Patent Application
20090304765
The invention relates to the field of vascular regulation and to the role of R-Ras in vascular regulation. The invention in particular relates to means and methods for promoting a quiescent state of a vascular cell and to means and methods for diagnosing a condition of vasculature of an individual. More in particular the invention relates to RAS proteins or functional equivalents thereof, as well as their genes, particularly in gene therapy settings for providing RAS-activity to vascular cells.
The Ras family of small GTPases comprises intracellular signalling molecules that function as binary switches regulated by GTP. The oncogenic Ras proteins, H-Ras, and K-Ras in particular, are central players in cellular signaling networks; they are activated by most growth factors as well as by integrins. R-Ras is a member of the Ras protein family that antagonizes H-Ras signaling1,2. Due to its sequence homology with the prototypic Ras proteins, R-Ras has been often described as a transforming oncogene3. The transforming activity of R-Ras in vitro is, however, quite low compared with that of H-Ras and K-Ras4, and there is no evidence that activating mutations would occur in spontaneous malignancies. Moreover, R-Ras differs from the other members of the Ras family in that it contains a proline-rich SH3 domain binding site, within which it can be phoshorylated by Eph receptors and Src; both SH3 domain binding and phosphorylation regulate R-Ras activity5-7.
The present invention makes use of the finding that R-Ras has different properties than the more conventional Ras proteins/genes. R-Ras and H-Ras for instance exert opposite effects on cell-extracellular matrix adhesion; R-Ras enhances integrin-mediated cell adhesion by elevating the affinity and avidity of integrins8, whereas H-Ras inhibits integrin activities9. There is also a striking contrast in R-Ras and H-Ras activities in cell differentiation. R-Ras promotes the differentiation of myoblasts and the fusion of these cells to myotubes, a process that requires cell cycle arrest and entry to GO state10, whereas H-Ras inhibits these processes11. The in vivo function of R-Ras, however, has formerly remained unclear. The invention surprisingly discloses that R-Ras is a vascular regulator. In particular, R-Ras is a regulator of vascular differentiation that affects the remodeling of blood vessels.
The invention in one embodiment provides a method for promoting a quiescent state of a vascular cell, comprising providing said cell with additional R-Ras activity. A quiescent state of a vascular cell as used in the invention is a state wherein the cell cycle of a vascular cell is arrested and/or wherein a cell is in the GO state. Promoting means that the process of cell cycle arrest is enhanced and/or that the entrance of a cell into the GO state is stimulated. Further, promoting means that a cell that is in a quiescent state is stimulated to maintain the quiescent state. A quiescent state of a vascular cell means that cell proliferation and/or migration is inhibited and further that the cell is in a state that facilitates differentiation. There are many instances wherein it would be very favourable to promote a quiescent cell state of a vascular cell. For instance, in tumors, in hyperplastic arterial lesions, in pulmonary hypertension, pulmonary vascular diseases, organ transplant rejection, peripheral artery disease, coronary artery disease, collagen vascular diseases such as lupus and scleroderma, vasculitis and autoimmune diseases. An additional R-Ras activity is any amount of activity that raises the activity of a cell above the initial level. This can be measured in the same cell or in a comparable cell. Thus, in a cell wherein R-Ras activity was for example absent, or alternatively, was present but at a lower level. A vascular cell as used in the invention is preferably an endothelial cell or a smooth muscle cell. R-Ras activity can be added by any means or method that enhances the presence or activity of R-Ras or a functional equivalent thereof in a vascular cell. This can be achieved directly by providing R-Ras protein or a functional equivalent thereof or indirectly by providing an expressible nucleic acid which can express R-Ras activity and/or by activating a pathway in a vascular cell that leads to expression of R-Ras activity (in particular of a R-Ras protein), or activation of pre-existing R-Ras.
In one embodiment of the invention additional R-Ras activity is provided through activation of inactive R-Ras in a cell. The activity of Ras proteins is in one aspect controlled by the balance of GTP and GDP bound to the protein, wherein the GTP-bound form is active and the GDP-bound form is inactive. Activation is for instance established by stimulating the binding of GTP to the R-Ras protein. In one embodiment of the invention additional R-Ras activity is provided by converting GDP-bound R-Ras protein into GTP-bound R-Ras protein, by phosphorylating said GDP. As the R-Ras protein itself is a GTPase, its activity is further regulated by accessory proteins that influence the activity of the GTPase functionality. In a further embodiment of the invention additional R-Ras activity is thus provided by inhibiting or enhancing presence and/or activity of accessory proteins. In an alternative embodiment of the invention post-translational modification by lipid attachment is enhanced to provide additional R-Ras activity or post-translational modification by lipid attachment is inhibited to reduce R-Ras activity. Post-translational modification of R-Ras protein in order to make the protein functional is, for instance, farnesyl or geranyl attachment. R-Ras has a further regulation mechanism that is shared by all R-Ras but not by other Ras proteins. R-Ras differs from the other members of the Ras family in that it contains a proline-rich SH3 domain binding site, within which it can be phosphorylated by Eph receptors and Src. Eph receptors and Src are both protein tyrosine kinases (PTKs). Protein tyrosine kinases (PTKs) are enzymes which catalyze the phosphorylation of tyrosine residues. PTKs are involved in cellular signaling pathways and regulate key cell functions such as proliferation, differentiation, anti-apoptotic signaling and neurite outgrowth. There are two main classes of PTKs: receptor PTKs and cellular, or non-receptor, PTKs. Cellular PTKs, such as Src, are located in the cytoplasm, nucleus or anchored to the inner leaflet of the plasma membrane. Receptor PTKs possess an extracellular ligand binding domain, a transmembrane domain and an intracellular catalytic domain. Eph receptors form the largest subfamily of receptor tyrosine kinases (RTKs). Both SH3 domain binding and phosphorylation regulate R-Ras activity5-7. Thus in one embodiment of the invention R-Ras activity is added by enhancing binding of SH3 domains to a proline-rich site of R-Ras. In a preferred embodiment, regulation of R-Ras activity is provided by enhancing or inhibiting presence of R-Ras protein, as the amount of protein is a stronger regulator of R-Ras activity in a cell than internal activating or inhibiting mechanisms of R-Ras protein.
The invention in a preferred embodiment provides a method according to the invention, wherein said activity is provided by a R-RAS protein and/or a functional equivalent thereof. An R-Ras protein is defined by its nucleotide sequence. An R-Ras protein is either a wild type R-Ras protein or a mutant R-Ras protein. A sequence of wild type R-Ras protein is given in supplementary
In an alternative embodiment of the invention R-Ras activity in a vascular cell is reduced. Reduction of R-Ras activity in a vascular cell is for example established by down-regulating R-Ras expression in the cell or by degrading R-Ras protein and/or R-Ras mRNA in a cell, and by promoting hydrolysis of GTP bound to R-Ras to GDP or by preventing the modification of R-Ras by lipid attachment. Down-regulating is for example established by an antisense-technique. Non-limiting examples of some of the newer antisense approaches are interference RNA (RNAi), microRNA and splice interference techniques such as exon-skipping. Reducing R-Ras activity in a cell stimulates proliferation and migration and diminishes differentiation of the cell. Angiogenesis is thus stimulated by down-regulation of R-Ras activity. Indications for down-regulating R-Ras activity in a cell are for example wound healing or rehabilitation of an infarct, a stroke or of organ or limb damage. Balancing down- and up-regulation of R-Ras activity is a method for structuring modelling of vasculature. The invention in one embodiment provides a method for structuring modelling of vasculature, comprising providing said cell with additional R-Ras activity or reducing R-Ras activity in said cell. Modelling herein under more proceeds through stimulation/inhibition of cell growth, differentiation, proliferation and migration.
In one embodiment of the invention, R-Ras activity is added or reduced by intervening in a R-Ras pathway. A preferred R-Ras pathway involves Raf-1.The results of examples of the invention demonstrate that the effect of R-Ras on vascular cell activities is mediated, at least in part, by inhibitory phosphorylation of Raf-1. Other pathways do, however, also play a role. In a preferred embodiment of the invention R-Ras activity is added by enhancing inhibitory phosphorylation of Raf-1, or reduced by diminishing inhibitory phosphorylation of Raf-1. Raf-1 is at least part of a PI3-kinase-Akt signaling pathway. In one embodiment of the invention R-Ras activity is enhanced by activating at least a subset of Akt molecules or reduced by inhibiting at least a subset of Akt molecules.
An R-Ras protein can be produced in any prokaryotic or eukaryotic cell. Delivering an R-Ras protein to a cell is done by any means that transports the protein/peptide into a cell. For example by electrotransfection, electroporation or through delivery by a cell penetrating peptide. Alternatively, R-Ras protein is delivered comprised in a nanoparticle. In a preferred embodiment the invention provides a method according to the invention, wherein said R-Ras protein or said functional equivalent thereof is provided with a cell penetrating peptide. There are many types of cell penetrating peptides available in the art. For references, see for example ref. 52-55. Any type of cell penetrating peptide that delivers R-Ras protein or a functional equivalent thereof can be used according to the invention. A cell penetrating peptide is for example a protein derived peptide, such as penetratin, a Tat-derived peptide, a signal-sequence-based peptide (I or II), or a synthetic and/or chimeric cell-penetrating peptide such as transportan or an amphiphilic model peptide. Specific preferred examples of such cell penetrating peptides are Tat, a cell penetrating peptide (refs. 96-98); F3 (ref. 95), an internalizing tumor specific homing peptide; and Arginine Heptamer (refs. 99-104), which is a dermis penetrating peptide.
In a further preferred embodiment the invention provides a method according to the invention, wherein said R-Ras protein or said functional equivalent is provided with a homing peptide. A homing peptide is any peptide that targets a cell of a selected tissue. In the art many homing peptides are available. In a preferred embodiment the homing peptides are lung homing peptides, heart homing peptides or tumor homing peptides. Heart homing peptides are for example a CRPPR peptide that at least binds to a Cystein-rich protein 2 receptor, and a CPKTRRVPC peptide that at least binds to a bc10 receptor. For further references for heart homing peptides see for instance ref 56, specifically the table on page 1605. Lung homing peptides are for example Metadherin or GFE-1 (CGFECVRQCPERC). For further references for lung homing peptides see for instance refs 57, 58 and 105-110. Preferred tumor-homing peptides are for example F3, a 34-amino acid basic peptide (see further refs. 59, 60); CGKRK (see under more refs. 61 and 62); and LyP-1, sequence CGNKRTRGC (see for instance refs. 63 and 64). Relevant tumor homing peptides are further for instance described in refs 59-64. Preferably a homing peptide homes to lung, heart, especially a coronary artery, or any vasculature. The cell penetrating peptides and/or homing peptides can be attached to the protein providing R-Ras activity by any conventional means. An easy way of preparing the complex is through producing a vector encoding a fusion of R-Ras protein with a cell penetrating peptide and/or a homing peptide and expressing said vector in a suitable host as disclosed in the examples. R-Ras is provided as a sequence encoding R-Ras or a functional equivalent thereof.
In one embodiment the invention provides R-Ras or a functional equivalent thereof for use as a medicine. The medicine is used for any occasion wherein it is desired to promote a quiescent state of a vascular cell. In a preferred embodiment R-Ras or a functional equivalent thereof is used in the preparation of a medicament to promote a quiescent state of a vascular cell. A medicament is any pharmaceutical composition comprising R-Ras or a functional equivalent thereof. Such a pharmaceutical composition optionally further comprises a pharmaceutically acceptable carrier and any additive. A medicine is for example produced as a pill, a capsule, a tablet, a medicinal gum, a solution, a dry powder form, an inhaler, transdermal patch, microsphere, nanocrystal, or as a coating for a device that is introduced in a body within contact of vasculature.
In one embodiment the invention provides a protein complex comprising R-Ras or a proteinaceous functional equivalent thereof and a cell penetrating peptide and/or a homing peptide. Such a protein complex is for example a fusion protein or a complex which is held together by disulfide bonds. In a preferred aspect of the invention a protein complex according to the invention is a fusion protein. There are diverse chemical coupling methods available in the art for the production of a fusion protein, a fusion protein of the invention is produced by any of these methods. An example of such a fusion protein is a fusion protein of R-Ras or R-Ras mutant 38V or 87L and a Tat-derived peptide. In one embodiment of the invention, a homing peptide is a part of a fusion protein. A functional equivalent as used in the invention is at least functionally the same in kind as R-Ras, though not necessarily the same in amount. The invention further provides a nucleic acid molecule comprising a nucleic acid sequence coding for a fusion protein according to the invention. A nucleic acid is for example a DNA, RNA or PNA. In an alternative embodiment of the invention, R-Ras is comprised in a nanoparticle. For this embodiment, R-Ras does not have to be modified, but can be if desired. A homing peptide is optionally attached to the particle core.
In one aspect the invention provides a gene delivery vehicle encoding R-Ras or a functional equivalent thereof. In a further embodiment the invention provides a gene delivery vehicle encoding a fusion protein according to the invention, or comprising a nucleic acid molecule according to the invention. A gene delivery vehicle is any vehicle that delivers a gene encoding an R-Ras or functional equivalent thereof to a cell. Preferably said cell is a vascular cell, more preferably an endothelial or a smooth muscle cell. A gene delivery vehicle is in a preferred embodiment a vector encoding R-Ras or a functional equivalent thereof A vector is preferably an expression vector wherein R-Ras or a functional equivalent thereof is operatively linked to an enhancer sequence and/or a promotor sequence. Transduction of a vector according to the invention is for example mediated through a virus. Non-limiting examples of such viruses are an adenovirus, an adeno associated virus, an alphavirus such as sindbis or SFV a lentivirus and/or a retrovirus. Non-viral delivery vehicles such as polymers and/or liposomes or other nanoparticles (based on e.g. biodegradable polymers).
In a preferred embodiment the invention provides an article of manufacture to be introduced into an environment comprising vascular cells, coated with a composition comprising R-Ras or a functional equivalent thereof, or a composition comprising a protein complex according to the invention, or a composition comprising a gene delivery vehicle according to the invention. Coated as used in the invention means that the additional R-Ras activity is available to the environment comprising vascular cells. Coated means preferably, but not necessarily, that the layer of the article of manufacture exposed to the environment is provided with additional R-Ras activity. Such an article of manufacture is introduced in any environment wherein additional R-Ras activity is beneficial. Additional R-Ras activity is for example beneficial in an environment where cell migration and proliferation, in particular of vascular cells, is undesirable. Providing additional R-Ras activity is for example beneficial in order to prevent a hyper-reactive response of an organism to an external device that is inserted in said organism. Adding R-Ras activity at least partially inhibits vascular cell proliferation and migration as a response to an external stimulus. Another example of a circumstance wherein adding R-Ras activity is beneficial, is in the case of tumor growth. R-Ras activity then prevents angiogenesis in and adjacent to the tumor, thereby inhibiting the blood supply to the tumor tissue. In addition, adding R-Ras activity increases cell adhesion. In one embodiment the invention provides use of R-Ras or a functional equivalent thereof, for stimulating adhesion of vascular endothelial cells to a vessel wall. Increasing cell adhesion can further, for instance, convert a leukemia into a lymphoma or inhibit metastasis. Increasing cell adhesion through R-Ras is at least partially mediated by integrins. In a preferred embodiment the invention provides an article of manufacture to be introduced into an environment comprising vascular cells, coated with a composition comprising R-Ras or a functional equivalent thereof, or a composition comprising a protein complex according to the invention, or a composition comprising a gene delivery vehicle according to the invention, further comprising a Tat peptide and/or a homing peptide.
An article of manufacture of the invention is any article of manufacture that is in any instance introduced into an environment comprising vascular cells. Such an article is for example a device that forms a portal in a mammal. Such a portal is for instance a portal to a bowel, a stomach, a bladder, an oesophagus or a trachea. Alternatively, an article of the invention is a device that is placed internally in an environment that comprises vascular cells. An internally placed article is for example an artificial heart-valve. In a preferred embodiment of the invention an article of manufacture according to the invention is a stent. A stent as used in the invention is a device that is open in that there is passage possible for bodily fluids and that is inserted in an organism, preferably a mammal. A stent is preferably a small tube that is inserted for more than 6 hours, preferably for longer, in a preferred embodiment permanently, in an organism. In a preferred embodiment the stent is introduced in an artery. The stent at least assists in holding open an artery so that blood can flow through it. In a preferred aspect a stent is an intraluminal coronary artery stent. Such a stent is typically used in angioplasty, which is a procedure to reduce or eliminate blockages in coronary arteries.
In one embodiment the invention provides a composition for promoting a quiescent state of a vascular cell, comprising R-Ras or a functional equivalent thereof, a protein complex according to the invention, or a gene delivery vehicle according to the invention. Such compositions can be pharmaceutical compositions, cosmetic compositions or compositions for use on cells in vitro. These compositions can therefore comprise the conventional usual excipients, buffers and other constituents for such compositions. The invention in another embodiment provides a composition according to the invention, further comprising a 2nd/3d active agent. In a further embodiment the invention provides such a composition whereto a 4th, 5th, or any further active agent is added. An active agent is any active agent that is beneficially combined with R-Ras activity in a composition. Such a composition is for instance a pharmaceutical that is intended for treatment and/or prevention of vascular narrowing. Further active agents in the composition are, for that indication, for instance heparin or another anticoagulant or acetylsalicylic acid. Vascular narrowing is for example a problem in a patient with postangioplasty restenosis or artherosclerosis. In a further preferred embodiment the invention provides a composition according to the invention, further comprising sildenafil citrate, sirolimus, and/or Gleevec.
Preferred active agents in a composition of the invention are:
Active agents that are specifically preferred in a composition of the invention for use in case of pulmonary hypertension are: epoprosternol, trepostinil, bosentan, sildenafil, siloprost, sitaxsentan, en Ambrisentan, or functional analogues or derivatives thereof. Preferred active agents in a composition of the invention for use in case of need of an anti-proliferative, for instance in cancer, are: Methotrexate, Fluorouracil, cisplatin, doxorubicin, 5-fluotacil; VEGF inhibitors such as bevacizumab (Avastin); Protein kinase inhibitors such as imantinib (Gleevac); and epidermal growth factor receptor tyrosine kinase inhibitors such as erlotinib (Tarceva) and gefitinib (Iressa), or functional analogues or derivatives of these agents. Preferred agents in a composition of the invention for use in stent coatings are: Sirolimus, Tacrolimus, Paclitaxel, Everolimus, or functional analogues or derivatives thereof.
In one embodiment a composition according to the invention is a pharmaceutical composition optionally further comprising additives, such as a pharmaceutically acceptable carrier or a colorant.
In a preferred embodiment the invention provides a composition, which is a controlled release composition. A controlled release composition as used in the invention is any composition that does not release all activity at one point in time. A controlled release composition of the invention preferably constantly releases approximately even amounts of active agent for a longer period of time. A longer period of time are preferably hours, more preferably days, and most preferred weeks, months or years. A controlled release composition of the invention is typically a biodegradable composition. A controlled release composition is for example produced in the form of a biodegradable microsphere (see for example ref. 65). A controlled release composition of the invention is for example used in a coated article of manufacture of the invention, such as a stent. Adding a controlled release composition of the invention to for instance a heart atrio-ventricular valve, will reduce vessel proliferation on implantation of the valve in for example a mammal. Thereby adding such a composition at least partially helps to prevent the forming of an obstructive mass in a blood stream.
In one embodiment the invention provides a method for diagnosing a condition of vasculature of an individual, comprising obtaining a sample from said individual and measuring an expression level of R-Ras expression in said sample. A condition of vasculature as used herein is a status of health or development of vasculature of an individual, such as a pathological or a physiological status. Furthermore the status refers to characteristics of the vasculature such as the differentiation state of a cell or of multiple cells. A sample as used in the invention is for example a sample of a bodily fluid, such as blood or lymph. A sample is alternatively obtained from a bronchoalveolar lavage (BAL), Transbronchial biopsy (TBB), or Endomyocardial heart biopsy. In a preferred embodiment a sample is a vascular cellular sample. A vascular cellular sample of the invention is any sample comprising cells that were located adjacent to or part of a vascular tissue. A vascular cellular sample is preferably derived from one of the larger vessels in an animal. Preferably said larger vessel is an artery. In a preferred embodiment a vascular cellular sample is obtained from a mammal, preferably a human being. A vascular sample as used in the invention is obtained with any method for taking a vascular cellular sample. Such a method is for example a surgical method or a minimal invasive method such as a cheek mucosal tissue sampling technique. In a preferred embodiment of the invention a sample in a method for diagnosing a condition of vasculature of an individual is obtained with a percutaneous endoarterial biopsy, a percutaneous atherectomy, or another surgical method.
An expression level of R-Ras can be measured in alternative ways. An expression level of R-Ras can be measured from any product of a R-Ras mRNA. For example the level of R-Ras protein, or the level of a derivative of R-Ras protein is measured. An expression level of R-Ras is for example performed through an immunodetecting technique, such as immunohistochemistry, immunofluorescence or immunoblotting. Alternatively expression levels are determined with a PCR technique, for instance quantitative real time PCR. In the art many other techniques for determining an expression level are available, such as multiple microarray techniques. In a preferred embodiment a method according to the invention is provided, wherein measuring is performed through PCR, a microarray technique, immunohistochemistry, immunofluorescence or immunoblotting.
In one embodiment the invention provides a method for diagnosing a condition of vasculature of an individual, wherein said condition of vasculature of said individual is associated with a disorder in said individual and wherein said disorder is a vascular proliferative disease. Diagnosis is either directed to a local, a regional or a systemic condition of vasculature of an individual. A vascular proliferative disorder is any disease wherein vasculature of an individual proliferates. Proliferation typically refers to cell multiplication, but generally, as in most vascular proliferative disorders, it also involves growth of at least some individual cells. Non-limiting examples of vascular proliferative disorders are: idiopathic pulmonary hypertension, chronic hypoxic pulmonary hypertension, systemic hypertension, artherosclerosis, postangioplasty restenosis, vasculopathy, diabetic vasculopathy, vascular injury, vasculitis, arteritis, capillaritis or carcinoma. In a preferred embodiment, the invention provides a method for diagnosing a condition of vasculature of an individual, wherein said vascular proliferative disease is selected from the following: pulmonary hypertension, carcinoma or vascular injury.
The invention provides a kit for diagnosing a condition of vasculature of an individual, at least comprising a means for measuring an expression level of R-Ras in a sample and an apparatus for obtaining a sample. An apparatus for obtaining a sample is any device that can be used to derive a sample as defined by the invention. A non-limiting example of such an apparatus is a syringe or syringe-like device or a catheter or biopsy device. In a preferred embodiment of the invention, an apparatus for obtaining a sample is an atherectomy catheter or an endoarterial biopsy device.
The invention further provides a kit according to the invention, wherein the means for measuring an expression level comprises a binding body and a detection reagent. Many different specific binding bodies are available. Of old, antibodies are used. However, currently many different parts, derivatives and/or analogues of antibodies are in use. Non-limiting examples of such parts, derivatives and/or analogues are, single chain Fv-fragments, monobodies, VHH, Fab-fragments and the like. A common denominator of such specific binding bodies is the presence of an affinity region (a binding peptide) that is present on a structural body that provides the correct structure for presenting the binding peptide. Binding peptides are typically derived from or similar to CDR sequences (typically CDR3 sequences) of antibodies, whereas the structure providing body is typically derived from or similar to framework regions of antibodies. A detection reagent is any reagent that provides detection of binding or non-binding of an antibody. A detection reagent for example comprises a reporter group or is a fluorescent marker.
The invention for the first time disclosed a role of R-Ras expression in vascular regulation. In an example of the invention the in vivo function of R-Ras is demonstrated using R-Ras-null mice. Heterozygous intercrosses produced viable offspring with Mendelian distribution of the genotypes. The R-Ras-null mice were fertile and displayed no obvious abnormalities, and their tissues appeared normal upon histological examination. The disruption of R-Ras mRNA and the absence of protein product in null mice were confirmed by RT-PCR, real time RT-PCR, and anti-R-Ras immunoblotting (Supplementary
Previously performed mRNA studies indicated that R-Ras is widely expressed throughout various tissues and organs11. The invention surprisingly discloses that whereas mRNA of R-Ras is widely present, R-Ras itself has actually a restricted tissue distribution. Antibody staining revealed a restricted tissue distribution for R-Ras, which was primarily confined to smooth muscle in various tissues and organs (Supplementary
The invention further determined the developmental pattern of R-Ras expression. All embryonic tissues were negative for R-Ras from E8 (embryonic day 8; the earliest time tested) to E16 (Supplementary
The observed spacio-temporal pattern of R-Ras expression demonstrates the regulatory role in the growth and/or homeostasis of the adult vasculature. The model used in the invention to study R-Ras, is commonly used to mimic the restenotic lesions that can develop after angioplasty15. Neointimal thickening was found greatly increased in R-Ras-null mice 5 to 6 weeks post injury (P<1×10−8) (
In a non-limiting example of the invention, the effect of absence of R-Ras on angiogenesis was tested. Neovascularization of mouse melanoma B16F10 tumor implants was greatly enhanced in R-Ras-null host mice compared to littermate controls (P=0.001) (
The effects of R-Ras expression were further demonstrated in primary cultures of human coronary artery SMC(CASMC). These cells exhibit dedifferentiated features in the presence of growth factor supplements (bFGF, EGF, and IGF), while their growth and migratory behaviours mimic those of VSMC in an atherosclerotic environment21. R-Ras expression in cultured VSMC and human umbilical vein EC (HUVEC) was 2˜5% of the levels in intact tissues (Supplementary
Transduction of activated R-Ras also induced changes in HUVEC (
In order to demonstrate that restored R-Ras signaling inhibits angiogenesis, a lentiviral vector was incorporated, carrying an activated R-Ras gene to matrigel plugs. The R-Ras-expressing plugs showed greatly reduced microvessel infiltration (
PI3-kinase-Akt signaling can inhibit Raf activity through phosphorylation of Raf-1 on Ser259 in differentiated myotubes, but not in their myoblast precursors22. PI3-kinase is the sole known effector of R-Ras21. As R-Ras was in the invention found to be differentially expressed during phenotypic transitions of VSMC and EC, Raf-1 and phosphorylation of its Ser259 residue were found to be central to the R-Ras activities. Transduction of activated R-Ras, but not inactive R-Ras43N or mock transduction, significantly increased Raf-1 Ser259 phosphorylation in both proliferating and quiescent HUVEC and counteracted the transient reduction of Ser259 phosphorylation in mitogen-activated cells (
Function of R-Ras/Akt-mediated Raf-1 phosphorylation was further studied in the invention, using a serine to alanine substitution at residue 259 (S259A) to prevent inhibitory phosphorylation at this residue24. R-Ras effectively inhibited the tube formation of HUVEC expressing wild-type Raf-1, but this effect was significantly reduced in the cells transduced with the S259A Raf-1 mutant (
R-Ras signaling activates a PI3-kinase-Akt pathway that is functionally distinct from the classic growth factor induced PI3-kinase-Akt pathway, this signaling pathway produces an effect on vascular responses. In addition to the classic pathway, which mediates growth stimulatory signaling, PI3-kinase and Akt can also engage in inhibitory regulation.
Mice. R-Ras-null mice OST24882 were generated by Lexicon Genetics (Woodlands, Tex.) as described before26. The inactivation of the R-Ras gene in these mice is caused by an insertion of a gene trap vector VICTR2026 between exons 4 and 5 of the R-Ras gene on chromosome 7. The insertion disrupted R-Ras gene expression as determined by RT-PCR, real time RT-PCR, and anti-R-Ras immunoblotting of tissue extracts (Supplementary
Immunological detection. R-Ras expression was determined by standard immunohistochemistry, immunofluorescence, and immunoblotting methods. Rabbit polyclonal antibodies were raised against full-length human R-Ras protein prepared as a GST fusion protein in bacteria6. Antibodies were affinity-purified by positive and negative selection on R-Ras and H-Ras affinity columns, respectively. Rabbit antiserum against amino acid 11-31 of mouse R-Ras27 was a gift from Dr. J. C. Reed of the Burnham Institute for Medical Research. Mouse tissues were fixed with Bouin's solution (Sigma), paraffin embedded, and sectioned for histological analyses. Sections of 8-16 d mouse embryo were obtained from Novagen. Lung metastases were produced by intra-tail vein injection of 1×105 mouse mammary tumor 4T1 cells into the syngeneic (BALB/c) mice. The metastasis-bearing lung was collected 12 d later and fixed in Bouin's solution. Rat monoclonal antibody against pan-endothelial cell antigen, MECA32, (Pharmingen) was used to visualize microvessels in tumor. To examine R-Ras expression in cultured cells, cells were cultured at different conditions, and the expression levels were determined by immunoblot analysis and densitometry. Anti-pan-actin or GAPDH (Chemicon) blot was used to normalize total protein loading. Heparin (Sigma) was used to promote differentiation of VSMC at 200 μg/ml in 5% FCS supplemented media for 3 d. Preparation of histological samples was performed at the Institutional Histological Facility
Experimental restenosis model. Neointimal hyperplasia was produced in mouse femoral artery as described before17. After the induction of the vascular injury, the injured vessels were collected at the times indicated. Areas of the media and neointima in the cross-sections of restenotic arteries were calculated by image analysis using Spot System (Universal Imaging Corporation, Downingtown, Pa.). Levels of intimal hyperplasia were presented as neointima:media area ratio. Statistical analysis was performed using Student's t-test (n=14). Proliferation of neointimal cells was assessed by anti-Ki-67 or Brd-U staining after the animals received 1 mg Brd-U/day for two days through i.p. injection prior to collecting the arterial lesion.
Angiogenesis assays. Tumor angiogenesis assay, Matrigel plug assay, and ex vivo microvessel outgrowth assay were performed as described elsewhere. Briefly, for subcutaneous tumor implantation, 1×106 B16F10 mouse melanoma cells were implanted into the flank of the mice, and the tumors were excised at 10 d post implantation for analyses. CD31-positive infiltrating microvessels were counted in five different fields per section from six animals per group. For Matrigel plug assay, growth factor reduced Matrigel® (Becton Dickinson) containing VEGF and heparin was implanted into the flank of the mice. Seven days later, the Matrigel plugs were harvested and the microvessel infiltration was quantified. Assay for endothelial tube formation on Matrigel was carried out using HUVEC as described elsewhere. The efficiency of tube formation was quantified by measuring the total length of the tubes in triplicate wells.
Preparation of lentivirus for in vivo gene delivery. The Lentiviral Vector carrying activated R-Ras (R-Ras38V and R-Ras87L), and the virus with an empty vector (control) were produced according to the manufacturer's manual (Invitrogen). Concentrated virus stocks were prepared by ultracentrifugation of conditioned medium from transfected packaging cells at 50,000×g for 2 h and re-suspending the pellets in PBS using 0.5% of the starting volume. To improve the infection and expression of the transgene, half of the virus stock was incubated with dNTPs for in vitro reverse transcription as described by Naldini et al.28. The reverse-transcribed and the original virus stocks were combined and concentrated by a second ultracentrifugation (50,000×g for 90 m). The final pellet was re-suspended in 0.1% of the starting volume of PBS containing 0.5% BSA and 4 μg/ml of polybrene, the titer of the virus stock was titrated, and 5×106 TU was used for in vivo experiments.
For the results of example 1 is referred to the detailed description of the invention.
This example demonstrates amongst others use of R-Ras as a lung homing vascular anti-proliferative agent for the treatment of patients diagnosed with pulmonary hypertension.
A medicament is formulated by linking R-Ras to Tat (GRKKRRQRRRPPQ), a cell penetrating peptide (ref. 66), and to CGFECVRQCPERC, a lung homing peptide (ref. 67). The medicament is administered by injection to patients who meet the criteria for diagnosis of pulmonary hypertension. Such patients show indications of elevated pulmonary artery pressure, a strong indication of blood vessel remodeling due to pulmonary hypertension and its vascular cell proliferative effects.
This example demonstrates amongst others use of R-Ras as a vascular anti-proliferative agent in the treatment of patients diagnosed with systemic hypertension and peripheral artery disease secondary to diabetes.
Patients in this example class are in imminent peril of amputation of distal limbs, especially feet and lower legs. On angiographic diagnosis, systemic thickening of arteries appears to be the cause of poor distal circulation. Discrete point blockages which are found in many PVD patients, not just diabetics, are rarely the final cause of diabetic amputation. Discrete point blockages can be successfully removed through a variety of interventional techniques including balloon angioplasty, atheroectomy, vein graft bypass, implanted vascular prosthesis, etc. However, these techniques are sometimes contraindicated in severely complicated diabetes patients. A systemically effective medicament is indicated since the distal blockages are smooth, widespread and inoperable. See further ref. 68.
A medicament for the treatment of patients suffering from systemic hypertension and peripheral artery disease secondary to diabetes, said medicament consisting of R-Ras linked to Tat, a cell penetrating peptide. The said medicament is administered at a dose which is effective at reducing vascular cell proliferation of all arteries and restoring natural blood flow.
Alternately, this medicament is used for patients suffering from atherosclerosis and systemic hypertension from unknown causes, or causes other than diabetes
A method of treating solid organ tumors consisting of controlled release bioabsorbable polymeric microspheres made of poly(lactic-co-glycolic acid) (PLGA) and impregnated with R-Ras linked with F3, an internalizing, tumor-specific homing peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) (See further refs. 69 and 70). Said microspheres are 10-50 microns in diameter, and are administered by a physician interarterially into tumor neovasculature by means of percutaneous catheter based injection (see supplementary
Alternately, the bioabsorbable polymeric microspheres is made of polycaprolactone or lactide-coglycolide polymer impregnated with R-Ras linked with F3.
A method of treating pulmonary hypertension consisting of controlled release bioabsorbable polymeric microspheres made of poly(lactic-co-glycolic acid) (PLGA) and impregnated with R-Ras linked with Tat (GRKKRRQRRRPPQ), a cell penetrating peptide and CGFECVRQCPERC, a lung homing peptide. Said microspheres are 10-50 microns in diameter, and are administered by a physician interarterially into distal lung vasculature by means of percutaneous catheter based injection (see supplementary
Alternately, the bioabsorbable polymeric microspheres are made of polycaprolactone or lactide-coglycolide polymer impregnated with R-Ras linked with F3.
A chewing gum with composition containing proportions of filler, chewing gum base, emulsifier, gum chicle or SBR synthetic rubber, flavoring, sweetener and the medicament of R-Ras linked with the tumor homing, internalizing peptide F3 to ameliorate medical conditions such as carcinomas of the lips, mouth and tongue due to tobacco use by controlling release of the R-Ras/F3 conjugate during chewing thereof.
A topical paste formulated in a hydrophilic petrolatum-based ointment containing R-Ras conjugated to arginine heptamer (RRRRRRRR), a transporter molecule for the outermost layer of the epidermis, the stratum corneum, for the treatment of spider veins, a disfiguring form of capillaritis. See further reference 71.
The topical paste optionally comprises additional components such as (a) petrolatum or mineral oil, (b) fatty alcohols, (c) fatty ester emollients, (d) silicone oils or fluids, and (e) preservatives. These components must in general be safe for application to the human skin and must be compatible with the other components of the formulation.
Alternately, this topical paste is formulated into a transdermal patch. A transdermal patch is a medicated adhesive patch that is placed on the skin to deliver a time released dose of R-Ras/arginine heptamer conjugate through the skin and into the bloodstream
This example describes a method of treating restenosis in the coronary arteries after angioplasty or atheroectomy procedures with the goal of improving blood flow through the vessels.
This example includes a drug-burdened vascular prosthesis in the tubular form of a stent, coated with bioabsorbable polyglycolic acid which is fused with and contains the following compound: R-Ras linked to Tat, a cell penetrating peptide (see ref. 72), and to a heart homing peptide such as CRPPR (see ref. 73).
Alternately, the stent coatings containing the R-Ras/Tat/CRPPR conjugate are fused into cellulose, polylactic acid, methacryloyl phosphorylcholine laurylmethacrylate, or a 50/50 mixture of polyethylene vinylacetate/polybutyl methacrylate.
To deploy the stent, standard angioplasty procedures are used to deliver the stent through the femoral artery or other arterial point of entry. In summary, following identification of the target tissue comprising atheromatous plaque on the wall of the vessel, the stent should be deployed in the area of injury and utilized according to the following steps:
a. the stent-deploying balloon catheter is positioned at the lesion site immediately after the completion of the angioplasty/atherectomy (the protective sheath should be pulled back to expose the drug-laden stent at this point if a sheath was used);
b. the balloon is expanded until the surface of the drug-ladened stent implant fully engages the arterial wall for the full 360.degrees;
d. the balloon is then deflated and all catheters are removed from the body; and
e. time released elution of R-Ras/Tat/CRPPR conjugate.
In this example, patients with collagen vascular disease are treated in a gene therapy protocol.
Patients suffering from collagen vascular disease experience hyperproliferation of systemic vascular cells causing a variety of debilitating and deadly symptoms. The lack of sufficient R-Ras activity in the vascular cells of the patients is the targeted dysfunction in the gene therapy protocol.
In a first step, an adeno-associated virus, from the parvovirus family, is selected for infection with R-Ras and appropriate enzymes and promoters. Working with a standard protocol for transfection of AAV vectors with desired DNA strands and appropriate joint elements, a gene therapy product is developed.
Patients are provided the gene therapy periodically by percutaneous infusion catheter in a controlled drip application under direct supervision by a physician. As the patient responds to medication, the periods of time between applications lengthen, but do not end.
Alternately, this gene therapy protocol is used for patients suffering from pulmonary hypertension.
In this example, R-Ras linked by protein fusion with Tat, an internalizing peptide and a vascular homing peptide are prepared as an aqueous solution for delivering to distal pulmonary capillaries via liposomes for pulmonary dysfunction secondary to Systemic Lupus Erythematosus.
In the example, liposomes are prepared according to standard procedures by dissolving in chloroform the following lipids: 85 mole percent distearyl phosphatidylglycerol (DSPG), 10 mole percent of the disulfide-linked PEG-DSPE conjugate prepared as described in Example 1, 1 mole percent of an affinity moiety-DSPE conjugate, and 4 mole percent cholesterol. The lipids are dried as a thin film by rotation under reduced pressure. The lipid film is hydrated by addition of an aqueous phase to form liposomes which are sized by sonication or by sequential extrusion through Nucleopore polycarbonate membranes with pore sizes of 0.4.mu.m, 0.2.mu.m, 0.1.mu.m and 0.5.mu.m to obtain liposomes of 100-150 nm in size.
Completed liposomes are introduced into lungs by means of a percutaneous catheter inserted via standard interventional technique to the primary pulmonary artery, where a saline solution containing liposome spheres is released at a controlled rate.
On contacting pulmonary capillary walls, the liposomes dissolve according to a predetermined half-life in normal blood, whereby the local administration of aqueous based drug is absorbed by appropriate hyperproliferated tissues for the relief of SLE related dyspnea and related pulmonary dysfunction.
In this example, R-Ras is formulated with the tumor specific, internalizing peptide F3 into nanoparticles for the treatment of solid organ tumors.
Tri-n-octylphosphine oxide-coated ZnS-capped CdSe qdots are synthesized and their surface chemistry modified to render them water soluble. After this, the surface of qdots are coated with mercaptoacetic acid.
R-Ras and F3 (YDEPQRRSARLSAICPAPPKPEPKPKKAPAKK) peptide are used to coat the qdots
The R-Ras and F3 are thiolated by using 3-mercaptopropionimidate hydrochloride (a.k.a. iminothiolane), an imidoester compound containing a sulfhydryl group. R-Ras and F3 are incubated with iminothiolane for 1 h in 10 mM PBS, pH 7.4, at a 1:1 molar ratio. Afterward, mercaptoacetic acid-coated qdots are added to the solution to exchange some of the mercaptoacetic acid groups with the thiolated peptide incubated overnight at room temperature. For coadsorption of polyethylene glycol (PEG), R-Ras and F3 peptide, amine-terminated PEG (Shearwater Polymers, Huntsville, Ala.) was thiolated with iminothiolane. Thiolated PEG was directly added to a solution of mercaptoacetic acid-coated qdots in 10 mM PBS, pH 7.4, and allowed to incubate overnight at room temperature. PEG helps the qdots maintain solubility in aqueous solvents and minimize nonspecific binding. Afterward, the thiolated peptide are added to the PEG/qdot solution and incubated overnight at room temperature. The coated qdots are purified with Microspin G-50 columns (Amersham Pharmacia) before intravenous injection into the patient.
See further ref. 74.
In this example, R-Ras is formulated with the cell penetrating peptide Tat, and lung homing peptide CGFECVRQCPERC into nanoparticles for the treatment of pulmonary hypertension.
Tri-n-octylphosphine oxide-coated ZnS-capped CdSe qdots are synthesized and their surface chemistry modified to render them water soluble. After this, the surface of qdots are coated with mercaptoacetic acid.
R-Ras, cell penetrating peptide Tat, and lung homing peptide CGFECVRQCPERC are used to coat the qdots
The R-Ras, Tat and CGFECVRQCPERC are thiolated by using 3-mercaptopropionimidate hydrochloride (a.k.a. iminothiolane), an imidoester compound containing a sulfhydryl group. R-Ras, Tat and CGFECVRVQCPERC peptides are incubated with iminothiolane for 1 h in 10 mM PBS, pH 7.4, at a 1:1 molar ratio. Afterward, mercaptoacetic acid-coated qdots are added to the solution to exchange some of the mercaptoacetic acid groups with the thiolated peptide incubated overnight at room temperature. For coadsorption of polyethylene glycol (PEG), R-Ras, Tat and CGFECVRQCPERC peptides, amine-terminated PEG (Shearwater Polymers, Huntsville, Ala.) was thiolated with iminothiolane. Thiolated PEG was directly added to a solution of mercaptoacetic acid-coated qdots in 10 mM PBS, pH 7.4, and allowed to incubate overnight at room temperature. PEG helps the qdots maintain solubility in aqueous solvents and minimize nonspecific binding. Afterward, the thiolated peptide are added to the PEG/qdot solution and incubated overnight at room temperature. The coated qdots are purified with Microspin G-50 columns (Amersham Pharmacia) before intravenous injection into the patient.
In this example, R-Ras is formulated with the cell penetrating peptide Tat, and heart homing peptide CRPPR into nanoparticles for the treatment of coronary artery disease.
Tri-n-octylphosphine oxide-coated ZnS-capped CdSe qdots are synthesized and their surface chemistry modified to render them water soluble. After this, the surface of qdots are coated with mercaptoacetic acid.
R-Ras, cell penetrating peptide Tat, and heart homing peptide CRPPR are used to coat the qdots
The R-Ras, Tat and CRPPR are thiolated by using 3-mercaptopropionimidate hydrochloride (a.k.a. iminothiolane), an imidoester compound containing a sulfhydryl group. R-Ras, Tat and CRPPR peptides are incubated with iminothiolane for 1 h in 10 mM PBS, pH 7.4, at a 1:1 molar ratio. Afterward, mercaptoacetic acid-coated qdots are added to the solution to exchange some of the mercaptoacetic acid groups with the thiolated peptide incubated overnight at room temperature. For coadsorption of polyethylene glycol (PEG), R-Ras, Tat and CRPPR peptides, amine-terminated PEG (Shearwater Polymers, Huntsville, Ala.) was thiolated with iminothiolane. Thiolated PEG was directly added to a solution of mercaptoacetic acid-coated qdots in 10 mM PBS, pH 7.4, and allowed to incubate overnight at room temperature. PEG helps the qdots maintain solubility in aqueous solvents and minimize nonspecific binding. Afterward, the thiolated peptide are added to the PEG/qdot solution and incubated overnight at room temperature. The coated qdots are purified with Microspin G-50 columns (Amersham Pharmacia) before intravenous injection into the patient.
R-Ras medicaments in an inhaler for the treatment of pulmonary hypertension. In a solution composition for use in an aerosol inhaler which comprises a IR-Ras/Tat/lung homing peptide CGFECVRQCPERC conjugate solution, a propellant such as 1,1,1,2-tetrafluoroethane (EIFA 134a), a cosolvent such as alcohol and optionally a low volatility compound the use of a mixture of HFA 134a and HFA 227 allows to modulate the mass median aerodynamic diameter (MAD) of the aerosol particles on actuation of the inhaler to target specific regions of the respiratory tract. Moreover the fine particle dose (FPD) of the active ingredient in the composition increases by reducing the metering chamber volume. HFAs and in particular 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane SOFA 227) have been acknowledged to be the best candidates for non-CFC propellants. Cosolvents which are optionally used in these formulations are for instance alcohols such as ethanol and polyols such as propylene glycol.
For aerosol suspension compositions, a surfactant is often added to improve the physical stability of the suspension. Surfactants also lubricate the valve components in the inhaler device.
The R-Ras conjugate is stored in solution in a pressurized cansiter. The canister is attached to a plastic, hand-operated pump. When pumped, a certain dose of the R-Ras conjugate is released in aerosol form. The operator puts the release end of the pump into his mouth, and depresses the canister to release the medicine, while simultaneously inhaling deeply. The aerosolized medicine is drawn into the lungs, where it is absorbed into the bronchial alveoli. Peripheral pulmonary deposition of R-Ras conjugates can easily be directly absorbed from the alveoli into the pulmonary circulation.
Alternately, a different approach to aerosol-inhalers is the use of finely divided powdered devices containing standardized amounts (dose) of R-Ras conjugate powder ready for the patient to take.
Real time PCR methods to determine R-Ras mRNA levels in vascular tissue samples
This example shows a method and features of a clinical laboratory Real Time Polymerase Chain Reaction protocol for the detection and quantification of R-Ras in solid tumor biopsy samples obtained from patients undergoing curative treatment.
R-Ras evaluation can take place in many bodily fluids or tissues; however, in the example endoarterial tissue is selected from either percutaneous or open biopsy.
In an earlier set-up protocol, probe sequences for RNA detection of R-Ras, and common mutations from wild type, are crafted using a reverse DNA transcription.
When biopsy samples arrive at the clinical laboratory, they are prepared for evaluation in AmpliTaq® DNA polymerase (GeneAmp RNA PCR kit, Perkin-Elmer Cetus) according to manufacturer's directions.
As R-Ras levels are detected in patients, reports recommending new treatment decisions are generated for attending physicians that reflect, for example, the need for supplementation of R-Ras in effected tissues, thereby controlling hyperproliferation typical in growing tumors and R-Ras depleted tissues.
See for further details refs. 75 and 76.
Production of fusion proteins containing R-Ras in Prokaryotic cells genetically transfected to express R-Ras
This example details a method by which R-Ras fusion proteins, with small variations in their structure for various medicinal purposes, are created in quantity using Prokaryotic cells by an inexpensive commercial method.
A fusion protein is a protein created through genetic engineering from two or more proteins/peptides. This is achieved by creating a fusion gene: removing the stop codon from the DNA sequence of the first protein, then appending the DNA sequence of the second protein in frame. That DNA sequence will then be expressed by a cell as a single protein.
After assembling the desired transcript of R-Ras, linkers, homing peptide(s), cell-penetrating peptides, and/or internalizing peptides, the cloned gene is inserted into a pMAL vector down-stream from the malE gene, which encodes maltose-binding protein (MBP). The technique uses the strong Ptac promoter and the translation initiation signals of MBP to express large amounts of the fusion protein. The fusion protein is then purified by one-step affinity purification specific for MBP.
The system uses the pMAL vectors which are designed so that insertion interrupts a lacZα gene allowing a blue-to-white screen for inserts on X-gal (5). The vectors include a sequence coding for the recognition site of a specific protease. This allows the protein of interest to be cleaved from MBP after purification, without adding any vector-derived residues to the protein (6). For this purpose, the polylinker includes a restriction site superimposed on the sequence coding for the site of the specific protease. This is where the gene encoding the fusion protein containing R-Ras is inserted.
Expression from the pMAL vectors yields up to 100 mg fusion protein from a liter of culture. In most cases, the expressed protein is soluble, as fusion to MBP has been proven to enhance the solubility of proteins expressed in E. coli (7). In this manner an ideal fusion of R-Ras with both medicinally effective and manufacturable combinations of internalizing peptides and homing peptides can be achieved. See further references 77-83.
In this example, patients presenting unexplained pulmonary hypertension are examined for R-Ras and other protein levels in the smooth muscle cells of their pulmonary arteries.
General diagnosis of suspected pulmonary arterial hypertension is effected by a combination of patient reports of dyspnea, exercise fatigue, and elevated pulmonary artery pressure.
Before directing the patient to an expensive and possibly ineffective regime of pharmacological vasodilatation, antiproliferatives or surgery, the attending physician in this example orders a histological and molecular diagnosis of the patient's pulmonary arterial wall to assess its health and possible contribution to symptoms.
Using standard cardiovascular intervention technique, tissue is obtained from the patient's arteries using endoarterial biopsy (as described by Rothman A. et al., ref. 84). A portion of the tissue is prepared for histological examination, while another portion is prepared for ELISA testing using a custom pre-fabricated instant ELISA kit, supplied commercially by Bender Medsystems.
In the pre-fabrication of the instant ELISA kit, a panel of proteins with direct or suspected effect on pulmonary hypertension is selected for antibody cloning and testing. Importantly, one of the proteins in the ELISA kit is R-Ras. A sensitive ELISA test for R-Ras determines its level, not simply its presence.
In the clinical laboratory, prepared samples of a patient's pulmonary artery tissue are tested using the instant ELISA kit, and R-Ras levels for that individual patient's artery wall are determined. Once complete, a report of the test is relayed to the attending physician who determines a treatment regime based on the information. In particular, low or absent R-Ras levels indicate to the physician that R-Ras must be supplemented.
Detection of diminished R-Ras activity in pulmonary hypertension by endoarterial biopsy and gene chip microarray.
In this example a pig model of pulmonary hypertension was created via a surgical anastomosis of the left pulmonary artery to the descending aorta (as described by Corno A. et al., ref. 85). After 21 days, endoarterial biopsies were obtained (as described by Rothman A. et al., ref. 86) from the left, hypertensive pulmonary arteries and the right, normotensive pulmonary arteries. RNA was isolated from the endoarterial biopsy samples using an Agilent Total RNA Isolation Micro Kit, and R-Ras gene expression levels in the samples were determined using Affymetrix porcine gene chip microarrays. R-Ras gene expression levels were diminished 58% in the hypertensive endoarterial biopsy samples as compared to the normotensive endoarterial biopsy samples, demonstrating that R-Ras activity is lower in pulmonary hypertensive arteries than in normotensive arteries.
Alternately, this diagnostic technique can be used to detect diminished R-Ras activity in percutaneously obtained atherectomy samples from coronary artery and peripheral artery disease patients.
The diagnostic kit used to perform the endoarterial biopsy described in: “A Kit for Obtaining an Endoarterial Biopsy Sample”, WIPO International Publication WO 02/36017, ref. 87; European Patent Application No. 01992532.0, ref. 88; U.S. patent application Ser. No. 10/415,719, ref. 89.
The endoarterial biopsy catheter and endoarterial biopsy method are described in:
U.S. Pat. No. 5,287,857 issued Feb. 22, 1994, Apparatus and Method for Obtaining an Arterial Biopsy, ref. 90; U.S. Pat. No. 5,406,959 issued Apr. 18, 1995, Method and Apparatus for Obtaining an Arterial Biopsy and Diagnosing Diseases of the Vascular System, ref. 91; and European Patent No. 0647121, ref. 92.
This example demonstrates the application of R-Ras medicaments to treat or prevent intimal hyperplasia in vascular grafts after surgery (e.g., coronary artery bypass grafts, peripheral artery bypass grafts, organ transplantation, heart valves, etc.).
An isotonic saline solution with a therapeutic dose of R-Ras/Tat conjugate is prepared in advance and made available in the operating theater during transplant operations.
Immediately after blood vessel or organ harvest, or peri-operative preparation of tissue-engineered vessels or organs, and prior to surgical implantation, the saline solution is injected into the vessel lumen or lumens so that the inner blood vessel layers are wetted with the solution.
After implantation, the anastomosed vessels are observed according to standard medical practice. If stenosis emerges, several therapeutic options are available to the treating physician.
First is a course of R-Ras pharmacological therapy (see example 2), which is for instance recommended prophylactically prior to observed vessel dysfunction.
Secondly, for example, is atheroectomy, followed by an assessment of R-Ras levels in the removed stenotic tissue (see example 15—ELISA), followed, if appropriate, by additional R-Ras pharmacological therapy.
Thirdly, at post-operative follow-up, diagnosis of the cleared artery by endoarterial biopsy to assess levels of R-Ras in the sampled tissue, and possibly followed up by R-Ras pharmacological therapy.
In every case of pharmacological therapy, if possible, R-Ras is conjugated with the appropriate homing peptides for the dysfunctional vessels.
In this example, pulmonary hypertension is treated by ex vivo gene therapy; specifically, dysfunctional pulmonary artery endothelial and smooth muscle cells are modified to produce R-Ras, thereby reducing their dysfunctional hyperplasia, and are then transplanted into original pulmonary arteries.
Endothelial and smooth muscle cells are harvested from a pulmonary hypertension patient by either open or percutaneous biopsy. Harvested cells are modified by adenovirus vector gene therapy protocol to express R-Ras, initially ex vivo.
Once modified, and tested for consistent R-Ras expression, the autologous cells are then reintroduced into the pulmonary arteries by means of percutaneous catheter based injection.
This example is a suppository for treatment of ano-rectal vasculopathies and malignancies. This example demonstrates the embedment of R-Ras linked with Tat, a cell-penetrating peptide, in polycaprolactone (PCL) microspheres for slow-release delivery of R-Ras across mucosal tissues, an application for which the long lasting hydrophobic polymer is particularly suited.
Polycaprolactone (PCL) is a biodegradable, biocompatible and semicrystalline polymer having a very low glass transition temperature. Due to its slow degradation, PCL is ideally suitable for long-term delivery.
Various categories of drugs have been encapsulated in PCL for targeted drug delivery and for controlled drug release. Microspheres of PCL either alone or of PCL copolymers have been prepared to obtain the drug release characteristics
A solution of R-Ras linked to Tat in ultra-pure water is emulsified with 6% (w/v) solution of PCL (Aldrich) in dichloromethane (UCB, Braine L'Alleud Belgium) using an Ultraturrax model T25 at high speed (8000 rmp) and room temperature. The resulting water-in-oil (w/o) emulsion is then emulsified with 5% (w/v) PVA solution (50 ml) in the same conditions that for the first emulsion to produce a water-in-oil-water (w/o/w) emulsion. The latter is then stirring magnetically at +−800 rpm overnight at room temperature and pressure, to allow the evaporation of the organic solvent and the formation of microparticles. Once their wall was hardened, the microparticles were collected by centrifugation (10 min at 4000×g) washed three times with pure water and freeze-dried. See for example ref. 93.
Microspheres are blended with suppository excipient, poured into plastic molds, and allowed to form. On removal, complete suppositories are wrapped in foil, and made available to treating physicians.
Alternately, these suppositories are used for vaginal and cervical carcinomas.
This example shows how to determine the proper dose for a R-Ras medicament:
a. Identify a reasonable starting dose, ideally with specific adjustments (or a firm basis for believing none is needed) for patient size, gender, age, concomitant illness, and concomitant therapy, reflecting an integration of what is known about pharmacokinetic and pharmacodynamic variability. Depending on circumstances (the disease, the drug's toxicity) the starting dose ranges from a low dose with some useful effect, to a dose that is at or near the full-effect dose.
b. Identify reasonable, response-guided titration steps, and the interval at which they should be taken, again with appropriate adjustments for patient characteristics. These steps would be based either on the shape of the typical individual's dose-effect curves (for both desirable and undesirable effects), if individual dose-response data were available, or if not, on the shape of the population (group)-average dose-response, and the time needed to detect a change in these effects. It should be noted that methodology for finding the population (group)-average dose-response is, at present, better established than is methodology for finding individual dose-response relationships.
c. Identify a dose, or a response (desirable or undesirable), beyond which titration should not ordinarily be attempted because of a lack of further benefit or an unacceptable increase in undesirable effects.
Source and reference: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use—10 March 1994
Supplementary
Supplementary
Supplementary
a, Wire-injured arteries were stained 5 weeks after the injury with smooth muscle □-actin. Positive staining of the neointimal thickening indicates accumulation of SMC in both wild-type and R-Ras-null lesions. Smooth muscle in the adventitia of small arteries and arterioles is also stained, while adventitial fibroblasts were negative. b, F4/80 staining of the arterial lesions at 3 weeks indicates lack of significant macrophage infiltration in this model. c, TUNEL staining of the lesions at 3 weeks after the injury shows negligible apoptosis of intimal and medial SMC in both wild-type and R-Ras-null lesions. Control, DNase-treated R-Ras-null section as a positive control for TUNEL staining.
Supplementary
Supplementary
R-Ras wildtype (R-Raswt) sequence and sequences of R-Ras mutants R-Ras 38V and R-Ras 87L.
Supplementary
R-Ras impregnated microparticle catheter directed infusion for lung cancer.
Supplementary
R-Ras impregnated microparticle catheter directed infusion for kidney cancer.
Supplementary
R-Ras impregnated microparticle catheter directed infusion for pulmonary hypertension.
Certain work described herein was supported, in part, by U.S. Federal Grant Nos. PO CA82713, RO1 CA79984, T32 CA09579, and P30 CA30199, awarded by the National Institutes of Health. The United States Government may have certain rights in the invention. All references noted herein are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL2005/000794 | 11/11/2005 | WO | 00 | 6/3/2009 |
