesis or a combination of both. Here, the synthesis typically bases on the stepwise coupling of amino acids bearing protected side chains (orthogonal protecting groups). Typically, during synthesis, the peptide strand grows from the C-terminus to the N-terminus. However, there are alternative methods wherein the peptide strand grows from the N-terminus to the C-terminus. Nowadays, the most common methods base on at least two different types of protecting groups that are cleavable under at least two different conditions, such as, e.g., the fluorenyl-9-methoxycarbonyl/tert-butanyl- (Fmoc/tBu) protecting group scheme (Sheppard Tactics) or the tert-butoxycarbonyl/benzyl- (Boc/Bzl) protecting group scheme (Merrifield Tactics). Alternatively or additionally, the peptide may be also provided by conjugating two or more peptide strand(s) with another by any conjugation method known in the art such as, e.g., Native Chemical Ligation (NCL), Click Chemistry, Maleimide-Thiol Conjugation, enzymatic conjugation, biochemical protein ligation and/or soluble handling conjugation.
Alternatively, the bradykinin receptor agonist may be obtained from a biotechnological method. Today, numerous biotechnological methods are well-known in the art such as, e.g., overexpression and/or heterologous expression, in particular heterologous expression based on cloning of one or more gene(s) in bacteria, insect cells, mammalian cells, plant cells or yeast cells. The bradykinin receptor agonist may further be extracted by any means known in the art. Alternatively, the bradykinin receptor agonist may be extracted from a natural source by any means known in the art. Additionally, the bradykinin receptor agonist may be purified by any means known in the art, such as, e.g., one or more chromatographic method(s), one or more filtration method(s), one or more electrophoretic method(s), one or more precipitation-based method(s), one or more dialysis method(s) or a combination of two or more thereof. The natural source may be any biological material such as, e.g., bacterial material, plant material, animal material or fungal material, such as e.g. tissue, liquids or secretion(s). Also bee toxin comprises bradykinin. The bradykinin receptor agonist obtained from a natural source may also be digested or partly digested by one or more enzymes, such as protease(s).
It will be understood by a person skilled in the art, that the aforementioned methods for providing a bradykinin receptor agonist may also be combined with another. In particular, a bradykinin receptor agonist obtained from a biotechnological method or a natural source may further be purified and/or modified by chemical means known in the art.
Any bradykinin receptor agonist may be used as an unbound molecule or may be non-covalently formulated with and/or conjugated to any other molecules such as, e.g., high-molecular weight compounds. Optionally, the bradykinin receptor agonist of the present invention may be stabilized against degradation by conjugation to one or more high-molecular weight compound(s).
Exemplarily, the bradykinin receptor agonist may be conjugated to one or more hydrophilic polymer(s). The term “hydrophilic polymer” as used herein may be understood in the broadest sense as any polymeric molecule that has a hydrophilic surface. A polymeric molecule comprises more than one monomer. The hydrophilic polymer may have a molecular mass of more than 500 Da. The hydrophilic polymer may be a soluble polymer, a gel-like polymeric matrix, a bead, a surface, a bead coating or the coating of a surface. Preferably, the hydrophilic polymer of the present invention is a soluble polymer. Exemplarily, the hydrophilic polymer of the present invention may be polyethylene glycol (PEG) or derivatives thereof, polyethylene imine (PEI) or derivatives thereof, polyacrylic acid or derivatives thereof, in particular hydroxypropyl methacrylate (HPMA), polysaccharide(s) or derivatives thereof, preferably amino polysaccharide(s), aminoalkyl polysaccharide(s), hydroxyalkyl polysaccharide(s) and/or alkyl polysaccharide(s), lipopolysaccharide(s), hydrophilic polypeptide(s) and/or conjugate(s) or blockpolymer(s) comprising two or more of the aforementioned. Alternatively, the hydrophilic polymer may be a bead having a hydrophilic surface or a bead having a coating comprising a hydrophilic polymer, in particular wherein the bead is a micro- or a nanobead. The hydrophilic polymer of the present invention may be conjugated with the bradykinin receptor agonist of the present invention via any functional group of the bradykinin receptor agonist. In particular when the bradykinin receptor agonist is a peptide, the hydrophilic polymer may be conjugated with the N-terminus of the peptide, an amino acid residue side chain, or the C-terminus of the peptide.
Conjugation to any other molecular structure may be facilitated by any means known in the art, such as the activation of one or more active groups(s). As used throughout the invention, the terms “active group” may be understood in broadest sense as a molecular moiety facilitating the formation of a bond, in particular an amide bond. Exemplarily, an active group may be an active ester (e.g., succinimidyl ester, N-hydroxysuccinimidyl (NHS) ester, carboxylic acid halogenide (acyl halide), pentafluorophenylester, thiophenyl ester, cyanomethyl ester, a nitrophenyl ester (e.g., 2-nitrophenyl ester, 4-nitrophenyl ester, 2,4-dinitrophenyl ester), a chlorophenyl ester (e.g., 2,4,5,-trichlorophenyl ester, pentachlorophenyl ester), pentafluorophenyl ester, N-hydroxypiperidinyl ester, 8-quinolyl ester, 1-hydroxybenzotriazolyl (HOBt) ester, 7-aza-1-hydroxybenzotriazolyl, N-norbornene-2,3-dicarboximidooxy ester, ethyl-1-hydroxy-1H-1,2,3-triazole-4-carboxylate), formation of an acid anhydride, and/or one or more coupling agent(s) (e.g., a carbodiimide (e.g., dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC), a phosphonium reagent (e.g., benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP)) or an uranium-type reagent (e.g., HBTU, TBTU, HATU, HAPyU, HAMDU, HAPipU, HAMTU). Alternatively, the bradykinin receptor agonist may be conjugated to the hydrophilic polymer by any other means known in the art such as, e.g., by Native Chemical Ligation (NCL), Click Chemistry, Maleimide-Thiol Conjugation, and/or employing two or more different types of orthogonal protecting groups (e.g., convergent SPPS).
The bradykinin receptor agonist of the present invention may also be a compound inhibiting the degradation of any other bradykinin receptor agonist. As mentioned above, many bradykinin receptor agonists, such as, e.g., the kinins bradykinin and kallidin, are inactivated by proteolytic cleavage, in particular proteolytic cleavage of the carboxy terminal amino acids. Cleavage may be mediated by the angiotensin-converting enzyme (ACE) or related enzymes. Therefore, any ACE inhibitor known in the art may also be used as a bradykinin receptor agonist in the context of the present invention. Further, inhibitors of the amino peptidase P (APP) and the carboxypeptidase N(CPN) may be used as a bradykinin receptor agonist in the context of the present invention.
It will be understood by a person skilled in that art that optionally two or more bradykinin receptor agonist(s) may be combined with another. Particularly, one or more compound(s) inhibiting the degradation of any other bradykinin receptor agonist may be combined with one or more bradykinin receptor agonist(s) of other type(s).
The term “preventing” or “prevention” or “prophylactic treatment” as used in the context of the present invention may be understood in the broadest sense as any means for decreasing the risk of developing a particular condition, such as a cardiovascular ischemic disease or condition or diminishing the upcoming implication of a cardiovascular ischemic disease or condition.
As used herein, the term “disorder associated with defective blood flow” may be understood in the broadest sense as any condition where the blood flow of a patient is in any way aberrant from normal or healthy blood flow. Such conditions include conditions where blood flow is restricted, such as cardiovascular ischemic disease, in addition to conditions where blood vessels are malformed, which may lead to unwanted increases in or rates of blood flow.
As used herein, the term “cardiovascular ischemic disease” may be understood in the broadest sense as any disease or condition associated with a restriction of blood flow, wherein said restriction of blood flow is generally due to factors in the blood vessels.
The term “patient at risk” may refer to any subject who is at risk of developing a disease condition in the sense of the present invention. Preferably, the risk of developing a disease condition for this subject is at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, or more than 500% higher compared to the average risk throughout the population. The patient in the sense of the present invention may be any subject who can develop a cardiovascular ischemic disease or condition in the sense of the present invention. Preferably, the patient is a mammal, in particular a human.
The bradykinin receptor agonist of the present invention may be administered by any means known in the art. Exemplarily, administration may be oral administration, injection (e.g., intravenous, intraperitoneal, subcutaneous or intramuscular injection), intranasal administration, topical administration or intradermal administration. In case of peptides or peptidomimetics, administration is preferably enabled via an injection in order to circumvent the first pass effect. The dose of the bradykinin receptor agonist administered evidently depends on numerous factors well-known in the art such as, e.g., the chemical nature and pharmaceutical formulation of the bradykinin receptor agonist, and of body weight, body surface, age and sex of the patient, as well as the time and route of administration. For an adult, the dose may exemplarily be between 0.5 μg and 1 g per day, preferably between 0.5 μg and 100 mg per day, more preferably between 1 μg and 100 mg per day, even more preferably between 1 μg and 100 mg per day, even more preferably between 1 μg and 10 mg per day, even more preferably between 5 μg and 10 mg per day. In a continuous infusion, the dose may exemplarily be between 0.5 μg and 100 mg, preferably between 1 μg and 10 mg per kilogram per minute. Should the bradykinin receptor agonist be a nucleic acid approximately between 106 copies and 1012 copies may be administered to the patient, depending on the used vector.
The present invention also relates to a method for the prevention of a cardiovascular ischemic disease in a patient at risk thereof by administering a pharmaceutically active amount of a bradykinin receptor agonist to said patient.
In a preferred embodiment, the bradykinin receptor agonist of the present invention induces arteriogenesis.
As described above, the term “arteriogenesis” as used in the context of the present invention may be understood in the broadest sense as the growth of arteries from preexisting arteriolar connections. In particular, arteriogenesis is the blood flow stimulated in situ growth of arteries by proliferation of endothelial and smooth muscle cells from preexisting arteriolar connections supplying blood to ischemic tissue. Therefore, arteriogenesis is induced by flow, namely by an increased shear stress which act on endothelial cells. This activates the bradykinin receptor signal pathway, through which arteriogenesis is enhanced. Collateral blood vessel growth can be modified (inhibition or stimulation) through modulation of the bradykinin receptor signal pathway. This enables methods involving the modification of this mechanism for the prevention of circulatory disorders, vascular diseases and/or ischemic arterial blockages or occlusions (KHK, stroke, PAVK). Modulation of arteriogenesis via the bradykinin signaling pathway can also be applied to other illnesses or disorders, for example AV-shunt malformation and the Moja Moja disease. The vessels largely growth outside from the vascular occlusion affected tissue but are much more important for the delivery of nutrients to ischemic territory, than capillaries sprouting in the diseased tissue by angiogenic processes. Therefore, arteriogenesis may function like a physiological bypass.
Arteriogenesis is mechanically linked to an elevated pressure in the vessels, which increases radial wall stress, and elevated flow, which may increase endothelial surface stress. In response, the vessels may increase in diameter until the stress is normalized.
The arteriogenesis of the present invention differs from angiogenic processes as the latter refer to the induction of capillary sprouting. Therefore, arteriogenesis decreases the flow resistance due to an increase of the diameter of collateral vessels from existing arterial networks, whereas angiogenesis may eventually even increase the flow resistance of the blood stream as, according to the Hagen-Poiseuille rule, the relationship between blood flow and artery diameter is enhanced by an exponential factor of 4 in relationship to the radius and the capillary diameter is typically small (often between 5 and 7 μm). Therefore, the present intervention relates to the enhancement of collateral artery growth with one or more bradykinin receptor agonist(s) by inducing arteriogenesis.
Accordingly, the present invention also refers to the bradykinin receptor agonist for use in a method for enhancing arteriogenesis for the treatment or prevention of cardiovascular ischemia.
In a further preferred embodiment, the patient is tested for arteriogenesis.
As used herein, the term “tested for arteriogenesis” may be understood in the broadest sense as any method showing the rate of arteriogenesis of the patient. The testing of arteriogenesis may be in vivo testing or in vitro (ex vivo) testing. Testing may be conducted by any means known in the art such as, e.g., testing of a blood sample, microscopic examination of the tissue, magnetic resonance tomography (MRT), computer tomography (CT), positron emission tomography (PET) scanning, diagnostic sonography, doppler sonography, examination of an extracted tissue sample, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), quantitative PCR and/or real-time PCR. It will further be understood by a person skilled in the art that different testing methods may also be combined with another and with staining methods such as, e.g., immunostaining and/or histolytic staining, and staining agents such as, e.g., fluorescence dyes and/or contrast agents.
In another preferred embodiment, the bradykinin receptor agonist of the present invention is a bradykinin receptor 1 agonist and/or a bradykinin receptor 2 agonist.
As used herein, the term “bradykinin receptor 1 agonist” may be understood as a compound that, at a given concentration, activates the bradykinin receptor 1 more than other bradykinin receptors such as the bradykinin receptor 2. Preferably, the bradykinin receptor 1 agonist activates the bradykinin receptor 1 more than 1.2 times, more than 1.5 times, more preferably more than 2 times, even more preferably more than 3 times, even more preferably more than 5 times more than the bradykinin receptor 2.
As used herein, the term “bradykinin receptor 2 agonist” may be understood as a compound that, at a given concentration, activates the bradykinin receptor 2 more than other bradykinin receptors such as the bradykinin receptor 1. Preferably, the bradykinin receptor 2 agonist activates the bradykinin receptor 2 more than 1.2 times, 1.5 times, more preferably more than 2 times, even more preferably more than 3 times, even more preferably more than 5 times more than the bradykinin receptor 1.
In a preferred embodiment, the bradykinin receptor agonist of the present invention is a bradykinin receptor 1 agonist.
Angina pectoris is a well-known disease condition characterized in that the patient has pain in the breast, in particular when exerting. Angina pectoris in the sense of the present invention may be stable angina or unstable angina. Stable angina is characterized in that it regularly occurs when exerting. Unstable angina is characterized in that it occurs irregularly, thus, sometimes when the patient exerts.
Further, a cardiovascular ischemic disease in the sense of the present invention may also be ischemic organ failure, in particular renal ischemia, renal occlusive disease, renal retinopathy, neuropathy, gut ischemia, bowel ischemia, mesenteric ischemia, mesenterial artery insufficiency, ischemic colitis, cutaneous ischemia and/or mottling.
As used herein, the terms “myocardial infarction” and “myocard infarct” may be understood interchangeably.
The term “particular periphery arterial occlusive disease” may be understood in the broadest sense as any disease state wherein the blood stream in a periphery arterial is disturbed. Typically, the patient will suffer from muscle pain when taking a rest.
The symptoms of being at risk of developing a cardiovascular ischemic disease may be any symptoms that are known in the art. Many symptoms may be observable when examining the patient by routine methods. For instance, high blood pressure is a typical symptom of being at risk. The symptoms may also include behavior such as motor movements. A smoking subject, in particular a heavily smoking subject may also be at particular risk.
A risk marker may be any risk marker known in the art. In particular, blood values may play an important role. For instance, a particularly high cholesterol level shows that the patient is at risk. A person skilled in the art will immediately see that it has to be up to the medical experts to determine exact concentrations and further risk markers showing that a patient is at risk.
A person who previously had a cerebral or myocardial ischemia is typically at risk.
A genetic predisposition in the sense of the present invention may be any genetic predisposition associated with ischemic diseases such as, e.g., diabetes type I or II, a congenital heart disease, lack of the expression of one or more bradykinin receptor(s), lack of the expression of one or more bradykinin precursor(s), or failure of cleavage of one or more bradykinin precursor(s).
Further, a person who had been subjected to a surgical procedure may be at particular risk.
In another preferred embodiment, the aforementioned symptoms of the patient at risk of developing a cardiovascular ischemic disease are selected from the group consisting of neurological malfunctions, transitory ischemic attack, congestive heart failure, transitory ischemic attack, angina pectoris, valvular heart disease, cardiomyopathy, pericardial disease, congenital heart disease, coarctation, atrial or ventricular septal defects and combinations of two and more thereof.
In another preferred embodiment, the patient further exhibits in particular, sclerosis, in particular atherosclerosis and/or transplantation-induced sclerosis; a cerebral occlusive disease, renal occlusive disease, a mesenterial artery insufficiency or an ophthalmic or retinal occlusion, post-operative or post-traumatic condition play an important role. Every injury may increase the risk of developing a cardiovascular ischemic disease.
In a preferred embodiment, the patient previously had been exposed to or is going to be exposed to a pharmaceutical or medical treatment damaging one or more arteries; a radiation treatment damaging one or more arteries; or a surgical treatment damaging one or more arteries.
A pharmaceutical or medical treatment may be any treatment that damages or harms arteries. Preferably, the treatment may refer to a local injection of a pharmaceutical compound, in particular a permanent local injection such as a drip injection. Here, the local concentration is very high. The pharmaceutical compound may, e.g., be a chemotherapeutic such as, e.g., a cancer agent (e.g., doxorubicin), that can lead to local necrosis. A medical treatment may be, e.g., dialysis. Here, the vessels may show an occlusion as platelets, calcium and other factors can form local plaques near the spot where the needle is regularly placed. A radiation treatment may be any treatment using radiation such as, e.g., X-ray radiation, radioisotope radiation, thermal radiation. Radiation therapy may also be combined with chemotherapy and/or micro- or nanobeads. A surgical treatment may refer to any surgical treatment known in the art, in particular an intracranial surgical treatment.
It will be apparent to a person skilled in the art that the pharmaceutical or medical treatment, the radiation treatment and the surgical treatment damaging one or more arteries itself is not encompassed by the present invention, but has been conducted previously or subsequently.
As used herein, arterial occlusive disease may also be intermittent claudation. The invention further relates to a method for treating a cardiovascular ischemic disease in a patient in need thereof, wherein a pharmaceutically effective amount of a bradykinin receptor agonist is administered to said patient and wherein said cardiovascular ischemic disease is a peripheral limb disease, in particular periphery arterial occlusive disease.
In a preferred embodiment, the bradykinin receptor agonist of the present invention further induces migration of leukocyte cells and/or leucocyte precursor cells.
As used herein, the term “leucocyte cell” may be understood interchangeable with the terms “leucocyte” and “white blood cells” as any leucocyte cell known in the art. Exemplarily, the leucocyte cell may be a myeloid cell or a lymphoid cell. Preferably, a leucocyte cell may be a granulocyte (e.g., a neutrophil granulocyte, an eosinophil granulocyte, a basophil granulocyte), a lymphocyte (e.g., a B-cell lymphocyte, a T-cell lymphocyte (e.g., a CD4+ T cells (T helper cell), a CD8+ T cell (cytotoxic T cell), a yδ T cell)), a monocyte, or a fixed leukocyte (e.g., a fixed macrophage (e.g. a Kupffer cell), a histiocyte, a dendritic cell, a mast cell, microglia). More preferably, a leucocyte cell of the present invention is a neutrophil granulocyte and/or a monocyte.
The term “leucocyte precursor cell” may be understood interchangeably with the term “progenitor cell” in the broadest sense as any cell known in the art that can mature into a leucocyte cell. The precursor cell may be a pluripotent precursor cell. AS used herein, the term “pluripotent” may be understood interchangably with “multipotent”. The precursor cell may be a stem cell, in particular a pluripotent stem cell. Preferably, the leucocyte precursor cell is not a progenitor cell of endothelial cells. Highly preferably, the leucocyte precursor cell is a hematopoietic progenitor cell. Said hematopoietic progenitor cell may be any hematopoietic progenitor cell known in the art such as, e.g., a myoloblast, Mo-blast, L-blast, myelocyte or megakaryocyte. Preferably, said hematopoietic progenitor cell is a myoloblast or Mo-blast. A hematopoietic progenitor cell may also be a hematopoietic stem cell (HSC).
Preferably, the leukocyte cells and/or leucocyte precursor cells migrate due to chemotactic signals. Preferably, the leukocyte cells and/or leucocyte precursor cells modulate arteriogenesis by chemotactic secretion of growth factors and inflammatory cytokines that may be important for the remodeling of an artery, in particular wherein the leukocyte cells and/or leucocyte precursor cells induce arteriogenesis. Typically, said secretion is a paracrine secretion that influences arteriogenesis locally.
In another preferred embodiment, the bradykinin receptor agonist of the present invention is administered in a long-term application. Preferably, said long-term application begins before the patient suffers from a cardiovascular ischemic disease or condition. Alternatively, said long-term application begins after at least two days, after at least four days, after at least at least one week, at least two weeks, at least one months, at least two months, at least half a year or at least one year after the occurrence of a cardiovascular ischemic disease or condition, wherein said cardiovascular ischemic disease or condition is in particular stroke or a myocard infarction. Alternatively, said long-term application begins after at least two days, after at least four days, after at least at least one week, at least two weeks, at least one months, at least two months, at least half a year or at least one year after the patient previously had been exposed a pharmaceutical or medical treatment, a radiation treatment or a surgical treatment damaging one or more arteries. Alternatively, said long-term application begins at least one day, two days, three days, at least one week, at least two weeks, at least one months or at least two months before the patient is going to be exposed to a pharmaceutical or medical treatment, a radiation treatment or a surgical treatment damaging one or more arteries. In a more preferred embodiment, the long-term application is administrating said bradykinin receptor agonist of the present invention to the patient for at least one week, at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least two years, at least three years or enduringly. Preferably, a long-term application is an application for a time span of between one week and two years, more preferably of between one week and one year, even more preferably of between one week and 26 weeks and most preferably of between one week and twelve weeks.
Especially with regards to enzyme inhibitors, the term “inhibitor” refers to compounds that diminish the functionality, thus, typically the rate of conversion catalyzed by an enzyme, of the enzyme they inhibit. Preferably, the rate of conversion is less than 0.5 times, more preferably less than 0.25 times, even more preferably less than 0.1 times, more preferably less than 0.05 times as active as the enzyme without an inhibitor. Herein, the peptidase is preferably a carboxy peptidase.
As used in the context of the present invention, the term “bradykinin receptor antagonist” may be understood in the broadest sense as any compound that can directly or indirectly inhibit the function of a bradykinin receptor or the signal transduction cascade related to a bradykinin receptor. The bradykinin receptor agonist may be a peptidomimetic, a non-peptide antagonist, a nucleic acid molecule inhibiting bradykinin precursor expression or bradykinin receptor expression, or a compound stimulating the degradation of an intrinsic bradykinin receptor agonist.
Preferably, the bradykinin receptor antagonist is a peptidomimetic, in particular a peptidomimetic consisting of nine or ten amino acids and amino acid analogues. Exemplarily, the peptidomimetic bradykinin receptor antagonist is icatibant (HOE140, H157); CP0597; D-Arg[Hyp3,Thi5,HypE(trans-propyl)7,Oic8]-bradykinin (NPC17731). The bradykinin receptor antagonist may also be a non-peptide antagonist such as, e.g., FR167344 or FR173657.
Said siRNA, microRNA or a nucleic acid analogue may be complementary to a part of the sequence of a gene encoding a bradykinin precursor (kininogen), such as, e.g., KNG1 kininogen 1 (SEQ ID NO:9); a gene encoding a bradykinin receptor, such as, e.g., BDKRB1 bradykinin receptor B1 (SEQ ID NO:10) or BDKRB2 bradykinin receptor B2 (SEQ ID NO:11); or a gene encoding a protease processing a bradykinin precursor to a kinin such as, e.g., bradykinin or kallidin, wherein said protease may be, e.g., a kallikrein, such as, e.g., KLK1 kallikrein 1 (SEQ ID NO:12).
The term “part of the sequence” as used herein mean a sequence of at least 6, at least 10, at least 15, at least 20, at least 25, at least 30 or at least 35 nucleotides in length.
As used herein, a vessel malformation is preferably the overproduction of vessels, in particular the overproduction of connections between arteries and arterioles and veins and venules (arteriovenous malformation). These malformations typically occur upon surgical proceedings, in particular upon intracranial surgical proceedings.
The bradykinin receptor antagonist of the present invention may be administered by any means known in the art. Exemplarily, administration may be oral administration, injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal injection), topical administration or intradermal administration. In case of peptides or peptidomimetics, administration is preferably enabled via an injection in order to circumvent the first pass effect. The dose of the bradykinin receptor antagonist administered evidently depends on numerous factors well-known in the art such as, e.g., the chemical nature and pharmaceutical formulation of the bradykinin receptor antagonist, and of body weight, body surface, age and sex of the patient, as well as the time and route of administration. For an adult, the dose may exemplarily be between 0.5 μg and 1 g per day, preferably between 0.5 μg and 100 mg per day, more preferably between 1 μg and 100 mg per day, even more preferably between 5 μg and 10 mg per day, even more preferably between 1 μg and 10 mg per day. In a continuous infusion, the dose may exemplarily be between 0.5 μg and 100 mg, preferably between 1 μg and 10 mg per kilogram per minute. Should the bradykinin receptor antagonist be a nucleic acid approximately between 106 copies and 1012 copies may be administered to the patient, depending on the used vector.
The present invention further relates to a method for treating or preventing the formation of vessel malformation in a patient, wherein a pharmaceutically active amount of a bradykinin receptor antagonist is administered to said patient. Therefore, in a preferred embodiment, the patient has previously been subjected to a surgical procedure and is at risk of developing an arteriovenous malformation. Alternatively, the patient may also be subjected to physical stress or trauma via an accident. In another preferred embodiment, the bradykinin receptor antagonist of the present invention is used in a method for treating a patient suffering from the Moja Moja Syndrome.
The terms “Moja Moja Syndrome”, “MojaMoja Syndrome” and “MoyaMoya Syndrome” may be understood interchangeably as a disease in which certain arteries in the brain are constricted. Blood flow is blocked by the constriction, and also by blood clots (thrombosis). The clinical features are cerebral ischemia (in particular strokes), recurrent transient ischemic attacks, sensorimotor paralysis (e.g., numbness and paralysis of the extremities), convulsions and/or migraine-like headaches. After an ischemic stroke, a hemorrhagic reperfusion stroke may occur. A hemorrhagic stroke may also stem from a rupture of the weak neovascular vessel walls.
In another preferred embodiment, the bradykinin receptor antagonist of the present invention is administered in a long-term application. The term long term application in relation to the bradykinin receptor antagonist can be understood in the same manner as long term application as described above for the agonist.
In a more preferred embodiment, the bradykinin receptor antagonist of the present invention is selected from the group consisting of HOE140, CP0597, NPC17731, FR167344; FR173657; a nucleic acid molecule inhibiting the formation of an intrinsic bradykinin receptor agonist or a bradykinin receptor and a combination of two or more thereof.
In a preferred embodiment, the aforementioned nucleic acid molecule that may serve as a bradykinin receptor agonist of the present invention is siRNA, microRNA or a nucleic acid analogue. Preferably, the siRNA or microRNA is complementarity to mRNA of components of the kallikrein-kinin system (e.g., a bradykinin precursor (e.g., kininogen), bradykinin receptor 1 and/or bradykinin receptor 2). Alternatively, the nucleic acid molecule may be DNA encoding said siRNA or microRNA.
An intrinsic bradykinin receptor antagonist may be any molecule decreasing the activity of a bradykinin receptor, wherein said molecule is produced by the patient's body itself. Exemplarily, an intrinsic bradykinin receptor antagonist may be an enzyme degrading bradykinin or kallidin. Preferably said enzyme is a protease, more preferably a carboxy peptidase, in particular ACE. The intrinsic bradykinin receptor antagonist may be given into the patient directly or a nucleic acid molecule encoding said intrinsic bradykinin receptor antagonist is given to the patient and expressed in said patient.
It will be understood by a person skilled in the art that two or more bradykinin receptor antagonist of the present invention may be combined with another. Further, one or more bradykinin receptor agonist(s) and one or more bradykinin receptor antagonist(s) of the present invention may be combined with another.
Moreover, one or more bradykinin receptor agonist(s) and/or one or more bradykinin receptor antagonist(s) of the present invention may be combined with other potential modulators or arteriogenesis such as, e.g., one or more colony stimulating factor(s) (CSF(s)) and/or one or more eicosanoid(s) (e.g., prostaglandin(s), prostacycline(s), leukotriene(s), thromboxane(s)).
In a further aspect, the present invention refers to a pharmaceutical composition comprising one or more bradykinin receptor agonists(s) of the present invention and/or one or more bradykinin receptor antagonists(s) of the present invention and a pharmaceutical acceptable carrier.
Preferably, the pharmaceutical composition comprises one or more bradykinin receptor agonists(s) of the present invention or one or more bradykinin receptor antagonists(s) of the present invention and a pharmaceutical acceptable carrier.
A pharmaceutical acceptable carrier in the sense of the present invention may be any non-toxic material that does not interfere with the effectiveness of the biological activity of the bradykinin receptor agonists(s) and/or bradykinin receptor antagonists(s) of the present invention. Evidently, the characteristics of the carrier will depend on the route of administration. Such a composition may contain, in addition to the active substance and carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intra-muscular administration and formulation.
The medicament, otherwise known as a pharmaceutical composition, containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated.
The present invention refers to a pharmaceutical composition for topical application, oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Topical application is one preferred embodiment, especially via a cream or gel. Topical application forms relate to gels, creams, suppositories, or films. The pharmaceutical composition of the invention may be in the form of a liposome in which the active substance of the invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art.
When a therapeutically effective amount of the active substance of the invention is administered by intravenous, cutaneous or subcutaneous injection, the active substance will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. As used herein, the term “therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit. The amount of active substance in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Larger doses may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the active substance, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
Further, a pharmaceutical acceptable carrier in the sense of the present invention may be any molecule(s) known in the art to facilitate uptake or to enhance the rate of uptake such as, e.g., positively charged polymer(s) (e.g., polyethylene imine (PEI)), neutral lipid(s), positively charged lipid(s), viral vector(s), liposome(s), micro- and/or nanobead(s), cell-penetrating peptide(s) (=protein-transduction domains, e.g., polyarginines (e.g., R8, R9, R10), HIV Tat peptide, antennapedia/penetratin peptide, a Lactoferrin-derived peptide), antimicrobial peptides and/or a Chariot peptide.
The present invention also relates to the pharmaceutical composition of the present invention for use in a method as described above. In particular, the pharmaceutical composition of the present invention is used in a method for enhancing collateral artery growth and for preventing a cardiovascular ischemic disease in a patient at risk thereof or for treating or preventing a cardiovascular ischemic disease in a patient in need thereof, wherein said cardiovascular ischemic disease is a peripheral limb disease, in particular periphery arterial occlusive disease.
The present invention further relates to modulation of flow induced collateral vessel or artery growth and the outgrowth of collateral arterioles from pre-existing arterial networks. Further subject matter of the invention is the induction or reduction of collateral vessel growth through the stimulation (agonists) or inhibition (antagonist) of the bradykinin receptor 1 and/or bradykinin receptor 2 signaling pathways in the artery or blood vessel wall (modulation of the bradykinin receptors of the endothelial cells, the smooth muscle cells or the leucocytes that have migrated into the vessel or artery wall).
Collateral vessel growth or artery growth according to the present invention are terms than can be used to describe arteriogenesis. Arteriogenesis is characterized by the outgrowth of collateral arterioles from pre-existing arterial networks. The term “pre-existing arterial networks” can also be described as arterial networks with pre-existing arteriolar connections, such as pre-existing capillaries, arteries, vessels or other conduits of blood found in a mammalian organism. Therefore the present invention further relates to the use of a bradykinin receptor 1 (B1R) and/or bradykinin receptor 2 (B2R) stimulating substance for modulating collateral growth of collateral arteries and/or other arteries from pre-existing arteriolar connections.
Agonists can be used for the induction of collateral vessel or artery growth (arteriogenesis) via the stimulation of the bradykinin receptor signaling pathways. Various BR-agonists can be used for the modulation of arteriogenesis, such as the nonapeptide bradykinin, structurally related kinins (T kinin, kallidin, Des-Arg9-bradykinin and Des-Arg10-kallidin) in addition to further structurally related substitutes with modified amino acid sequences. The invention further relates to an induction of arteriogenesis through the stimulation of bradykinin receptors with non-peptide molecules.
The invention further relates to the inhibition of arteriogenesis via antagonists of the bradykinin receptors. As antagonists the following molecules are intended as examples: icatibant (BR2 antagonists) or (Des-Arg9, Leu8)-bradykinin (BR1 antagonists), in addition to related substitutes or alternatives with modified amino acid sequences. The inhibition of arteriogenesis via non peptide molecules, which preferably bind to the bradykinin receptor, is a further aspect of the invention. The invention also includes modulation of collateral blood vessel or artery growth by means of nucleic acid molecules, for example through nucleic acid molecules (siRNA, microRNA) with homology to the mRNA of components of the kallikrein-kinin systems (for example kininogen, BR1, BR2).
The present invention is relevant for the medical and pharmaceutical industries. A stimulation of arteriogenesis via the bradykinin receptor signaling pathways, particularly via a long-term application of a stimulant for the prevention of ischemic insults, is subject matter of the present invention. Further subject matter of this invention is therefore the prevention of strokes, coronary heart conditions or PAVKs via a stimulation of the bradykinin signaling pathways. Patients which have already exhibited the first warning signal of a stroke (the so called transitory ischemic attacks [TIA]), are one example of a patient group that could benefit from the therapeutic induction of arteriogenesis. Patients with such vessel or artery constriction could avoid the threatening stroke or coronary heart condition by an induction of a naturally occurring biological bypass via bradykinin receptor agonists, when applied at an early time point.
The present invention relates therefore to the prevention and/or therapy of circulatory disorders, cardiovascular disease, artery or blood vessel conditions and/or ischemic obstructive or occlusive diseases or conditions.
Circulatory disorders, cardiovascular disease, artery or blood vessel conditions and/or ischemic obstructive or occlusive diseases or conditions are terms that encompass a variety of diseases and conditions. The diseases treated and/or prevented by the present invention are vascular diseases of mammals. The word mammal means any mammal. Some examples of mammals include, for example, pet animals, such as dogs and cats; farm animals, such as pigs, cattle, sheep, and goats; laboratory animals, such as mice and rats; primates, such as monkeys, apes, and chimpanzees; and humans. In some embodiments, humans are preferably treated by the methods of the invention.
The terms circulatory disorders, cardiovascular disease, artery or blood vessel conditions and/or ischemic obstructive or occlusive diseases or conditions refer to states of vascular tissue where blood flow is, or can become, impaired or altered from normal levels. Many pathological conditions can lead to vascular diseases that are associated with alterations in the normal vascular condition of the affected tissues and/or systems. Examples of vascular conditions or vascular diseases to which the methods of the invention apply are those in which the vasculature of the affected tissue or system is senescent or otherwise altered in some way such that blood flow to the tissue or system is reduced or in danger of being reduced or increased above normal levels. It refers to any disorder in any of the various parts of the cardiovascular system, which consists of the heart and all of the blood vessels found throughout the body. Diseases of the heart may include coronary artery disease, CHD, cardiomyopathy, valvular heart disease, pericardial disease, congenital heart disease (e.g., coarctation, atrial or ventricular septal defects), and heart failure. Diseases of the blood vessels may include arteriosclerosis, atherosclerosis, hypertension, stroke, vascular dementia, aneurysm, peripheral arterial disease, intermittent claudication, vasculitis, venous incompetence, venous thrombosis, varicose veins, and lymphedema.
Examples of vascular conditions that can be treated or prevented with the compositions and methods of the invention include atherosclerosis, preeclampsia, peripheral vascular disease, erectile dysfunction, cancers, renal failure, heart disease, and stroke. Vascular, circulatory or hypoxic conditions to which the methods of the invention apply also include those associated with, but not limited to, maternal hypoxia (e.g., placental hypoxia, preeclampsia), abnormal pregnancy, peripheral vascular disease (e.g., arteriosclerosis), transplant accelerated arteriosclerosis, deep vein thrombosis, erectile dysfunction, cancers, renal failure, stroke, heart disease, sleep apnea, hypoxia during sleep, female sexual dysfunction, fetal hypoxia, smoking, anemia, hypovolemia, vascular or circulatory conditions which increase risk of metastasis or tumor progression, hemorrhage, hypertension, diabetes, vasculopathologies, surgery (e.g., per-surgical hypoxia, post-operative hypoxia), Raynaud's disease, endothelial dysfunction, regional perfusion deficits (e.g., limb, gut, renal ischemia), myocardial infarction, stroke, thrombosis, frost bite, decubitus ulcers, asphyxiation, poisoning (e.g., carbon monoxide, heavy metal), altitude sickness, pulmonary hypertension, sudden infant death syndrome (SIDS), asthma, chronic obstructive pulmonary disease (COPD), congenital circulatory abnormalities (e.g., Tetralogy of Fallot) and Erythroblastosis (blue baby syndrome). In particular embodiments, the invention is a method of treating loss of circulation or endothelial dysfunction in an individual.
Thus, the invention is directed to compositions useful in a method of treating and/or preventing diseases such as stroke, atherosclerosis, acute coronary syndromes including unstable angina, thrombosis and myocardial infarction, plaque rupture, both primary and secondary (in-stent) restenosis in coronary or peripheral arteries, transplantation-induced sclerosis, peripheral limb disease, intermittent claudication and diabetic complications (including ischemic heart disease, peripheral artery disease, congestive heart failure, retinopathy, neuropathy and nephropathy), or thrombosis.
Circulatory disorders relate to various organs such as brain stem, heart, kidney, adrenal and lung. In addition, the present invention is useful for the prevention and treatment of the diseases induced by peripheral circulation failure in mammals, such as Raynaud's syndrome, arteriosclerosis obliterans, romsoongitis obliterans, Buerger disease, diabetic microvascular disorders and peripheral arterial obstruction (e.g. diabetic gangrene). Moreover, the present invention is useful for the prevention and treatment of hypertensive organ circulatory disorders, as well as for the treatment of cerebrovascular disorders both in acute stages and chronic stages.
Ischemic conditions include coronary arteriosclerosis, angina pectoris, and acute and old myocardial infarction and refers to the serious disorder of the vital heart that mostly affects males and females in late middle age or older. Coronary arteriosclerosis is characterized by arteriosclerosis in the coronary artery that supplies nutrients to the heart. Angina pectoris is characterized by attacks of chest pain caused by impaired blood flow in the coronary artery. Myocardial infarction is characterized by myocardial necrosis caused by impaired blood flow in the coronary artery and by fatal complications coming there with such as arrhythmia, cardiac failure, cardiac rupture, and pump failure. Impaired blood flow to the heart, a vital organ, is an essential characteristic of these ischemic heart diseases
Peripheral arterial occlusive disease (PAOD; also referred to as peripheral arterial disease (PAD)) results either from atherosclerotic or inflammatory processes producing arterial stenosis, or from thrombus formation associated with underlying atherosclerotic disease. A common site for PAOD is in the lower limbs. This process of atherosclerosis causes initial thickening and plaque formation encroaching the arterial lumen, decreasing the effective luminal radius of afflicted arterial segments, producing an anatomic and sometimes functional obstruction to blood flow.
Conditions such as the Moja Moja syndrome can however be treated through modulation of arteriogenesis. The Moja Moja condition is characterized by closing of the internal carotid artery, whereby the blood flow can however be maintained over collateral arteries. A stimulation of the collateral blood flow can in such cases lead to an enhanced perfusion in a hypoperfusion area.
Flow induced collateral blood vessel growth has not only a physiological importance but can also lead to a pathological formation of collateral vessels and arteries, especially in cases of etiologically unclear brain diseases involving the arterio-venous vessel malformation. An AV-shunt can usually be treated using a medical gel in an operative situation in order to close the shunt. This treatment of arterio-venous malformation can however lead to the induction of collateral blood vessels and arteries, which after the operation can however lead to the further growth of collateral arteries and therefore formation of a new shunt. In such cases the inhibition of arteriogenesis through the bradykinin receptor antagonists (for example as part of an AV-shunt gel) would be useful.
Ligand B1 and B2 receptor agonism plays beneficial and protecting roles in cardiovascular-related pathologies such as ischaemic heart diseases, periphery artery diseases and stroke. The present invention indicates that a strong cardioprotective effect is provided by bradykinin receptor signaling to therapeutically enhance arteriogenesis. Therefore it is suggested to therapeutically stimulate arteriogenesis via the B2 receptors and preferably via the B1 receptor pathway (see FIG. 6). Stimulation of collateral artery growth in case of obstruction or occlusion of a major artery, enhancing blood flow into the hypoperfused area distal of the occlusion, can prevent ischemia.
Interestingly, both bradykinin receptors exhibit different biological features, such as that B2 receptors are considered to be constitutively expressed and ready to respond to newly formed kinins. Upon ligand binding B2 receptors are rapidly desensitized and degraded. In contrast, Bradykinin receptor 1 is expressed upon induction. B1 receptors generally exhibit long term effects. Since signaling via B1 or B2 receptors will result in different signaling pathways, stimulation of bradykinin receptor 2 or bradykinin receptor 1 by selective compound selection can enable distinct options in therapeutic-induced collateral growth. Here, B1 or B2 receptors show distinct affinity for different bradykinin compounds. Naturally, Bradykinin B2 receptors have high affinity for bradykinin and kallidin (lys-bradykinin). In contrast, the Bradykinin receptor 1 exhibits a high affinity for des-Arg9-bradykinin and des-Arg9-kallidin. These natural bradykinin receptor ligands are subjected to rapid inactivation by tissue and plasma enzymes, which limit their stability. However, many bradykinin compounds have been designed with improved properties for clinical intervention. For example, stimulation by orally active and selective bradykinin 2 receptor ligands for clinical intervention in different disease models was tested for the non-peptide compounds FR-190997 and FR-191413 (Fujisawa). Such studies might encourage the required efforts to identify more suited candidates.
The best-characterized ligand for the Bradykinin receptor 1 signaling peptide, Sar[DPhe8]desArg9BK, exhibits a lower susceptibility toward proteolysis and a moderate affinity and potency (about 11- and 2-fold less, respectively) relative to the natural ligand Lys-desArg-bradykinin (Coté, 2009). FIG. 6 shows how cerebral arteriogenesis can be therapeutically enhanced by stimulation with Sar[DPhe8]desArg-bradykinin. Here it is demonstrated that B1R agonists bearing optimized pharmacological features (high potency, selectivity and stability toward proteolysis) hold promise as valuable therapeutic agents in the treatment of ischemic diseases.
Tables 1 and 2 provide a list of bradykinin receptor ligands for use in modulating (inhibit/activate) arteriogenesis. Table 3 provides a list of publications, which are incorporated by reference in their entireties, in which bradykinin receptor modulators are disclosed that can be used for modulating arteriogenesis via bradykinin receptor modulation.







TABLE 1







List of bradykinin receptor signaling agonists


that can be used to modulate arteriogenesis.









Agonist
B-Receptor
Substance





Bradykinin/H-1970
B2
Peptide


natural peptide


Des-Arg9-Bradykinin
B1
Peptide


natural peptide


Kallidin/Lys-Bradykinin/H-2180
B2
Peptide


natural peptide


(Des-Arg10)-Kallidin/Lys-(Des-Arg9)-Bradykinin
B1
Peptide


natural peptide


Sar-[D-Phe8]-des-Arg9-Bradykinin
B1
Modified Peptide


potent, selective, resistant to degradation


SarLys[Hyp3, Cha5, DPhe8]desArg9BK
B1
Modified Peptide


potent, selective, resistant to degradation


SarLys[Hyp3, Igl5, DPhe8]desArg9BK
B1
Modified Peptide


potent, selective, resistant to degradation


SarLys[Hyp3, Cpg5, DPhe8]desArg9BK
B1
Modified Peptide


potent, selective, resistant to degradation


RMP-7 lobradamil/Cereport
B2
Modified Peptide


potent, selective, resistant to degradation


JMV-1116
B2
Modified Peptide


potent, selective, resistant to degradation


Ile-Ser-BK (B 1643)
B2
Modified Peptide


[Phe8-(ψ-CH2NH)-Arg9]-BK
B2
Modified Peptide


Hyp3,Tyr(Me)8]-BK
B2
Modified Peptide


JMV1116 and analogues
B2
Modified Peptide


[Hyp3]-BK (B 7775)
B2
Modified Peptide


FR-190997
B2
Non-Peptide


4-(2-pyridylmethoxy)-substituted quinoline


FR-191413
B2
Non-Peptide


3-(2-pyridylmethyl)-substituted benzimidazole
















TABLE 2







List of bradykinin receptor signaling antagonists


that can be used to modulate arteriogenesis.









Antagonist
B-Receptor
Substance





AC-Lys-Arg-Pro-Pro-Gly-Phe-Ser-DβNal-Iie
B1
Modified Peptide


R 715


AC-Lys-Arg-Pro-Pro-Gly-(αMe)Phe-Ser-DβN al-Iie
B1
Modified Peptide


R892


(Des-Arg9,Leu8)-Bradykinin
B1
Peptide


[Leu8,des-Arg9]-BK
B1
Modified Peptide


[Leu9,des-Arg10]-Kallidin (CP0298)
B1
Modified Peptide


[des-Arg10]-HOE140
B1
Modified Peptide


Lys-[Leu8,desArg9]-BK
B1
Modified Peptide


(B9858: H-Lys-Lys-Arg-Pro-
B1
Modified Peptide


Hyp-Gly-Igl-Ser-D-Igl-Oic-OH)


B-9430 (H-D-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-
B2 + B1
Modified Peptide


D-Igl-Oic-Arg-OH)


Icatibant/Hoe140/H157
B2
Modified Peptide


FIRAZYR


FR-167344
B2
Non-Peptide


FR-173657
B2
Non-Peptide


D-Arg[Hyp3,Thi5,HypE(trans-propyl)7,Oic8]-BK
B2
Modified Peptide


NPC17731


WIN 64338
B2
Non-Peptide


Bradyzide
B2
Non-Peptide
















TABLE 3







List of bradykinin receptor modulators that


can be used in the in the present invention.











B receptor


Inventor
Patent; year
Antagonist





Boehringer Ingelheim Int
WO2006048209; 2006
B1R


Boehringer Ingelheim Int
WO2008022945; 2008
B1R


Boehringer Ingelheim Int
WO2009013299; 2009
B1R


Elan Pharmaceutical, Inc
WO2006071775; 2006
B1R


Elan Pharmaceutical, Inc
WO2006113140; 2006
B1R


Evotec Neurosciences GMBH
WO2008125570; 2008
B1R


Richter Gedeon Nyrt
WO2007072092; 2007
B1R


Richter Gedeon Nyrt
WO2008050167; 2008
B1R


Richter Gedeon Nyrt
WO2008050168; 2008
B1R


Richter Gedeon Nyrt
WO2008068540; 2008
B1R


Grünenthal GMBH
WO2008040492; 2008
B1R


Grünenthal GMBH
WO2008046573; 2008
B1R


Grünenthal GMBH
WO2008131946; 2008
B1R


Jerini AG
WO2007003411; 2007
both


Jerini AG
WO2008116620; 2008
B2R


Menarini
WO2006040004; 2006
B2R


Menarini
WO2003103671; 2003
B2R


Merck & Co
US20050261327; 2005
B1R


Merck & Co
US20060122236; 2006
B1R


Merck & Co
US20060173023; 2006
B1R


Merck & Co
WO2005016886; 2005
B1R


Merck & Co
US20060106011; 2006
B1R


Merck & Co
US20060111392; 2006
B1R


Merck & Co
US20060128765; 2006
B1R


Merck & Co
WO2005004810; 2005
B1R


Neurogen Corporation
WO200701007; 2007
B1R


Neurogen Corporation
WO2007140383; 2007
B1R


Neurogen Corporation
WO2008002849; 2008
B1R


Neurogen Corporation
WO2008024692; 2008
B1R


Neurogen Corporation
WO2008033739; 2008
B1R


Sherbrooke University
WO2006017938; 2006
B1R


Sherbrooke University
U.S. Pat. No.
B1R



7,211,566; 2007


Park Choo
WO2007108566; 2007
B1R


Park Choo
WO2007142431; 2007
B1R


University of Colorado
WO2008124756; 2008
B1R









Bradykinin is rapidly hydrolyzed and degraded by ACE. Locally increased kinin concentrations, arising as a consequence of the blockade of ACE as the major kinin-degrading enzyme, significantly contribute to the apparent beneficial effects of ACE inhibitors, including their infarct size-reducing effects. In addition to direct Bradykinin receptor agonists and antagonists, Table 4 provides a comprehensive list of angiotensin converting enzyme (ACE) inhibitors.







TABLE 4







Sample list of ACE-Inhibitors for enhancing modulation


of arteriogenesis via bradykinin receptor signaling








ACE-Inhibitors
Obtained from





Ramipril
Roche, AwD-Pharma, Schwarz-Pharma/Sanol,


Quinapril
R.A.N.-Pharm, ratiopharm, TAD Pharma,


Cilazapril
betapharm, Hennig, Abott, CT


Spirapril-HCl 1H2O
Arzneimittel, Wörwag, HEXAL, Winthrop,


Catopril
1 A Pharma, AbZ-Pharma, ALIUD PHARMA,


Enalaprilmaleat
Sandoz, STADApharm, Schwarz Pharma


Lisinopril


Bencapril-HCl


Perindopril-Erbumin


Trandolapril


Fosinopril-Natrium












FIGURES
FIG. 1: Schematic of the flow induced collateral arterial system (arteriogenesis).
FIG. 2a: Relationship between blood flow and artery radius (The Law of Hagen-Poiseuille).
FIG. 2b: Differences between angiogenesis and arteriogenesis
FIG. 3: Schematic representation of the kallikrein-kinin system.
FIG. 4a: Measurement of collateral perfusion in the femoral ligature model in the mouse.
FIG. 4b: Hindlimb perfusion recovery—3 weeks post Femoral Artery Ligation.
FIG. 5: Pharmacological inhibition of bradykinin receptor 1 and 2 signaling in the 3-vessel occlusion (3-VO) cerebral arteriogenesis model in rat.
FIG. 6: Therapeutic stimulation of arteriogenesis with a selective BR1 agonist in the 3-vessel occlusion (3-VO) cerebral arteriogenesis model in rat.



DETAILED DESCRIPTION OF THE FIGURES
FIG. 1: Schematic of the Flow Induced Collateral Arterial System (Arteriogenesis).
An artery occlusion of a major vessel leads to redistribution of blood flow by the recruitment of pre existing collateral vessels. Upon enhanced blood flow small arteries are remodeled into functional conductance arteries to supply hypoperfusion area with blood (Arteriogenesis—biological bypass).
FIG. 2a: Relationship Between Blood Flow and Artery Radius (the Law of Hagen-Poiseuille).
Equation clarifies the relationship between flow and vessel radius to the power of 4. Flow volume v, viscosity η, pressure differences ΔP, distance differences (vessel length) Δl, radius r. A small increase in vessel radius leads to a significant enhancement of blood flow.
FIG. 2b: Differences Between Angiogenesis and Arteriogenesis
Angiogenesis refers to the growth of new small capillaries in the ischemic region downstream of the obstruction of a conductance vessel (driving force ischemia). Arteriogenesis refers to the redistribution of blood flow via the collateral artery network outside the ischemic region in oxygen-rich tissue that bypass the site of occlusion (driving force blood shear stress on vessel). Stimulation of arteriogenesis increases vessel diameter of the bypassing arteries, which produces a new functional conductance vessel to supply the endangered territory with blood.
FIG. 3: Schematic Representation of the Kallikrein-Kinin System.
Kininogen is processed by kallikrein to cleave bradykinin, kallidin and other kinins. Kinins signal via the constitutively expressed B2 receptor and the inducible B1 receptor. Stimulation of bradykinin receptors may activate intracellular signaling pathways in endothelial cells, smooth muscle cells and leucocytes, such as monocytes. This results in cell proliferation, cell migration and leucocyte recruitment, which are features of arteriogenesis. Collateral growth can be modulated by stimulation/inhibition of the bradykinin receptor signaling.
FIG. 4a: Measurement of Collateral Perfusion in the Femoral Ligature Model in the Mouse (7 Days Post Femoral Artery Ligation)
Arteriogenesis was measured in bradykinin receptor 1 knock out (KO) mice, bradykinin receptor 2 KO mice, bradykinin receptor 1 and 2 double KO mice and in wild type (WT) mice. All bradykinin receptor knock out mice exhibit a markedly reduced arteriogenesis. The B1 KO mice have the strongest phenotype and arteriogenesis is severely reduced in comparison with WT mice (more than 50% reduction in blood perfusion rate).
FIG. 4b: Hindlimb Perfusion Recovery—3 Weeks Post Femoral Artery Ligation
(A) Schematic representation of the mice hindlimb collateral network. (FA: femoral artery, LCFA: lateral caudal femoral artery, SA: saphenous artery). (B) Occlusion of the femoral artery results in a positive outward remodeling of collateral arteries and recovery of blood flow within 7 days to 21 days. 3 weeks post FAO Hindlimb perfusion (measured by microspheres—in arbitrary units) is still significantly reduced in B1R KO (C) and blood flow does not recover. In contrast 3 weeks post FAO B2R KO mice (D) blood flow recovers and is comparable to WT mice 3 weeks post FAO. *, p<0.05; **, p<0.01.
FIG. 5: Pharmacological Inhibition of Bradykinin Receptor 1 and 2 Signaling in the 3-Vessel Occlusion (3-VO) Cerebral Arteriogenesis Model in Rat.
A. project design I. H-1960 is used as bradykinin receptor 1 (BR1) antagonist, Icatibant is used as BR2 antagonist. B,C. Diameters of the collateral pathway (posterior cerebral artery) ipsilateral (B) and contralateral (B) of the carotid artery ligation. D. Cerebrovascular reactivity. Collateral diameters (B,C) and cerebrovascular reactivities (D) are given for untreated animals (without 3-VO surgery), for control animals (3 weeks post 3-VO surgery without plus vehicle), for BR1 inhibition (3 weeks post 3-VO plus bradykinin receptor 1 antagonist treatment) and for BR2 inhibition (3 weeks post 3-VO bradykinin receptor 2 antagonist treatment). E. Brain vessel angiographies of untreated animals, post 3-VO and sham controls.
FIG. 6: Therapeutic Stimulation of Arteriogenesis with a Selective BR1 Agonist in the 3-Vessel Occlusion (3-VO) Cerebral Arteriogenesis Model in Rat.
A. project design II. R838 is used as bradykinin receptor 1 (BR1) agonist. B,C. Diameters of the collateral pathway (posterior cerebral artery) ipsilateral (B) and contralateral (B) of the carotid artery ligation. B,C. Diameters of the collateral pathway (posterior cerebral artery) ipsilateral (B) and contralateral (B) of the carotid artery ligation. D. Cerebrovascular reactivity. Collateral diameters (B,C) and cerebrovascular reactivities (D) are given for untreated animals (without 3-VO surgery), for control animals (1 weeks post 3-VO surgery plus vehicle), and for BR1 stimulation (1 weeks post 3-VO plus R838 treatment) E. Brain vessel angiographies of untreated animals, post 3-VO and sham controls.
EXAMPLES
Example 1
Measurement of Collateral Perfusion in the Femoral Ligature Model in the Mouse
Arteriogenesis was examined in an arteriogenesis model (ligation of the femoral artery) on the hind leg of C57/Bl6 mice. The ligature of the femoral artery in hind leg of a mouse is an established model for examining arteriogenesis (Hoefer, van Royen et al. 2001). Here arteriogenesis is measured through the collateral perfusion rate of the hind leg arteries by pressure controlled perfusion with fluorescent microspheres (Molecular Probes) of various sizes (Bergmann, Hoefer et al. 2006). In brief, a number of fluorescent microspheres where normalized against a reference number of blue microspheres. Values are expressed as percent perfusion by comparing the ligated vs. the unligated leg. Calculating collateral perfusion rate is a functional measurement to evaluate collateral growth (arteriogenesis). Finally arteriogenesis can be estimated by a count of microspheres using flow cytometry (MACS Quant, Miltenyi). Of all the functional measurements, the measurement of collateral growth by microsphere perfusion is considered to be the gold standard in evaluating arteriogenesis (Schaper 2009).
Collateral blood vessel growth is inhibited in BR1, BR2 and BR1/BR2 double-receptor knock out (KO) mice in comparison to wild type mice (WT) (FIGS. 4a and 4b). Arteriogenesis is significantly inhibited in BR2-receptor knock out and particular, bradykinin receptor BR1 KO mice (22.41 units) exhibit a more than 50% reduced collateral perfusion rate as compared to WT mice (56.17 units). In conclusion it was shown that the bradykinin receptor signaling pathway has a significant relevance for the modulation of arteriogenesis (molecular biological demonstration).
Example 2
Pharmacological Inhibition of Bradykinin Receptor 1 and 2 Signaling in the 3-Vessel Occlusion (3-VO) Cerebral Arteriogenesis Model in Rat
Pharmacological modulation of arteriogenesis was analyzed using a 3 vessel occlusion (3-VO) model for cerebral arteriogenesis in the rat (Occlusion of both vertebral arteries and one carotid artery). Upon occlusion of three of four blood-supplying arteries to the brain, blood flow is redistributed by the recruitment of collateral pathways such as the posterior cerebral artery to supply the hypoperfusion area with blood. The 3-VO model in the rat has been established since 2003 and is acknowledged as a hypoperfusion model for testing cerebral arteriogenesis (Busch, Buschmann et al. 2003). By using the 3-VO model, relevant therapeutic stimulation of arteriogenesis was shown for instance by the application of GM-CSF (Buschmann, Circulation 2003).
Either by inhibition or stimulation of the bradykinin receptor signaling pathways, arteriogenesis was determined and physiological parameters of the brain were tested. Here, pharmacological modulation of collateral growth in the 3-VO model demonstrate that arteriogenesis can be inhibited through the selective B1 antagonists (Des-Arg9, Leu8)-bradykinin (H1960) and the selective B2-receptor antagonist HOE-140 (Icatibant) (FIG. 5). It is additionally demonstrated that cerebral arteriogenesis can be stimulated the selective BR1 agonist Sar-[D-Phe]des-Arg9-bradykinin (R838) (FIG. 6).
The characterization of arteriogenesis was carried out via measurement of the artery or blood vessel diameter (posterior cerebral artery—PCA) in addition to via a measurement of the cerebrovascular reactivity (perfusion reserves in the brain). Cerebrovascular anatomy was studied after maximal vasodilation by a modification of the postmortem latex perfusion method of Maeda et al (Maeda, Hata et al. 1998). Through a measurement of the cerebrovascular reactivity the CO2 partial pressure in the blood was artificially increased (by means of a carboanhydrase-inhibitor acetazolamide) and the reaction of the artery was measured via a measurement of blood flow via laser-doppler cytometry.
FIGS. 5B and 5C demonstrate that collateral arterial growth is strongly reduced in response to the selective inhibition of the bradykinin receptor 1 signaling pathway and slightly in response to the selective inhibition of the bradykinin receptor 2 signaling. In the case of previous treatment with a bradykinin receptor B1 antagonist, it is demonstrated that the arteries increase in diameter to a lesser extent than in the untreated controls. Here collateral growth is reduced in both collateral pathways (ipsilateral and contralateral of the occluded carotide artery). Experiments which examine the diameter changes after application of a bradykinin receptor B2 antagonist demonstrate reduced collateral growth.
CVR is reduced through selective inhibition of the bradykinin B1 and B2 signaling pathways. Negative CVR values are based on the Steal phenomenon (Kuwabara, Ichiya et al. 1995). (FIG. 5D). Upon increase of the CO2 partial pressure under normal conditions, vessels in the brain are capable of increasing blood flow to around 20% of the normal blood flow. Directly after the 3-VO experiment no cerebrovascular reactivity is detectable anymore. Blood flow recovers within 3 weeks post 3-VO and the perfusion reserve is restored to around 10%. In contrast, three weeks after 3-VO, no increase in blood flow is detected in animals treated with bradykinin receptor inhibitors (BR1i, BR2i); thereby demonstrating that arteriogenesis has been effectively inhibited.
Example 3
Therapeutic Stimulation of Arteriogenesis with a Selective BR1 Agonist in the 3-Vessel Occlusion (3-VO) Cerebral Arteriogenesis Model in Rat
FIG. 6 demonstrates therapeutic stimulation of arteriogenesis by using a selective bradykinin receptor 1 agonist Sar-[D-Phe]des-Arg9-bradykinin (R838). In the case of simultaneous application of the bradykinin receptor 1 agonist R838, it is demonstrated that seven days after 3-VO the diameter of the posterior cerebral artery (PCA) is significantly greater than the diameter in untreated rats after 3-VO (FIG. 6B,C). Furthermore FIG. 6D. shows that CVR is significantly increased through stimulation with a selective bradykinin receptor 1 agonist R838 (Sar-[D-Phe]des-Arg9-bradykinin). Arteriogenesis can by stimulated therapeutically by application of R838 for 1 week.
In summary the examples demonstrate that cerebral arteriogenesis is significantly inhibited by antagonists via blocking the bradykinin receptors (FIG. 5). Stimulation with a selective B1R agonist demonstrates that the cerebral arteriogenesis can also be stimulated (FIG. 6). Rats have been used in the present examples as a model for mammals. The present invention including products and methods described herein therefore also encompass the application of said products and methods in the treatment of all mammals, especially humans.

## Claims

1. A method for modulating collateral blood vessel growth of collateral arteries and/or non-collateral blood vessels of pre-existing arterial networks comprising
administering, to a patient at risk of cardiovascular ischemic disease a bradykinin receptor 1 (B1R) agonist in an amount effective to modulate collateral blood vessel growth of (a) collateral arteries and/or (b) non-collateral blood vessels of pre-existing arterial networks, wherein said patient:
(a) shows symptoms of being at risk of developing said cardiovascular ischemic disease, wherein said symptoms are:
a blood pressure and/or a cholesterol level that affords an at least 10% higher risk of developing said cardiovascular ischemic disease than an average risk of developing said cardiovascular ischemic disease throughout a population,
smoking,
atherosclerosis,
thrombosis,
embolism, and/or
restenosis;
(b) shows a risk marker for cardiovascular ischemic disease in ex vivo tests; and/or
(c) has a genetic predisposition of developing a cardiovascular ischemic disease.

2. The method of claim 1, wherein said cardiovascular ischemic disease is
myocardial ischemia.

3. The method of claim 1, wherein said patient previously had been exposed to or is going to be exposed to
(a) a pharmaceutical or medical treatment damaging one or more arteries;
(b) a radiation treatment damaging one or more arteries; and/or
(c) a surgical treatment damaging one or more arteries.

4. The method of claim 1, wherein said patient is tested for arteriogenesis.

5. The method of claim 1, wherein said B1R agonist is administered in a long-term application.

6. The method of claim 5, wherein said long-term application is administrating said B1R agonist to the patient for at least one week, at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least two years, at least three years or enduringly.

7. The method of claim 1, wherein said B1R agonist is selected from the group consisting of
Sar-[D-Phe8]-des-Arg9-bradykinin (R838);
Lys-(Des-Arg9)-bradykinin;
T-kinin;
kallidin;
des-Arg9-bradykinin;
des-Arg10-kallidin;
Bradykinin;
bradykinin/H1970;
kallidin/Lys-bradykinin/H2180;
SarLys[Hyp3,Cha5,D-Phe8]desArg9-bradykinin;
SarLys[Hyp3,Igl5,D-Phe8]desArg9-bradykinin;
SarLys[Hyp3,Cpg5,D-Phe8]desArg9-bradykinin;
Ile-Ser-bradykinin (B1643);
[Phe8-ψ-CH2NH)-Arg9]-bradykinin;
[Hyp3,Tyr(Me)8]-bradykinin;
[Hyp3]-bradykinin (B7775); and
a combination of two or more thereof
and is administered optionally together with an agent increasing a concentration or a half-life of a bradykinin receptor agonist.

8. The method of claim 7, wherein said bradykinin receptor agonist is selected from the group consisting of R838, T-kinin, kallidin, des-Arg9-bradykinin and des-Arg10-kallidin, and is administered together with an agent increasing a concentration or a half-life of the bradykinin receptor agonist.

9. The method of claim 7, wherein said agent increasing the concentration or the half-life of a bradykinin receptor agonist is an enzyme inhibitor.

10. The method of claim 9, wherein said enzyme inhibitor is an angiotensin-converting enzyme (ACE) inhibitor, an amino peptidase P (APP) inhibitor and/or a carboxypeptidase N (CPN) inhibitor.

11. The method of claim 10, wherein said ACE inhibitor is selected from the group consisting of ramipril, quinapril, ciiazapril, spirapril-HCl 1H20, catopril, enalaprilmaleat, lisinopril, perindopril-erbumin, trandolapril, fosinopril-sodium and a combination of two or more thereof.

12. The method of claim 1, wherein said B1R agonist further induces migration of leukocyte cells and/or leucocyte precursor cells.

13. The method of claim 1, further comprising administering
(a) one or more colony stimulating factor(s) (CSF(s)); or
(b) one or more eicosanoid(s).

14. The method of claim 13, wherein the one or more eicosanoid(s) is one or more prostaglandin(s).

15. The method of claim 2, wherein
the myocardial ischemia is a coronary heart disease and/or myocardial infarction.

16. The method of claim 8, wherein the bradykinin receptor agonist is R838, T-kinin and/or kallidin.