Patent Grant
7727971
The present invention relates to prevention and treatment of strokes and ischemic diseases and to post-ischemic therapeutic treatment. The invention furthermore relates to the use of a growth factor for treating, more particularly restoring the function of ischemic tissue, in particular muscles such as myocardium and skeletal muscles. The present invention also relates to a method of curing a reduced (e.g. muscular) performance in a mammal, in particular a human being, after an ischemic event. Advantageously the present invention is concerned with the use of a hemodynamically stable medicament which is devoid of side effects associated with other similar growth factors, thus providing specific advantages for hemodynamically compromised patients.
Stroke, defined as a sudden weakening or loss of consciousness, sensation and voluntary motion caused by rupture or obstruction of an artery of the brain, is the third cause of death in the United States. Worldwide, stroke is the number one cause of death due to its particularly high incidence in Asia. Ischemic stroke is the most common form of stroke, being responsible for about 85% of all strokes, whereas hemorrhagic strokes (e.g. intraparenchymal or subarachnoid) account for the remaining 15%. Due to the increasing mean age of the population, the number of strokes is continuously increasing. Because the brain is highly vulnerable to even brief ischemia and recovers poorly, primary prevention in ischemic stroke prevention offers the greatest potential for reducing the incidence of this disease.
Focal ischemic cerebral infarction occurs when the arterial blood flow to a specific region of the brain is reduced below a critical level. Cerebral artery occlusion produces a central acute infarct and surrounding regions of incomplete ischemia (sometimes referred to as ‘penumbra’), that are dysfunctional-yet potentially salvageable. Ischemia of the myocardium, as a result of reduced perfusion due to chronic narrowing of blood vessels, may lead to fatal heart failure and constitutes a major health threat. Acute myocardial infarction, triggered by coronary artery occlusion, produces cell necrosis over a time period of several hours. In the absence of reflow or sufficient perfusion, the cerebral or myocardial ischemic regions undergo progressive metabolic deterioration, culminating in infarction, whereas restoration of perfusion in the penumbra of the brain infarct or in the jeopardized but salvageable region of the myocardium may ameliorate the tissue damage.
Growth factor mediated improved perfusion of the penumbra in the brain or of the jeopardized myocardium of patients suffering ischemic events, either via increased vasodilation or angiogenesis (the formation of endothelial-lined vessels), may be of great therapeutic value according to Isner et al. in J. Clin. Invest. (1999) 103(9):1231-6 but many questions yet remain to be answered in this respect. This is also true for ischemic conditions related to peripheral limbs (eg peripheral arterial disease, peripheral ischemia). For instance, an outstanding question is whether formation of new endothelial-lined vessels (i.e. angiogenesis) alone is sufficient to stimulate sustainable functional tissue perfusion. Indeed, coverage of endothelial-lined vessels by vascular smooth muscle cells (i.e. arteriogenesis) provides vasomotor control, structural strength and integrity and renders new vessels resistant to regression.
Capillary blood vessels consist of endothelial cells and pericytes, which carry all the genetic information required to form tubes, branches and entire capillary networks. Specific angiogenic molecules can initiate this process. A number of polypeptides which stimulate angiogenesis have been purified and characterized as to their molecular, biochemical and biological properties, as reviewed by Klagsbrun et al. in Ann. Rev. Physiol. (1991) 53:217-239 and by Folkman et al. in J. Biol. Chem. (1992) 267:10931-4. One factor that can stimulate angiogenesis and which is highly specific as a mitogen for vascular endothelial cells, is termed vascular endothelial growth factor (hereinafter referred as VEGF) according to Ferrara et al. in J. Cell. Biochem. (1991) 47:211-218. VEGF is also known as vasculotropin. Connolly et al. also describe in J. Biol. Chem. (1989) 264:20017-20024, in J. Clin. Invest. (1989) 84:1470-8 and in J. Cell. Biochem. (1991) 47:219-223 a human vascular permeability factor that stimulates vascular endothelial cells to divide in vitro and promotes the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The term vascular permeability factor (VPF for abbreviation) was adopted because of increased fluid leakage from blood vessels following intradermal injection and appears to designate the same substance as VEGF. The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in chloroid plexus and kidney glomeruli, which is consistent with its role as a multifunctional regulator of endothelial cell growth and differentiation as disclosed by Breier et al. in Development (1992) 114:521-532. VEGF shares about 22% sequence identity, including a complete conservation of eight cysteine residues, according to Leung et al. in Science (1989) 246:1306-9, with human platelet-derived growth factor PDGF, a major growth factor for connective tissue. Alternatively spliced mRNAs have been identified for both VEGF and PDGF and these splicing products differ in their biological activity and receptor-binding specificity. VEGF is a potent vasoactive protein that has been detected in and purified from media conditioned by a number of cell lines including pituitary cells, such as bovine pituitary follicular cells (as disclosed by Ferrara et al. in Biochem. Biophys. Res. Comm. (1989) 161:851-858 and by Gospodarowicz et al. in Proc. Natl. Acad. Sci. USA (1989) 86: 7311-5), rat glioma cells (as disclosed by Conn. et al. in Proc. Natl. Acad. Sci. USA (1990) 87:1323-1327) and several tumor cell lines. Similarly, an endothelial growth factor isolated from mouse neuroblastoma cell line NB41 with an unreduced molecular mass of 43-51 kDa has been described by Levy et al. in Growth Factors (1989) 2:9-19.
Recent data show that VEGF has a modest effect on collateral growth and limb perfusion measured by microspheres (Deindi et al. in Circ. Res. (2001) 89:779-86), but increases perfusion measured by laser doppler (Isner et al. in Circulation (1999) 99:3188-98). Thus, despite a slight increase in collateral growth and hind limb perfusion, the overall functional reserve after VEGF treatment was impaired, presumably because of unfavorable hemodynamic effects. In other words, it is known that despite vessel formation by means of VEGF, the functionality of the vessels formed is very low. Despite promising initial clinical trials, a large double-blind placebo-controlled trial testing the efficacy of intracoronary and intravenous VEGF for therapeutic angiogenesis in patients with chronic myocardial ischemia not amenable to standard revascularization techniques, indicated that even high dose treatment with VEGF did not lead to a significant improvement in ETT time (Henry et al. 2003, Circulation, 107:1359-1365).
Placental growth factor (hereinafter referred as PIGF) was first disclosed by Maglione et al. in Proc. Natl. Acad. Sci. USA (1991) 88(20):9267-71 as a protein related to the vascular permeability factor. U.S. Pat. No. 5,919,899 discloses nucleotide sequences coding for a protein, named PIGF, which can be used in the treatment of inflammatory diseases and in the treatment of wounds or tissues after surgical operations, transplantations, burns of ulcers and so on. Soluble non-heparin binding and heparin binding forms, built up of 131 and 152 amino-acids and known as PIGF-1 and PIGF-2 respectively, have been described for PIGF and were found to be differentially expressed in placenta, trophoblastic tumors and cultured human endothelial cells (Maglione et al., 1993).
Carmeliet et al. Nat. Med. (2001) 7:575-583 provides an extensive and comprehensive review of the state of the art related to PIGF as follows. VEGF stimulates angiogenesis by activating the VEGF tyrosine kinase receptor-2 (VEGFR-2). Recombinant PIGF stimulates angiogenesis in particular conditions and induces vascular permeability when co-injected with VEGF, but the role of endogenous PIGF remains unknown. Both PIGF and VEGF bind to VEGF receptor-1 (VEGFR-1). PIGF has been proposed to stimulate angiogenesis by displacing VEGF from the ‘VEGFR-1 sink’, thereby increasing the fraction of VEGF available to activate VEGFR-2. Alternatively, PIGF might stimulate angiogenesis by transmitting intracellular signals through VEGFR-1. PIGF might also affect angiogenesis by forming heterodimers with VEGF, but their role is controversial.
The role of PIGF in angiogenesis was evaluated by inactivating the gene expressing PIGF (Pgf) in mice. Unexpectedly, the absence of PIGF had a negligible effect on vascular development. However, PIGF deficiency reduced pathological angiogenesis, permeability and collateral growth in ischemia, inflammation and cancer.
Carmeliet et al. (cited supra) studied the growth of collateral arteries after ligation of the femoral artery in mice and found that PIGF levels were 45% higher in ligated vessels than in control vessels. On the other hand macrophages, known to play an essential role in collateral growth, infiltrated significantly more collaterals in wild-type mice than in Pgf−/− mice. In another experiment, mice were transplanted with congenic bone marrow and capillary ingrowth in matrigel, supplemented with the VEGF165 isoform, and angiogenesis was quantified by measuring the hemoglobin content per implant. Matrigel angiogenesis was abundant when both the donor bone marrow and the host vessels produced PIGF but minimal when the bone marrow and host lacked PIGF. Capillaries still infiltrated the matrigel in wild-type mice transplanted with Pgf−/− bone marrow, indicating that PIGF production by vessel-wall-associated endothelial cells was sufficient for angiogenesis. When a Pgf−/− recipient was transplanted with wild-type bone marrow, matrigel angiogenesis still occurred, indicating that production of PIGF by bone-marrow cells can stimulate angiogenesis at distant sites. Transplantation of wild-type bone marrow in Pgf−/− mice also rescued the impaired collateral enlargement after ligation of the femoral artery, possibly by mobilizing PIGF-producing monocytes/macrophages to the collaterals. In still another experiment, in order to determine whether the amplification of the VEGF response by PIGF resulted from direct stimulation of endothelial cells, Carmeliet et al. (cited supra) studied capillary outgrowth in intact aortic rings and found that, because PIGF alone (in the absence of VEGF) did not stimulate isolated endothelial cells, PIGF probably stimulated capillary outgrowth by amplifying endogenous VEGF in aortic rings. Carmeliet et al. (cited supra) further observed that even at low doses PIGF dose-dependently restored the impaired VEGF response of Pgf−/− endothelial cells. Further, PIGF activated VEGFR-1. These findings altogether indicate that PIGF, via activation of VEGFR-1, specifically potentiates the angiogenic response to VEGF and that by affecting vascular growth and remodeling, PIGF contributes to the pathogenesis of several disorders with high morbidity.
One problem to be solved by the present invention is to provide a pharmaceutical composition and methods particularly suited for improving perfusion of tissues of patients suffering ischemic events, which will prove to be useful for the prevention and treatment of strokes and ischemic diseases. Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods for reducing or suppressing infarct expansion of the penumbra during ischemic cerebral infarction and for enhancing revascularization of acute myocardial infarcts, making them useful for preventing and treating such events. Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods for enhancing revascularization of tissues in limb-threatening ischemia making them useful for treating such a disease.
Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods not only for enhancing vascularization but for restoring the functionality of ischemic tissue, in particular ischemic muscles such as the cardiac muscle and skeletal muscles in the head, neck, thorax, abdomen, back and upper and lower limbs. There is also a need in the art for curing a reduced physical muscular performance, especially that of heart and the aforesaid muscles after an ischemic event in a mammal, in particular a human being. Another problem to be solved by the present invention is providing a pharmaceutical and methods which can be used as described above without incurring the well known disadvantages of some growth factors such as VEGF, e.g. hypotension after systemic administration of high doses over short periods of time (see for instance Hariawala et al. in J. Surg. Res. (1996) 63(1):77-82).
The various above-mentioned goals of the present invention have been successfully and unexpectedly satisfied by a suitable use of placenta growth factor or a fragment, derivative or homologue thereof or alternatively the use of a combination of PIGF and VEGF such as disclosed hereinafter.
The present invention is based on a first unexpected finding that PIGF increases VEGF expression in places where VEGF is already up-regulated, due to ischemic disease conditions, i.e. in places where VEGF is needed. The present invention is also based on a second unexpected finding that PIGF simultaneously stimulates both smooth muscle cells and endothelial cells, whereas VEGF preferentially stimulates endothelial cells. Other observations of the present inventors are that PIGF significantly increases capillary vessel density in muscles, that it is much more efficient than VEGF in stimulating collateral arteriolar growth (for instance in ischemic myocardium or in an ischemic limb). This results in an improved function of the ischemic tissue, more particularly, strongly increased limb motoric function (as could be measured in an endurance swim test). This is corroborated by limb perfusion data showing that, contrary to laser Doppler measurements, perfusion measured by microspheres is quite different in ischemic limbs treated with VEGF and PIGF respectively. PIGF efficiency is thus associated with improved perfusion as a result of constructive angiogenesis, arteriogenesis and collateral vessel growth. These qualities are of particular interest for the restoration of ischemic tissue.
The present invention is further based on the finding that administration of PIGF and VEGF has a synergistic effect in the treatment of in an animal ischemic myocardium and in suppressing infarct expansion of the penumbra.
The present invention is further based on the important finding that treatment with PIGF has less systemic hemodynamic side effects (e.g. loss of blood pressure, edema, hemangioma) than VEGF.
In view of these observations, the present invention first provides the use of PIGF for the treatment and prevention of ischemic disease, and for restoring the function of ischemic tissue in a mammal. The present invention also provides method for curing a reduced physical muscular performance in a mammal after an ischemic event, the said method comprising administering to the said mammal a therapeutically effective amount of a placental growth factor.
The present invention also relates to a method of treatment or prevention of a stroke (including a hemorrhagic stroke) or an ischemic disease in a mammal, comprising administering to the mammal in need of such treatment or prevention a therapeutically effective amount of placental growth factor, a fragment, a derivative or a homologue thereof or a combination of PIGF with VEGF.
The present invention also relates to a method of reducing infarct size during treatment of an ischemic disease in a mammal, comprising administering to the mammal in need of such treatment or prevention a therapeutically effective amount of placental growth factor, a fragment, a derivative or a homologue thereof.
A further aspect of the invention provides for methods of treatment or prevention of a stroke (including a hemorrhagic stroke) or an ischemic disease in a mammal, using gene transfer. More particularly the invention provides for methods of treatment or prevention of ischemia in a muscle of a mammal, which comprise administering to a mammal in need thereof, a nucleic acid which is capable of increasing expression of PIGF in the mammal upon administration thereto. Most particularly, the methods of the invention relate to the treatment or prevention of ischemia in skeletal muscle.
According to one embodiment of this aspect of the invention an increase in expression of PIGF is achieved by administering a nucleic acid comprising a sequence encoding PIGF. The sequence encoding PIGF is most particularly under control of a promotor directing expression, more particularly a promoter that is strongly expressed in skeletal muscle. The present application envisages different methods of gene transfer which ensure appropriate expression of PIGF, such as, but not limited to methods wherein one or more nucleic acids capable of increasing expression of PIGF are administered by electroporation and methods wherein the nucleic acid administered is an adenoviral vector.
Particular embodiments of this aspect of the invention relate to methods of therapy and prevention of ischemic muscle tissue in humans. One particular embodiment relates to the treatment and prevention of ischemic muscles in hemodynamically compromised mammals, for which the methods of the invention are of particular interest, in view of the limited side-effects of PIGF.
The invention further provides methods for the treatment of ischemia in skeletal muscles, whereby the treatment results in functional improvement of the ischemic skeletal muscle. Such improvement can be evaluated by means of an endurance exercise test wherein the ischemic skeletal muscle under treatment is put under a physical stress condition, and whereby the function of the muscle is evaluated after treatment using the methods of the present invention. According to one embodiment functional improvement is considered relative to the function of the muscle (immediately) after the ischemic event. Additionally or alternatively, functional restoration can be considered relative to the function of the muscle prior to the ischemic event.
Particular embodiments of the invention relate to methods of treatment and prevention of ischemic muscles, which comprise administering an amount of one or more nucleic acids which ensure increased expression of PIGF, whereby the increased expression results in a concentration of at least 10 ng PIGF/ml blood between day 3 and day 10 after administration of the nucleic acid.
The present invention also relates to a method of reducing infarct size during treatment of an ischemic disease in a mammal, comprising administering to the mammal in need of such treatment or prevention a therapeutically effective amount of one or more nucleic acids which ensure increased expression of PIGF.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
In the present invention “Placental growth factor” or “PIGF” should be understood as including all naturally-occurring PIGF isoforms, including the isoforms PIGF-1, PIGF-2 and PIGF-3, as well as PIGF-4, which consists of the sequence of PIGF-3, plus the heparin binding domain present only in PIGF-2. General reference to PIGF also includes variants having one or more internal deletions or mutations of one or more amino-acids, provided that the said variants have at least 75%, preferably at least 80% and more preferably at least 90%, sequence identity with one or more of the aforesaid isoforms. It includes both the full-length protein and modified versions such as N-terminally and/or C-terminally truncated versions of PIGF. The term PIGF relates to both natural and synthetic or recombinant versions of the protein.
According to one embodiment, PIGF according to the present invention is placental growth factor originating from a mammal or corresponding to a mammalian, most particularly a human PIGF. According to a further embodiment the PIGF envisaged for use as a medicament or for prophylactic use on a species preferably originates from that same species or corresponds to a PIGF of that same species (e.g. use of human PIGF for treating humans), although the development of a pharmaceutical of PIGF from one species for treatment of another species is not excluded (e.g. administration of murine, porcine, bovine or primate PIGF to humans).
The terms “growth factor encoding polynucleotide (or nucleic acid)” and “polynucleotide (or nucleic acid) encoding PIGF or PIGF-encoding polynucleotide” as used herein refer to a nucleotide sequence, which, when expressed in an appropriate environment, results in the generation of the relevant growth factor or PIGF protein sequence or a biologically active fragment or derivative thereof. Such polynucleotides or nucleic acids include the natural sequences encoding the growth hormones (the natural sequences encoding PIGF) as well as derivatives and fragments of these nucleic acids capable of expressing a biologically active protein.
According to one embodiment, the nucleic acid encoding PIGF according to the present invention is a sequence encoding placental growth factor originating from a mammal or corresponding to a mammalian, most particularly a human PIGF. Most particular embodiments of the present invention relate to sequences corresponding to or derived from human mRNA encoding for PIGF-1 (149AA; such as X54936), PIGF-2 (170AA, such as S72960), PIGF-3 (221AA; such as P49763) or PIGF-4. According to a further embodiment the nucleic acid encoding PIGF envisaged for use as a medicament or for prophylactic use on a species preferably originates from that same species or corresponds to a nucleic acid encoding PIGF of that same species (e.g. use of nucleic acid encoding human PIGF for treating humans), although the development of a pharmaceutical of PIGF from one species for treatment of another species is not excluded (e.g. administration of murine, porcine, bovine or primate nucleic acid encoding PIGF to humans).
The term “vascular endothelial growth factor” or “VEGF” as used herein refers, whether of human or animal origin, to all isoforms thereof such as disclosed in the section BACKGROUND OF THE INVENTION. A specific embodiment of the invention relates to the 165 amino-acid isoform of VEGF.
“bFGF”, as used herein or “basic Fibroblast Growth Factor”, refers to all isoforms of basic Fibroblast Growth factor, which is a wide-spectrum mitogenic, angiogenic and neurotrophic factor that is expressed at low levels in many tissues and reaches high concentrations in brain and pituitary.
“ischemia” and “ischemic” is defined herein as a local anemia due to mechanical (e.g. atherosclerotic) obstruction (mainly arterial narrowing or disruption of the blood supply) of the blood vessels. An ischemic organ is an organ to which, as a result of constriction or blockage of the blood vessels supplying it, there is inadequate flow of blood. Typical examples of ischemic organs are heart, kidney and brain.
The term “ischemic disease” thus generally relates to diseases whereby one or more organs or tissues are affected by ischemia. Examples of ischemic diseases, involving particular ischemic organs include, among others: ischemic stroke or focal ischemic cerebral infarction, acute myocardial infarction or coronary ischemia, chronic ischemic heart disease, ischemic disease of an organ other than myocardium or a region of the brain, for instance a peripheral limb (e.g. limb ischemia or peripheral arterial disease). Accordingly, the ischemic tissue concerned may be any tissue of the mammalian body.
The term “ischemic muscles” relates to muscles such as heart (myocardium)(also referred to as ischemic myocardium) and skeletal muscles (also referred to as ischemic skeletal muscles).
Peripheral ischemia as used herein refers to a condition of decreased blood supply to one or more limbs. Typically this is connected to Peripheral arterial disease (PAD). The term “myocardial ischemia” is used to refer to decreased blood supply to the heart.
Peripheral ischemia can be either acute or chronic. Typical causes of acute ischemia include embolism, thrombosis, dissection, venous occlusion, trauma. Typical etiologies of chronic ischemia include chronic atherosclerosis, aneurysm, entrapment syndromes, trauma, and a variety of non-atherosclerotic occlusive entities.
In all of the various aspects of the present invention, the term “mammal” is considered in its common meaning and includes namely humans, equines, felines, canines, porcines, bovines, ovines and the like.
“infarct” herein means any area of necrosis due to a sudden insufficiency of arterial or venous blood supply.
“spontaneous mobility” is defined herein as being the muscle mobility under normal conditions, i.e. without load or stress; examples include the walking at normal speed of a human before occurrence of the ischemic event.
The terms “functional improvement” and “functional restoration or recovery” refer to a partial and/or complete, but in any case significant improvement or restoration of at least one aspect of muscle function or of the muscle reserve of an ischemic tissue. This is determined, e.g. by comparing the functional efficiency of the tissue after treatment to the functional efficiency of the same tissue immediately prior to (restoration) and/or subsequent to (improvement) the ischemic event in the patient and optionally with reference to the functional efficiency of that tissue in a healthy population. Typically, the determination of one or more aspects of muscle function (e.g. mobility, strength, endurance, performance) will be indicative of overall functional improvement. Most tests combine one or more of these aspects of muscle function. Functional restoration or improvement of a muscle tissue can for instance be evaluated and/or quantified by means of an endurance exercise test. Optionally, the ischemic tissue or muscle is thereby subjected to a normal physical effort (i.e. a physical activity) optionally combined with load stress (i.e. stress induced by a load) condition. Additionally or alternatively, the degree of functional improvement or restoration is measured by means of an exercise test which reflects the function of the ischemic tissue (e.g. exercise treadmill test for heart function). Most preferably, according to the present invention, improvement or restoration obtained by PIGF treatment is at least 30%, more preferably at least 50%, most preferably at least 60%.
“stress condition” refers to a condition significantly more stringent than spontaneous mobility, such as the practice of a sport or physical work involving an effort such as running (as opposed to normal walking), jumping, swimming and the like. The test involved may also include one or more conditions such as load, torsion or similar.
The “hemodynamic side effects” referred to herein relate to disturbances of different aspects of the normal blood flow. More particularly, in the context of the present invention, this relates to (systolic) hypotension, the formation of hemangioma's, edema, ectopic angiogenesis, anemia, neointimal proliferation and renal toxicity.
The feature “susceptible to hemodynamic side-effects” when referring to a patient as used herein refers to the feature whereby, due to circumstances not directly related to the disease (such as, but not limited to, age, genetic disposition, diet, medication, surgery), the patient is more susceptible to the hemodynamic side effects described above. Indeed, according to a particular aspect of the invention, the absence of one or more side effects upon treatment with a medicament is translated into a particularly beneficial use thereof for the treatment of particular disorders, for particular regimens and/or for the treatment of particular patient group, susceptible to the occurrence of such side effects.
A “hemodynamically compromised patient” as used herein refers to a patient with a sub-normal oxygenation of tissues caused for instance by hypotension or severe tachycardia. More particularly, according to one aspect of the present invention, the patient can be defined as having a systolic blood pressure of <100 mm Hg.
A “patient not responsive to VEGF treatment” as used herein refers to a patient in which treatment with VEGF alone (not combined with other growth factors), at doses which do not cause hemodynamic side effects, does not result in significant clinical improvement, more particularly does not result in a significant (and preferably long-term) improvement of the parameters used to measure angiogenic efficacy (such as but not limited to ETT, angina time and angina frequency). An example of such a patient group is described by Henry et al. (2003, Circulation, 107:1359-1365).
By “therapeutically effective amount” is meant an amount of PIGF which produces the desired therapeutic effect in a patient. For example, in reference to a disease or disorder, it is the amount which reduces to some extent one or more symptoms of the disease or disorder, and preferably returns to normal, either partially or completely, physiological or biochemical parameters associated or causative of the disease or disorder. Preferably, according to one aspect of the present invention, the therapeutically effective amount is the amount of PIGF which will lead to an improvement or restoration of function of the ischemic tissue. For instance, when used to therapeutically treat a mammal affected by reduced physical muscular performance, it is a daily amount between about 2 μg/kg and 50 mg/kg, preferably between about 0.05 mg/kg and about 2 mg/kg or less than 10 mg/kg, more preferably less than 1 mg/kg body weight of the said mammal. These dosages can be administered either directly. Alternatively, where the administration is through gene-therapy, the amount of naked DNA or viral vectors is adjusted to ensure the local production of the relevant dosage of PIGF.
According to the present invention, dosage of PIGF may be significantly higher, can be administered at a higher rate and/or may be administered to a patient for longer periods of time, than can be administered of VEGF without the occurrence of hemodynamic side effects. More particularly, such a dose can be higher than 15 μg/kg/day, up to 100 μg/kg/day and higher. Alternatively, such a dose can be significantly higher than 50 ng/kg/min, including 100 ng/kg/min and higher.
The term “homologue” as used herein with reference to growth factors of the present invention refers to molecules having at least 50%, more preferably at least 70% and most preferably at least 90% amino acid sequence identity with the relevant growth factor.
As used herein, the term “biologically active” in reference to a protein, protein fragment or derivative thereof is defined as an ability of the nucleic acid or amino acid sequence to mimic a known biological function elicited by the wild type form of the protein. Unless specified, when referring to a derivative or fragment of PIGF, a biologically active protein is a protein having at least 50% of the activity of PIGF (angiogenic activity, more particularly the ability to induce the generation of collaterals) under comparable conditions in vitro. The term “fragment” as used herein with reference to growth factors of the present invention refers to molecules which contain the active portion of the growth factor, i.e. the portion which is functionally capable of improving perfusion or reducing or suppressing infarct expansion or otherwise enhancing revascularization of infarcts, and which may have lost a number of non-essential (with respect to angiogenesis and/or arteriogenesis) properties of the parent growth factor. Preferably the fragment used in the present invention is the angiogenetic and/or arteriogenetic fragment of the relevant growth factor.
The term “derivative” as used herein with reference to growth factors of the present invention refers to molecules which contain at least the active portion of the growth factor (as defined herein above) and a complementary portion which differs from that present in the wild-type growth factor, for instance by further manipulations such as introducing mutations. Derivatives of a growth factor thus include molecules having the essentially the same biological activity as the growth factor and having at least 80% amino acid sequence identity with the relevant growth factor, more particularly at least 90%, most particularly between 95 and 99% sequence identity at the amino acid level with the growth factor. Similarly derivatives of a fragment of a growth factor refer to biologically active fragments having a modified amino acid sequence compared to the native sequence of the growth factor (i.e. an amino acid sequence identity with the relevant fragment of between 80-99%). A derivative of a growth factor- or PIGF-encoding nucleic acid as used herein is a nucleic acid encoding a biologically active protein whereby the sequence of the nucleic acid has been modified with respect to the naturally occurring growth factor or PIGF.
The “sequence identity” of two sequences as used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, when the two sequences are aligned. In particular embodiments said sequence identity is higher than 70%-80%, more particularly 81-85%, more preferably 86-90%, especially preferably 91-95%, most preferably 96-100%, more specifically is 100%.
The present invention demonstrates that PIGF is capable of reducing infarct size caused by occlusion of the middle cerebral artery in a mammal and that in doing so, PIGF is as effective as VEGF. Furthermore, in an ischemic mammal, placental growth factor stimulates, better than VEGF, the formation of new endothelial-lined vessels and maturation of these vessels by coverage with vascular smooth muscle cells (arteriogenesis). Thus, according to a first aspect, the present invention provides the use of PIGF for the treatment and prevention of ischemic disease. The present invention thus relates to a method of treatment or prevention of a stroke (including a hemorrhagic stroke) or an ischemic disease in a mammal, comprising administering to the mammal in need of such treatment or prevention a therapeutically effective amount of placental growth factor, a fragment, a derivative or a homologue thereof.
According to this aspect of the invention, PIGF has significant arteriogenic activity upon administration to mammals suffering from ischemic disease. Thus, the present invention is particularly suited for the treatment of patients in need of improved collateral flow to ischemic tissues, e.g. patients with viable but underperfused myocardium as a result of chronic myocardial ischemia, more particularly those patients who are considered unsuitable (on the basis of coronary angiography) for standard revascularization techniques.
The present invention further demonstrates restoration or improvement of the function of an ischemic tissue of a mammal upon treatment with PIGF and that this restoration is significantly better than upon treatment with VEGF. Thus, a second aspect of the invention relates to the use of PIGF for restoring the function of ischemic tissue in a mammal. Within this aspect, the following particulars are preferred, but not limitative: the ischemic tissue may be a muscle such as cardiac muscle (myocardium) or a skeletal muscle. The latter may be selected from the musculature of head and neck, the musculature of thorax and abdomen, the musculature of back and the musculature of upper and lower limbs. A non-exhaustive list of skeletal muscles for which the treatment of the invention will be beneficial includes the following: sternocleidomastoid, trapezius, deltoid, pectoralis major, serratus anterior, external oblique abdominal, sartorius, platysma, risorius, depressor anguli oris, depressor labili inferioris, mentalis, orbicularis oris, zygomaticus minor, levator labili superioris, nasalis, zygomaticus major, orbicularis oculi, anterior scalene, levator scapulae, splenius capitis, masseter, auricularis posterior, auricularis superior, auricularis anterior, temporoparietalis, epicranius, semispinalis capitis, triceps brachii, teres major, teres minor, infraspinatus, latissimus dorsi, gluteus maximus, anconeus, extensor carpi ulnaris, extensor digitorum, extensor pollicis longus, extensor retinaculum, dorsal interosseus, adductor pollicis, extensor pollicis brevis, adductor pollicis longus, extensor carpi radialis, brachioradialis, brachialis, piriformis, obturator internus, adductor magnus, semimembranosus, gracilis, semitendinosus, gastronecmius, vastus medialis, rectus femoris, adductor longus, biceps femoris, vastus lateralis, quadriceps femoris, tibialis anterior and peroneus longus.
According to one embodiment of this aspect of the invention, the functional restoration or improvement of ischemic tissue occurs together with an increased transport of particles or oxygen through arteries and/or veins of the said ischemic tissue, as compared with the corresponding transport before use of the said medicament.
The (degree of) functional restoration or improvement of ischemic tissue can be evaluated in different ways, depending on the tissue concerned. The improvement of function in an ischemic limb is preferably evaluated by means of an endurance exercise test wherein the ischemic tissue under treatment is put under a physical stress or a load stress condition. Alternative assays for measuring function of ischemic tissue are described in the art.
According to this aspect of the invention, the use of PIGF results in a restoration or improvement of function. Thus, the present invention is particularly suited for the treatment of patients in need of improved tissue function, e.g. patients reduced heart function. More particularly the use of PIGF is suited for patients not responding to other angiogenic growth factors such as VEGF.
A third aspect of the invention is a method for curing a reduced physical muscular performance in a mammal after an ischemic event, the said method comprising administering to the said mammal a therapeutically effective amount of PIGF. According to this aspect of the invention, PIGF for use in post-ischemic therapeutic treatment is provided.
Preferably, the reduced physical muscular performance to be cured is the performance of a skeletal muscle (e.g. as listed above) or the cardiac muscle.
Preferably, according to this aspect of the invention, curing success is evaluated, preferably quantified by means of an endurance exercise test wherein the ischemic muscle under treatment is put under a physical or load stress condition or by measuring the performance in an exercise test which reflects the function of the ischemic muscle.
A fourth aspect of the invention relates to the use of compositions comprising PIGF and VEGF, as active ingredients in respective proportions such as to provide a synergistic effect in the prevention or treatment of strokes and ischemic diseases in mammals. In view of the hemodynamic side-effects observed with higher doses of VEGF, the combination of VEGF with a growth factor which not only provides a synergistic effect but moreover does not to the same extent cause these side effects may be of particular value for the treatment or prevention of particular diseases, or for a particular patient group.
According to the present invention, a medicament is provided for the treatment and prevention of ischemic disease, which does not significantly cause the hemodynamic side-effects of other known growth factors. More particularly, it is demonstrated that the treatment of an ischemic mammal with PIGF has a much more limited effect on systolic blood pressure and causes less edema than VEGF, which may be of use for the patients in need thereof. Thus, according to a fourth aspect of the present invention, the use of PIGF is provided for the treatment of patients who are particularly susceptible to hemodynamic side-effects or are hemodynamically compromised or who are not responsive to VEGF, most particularly those patients not responding positively to high doses of VEGF. Moreover, the present invention provides the use of placental growth factor, where prolonged treatment is required, e.g. for the treatment of chronic ischemic diseases.
The present invention furthermore relates to the manufacture of a medicament for the treatment and prevention of the conditions and diseases described above.
In the methods of treatment or prevention constituting various aspects of the present invention, the administration of active ingredient(s) may be chronic or intermittent, depending on the medical status and need of the mammal. According to one aspect of the present invention, the mammal particularly suited for the treatment with PIGF is a mammal suffering from an ischemic disease, particularly in cases where standard revascularization techniques are not advisable. Treatment of mammals with myocardial ischemia with PIGF in the context of the present invention is envisaged both immediately after myocardial infarction and up to or starting from a period thereafter, including 6 months after infarction.
In the various aspects of the present invention where both placenta growth factor and vascular endothelial growth factor are present as biologically active ingredients (a) and (b) respectively, they are preferably used as a combined preparation comprising from about 1 to about 99% by weight of ingredient (a) and from about 1 to about 99% by weight of ingredient (b) for simultaneous, separate or sequential use. In view of the fact that ingredients (a) and (b) do not necessarily bring out their joint therapeutic effect directly at the same time in the mammal to be treated or prevented, the pharmaceutical compositions of the present invention which comprise both ingredients (a) and (b) may be in the form of a medical kit or package containing the two ingredients in separate but adjacent form. In the latter context, each of ingredients (a) and (b) may therefore be formulated in a way suitable for an administration route different from that of the other ingredient.
The optimal formulation and mode of administration of PIGF (optionally combined with VEGF) to a patient depend on factors known in the art such as the particular disease or disorder, the desired effect, and the type of patient. Preferably, the therapeutically effective amount is provided as a pharmaceutical or veterinary composition. Suitable forms in part depend upon the mode of administration, for example oral, transdermal, or by injection into the blood stream. Such forms should allow PIGF to efficiently reach the target cells. For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which would prevent the composition from exerting its effect.
In the pharmaceutical compositions constituting various aspects of the present invention, the term “pharmaceutically acceptable carrier” means any material or substance with which the active ingredient(s) is formulated in order to facilitate its application or dissemination, for instance by dissolving, dispersing or diffusing the said ingredient, and/or to facilitate its storage, transport or handling without impairing its effectiveness. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, pellets or powders. However a formulation suitable for subcutaneous use is highly preferred.
Suitable pharmaceutical carriers for use in the present compositions are well known to those skilled in the art, and there is no particular restriction to their selection within the invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents may also be prepared by micronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 μm, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients.
Suitable surface-active agents to be used in the pharmaceutical compositions of the present invention are non-ionic, cationic and/or anionic materials having good emulsifying, dispersing and/or wetting properties. Suitable anionic surfactants include both water-soluble soaps and water-soluble synthetic surface-active agents. Suitable soaps are alkaline or alkaline-earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures obtainable form coconut oil or tallow oil. Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulphonates and sulphates; sulphonated benzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates or sulphates are usually in the form of alkaline or alkaline-earth metal salts, unsubstituted ammonium salts or ammonium salts substituted with an alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium or calcium salt of lignosulphonic acid or dodecylsulphonic acid or a mixture of fatty alcohol sulphates obtained from natural fatty acids, alkaline or alkaline-earth metal salts of sulphuric or sulphonic acid esters (such as sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are the sodium, calcium or alcanolamine salts of dodecylbenzene sulphonic acid or dibutyl-naphtalenesulphonic acid or a naphtalene-sulphonic acid/formaldehyde condensation product. Also suitable are the corresponding phosphates, e.g. salts of phosphoric acid ester and an adduct of p-nonylphenol with ethylene and/or propylene oxide, or phospholipids. Suitable phospholipids for this purpose are the natural (originating from animal or plant cells) or synthetic phospholipids of the cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin, dioctanylphosphatidyl-choline, dipalmitoylphoshatidyl-choline and their mixtures.
Suitable non-ionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides containing at least 12 carbon atoms in the molecule, alkylarenesulphonates and dialkylsulphosuccinates, such as polyglycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives preferably containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Further suitable non-ionic surfactants are water-soluble adducts of polyethylene oxide with poylypropylene glycol, ethylenediaminopolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ether groups. Such compounds usually contain from 1 to 5 ethyleneglycol units per propyleneglycol unit. Representative examples of non-ionic surfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolic ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethyleneglycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable non-ionic surfactants.
Suitable cationic surfactants include quaternary ammonium salts, preferably halides, having 4 hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy; for instance quaternary ammonium salts containing as N-substituent at least one C8-C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl and the like) and, as further substituents, unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy-lower alkyl radicals.
A more detailed description of surface-active agents suitable for this purpose may be found for instance in “McCutcheon's Detergents and Emulsifiers Annual” (MC Publishing Crop., Ridgewood, N.J., 1981), “Tensid-Taschenbuch”, 2ed. (Hanser Verlag, Vienna, 1981) and “Encyclopaedia of Surfactants (Chemical Publishing Co., New York, 1981).
Additional ingredients may be included in order to control the duration of action of the active ingredient in the pharmaceutical composition of the invention. Control release compositions may thus be achieved by selecting appropriate polymer carriers such as for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like. The rate of drug release and duration of action may also be controlled by incorporating the active ingredient into particles, e.g. microcapsules, of a polymeric substance such as hydrogels, polylactic acid, hydroxymethylcellulose, polymethyl methacrylate and the other above-described polymers. Such methods include colloid drug delivery systems like liposomes, microspheres, microemulsions, nanoparticles, nanocapsules and so on.
Carriers or excipients may be used to facilitate administration of PIGF. Examples of suitable carriers and excipients include fillers, various sugars such as lactose, glucose, sucrose, starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
PIGF may be formulated in solid form and dissolved or suspended immediately prior to use. Lyophilized forms for the extemporaneous preparation also included.
The pharmaceutical (or veterinary) composition of the present invention may be administered by different routes including intravenously, intraperitoneally, subcutaneously, intramuscularly, orally, topically or transmucosally. PIGF may be formulated for either systemic or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences (1990), 18th ed., Mack Publishing Co., Easton, Pa. A suitable administration form may best be determined by a medical practitioner for each patient individually.
For systemic administration, injection is preferred. Typical carriers for this purpose therefore include biocompatible aqueous buffers (such as Hank's solution or Ringer's solution) and excipients being generally accepted as safe as defined by USP standards (e.g., ethanol, glycerol, propylene glycol, polyethylene). It can e.g. be suspended in an inert oil, suitably a vegetable oil such as sesame, peanut, olive oil, or other acceptable carrier. Preferably, it suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 5.6 to 7.4. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. They may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents. Useful buffers include for example, sodium acetate/acetic acid buffers.
Systemic administration can also be by transmucosal or transdermal means or orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or suppositories. For oral administration, the therapeutically active substance is formulated into conventional oral administration dosage forms such as capsules, tablets or liquid preparations.
For topical administration, PIGF may be formulated into ointments, gels or creams as is generally known in the art. If desired the above compositions may be thickened with a thickening agent such as methylcellulose. They may be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant (such as Tween®), or an ionic surfactant (such as an alkali polyether alcohol sulfate or sulfonate, e.g. Triton®).
Pharmaceutical or veterinary compositions useful in the invention may be prepared by mixing the above-mentioned ingredients following generally accepted procedures. For example, the said ingredients may be simply mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final desired concentration and viscosity by the addition of water and/or a thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
PIGF may also be used according to gene therapy methods well known in the art. Different methods of achieving, by way of gene therapy, increased levels of PIGF in a mammal in vivo are envisaged within the context of the present invention. Accordingly, the present invention provides for the use of a nucleic acid which is capable of increasing expression of PIGF in vivo, for the treatment and/or prevention of ischemic tissue, more particularly ischemic muscle, such as ischemic skeletal muscle. According to one embodiment, the nucleic acid capable of increasing expression of PIGF in vivo is a sequence encoding PIGF, which is operably linked to a promoter. Such a sequence can be administered directly or indirectly. For instance, an expression vector containing the PIGF coding sequence may be inserted into cells, after which the said cells are grown in vitro and then injected or infused into the patient. Alternatively the nucleic acid capable of increasing expression of PIGF in vivo is a sequence which modifies endogenous expression of the cells. Thus, in another example, a DNA segment containing a promoter (e.g. a strong promoter) is transferred into cells containing an endogenous PIGF in such a manner that the promoter segment enhances expression of the endogenous PIGF gene. The gene therapy method may involve the use of an adenovirus vector including a nucleotide sequence coding for PIGF or a naked nucleic acid molecule coding for a PIGF subunit. Alternatively, engineered cells containing a nucleic acid molecule coding for a PIGF subunit may be injected.
Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, RNA viruses or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA) encoding recombinant PIGF subunit into the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing such coding sequences. Illustrative embodiments thereof are provided in the Examples section herein.
Alternatively, recombinant nucleic acid molecules encoding protein sequences can be used as naked DNA or in liposomes or other lipid systems for delivery to target cells. Other methods for the direct transfer of plasmid DNA into cells are well known to those skilled in the art for use in human gene therapy and involve targeting the DNA to receptors on cells by complexing the plasmid DNA to proteins. In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection. Once recombinant genes are introduced into a cell, they can be recognized by the cells normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been attempted for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with calcium phosphate and taken into cells by pinocytosis; electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane); lipofection/liposome fusion, wherein DNA is packed into lipophilic vesicles which fuse with a target cell; and particle bombardment using DNA bound to small projectiles. Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins. Adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. Mixing adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene. Adeno-associated virus vectors may also be used for gene delivery into vascular cells. As used herein, “gene transfer” means the process of introducing a foreign nucleic acid molecule into a cell, which is commonly performed to enable the expression of a particular product encoded by the gene. The said product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into mammals.
In another embodiment, a vector having nucleic acid molecule sequences encoding PIGF is provided in which the nucleic acid molecule sequence is expressed only in a specific tissue. Methods of achieving tissue-specific gene expression are well known in the art. According to one embodiment, this is achieved by placing the sequence encoding PIGF under control of a promoter which directs expression in one or more particular tissues. A most particular embodiment of the present invention, relates to the use of a nucleic acid sequence comprising a sequence encoding PIGF or a fragment or derivative thereof having biological activity under control of a promoter directing expression in skeletal muscle, most particularly a promotor which ensures strong expression in skeletal muscle. Examples thereof include the mammalian CMV promotor.
In yet another embodiment, a method of gene replacement is set forth. “Gene replacement” as used herein means supplying a nucleic acid molecule sequence which is capable of being expressed in vivo in a mammal and thereby providing or increasing the function of an endogenous gene which is missing or defective in the said mammal.
Where the administration of PIGF is ensured through gene transfer (i.e. the administration of a nucleic acid which ensures increased expression of PIGF in vivo upon administration), the appropriate dosage of the nucleic acid can be determined based on the amount of PIGF expressed as a result of the nucleic acid, such as e.g. by determining the concentration of PIGF in the blood after administration. According to one embodiment the therapeutic concentrations of PIGF in the blood envisaged are between 10 ng/ml-100 μg/ml.
The present invention will now be illustrated by means of the following examples which are provided without any limiting intention.
Experimental Methods
VEGF and PIGF protein levels were quantified by ELISA (R&D Systems, Abingdon, UK). rhVEGF165 was available from R&D Systems and rhPIGF-2 from Gaymonat, Spa, Anagni, Italy or from Reliatech (Braunschweig, Germany). It was observed that PIGF obtained from different sources had different specific activity. Nevertheless, the relative safety of PIGF over VEGF was always confirmed, indicating that a dosage of PIGF of more than 15 μg/kg/day of active ingredient up to 100 μg/kg/day or higher could be administered without the occurrence of hemodynamic side effects.
Animal experiments were conducted according to the guiding principles of the American Physiological Society and the International Committee on Thrombosis and Haemostasis as published by A. Giles in Thromb. Haemost. (1987) 58:1078-1084.
For isolation of smooth muscle cells (SMCs), media fragments of the aorta were incubated for 16 h at 37° C. in DMEM containing 0.15% collagenase, 5% fetal calf serum (FCS) and antibiotics. After incubation, SMCs were sedimented by centrifugation (400 g; 10 min), resuspended in DMEM+10% FCS and grown at 37° C. in a humidified atmosphere of 5% CO2 in air. Cells were routinely used from the third to the sixth passage. Proliferation of SMCs was studied. Generation of mice knock-outs for PIGF (Pgf−/−) was performed as described in Cameliet et al. 2001, Nature Med, 7(5):575-583.
Mouse Model of Focal Cerebral Ischemia
Focal cerebral ischemia was produced by persistent occlusion of the MCA according to Welsh et al. in J. Neurochem. (1987) 49:846-51. Briefly, mice of either sex weighing 20 to 30 g, with a genetic background of 50% Swiss/50% 129 were anesthetized by intraperitoneal injection of ketamine (75 mg/ml, available from Apharmo, Arnhem, Netherlands) and xylazine (5 mg/ml, available from Bayer, Leverkusen, Germany). Atropine (1 mg/kg, available from Federa, Brussels, Belgium) was administered intramuscularly and body temperature was maintained by keeping the animals on a heating pad. A “U” shape incision was made between the left ear and left eye. The top and backside segments of the temporal muscle were transsected and the skull was exposed by retraction of the temporal muscle. A small opening (1 to 2 mm diameter) was made in the region over the MCA with a hand-held drill, with saline superfusion to prevent heat injury. The meningae were removed with a forceps and the MCA was occluded by ligation with 10-0 nylon thread (available from Ethylon, Neuilly, France) and transsected distally to the ligation point. Finally, the temporal muscle and skin were sutured back in place. The animals were allowed to recover and were then returned to their cages. Infarcted mice were treated with saline (for control) or growth factors using an osmotic minipump (Alzet type 2001, Broekman Institute, Someren, Netherlands), subcutaneously implanted on the back, so that the growth factors were continuously delivered over a period of 7 days. After that period, the animals were sacrificed with an overdose of Nembutal (500 mg/kg, available from Abbott Laboratories, North Chicago, Ill.), perfusion fixed via the left ventricle with 4% formalin in phosphate buffer saline, and decapitated. The brain was removed, processed for histology as described by P. Carmeliet et al. in Nature (1996) 380:435439, Nature (1996) 383:73-75 and Nature (1998) 394:485490 and immune-stained for microtubule-associated protein-2 (hereinafter referred as MAP2), a structural protein that disappears early during irreversible ischemic death. MAP-2 negative infarct areas throughout the brain were morphometrically quantified at 420 [mu]m distances using a dedicated image analysis system (Quantimed 6000, available from Leica). The infarct volume was defined as the sum of the unstained areas of the sections multiplied with their thickness.
Mouse Model of Myocardial and Limb Ischemia
In order to induce limb ischemia, unilateral right or bilateral ligations of the femoral artery and vein (proximal to the popliteal artery) and the cutaneous vessels branching from the caudal femoral artery side branch were performed without damaging the nervus femoralis. Then a subcutaneously implanted osmotic minipump (Alzet, type 2001, available from Iffa Credo, Belgium) continuously delivered during 7 days a daily dose of growth factors or saline. Two superficial preexisting collateral arterioles, connecting the femoral and saphenous artery, were used for analysis.
Functional perfusion measurements of the collateral region were performed using a Lisca PIM II camera (available from Gambro, Breda, The Netherlands) and analyzed as decribed in Couffinhal et al., Am. J. Pathol. (1998) 152:1667-79. Perfusion, averaged from three images per mouse in the upper hind limb (adductor region where collaterals enlarge) or in total hind limb, was expressed as a ratio of right (ischemic) to left (normal) limb.
Spontaneous mobility was scored by monitoring the gait abnormalities, the position of right foot in rest and after manipulation, and the “tail-abduction-reflex”. Mice were scored 0 when one observation was abnormal and 1 when normal.
Restoration of muscle function was tested by way of a physical stress test. An endurance exercise swim test for mice was developed. Mice were conditioned to swim in a 31° C. temperature controlled swimming pool for 9 days in non-stressed conditions. At day 10, baseline exercise time for each mouse was determined using a counter-current swimming pool (flow at 0.2 m/s), as disclosed by Matsumoto et al. in J. Appl. Physiol. (1996) 81:1843-9. For determining maximal endurance exercise, the total swimming period until fatigue, defined as the failure to rise to the surface of the water to breathe within 7 seconds, was assessed. At day 11, the femoral artery was occluded, and at day 18, minipumps were removed under isoflurane anesthesia prior to endurance exercise. Recovery of functionality was expressed as a ratio to the baseline exercise time.
Hind-limb perfusion was measured as follows: fluorescent microspheres (yellow-green, 15 μm, 1×106 beads/ml, available from Molecular Probes) were administered after maximal vasodilation (sodium nitroprusside, 50 ng/ml, Sigma) and flow was calculated as described by Carmeliet et al. in Nat. Med. (1999) 5:495-502. Bismuth gelatino-angiography was performed as described by Carmeliet et al. (cited supra)
Collateral arterioles were identified by photo-angiographs (obtained with a Nikon D1 digital camera) analyzed in a blinded manner. Collateral side branches were categorized as follows: second generation collateral arterioles directly branched off from the main collateral, while third generation collateral arterioles were orientated perpendicularly to the second generation branches. The number of collateral branches per cm length of the primary collateral arteriole was counted.
Fluoroangiography was performed as follows. Images were reconstructed with a Zeiss LSM510 confocal laser microscope. After perfusion-fixation, the two superficial collateral arterioles were post-fixed in paraformaldehyde 1% and paraffin-embedded. Twelve 5 μm cross-sections per superficial collateral, starting from the mid-zone and ranging over 1.95 mm to each end were morphometrically analyzed. Collateral side branches were categorized as second generation (luminal area above 300 μm2) or third generation (below 300 μm2). Total perfusion area was calculated using the total sum of the side branch luminal areas.
Capillary density was determined by immuno-staining for thrombomodulin. Wall thickness of fully small muscle cells (SMC)-covered vessels was morphometrically measured on histological sections, after smooth muscle alpha-actin staining. For all treatment groups, six cross-sections (150 μm apart) were analyzed per main collateral. Only second-generation collateral arterioles larger than 300 μm2 were included in the analysis (see Table 4). At least ten measurements of wall thickness of the second generation collateral arterioles were obtained.
Infarcted mice were treated with saline (for control), PIGF (715 ng/day) or VEGF days (425 ng/day) or a combination of both. The data of these experiments, presented in Table 1 below, are the mean+-standard error of mean (SEM) values of the infarct size expressed in mm and used as a means for measuring cerebral infarction, including the number of observations between brackets and wherein an asterisk means p=0.001 vs. control. The significance of differences was determined by unpaired t-test. Intracerebral bleeding was not observed in any of the mice. These data indicate that PIGF is as effective as PIGF in suppressing infarct expansion of the penumbra. These data also indicate that there is a synergistic effect of VEGF and PIGF in suppressing infarct expansion of the penumbra
The therapeutic effect of PIGF and VEGF was compared in a murine model for acute myocardial infarction by delivering the growth factors continuously over 7 days. Treatment of infarcted mice with PIGF (715 ng/day or 3.5 μg/day) was more effective than VEGF (450 ng/day) in improving myocardial angiogenesis and arteriogenesis, as shown in Tables 2 and 3 Moreover, a synergistic effect of PIGF and VEGF can be observed, especially in the formation of medium and large vessels. Table 2 provides the number of vessels (mean±SEM values), identified by thrombomodulin staining of endothelial cells as a measure of angiogenesis, throughout the infarct in groups of 8 to 10 mice each. An asterisk means p<0.05 vs. control. Table 3 provides the number of vessels (mean±SEM values), identified by smooth muscle alpha-actin staining of endothelial cells as a measure of arteriogenesis, throughout the infarct in groups of 8 to 10 mice each. An asterisk means p<0.05 vs. control.
It was also found that PIGF stimulates myocardial angiogenesis by increasing VEGF expression by fibroblasts, which are abundant in the myocardial stroma. The following expression levels were detected:
The direct effect of PIGF on arteriogenesis i.e. the maturation of vessels via coverage with smooth muscle cells (SMC) leading to stabilization and durability of new vessels was also investigated. It was found that in the ischemic mouse model rhPIGF-2 stimulated arteriogenesis, since 30% of the new myocardial vessels stained positively for the SMC alpha-actin marker after treatment with all PIGF isoforms. A similar fraction (25%) of myocardial vessels is normally covered by SMC, indicating that PIGF did not (contrary to VEGF, see Carmeliet in Nat. Med. (2000) 6:1102-3) cause the formation of hemangiomas but created a new myocardial vasculature with normal characteristics. The functionality of the new vasculature was evidenced by the improved perfusion of the ischemic myocardium after PIGF treatment (1300 μl/min.g after saline versus 2100 μl/min.g after hPIGF-2 as determined by microspheres).
PIGF determined the responsiveness of SMC to VEGF, since PIGF−/− SMC only proliferated normally in response to VEGF when PIGF was present. As evidenced by
At the dose of 1.5 μg, administered daily for 7 days PIGF did not cause oedema or ectopic angiogenesis in the mouse model for acute myocardial infarction, while local signs of oedema were clearly present around the mini-pumps delivering VEGF. PIGF plasma levels up to about 50 ng/ml (achieved by the delivery of 7 μg hPIGF-2 per day for 7 days) were well tolerated without any sign of distress, while VEGF plasma levels above 10 ng/ml caused severe life threatening signs of oedema and circulatory shock. On the other hand, unlike VEGF, PIGF only minimally affected the blood pressure. Mean arterial blood pressure (measured using high fidelity pressure micromanometers, Miller Instruments, Houston, Tex.) was 93±5 mm Hg under baseline conditions (control mice). Intravenous bolus injection of 3 μg VEGF caused significant hypotension (68±3 mm Hg; p<0.05), while 5 to 10 μg hPIGF-2 did not significantly reduce arterial blood pressure (91±11 mm Hg). Thus, PIGF effectively stimulates angiogenesis in the ischemic myocardium, but with the absence of hemodynamic side effects such as oedema or hypoxia.
The therapeutic effect of PIGF and VEGF was further compared in a murine model for hind-limb ischemia by delivering the growth factors continuously over 7 days at a dose of 1.5 μg of rhVEGF165 or rhPIGF-2 (in quantities expressed as active dimer). In contrast to its angiogenic effect in the ischemic heart, VEGF has only a very limited arteriogenic activity in the ischemic limb and, unlike PIGF, did not induce the same favourable structural and perfusional changes as PIGF. Especially the microsphere measurements, which more reliably assessed perfusion in the affected muscle than laser doppler measurements and are not influenced by vasodilation in the skin, revealed that hind limb perfusion was not as much increased by VEGF treatment as by PIGF treatment, as shown in Table 4.
As can be seen from Table 4, perfusion with PIGF both significantly improved the spontaneous hind limb mobility and restored hind limb function to 67% (as opposed to 36% restoration by saline) as measured by the swim test. VEGF treatment improved the spontaneous hind limb mobility but VEGF-treated mice were much weaker and performed significantly worse in the swim endurance test (Table 4). Thus, despite a slight increase in collateral vessel growth and hind limb perfusion, the overall functional reserve after VEGF treatment was impaired, presumably because of unfavourable hemodynamic effects.
The ability of PIGF to stimulate the growth of pre-existing arterial collaterals and their second generation and third generation side branches (collateral growth) was evaluated by treating mice with PIGF after ligation of their femoral artery. The development of a primary collateral artery (C) with secondary (2) and tertiary (3) collateral side branches could be determined by fluoroangiography (
VEGF and PIGF have to bind as dimers to their cognate receptors. The activity of VEGF/VEGF-homodimers and PIGF/PIGF-homodimers is described above. However, VEGF and PIGF can also form heterodimers and have been documented in vivo (Cao, Y., Linden, P., Shima, D., Browne, F. & Folkman, J. In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor. J Clin Invest 98, 2507-11, 1996; DiSalvo, J. et al. Purification and characterization of a naturally occurring vascular endothelial growth factor placenta growth factor heterodimer. J Biol Chem 270, 7717-23, 1995). Their role in angiogenesis and arteriogenesis in vivo remains controversial, and no information is available whether VEGF/PIGF heterodimers can be used for therapeutic applications.
Using the same model of infarct revascularization as exemplified above, VEGF/PIGF heterodimer (from R&D, Abbingdon, UK) was administered via osmotic minipumps for a week at a dose of 10 microgram VEGF/PIGF heterodimer in wild type mice. The experimental data is reported in tables 6 and 7 as vessels/mm2 instead of as vessels/infarct. However the tables may be interpreted qualitatively in the same way as previous tables.
All values are statistically significant (p<0.05; treatment versus control)
Murine PIGF (NM—00827) from −19 at the start codon (ATG 323) until position 1324 including part of the 3′ UTR region was cloned inside the Not1 site in the pcDNA3 vector. The pcDNA3 vector is a commercial vector from INVITROGEN that carries the mammalian cytomegalovirus (CMV) promoter that binds the transcription factor GABP•p300/cbp thereby assuring high expression in skeletal muscle. The resulting vector was called pCDNA3mPLGF.
In vivo electroporation was used to deliver PIGF in skeletal muscles (SM). It was observed that SM-targeting via in vivo electroporation (EP) induces minimal damage of the SM fibers and a mild degree of influx of inflammatory cells. However, no dysfunction of the lower limb was observed. With skeletal muscle-targeted EP, gene expression up to 17 weeks after gene transfer has been described, which may be attributable to the high cytoplasmatic stability as a consequence of the low proliferation rate of this tissue. Moreover, it is known that myofibers have a high secretory capability. In addition, also the immune response of this approach is insignificant in contrast to other approaches like AAV, lentiviral or adenoviral vector delivery in skeletal muscle.
In a first set of experiments the expression of GFP after in vivo electroporation of PCDNA3-GFP in SM (in mouse tibialis and adductor muscle) was confirmed in five out of six mice at day 10 after EP. To evaluate the PIGF secretory capability of transduced SM, 50 μg of pcDNA3-PIGF was electroporated into mouse tibialis anterior muscle.
Concentrations of PIGF in plasma were determined using a “Quantikine” mouse PIGF immunoassay (R&D Systems, Inc., Minneapolis, Minn., USA). Concentrations of PIGF were expressed as pg/ml.
As shown in Table 7, PIGF electroporation produces detectable levels of PIGF in plasma at 10 days after injection.
To evaluate the therapeutic potential of PIGF administered through electroporation, experiments were performed using the hind limb ischemia model.
8 mice (4+4) were electroporated in the mouse adductor skeletal muscle with 14 μg of PIGF (pCDNA3mPIGF) in combination with a GFP reporter plasmid (3 μg) or with the empty pCDNA3 (14 μg) plus GFP (3 μg). The right adductor muscle was exposed, and 14 μg of the pCDNA3mPLGF vector (or empty pCDNA3 vector), dissolved in 0.9% saline at 1 μg/μl, was injected at three sites (in the form of a Mercedes star) in the muscle; a total of 14 μl of the plasmid DNA was administered in each animal. After the injection, a pair of electrode needles was placed onto the muscle. Electric pulses (100 V, 2 times 4 pulses) were delivered using an electric pulse generator.
Twenty-four hours later, limb ischemia was induced by femoral artery ligation, as described above. Seven days after the electroporation, as shown in
After perfusion with a contrast-solution of bismuth and gelatin and fixation, the blood vessels could be visualized macroscopically. Visual examination showed that the limbs that were electroporated with the PIGF plasmid had increased number of collateral vessels and increased number of small vessels (
Transverse cross-sections through the adductor SM at the start, the middle and the distal end were morphometrically analysed (
As shown in
The entire murine PIGF cDNA, including presequences and prosequences (as described by Maglione D et al. Growth Factors. 1993; 8(2):141-152, was cloned into the pACCMVpLpA vector (Gomez-Foix A M, et al., J Biol Chem. 1992; 267(35):25129-25134). Ten micrograms of this plasmid (pACCMV PIGF) was cotransfected into 293 cells together with 5 μg of the pJM17 plasmid that contains the full-length adenovirus serotype 5 genome (McGrory W J, et al. Virology. 1988; 163(2):614-617). Transfection efficiency was boosted after 6 hours by glycerol shock with 15% glycerol in Dulbecco's modified Eagle medium (DMEM, Gibco). Homologous recombination between pJM17 and pACCMV PIGF lead to the formation of a recombinant adenovirus (AdCMV PIGF) in which most of the early region 1 (E1) of the adenoviral genome is replaced by the expression cassette containing the PIGF cDNA under control of the cytomegalovirus (CMV) promoter. The recombinant adenovirus is defective because of the absence of the E1 region, which is provided in trans by the 293 cells (Graham F L et al. J Gen Virol. July 1977; 36(1):59-74). The cells were overlaid with 0.65% noble agar in modified Eagle medium (MEM, Gibco) supplemented with 4% fetal bovine serum (Gibco) 24 hours after transfection. Viral plaques, appearing within 7 to 10 days, were picked and used to infect 293 cells. Recombinant adenovirus was identified by measuring murine PIGF-2 concentration in the conditioned medium by ELISA (R&D systems). Large-scale production of recombinant adenovirus was performed by infecting confluent monolayers of 293 cells in 15-cm dishes. When 90% of the cells showed a cytopathic effect, Igepal CA-630 (Sigma) was added to a final concentration of 0.1% to lyse the cells. Cellular debris was removed by centrifugation at 12 000 g for 10 minutes. After addition of 0.5 volume of 20% polyethylene glycol in 2.5 mol/L NaCl, virus-containing extracts were incubated on ice for 1 hour, and virus was precipitated by centrifugation at 12 000 g for 20 minutes. The pellet was resuspended in 20 mmol/L Tris-HCl, pH 7.8, containing CsCl to obtain a relative density of 1.1. After CsCl step gradient, ultracentrifugation at 22 000 rpm for 2 hours, the virus was harvested from the 1.3 and 1.4 interface. The recombinant virus was desalted by chromatography on Sepharose CL4B in an isotonic saline buffer (10 mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2). The virus was stored at −80° C. until use. Viral titers were determined by plaque assay, and doses were expressed in plaque forming units (p.f.u.). The generation of the recombinant adenovirus AdRR5, which does not contain an insert after the CMV promoter is described in Alcorn J L et al. (Mol Endocrinol. 1993; 7(8):1072-1085).
To evaluate the therapeutic potential of adenovirally administered PIGF, the hind limb ischemia model was used. Mice were injected in the mouse adductor skeletal muscle with adenoviral PIGF expression constructs. Twenty-four hours later, limb ischemia was induced by femoral artery ligation, as described above. After perfusion with a contrast-solution of bismuth and gelatin and fixation, the blood vessels could be visualized macroscopically. Visual examination showed that the limbs that had been injected with the PIGF adenovirus had increased number of collateral vessels and increased number of small vessels (
Transverse cross-sections through the adductor SM at the start, the middle and the distal end were analysed (
A similar experiment was performed wherein adenoviral human hPLGF-1 and hPLGF-2 constructs were used (
Injection of hPIGF expression constructs shows an increase in both their number and perfusion area. Mainly the small side branches contribute to the increase of number of vessels and the perfusion area.
As a summary, the present invention reports that PIGF stimulates the formation of mature and durable vessels in the ischemic heart, and enlarges collateral arterioles in the ischemic limb with marked perfusional and functional improvement, independent of whether PIGF is administered directly or through gene therapy. PIGF treatment achieves this therapeutic goal by unexpectedly combining two desirable properties, i.e. stimulating smooth muscle cells (SMC) simultaneously with endothelial cells (EC). PIGF thereby induces vessel growth in a more balanced manner than VEGF which preferentially stimulates EC. Importantly, the PIGF-induced mature vessels persisted for prolonged periods of more than one year, even long after the arteriogenic stimulus had disappeared, indicating that it may suffice to stimulate new vessels with a short-term delivery of PIGF. Moreover, PIGF shows the advantage of causing less side effects than VEGF in situations where chronic sustained delivery is required. In particular, PIGF did not cause the undesirable side effects of hyperpermeability, oedema and hemangioma formation associated with VEGF.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0002527.0 | Feb 2000 | GB | national |
This application is a continuation-in-part of U.S. Ser. No. 10/454,242, filed Jun. 4, 2003, now U.S. Pat. No. 7,105,168, which claims the benefit of U.S. Ser. No. 60/386,116, filed Jun. 4, 2002, and which is a continuation-in-part of U.S. Ser. No. 10/182,359, filed Sep. 23, 2002, now U.S. Pat. No. 6,930,089; U.S. Ser. No. 10/182,359 is the U.S. National Stage of PCT/EP01/01208, filed Feb. 5, 2001, which, in turn, claims the benefit of U.S. Ser. No. 60/236,594, filed Sep. 29, 2000 and GB 0002527.0, filed Feb. 4, 2000; the disclosure of which is disclosures of which are hereby incorporated by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5919899 | Persico et al. | Jul 1999 | A |
| 7105168 | Carmeliet et al. | Sep 2006 | B1 |
| Number | Date | Country |
|---|---|---|
| 2 332 373 | Jun 1999 | GB |
| WO 9714307 | Apr 1997 | WO |
| WO 9800493 | Jan 1998 | WO |
| WO 9807832 | Feb 1998 | WO |
| WO 9849300 | Nov 1998 | WO |
| WO 9940197 | Aug 1999 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20070027100 A1 | Feb 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60386116 | Jun 2002 | US | |
| 60236594 | Sep 2000 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10454242 | Jun 2003 | US |
| Child | 11471823 | US | |
| Parent | 10182359 | US | |
| Child | 10454242 | US |
