WO2001072339A2

WO2001072339A2 - Method of treatment of conditions involving obstruction of blood flow

Info

Publication number: WO2001072339A2
Application number: PCT/IL2001/000289
Authority: WO
Inventors: Eli Keshet
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem
Priority date: 2000-03-30
Filing date: 2001-03-28
Publication date: 2001-10-04
Also published as: AU2001244509A1; WO2001072339A3

Abstract

The present invention concerns a medicament or diagnostic agent which is expressed only at regions of obstructed blood flow, such as artherosclerotic plaque, since the gene coding for the medicament or diagnostic agent is under the expression control of the Tie-1 promoter. The invention also concerns a medicament, or diagnostic moiety agent which is targeted to regions of obstruction of blood flow due to binding the medicament or diagnostic moiety and agent such as anti-Tie-1 antibody which can specifically target them to the Tie-1 product.

Description

METHOD OF TREATMENT OF CONDITIONS INVOLVING OBSTRUCTION OF BLOOD FLOW

FIELD OF THE INVENTION

The present invention is in the field of pharmaceutical compositions and diagnostic substances, as well as in the field of methods of treatment and diagnostic methods, generally for the treatment or detection of various conditions involving blood vessels and blood flow.

BACKGROUND OF THE INVENTION

Embryonic vascular system undergoes a series of complex, highly regulated events involving differentiation, migration and association of primitive endothelial cells. This process is termed vasculogenesis. A further remodeling of the primitive vascular system forms the mature cardiovascular system. This process is known as angiogenesis (sprouting of new capillary vessels from pre-existing vasculature). The development of primordia of the heart and large vessels, primary capillary networks, in the embryo and the extraembryonic structures in the yolk sac. Angiogenesis accounts for the formation of vasculature into previously avascular organs such as brain and kidney. Angiogenic activity in the adult is required during the normal tissue repair, and for the remodeling of the female reproductive organs (ovulation and placental development). Certain pathological conditions, such as tumor growth and diabetic retinopathy, also require angiogenesis. The genetic and molecular mechanism that influence angiogenesis has only recently begun to be studied and identified. One family of receptor tyrosine kinases termed "Tie-1 " and "Tie-2" or "Tek" have also been identified in vascular endothelium and hematopoietic cells. Mice lacking Tie-1 and Tie-2 die during embryonic development. Analysis of mice lacking either VEGF receptors or Tie-1 receptor have shown that Tie-1 is required at a latter developmental stage than VEGF signaling as the intravascular plexus is formed. However, it is extremely fragile and cannot maintain its functional integrity.

The structure and sequence of the Tie receptor has been disclosed and its extracellular, transmembrane and intracellular domains have been defined. Studies of the patterns of Tie expression during development established its endothelial specificity as well as its endothelial and hematopoietic progenitors.

In adults, expression pattern of Tie- 1 show an overall down-regulation, but indicate that it is capable of being reactivated at sites of ongoing neovascularization such as during periods of ovulation, and wound healing. Tie-1 has also been tied in the vasculator of certain tumors together with additional markers of activated endothelium. The Tie-1 promoter element has been studied in the publication of Alitalo et al. in //. J. In et al, FAFEB Journal, 13:377, 1999.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that a reporter gene, placed under the expression control of Tie-1 promoter elements, is selectively expressed in endothelial cells in regions of turbulent flow (i.e. non-laminar flow), such as at sites of blood vessel branching point, and in valves of folly-developed hearts, as well as in an experimentally-induced obstruction of blood flow caused by aortic coarctation. The present invention is further based on the finding that the Tie-1 expression (i.e. expression of the authentic endogenous Tie-1) is specifically up-regulated in endothelium over-laying atherosclerotic plaques.

By a first aspect termed the "Tie 1 -promoter aspect", the present invention concerns pharmaceutical compositions for the treatment of obstruction of blood flows comprising a pharmaceutically acceptable carrier and as an active ingredient, an expression vector comprising a gene coding for a therapeutically beneficial agent under the expression control of the Tie-1 promoter.

The term "obstruction in blood flow" refers to any situation in which laminar blood flow reverts to turbulent flow. Examples of such conditions are: atherosclerotic plaques; various benign and malignant tumors which penetrate the blood vessels and cause obstruction of blood flow; various diseases of the blood vessels which cause non-normal turbulent flow of blood at a specific site; and restenosis, which is the process of pathogenic proliferation of muscle cells from within the vessel wall which produce a so-called neointima which protrudes into the lumen and creates a severe narrowing of the blood vessel. Restenosis is a frequent complication of angioplasty vessel grafting procedures.

The term "expression vector" refers to any vector which is routinely used in gene therapy which can be safely administered to the body of the individual, penetrate into the individual cells and express its nucleic acid contents, under appropriate conditions, to produce a protein.

The term "Tie-1 promoter elements" refers to all control elements of the Tie-1 gene and include the promoter and optionally also other control elements such as enhancer and other expression control elements. An example of the promoter is given in the publication of Alitalo et al. (Supra).

The term "therapeutically beneficial agent" refers to proteins, coded by the gene which under the control of the Tie-1 promoter, which proteins can ameliorate, cure or prevent to some extent the condition which causes the obstruction in blood flow, and in particular proteins which can ameliorate, cure or prevent conditions associated with atherosclerotic plaques.

An example of such a therapeutically beneficial agent is a protein which is antagonistic for smooth muscle cell proliferation, such as the dominant-negative version of the PDGF-B/PDGF-β receptor system.

Another embodiment of the "Tie-1 promoter" aspect of the invention is intended for diagnostic purposes. In such a case, the present invention concerns a diagnostic preparation for the detection of obstruction of blood flow, comprising an expression vector comprising a reporter gene under the expression control of Tie-1 promoter. In particular, the diagnosis is for detection of regions of atherosclerotic plaques. The detection is for the identification of regions of obstruction of blood flows so that further treatment, such as surgery, angioplasty or vessel grafting may be directed precisely to said regions.

The term "reporter gene" refers to a gene capable of producing a protein which can be detected by means well known in the art, such as a gene capable of luminescence or any other gene capable of being detected by various visualization means, as well as genes capable of production of proteins detected by other modes of imagings such as MRI imaging, x-ray imaging, CT-scan and the like.

In both the pharmaceutical, and the diagnostic embodiments of the Tie-1 promoter aspect, the expression vector is administered to the circulation, for example, by intravenous administration or injection. The expression vector then penetrates, indiscriminately, endothelial cells overlying in the blood vessels, however the Tie-1 promoter ensures that the gene for the beneficial agent (in the pharmaceutical embodiment) or the reporter gene (for the diagnostic aspect) is expressed only in regions of obstruction of blood flow or in regions of artherosclerotic plaques. This ensures, in the pharmaceutical aspect, that the therapeutically beneficial agent is expressed selectively only at the site of obstruction of blood flow, or at artherosclerotic plaques and not in other endothelial cells where blood flow is laminar. In the diagnostic aspect the selective expression is only in regions of obscured blood flow such as artherosclerotic plaques, and this can ensure that further treatment can be directed to these sites alone, and in addition can help determine the severity and progression of said obstruction.

According to said aspect, the present invention further comprises a method for the treatment of obstruction of blood flow comprising administering to a subject in need of such treatment an expression vector comprising a gene coding for a therapeutically beneficial agent under the expression control of the Tie-1 promoter. In particular, the method of the invention is intended for the treatment of artherosclerotic plaques.

The invention further concerns an expression vector comprising a gene coding for a therapeutically beneficial agent under the expression control of a Tie-1 promoter.

It should be emphasized, that the pharmaceutical composition, the diagnostic composition, and the therapeutical method of this aspect are suitable for treatment, or detection of blood obstructions in adults, and are not limited to said treatment or detection of obstruction in embryos or newborns. According to a second aspect of the invention termed "The Tie-1 product aspect" the invention is based on the finding that the Tie-1 product itself is specifically up-regulated in regions of obstruction of blood flow, as can be determined by in situ hybridization with mRNA probes. According to this aspect the invention concerns a pharmaceutical composition for the treatment of obstruction in blood flow comprising a pharmaceutically acceptable carrier and as an active ingredient a chimeric molecule comprising a Tie-1 binding moiety, bound to a medicament. In particular the pharmaceutical compositions of the invention are for the treatment of artherosclerotic plaques

The Tie- 1 binding agent of the chimeric molecule may be, for example, an anti-Tie- 1 antibody, or may be the native ligand of the Tie-1 receptor, or an agonist of said receptor.

The medicament can be any substance which is known to be capable of curing, ameliorating or preventing at least some physiological phenomena associated with obstruction in blood flow inside blood vessels, and in particular a medicament known to be able to decrease artherosclerotic plaques.

For example, the medicament may be proteins which are antagonists for smooth muscle cell proliferation, such as dominant-negative version of the PDGF-β-/PDGF-β receptor system. The medicament may also be pro-fibronolytic moieties such as plasmogen activators well known in the art. Other medicaments known to be used for the treatment of obstruction in blood flow and in particular artherosclerosis can also be used. Where the blood obstruction is due to atherosclerotic plaque, the medicament may be for example a photo-synthesizable agent, intended for photodynamic therapy later which is to obstruct the atherosclerotic plaque. In addition, the anti-Tie- 1 molecule may be conjugated to a cytotoxic agent.

Other medicaments will be apparent to any man versed in the art.

It should be noted that the medicament may be a protein, an agent prepared by peptido-memetics, a chemical moiety, as well as an expression vector capable of expressing a therapeutically beneficial agent (as described above) which is targeted to the region of blood obstruction by said Tie-1 binding moiety.

By another embodiment of the Tie-1 product aspect, the present invention concerns a diagnostic preparation for detection of obstruction in blood flow comprising a chimeric molecule comprising Tie-1 binding agent bound to an imaging moiety. Imaging moieties may be any moiety well known in the art to be used within bodies of individuals for imaging purposes such as by luminescence, MRI, x-ray, CT-scan and the like. The Tie-1 binding agent assures that the medicament or the diagnostic agent, as the case may be, is targeted specifically to the site of blood obstruction. In particular the diagnosis is for the detection of artherosclerotic plaques.

By another embodiment of the Tie-product, the present invention concerns a method for the treatment of obstructions in blood flow comprising administering to a subject in need of such treatment a therapeutically effective amount of a chimeric molecule comprising a Tie-1 binding moiety bound to a medicament. The method is especially relevant for the treatment of artherosclerotic plaques.

The invention further concerns chimeric molecules comprising Tie-1 bound to an imaging moiety. BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: Fig. 1 A is the wholemount preparation of a segment of rat aorta stained for β-gal activity. Arrows highlight regions of up-regulated expression;

Fig. IB is the wholemount preparation of a rat retina stained for β-gal activity;

Fig. 2 is a section through an aortic valve (showing a section through one out of the three semilunar valves located downstream of the site where the dorsal aorta emerges from the ventricle). Arrows highlight regions of up-regulated expression;

Fig. 3A shows an abdominal aorta of transgenic mice at a site of narrowing by use of a band with a surgical suture; Fig. 3B shows β-gal activity in the zone of maximal narrowing. Arrows highlight endothelial cells showing up-regulated expression; and

Fig. 4 shows the result of in situ hybridization analysis for endogenous expression of Tie-1 mRNA artherosclerotic plaque spontaneously produced in ApoE-null mice. Brightened and dark field images are shown.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Example 1 A Tie-1 promoter elements directs expression of a reporter gene to areas of turbulent flow in vivo

Branching Points A. Methods:

A Tie-1 promoter element was ligated upstream of a promoter-less β-gal reporter gene. The construct was inserted to an expression vector which was used to produce transgenic mice and rats. Particular segments of the vasculature from postnatal and adult transgenic animals were isolated and analyzed for β-gal activity. B. Results:

A non-uniform vascular staining pattern was observed. Remarkably, high levels of reporter gene expression were detected at sites of blood vessel branching points known to produce significant disturbances in laminar flow. This was found in different anatomical regions and in vessels of different sizes.

Two representative examples are shown in Fig. 1.

In Fig. IA the up-regulated expression of the Tie-1 promoter-driven reporter at sites of branching from the aorta where a sharp change in flow direction is bound to produce a turbulent flow should be noted. In Fig. IB, the augmented expression of the reporter gene in endothelial cells immediately downstream of the branching point from major restinal arterioles should be noted.

Valves: Another major site of where Tie-1 promoter-driven expression is strongly up-regulated is in valves of the fully-developed heart. A representative example is shown in Fig. 2 which shows a section through an aortic valve (showing a section through the three semilunar valves located downstream of the site where the dorsal aorta emerges from the ventricle). Note that up-regulated expression of the reporter gene is asymetrical and confined to the aspect of the endothelium facing the turbulent back- flow of blood in aorta-to-ventricle direction.

Conclusion

In the adult animal activity of the Tie-1 promoter is up-regulated in a specific manner at sites known to experience turbulent flow. This means that genes under the expression control of this promoter will be selectively expressed in these sites. Example 2 A Tie-1 promoter element directs expression of a reporter gene to an experimentally-induced obstruction in blood flow (aortic coarctation)

A. Methods:

The abdominal aorta of transgenic rats carrying a Tie-1 promoter-β-Gal transgene obtained as described in Example 1 above, was coarctated using banding with a surgical suture. The degree of tightening was adjusted such that partial narrowing of the lumen was produced (see Fig. 3 A). The aorta was removed 4 days later and analyzed for reporter gene expression. The results are shown in Fig. 3B.

B. Results:

As can be seen increased β-Gal activity was detected in regions of aorta narrowing where turbulent blood flow occurred while not detected in regions of laminar blood flow.

Conclusion:

Introduced obstruction in laminar blood flow induces up-regulated Tie-1 expression.

Example 3 Tie-1 is specifically up-regulated in endothelium overlaying atherosclerotic plaques

A. Methods: Mice with a genetic predisposition for developing atherosclerotic plaques were used (ApoE-null mice). Following the spontaneous development of atherosclerotic plaques, the aorta was removed and subjected to in situ hybridization analysis for expression of endogenous Tie-1 mRNA.

B. Results:

Tie-1 mRNA was specifically up-regulated in the endothelium overlying atherosclerotic plaques (Fig. 4). Conclusion:

The Tie-1 product can be found at atherosclerotic plaques. Thus by utilizing agents capable of binding to the Tie- 1 , it is possible to target agents specifically to said plaques, and not to other regions which do not feature plaques.

Claims

CLAIMS:

1. A pharmaceutical composition for the treatment of obstructions of blood flows comprising a pharmaceutically acceptable carrier and as an active ingredient an expression vector comprising a gene coding for a therapeutically beneficial agent under the expression control of the Tie-1 promoter.

2. A pharmaceutical composition according to Claim 1 , where the obstruction in blood flow are due to a condition selected from: atherosclerotic plaques; benign or malignant tumors; restenosis; and neointima formation.

3. A diagnostic preparation for the detection of obstructions of blood flow comprising an expression vector comprising a reporter gene under the expression control of the Tie-1 promoter.

4. A diagnostic composition according to Claim 3, wherein the obstructions in blood flow are due to a condition selected from: atherosclerotic plaques; benign or malignant tumors; restenosis; and neointima formation.

5. A method for the treatment of obstructions of blood flow comprising administering to a subject in need of such treatment an expression vector comprising a gene coding for a therapeutically beneficial agent under the expression control of the Tie-1 promoter.

6. A method according to Claim 4, wherein the obstructions in blood flow are due to a condition selected from the group consisting of: atherosclerotic plaques; benign or malignant tumors; restenosis; and neointima formation.

7. An expression vector comprising a gene coding for a therapeutically beneficial agent under the expression control of the Tie-1 promoter element.

8. A pharmaceutical composition for the treatment of obstructions in blood flow comprising a pharmaceutically acceptable carrier and as an active ingredient a chimeric molecule comprising a Tie-1 binding-moiety bound to a medicament.

9. A phannaceutical composition according to Claim 8, wherein the obstructions in blood flow are due to a condition selected from: atherosclerotic plaques; benign or malignant tumors; restenosis; and neointima formation.

10. A pharmaceutical composition according to Claim 9, wherein the Tie-1 binding-agent is selected from: an anti-Tie- 1 antibody, a Tie-1 ligand and an agonist of the Tie-1.

11. A pharmaceutical composition according to Claim 8, wherein the medicament is selected: pro-fibronolytic moieties, photosensitizable agents and cytotoxic agents.

12. A diagnostic preparation for detection of obstructions in blood flow comprising a chimeric molecule comprising a Tie-1 binding-agent bound to an imaging moiety.

13. A diagnostic preparation according to Claim 12, wherein the Tie-1 binding-agent is selected from: an anti-Tie- 1 antibody, a Tie-1 ligand and an agonist of the Tie-1.

14. A method for the treatment of obstructions in blood flow comprising administering to a subject in need of such treatment a therapeutically effective amount of a chimeric molecule comprising a Tie-1 binding moiety bound to a medicament.

15. A method according to Claim 14, wherein the obstructions in blood flow are due to a condition selected from: atherosclerotic plaques; benign or malignant tumors; restenosis; and neointima formation.

16. A chimeric molecule comprising Tie-1 binding-agent bound to a medicament.

17. A chimeric molecule comprising Tie-1 binding-agent bound to an imaging moiety.

18. A chimeric molecule according to Claim 16 or 17, wherein wherein the Tie-1 binding-agent is selected from: an anti-Tie- 1 antibody, a Tie-1 ligand and an agonist of the Tie- 1.