WO1999031119A1 - Platelet derived growth factor (pdgf) nucleic acid ligand complexes - Google Patents
Platelet derived growth factor (pdgf) nucleic acid ligand complexes Download PDFInfo
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- WO1999031119A1 WO1999031119A1 PCT/US1998/009050 US9809050W WO9931119A1 WO 1999031119 A1 WO1999031119 A1 WO 1999031119A1 US 9809050 W US9809050 W US 9809050W WO 9931119 A1 WO9931119 A1 WO 9931119A1
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Definitions
- Described herein are high affinity ssDNA and RNA ligands to platelet derived growth factor (PDGF).
- SELEX an acronym for Systematic Evolution of Ligands by Exponential enrichment.
- a method for preparing a therapeutic or diagnostic Complex comprised of a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High
- the invention further includes Complexes comprised of one or more PDGF Nucleic Acid Ligands and a Non-Immunogenic, High Molecular Weight Compound or a Lipophilic Compound.
- the invention further relates to improving the Pharmacokinetic Properties of a PDGF Nucleic Acid Ligand by covalently linking the PDGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound to form a Complex.
- the invention further relates to improving the Pharmacokinetic Properties of a PDGF Nucleic Acid Ligand by using a Lipid Construct comprising a PDGF Nucleic Acid Ligand or a Complex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.
- This invention further relates to a method for targeting a therapeutic or diagnostic agent to a biological target that is expressing PDGF by associating the agent with a Complex comprised of a PDGF Nucleic Acid Ligand .and a Lipophilic Compound or Non-Immunogenic, High Molecula- r Weight Compound, wherein the Complex is further associated with a Lipid Construct and the PDGF Nucleic Acid Ligand is further associated with the exterior of the Lipid Construct.
- SELEX Systematic Evolution of Ligands by Exponential enrichment
- Patent Applications describes a fundamentally novel method for making a Nucleic Acid Ligand to any desired target molecule.
- the SELEX process provides a class of products which are referred to as Nucleic Acid Ligands, each ligand having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule.
- Each SELEX-identified Nucleic Acid Ligand is a specific ligand of a given target compound or molecule.
- SELEX is based on the unique insight that Nucleic Acids have sufficient capacity for forming a variety of two- and three-dimensional structures .and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
- the SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
- the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound Nucleic Acids from those Nucleic Acids which have bound specifically to target molecules, dissociating the Nucleic Acid-target complexes, amplifying the Nucleic Acids dissociated from the Nucleic Acid-t.arget complexes to yield a ligand-enriched mixture of Nucleic Acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity Nucleic Acid Ligands to the target molecule.
- Candidate Mixture of approximately 10 13 to 10 18 Nucleic Acids the present inventors postulate that at least one Nucleic Acid exists with the appropriate shape to facilitate each of a broad variety of physical and chemical interactions.
- the SELEX method encompasses the identification of high-affinity Nucleic Acid Ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified Nucleic Acid Ligands containing modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," abandoned in favor of United States Patent Application Serial No. 08/430,709, now United States Patent
- the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” now United States
- These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
- the SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in United States Patent Application Serial No.
- VEGF Nucleic Acid Ligands that are associated with a Lipophilic Compound, such as diacyl glycerol or dialkyl glycerol, in a diagnostic or therapeutic complex are described in United States Patent Application Serial No. 08/739,109, filed October 25, 1996, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes.”
- VEGF Nucleic Acid Ligands that are associated with a Lipophilic Compound, such as a glycerol lipid, or a Non-Immunogenic, High Molecular Weight Compound, such as polyethylene glycol are further described in
- VEGF Vascular Endothelial Growth Factor
- VEGF Nucleic Acid Ligands that are associated with a non-immunogenic, high molecular weight compound or lipophilic compound are also further described in PCT/US97/18944, filed October 17, 1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes.”
- Lipid Bilayer Vesicles are closed, fluid-filled microscopic spheres which are formed principally from individual molecules having polar (hydrophilic) and non-polar (lipophilic) portions.
- the hydrophilic portions may comprise phosphato, glycerylphosphato, carboxy, sulfato, amino, hydroxy, choline or other polar groups.
- lipophilic groups are saturated or unsaturated hydrocarbons such as alkyl, alkenyl or other lipid groups.
- Sterols e.g., cholesterol
- other pharmaceutically acceptable adjuvants including anti-oxidants like alpha-tocopherol
- Liposomes are a subset of these bilayer vesicles and are comprised principally of phospholipid molecules that contain two hydrophobic tails consisting of fatty acid chains. Upon exposure to water, these molecules spontaneously align to form spherical, bilayer membranes with the lipophilic ends of the molecules in each layer associated in the center of the membrane and the opposing polar ends forming the respective inner and outer surface of the bilayer membrane(s). Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium.
- These membranes may be arranged in a series of concentric, spherical membranes separated by thin strata of water, in a manner not dissimilar to the layers of an onion, around an internal aqueous space.
- MLV multilamellar vesicles
- UV Unilamellar Vesicles
- liposomes The therapeutic use of liposomes includes the delivery of drugs which are normally toxic in the free form. In the liposomal form, the toxic drug is occluded, and may be directed away from the tissues sensitive to the drug and targeted to selected areas. Liposomes can also be used therapeutically to release drugs over a prolonged period of time, reducing the frequency of administration. In addition, liposomes can provide a method for forming aqueous dispersions of hydrophobic or amphiphilic drugs, which are normally unsuitable for intravenous delivery.
- liposomes In order for many drugs and imaging agents to have therapeutic or diagnostic potential, it is necessary for them to be delivered to the proper location in the body, and the liposome can thus be readily injected and form the basis for sustained release and drug delivery to specific cell types, or parts of the body.
- Several techniques can be employed to use liposomes to target encapsulated drugs to selected host tissues, and away from sensitive tissues. These techniques include manipulating the size of the liposomes, their net surface charge, and their route of administration.
- MLVs primarily because they are relatively large, are usually rapidly taken up by the reticuloendothelial system (principally the liver and spleen). UVs, on the other hand, have been found to exhibit increased circulation times, decreased clearance rates and greater biodistribution relative to MLVs.
- Liposomes Passive delivery of liposomes involves the use of various routes of administration, e.g., intravenous, subcutaneous, intramuscular and topical. Each route produces differences in localization of the liposomes.
- Two common methods used to direct liposomes actively to selected target areas involve attachment of either antibodies or specific receptor ligands to the surface of the liposomes.
- Antibodies are known to have a high specificity for their corresponding antigen and have been attached to the surface of liposomes, but the results have been less than successful in many instances. Some efforts, however, have been successful in t-argeting liposomes to tumors without the use of antibodies, see, for example, U.S. Patent No. 5,019,369, U.S. Patent No. 5,441,745, or U.S. Patent No. 5,435,989.
- An area of development aggressively pursued by researchers is the delivery of agents not only to a specific cell type but into the cell's cytoplasm and, further yet, into the nucleus. This is particularly important for the delivery of biological agents such as DNA, RNA, ribozymes and proteins.
- a promising therapeutic pursuit in this area involves the use of antisense DNA and RNA oligonucleotides for the treatment of disease.
- one major problem encountered in the effective application of antisense technology is that oligonucleotides in their phosphodiester form are quickly degraded in body fluids and by intracellular and extracellular enzymes, such as endonucleases and exonucleases, before the target cell is reached.
- Intravenous administration also results in rapid clearance from the bloodstream by the kidney, and uptake is insufficient to produce an effective intracellular drug concentration.
- Liposome encapsulation protects the oligonucleotides from the degradative enzymes, increases the circulation half-life and increases uptake efficiency as a result of phagocytosis of the Liposomes. In this way, oligonucleotides are able to reach their desired target and to be delivered to cells in vivo.
- Antisense oligonucleotides are only effective as intracellular agents.
- Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor have been encapsulated into Liposomes linked to folate via a polyethylene glycol spacer
- Loading of biological agents into liposomes can be accomplished by inclusion in the lipid formulation or loading into preformed liposomes. Passive anchoring of oligopeptide and ohgosaccharide ligands to the external surface of liposomes has been described (Zalipsky et al. (1997) Bioconjug. Chem. 8:111:118).
- Both species contain five immunoglobulin-like extracellular domains, a single transmembrane domain and an intracellular tyrosine kinase domain separated by a kinase insert domain.
- the functional high affinity receptor is a dimer and engagement of the extracellular domain of the receptor by PDGF results in cross-phosphorylation (one receptor tyrosine kinase phosphorylates the other in the dimer) of several tyrosine residues.
- Receptor phosphorylation leads to a cascade of events that results in the transduction of the mitogenic or chemotactic signal to the nucleus.
- src-homology 2 SH2 domain-containing proteins including phospholipase C-g, phosphatidylinositol 3 '-kinase, GTPase-activating protein and several adapter molecules like She, Grb2 and Nek (Heldin (1995) Cell 80:213).
- SH2 src-homology 2
- the ⁇ -receptor homodimer binds all three PDGF isoforms with high affinity, the ⁇ -receptor homodimer binds only PDGF BB with high affinity and PDGF-AB with approximately 10-fold lower affinity, and the o -receptor heterodimer binds PDGF-BB and PDGF-AB with high affinity (Westermark & Heldin (1993) Acta Oncologica 32:101).
- the specificity pattern results from the ability of the A-chain to bind only to the ⁇ -receptor and of the B-chain to bind to both and ⁇ -receptor subunits with high affinity.
- PDGF proliferative diseases, such as cancer, restenosis, fibrosis, angiogenesis, and wound healing has been established.
- glioma The progression to high grade glioma was accompanied by the increase in expression of PDGF-B and the ⁇ -receptor in tumor-associated endothelial cells and PDGF-A in glioma cells.
- PDGF overexpression may thus promote tumor growth either by directly stimulating tumor cells or by stimulating tumor-associated stromal cells (e.g., endothelial cells).
- tumor-associated stromal cells e.g., endothelial cells.
- the proliferation of endothelial cells is a hallmark of angiogenesis.
- PDGF and/or PDGF receptors have also been observed in other malignancies including fibrosarcoma (Smits et al. (1992) Am. J. Pathol. 140:639) and thyroid carcinoma (Heldin et al. (1991) Endocrinology 129:2187).
- PTCA Percutaneous transluminal coronary angioplasty
- CAD occlusive coronary artery disease
- CAD occlusive coronary artery disease
- PTCA while it involves manipulations inside of coronary arteries, is not considered to be a cardiac surgical intervention.
- a balloon catheter is threaded through a femoral artery and is positioned within the plaque-laden segment of an occluded coronary vessel; once in place, the balloon is expanded at high pressure, compressing the plaque and increasing the vessel lumen.
- SMCs Smooth muscle cells
- medial vessel layer medial vessel layer
- SMCs proliferate and migrate into the intima, forming neointimal thickening characteristic of restenosis lesions.
- restenosis occurs subsequent to angioplasty, it is usually treated by repeat angioplasty, with or without placement of a stent, or by vascular graft surgery (bypass).
- a stent is a rigid cylindrical mesh that, once placed and expanded within a diseased vessel segment, mechanically retains the expanded vessel wall.
- the stent is deployed by catheter and, having been positioned at the desired site, is expanded in situ by inflation of a high pressure balloon.
- the expanded stent achieves and maintains a vessel lumen diameter comparable to that of adjacent non-diseased vessel; being pressed tightly into the overlying intima/media, it is resistant to migration within the vessel in response to blood flow.
- PTCA with stent placement has been compared with PTCA alone and shown to reduce restenosis to about half and to significantly improve other clinical outcomes such as myocardial infarction (MI) and need for bypass surgery.
- MI myocardial infarction
- PDGF-B The effect of PDGF-B on SMCs is mediated through PDGF ⁇ -receptor which is expressed at high levels in these cells after balloon injury (Lindner and Reidy (1995) Circulation Res. 76:951-957). Furthermore, the degree of neointimal thickening following balloon injury was found to be inversely related to the level of expression of PDGF ⁇ -receptor at the site of injury (Sirois et al. (1997) Circulation 95:669-676).
- United States Patent No. 5,171,217 discloses a method and composition for delivery of a drug to an affected intramural site for sustained release in conjunction with or following balloon catheter procedures, such as angioplasty.
- the drug may be selected from a variety of drugs known to inhibit smooth muscle cell proliferation, including growth factor receptor antagonists for PDGF.
- United States Patent No. 5,593,974 discloses methods for treating vascular disorders, such as vascular restenosis, with antisense oligonucleotides.
- the method is based on localized application of the antisense oligonucleotides to a specific site in vivo.
- the oligonucleotides can be applied directly to the target tissue in a mixture with an implant or gel, or by direct injection or infusion.
- United States Patent No. 5,562,922 discloses a method for preparing a system suitable for localized delivery of biologically active compounds to a subject. The method relates to treating polyurethane coated substrate with a coating expansion solution under conditions that will allow penetration of the biologically active compound throughout the polyurethane coating.
- Substrates suitable for this invention include, inter alia, metallic stents.
- Biologically active compounds suitable for use in this invention include, inter alia, lipid-modified oligonucleotides.
- PDGF-B-chain appears to have a central role in driving both of these processes given that 1) mesangial cells produce PDGF in vitro and various growth factors induce mesangial proliferation via induction of auto- or paracrine PDGF-B-chain synthesis; 2) PDGF B-chain and its receptor are overexpressed in many glomerular diseases; 3) infusion of PDGF-BB or glomerular transfection with a PDGF-B- chain cDNA can induce selective mesangial cell proliferation and matrix accumulation in vivo; and 4) PDGF-B-chain or ⁇ -receptor knock-out mice fail to develop a mesangium (reviewed in Floege and Johnson (1995) Miner.
- Electrolyte Metab. 21 :271-282 In addition to contributing to kidney fibrosis, PDGF is also believed to play a role in fibrosis development in other organs such as lungs and bone marrow and may have other possible disease associations (Raines et al. (1990) in Experimental Pharmacology, Peptide Growth Factors and Their Receptors, Sporn & Roberts, eds., pp. 173-262, Springer, Heidelberg).
- the present invention includes methods of identifying and producing nucleic acid ligands to platelet-derived growth factor (PDGF) and homologous proteins and the nucleic acid ligands so identified and produced.
- PDGF refers to PDGF-AA, -AB, and -BB isoforms and homologous proteins. Specifically included in the definition are human PDGF-AA, -AB, and -BB isoforms.
- Described herein are high affinity ssDNA and RNA ligands to platelet derived growth factor (PDGF).
- PDGF platelet derived growth factor
- SELEX an acronym for Systematic Evolution of Ligands by Exponential enrichment.
- a PDGF Nucleic Acid Ligand comprised of a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound by the method comprising identifying a Nucleic Acid Ligand from a Candidate Mixture of Nucleic Acids where the Nucleic Acid is a ligand of PDGF by the method of (a) contacting the Candidate Mixture of Nucleic Acids with PDGF, (b) partitioning between members of said Candidate Mixture on the basis of affinity to PDGF, and c) amplifying the selected molecules to yield a mixture of Nucleic Acids enriched for Nucleic Acid sequences with a relatively higher affinity for binding to PDGF, and covalently linking said identified PDGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular Weight Compound or a Lipophilic Compound.
- the invention further comprises a Complex comprised of a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or a Lipophilic Compound.
- the invention further includes a Lipid Construct comprising a PDGF Nucleic Acid
- the present invention further relates to a method for preparing a Lipid Construct comprising a Complex wherein the Complex is comprised of a PDGF Nucleic Acid Ligand and a Lipophilic Compound.
- this invention provides a method for improving the pharmacokinetic properties of a PDGF Nucleic Acid Ligand by covalently linking the PDGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecule Weight Compound or Lipophilic Compound to form a Complex and administering the Complex to a patient.
- the invention further relates to a method for improving the pharmacokinetic properties of a PDGF Nucleic Acid Ligand by further associating the Complex with a Lipid Construct. It is an object of the present invention to provide Complexes comprising one or more
- PDGF Nucleic Acid Ligands in association with one or more Non-Immunogenic, High Molecular Weight Compounds or Lipophilic Compounds and methods for producing the same. It is a further object of the present invention to provide Lipid Constructs comprising a Complex. It is a further object of the invention to provide one or more PDGF Nucleic Acid Ligands in association with one or more Non-Immunogenic, High Molecul-ar Weight Compounds or Lipophilic Compounds with improved Pharmacokinetic Properties. In embodiments of the invention directed to Complexes comprised of a PDGF Nucleic
- the Non-Immunogenic, High Molecular Weight Compound is Polyalkylene Glycol, more preferably, polyethylene glycol (PEG). More preferably, the PEG has a molecular weight of about 10-80K. Most preferably, the PEG has a molecular weight of about 20-45K.
- PEG polyethylene glycol
- the Lipophilic Compound is a glycerolipid.
- the Lipid Construct is preferably a Lipid Bilayer Vesicle and most preferably a Liposome.
- the PDGF Nucleic Acid Ligand is identified according to the SELEX method.
- the PDGF Nucleic Acid Ligand or Ligands can serve in a targeting capacity. Additionally, the PDGF Nucleic Acid Ligand can be associated through Covalent or
- Non-Covalent Interactions with a Lipid Construct without being part of a Complex are non-Covalent Interactions with a Lipid Construct without being part of a Complex.
- Lipid Constructs comprising a PDGF Nucleic Acid Ligand or a Non-Immunogenic, High Molecular Weight or Lipophilic Compound/PDGF Nucleic Acid Ligand Complex
- the Lipid Construct is of a type that has a membrane defining an interior compartment such as a Lipid Bilayer Vesicle
- the PDGF Nucleic Acid Ligand or Complex in association with the Lipid Construct may be associated with the membrane of the Lipid Construct or encapsulated within the compartment.
- the PDGF Nucleic Acid Ligand can associate with the interior-facing or exterior-facing part of the membrane, such that the PDGF Nucleic Acid Ligand is projecting into or out of the vesicle.
- a PDGF Nucleic Acid Ligand Complex can be passively loaded onto the outside of a preformed Lipid Construct.
- the Nucleic Acid Ligand is projecting out of the Lipid Construct
- the PDGF Nucleic Acid Ligand can serve in a targeting capacity.
- the PDGF Nucleic Acid Ligand of the Lipid Construct serves in a targeting capacity
- the Lipid Construct can have associated with it additional therapeutic or diagnostic agents.
- the therapeutic or diagnostic agent is associated with the exterior of the Lipid Construct.
- the therapeutic or diagnostic agent is encapsulated in the Lipid Construct or associated with the interior of the Lipid
- the therapeutic or diagnostic agent is associated with the Complex.
- the therapeutic agent is a drug.
- the therapeutic or diagnostic agent is one or more additional Nucleic Acid
- PDGF-mediated diseases include, but are not limited to, cancer, angiogenesis, restenosis, and fibrosis.
- PDGF-mediated diseases include, but are not limited to, cancer, angiogenesis, restenosis, and fibrosis.
- Figure 1 shows the consensus secondary structure for the sequence set shown in Table 3.
- R A or G
- Y C or T
- K G or T
- N and N' indicate any base pair.
- Figures 2A-2C show the minimal ligands 20t (SEQ ID NO:83), 36t (SEQ ID NO:84) and 41t (SEQ ID NO:85) folded according to the consensus secondary structure motif.
- [3'T] represents a 3 '-3' linked thymidine nucleotide added to reduce 3'-exonuclease degradation.
- Figures 3A-3C show the binding of minimal high affinity DNA ligands to PDGF- AA, PDGF-AB, and PDGF-BB, respectively.
- the fraction of 32 P 5' end-labeled DNA ligands bound to varying concentrations of PDGF was determined by the nitrocellulose filter binding method.
- Data points were fitted to equation 1 (for binding of the DNA ligands to PDGF-AA) or to equation 2 (for binding to PDGF-AB and -BB) using the non-linear least squares method. Binding reactions were done at 37°C in binding buffer (PBSM with 0.01% HSA).
- Figure 4 shows the dissociation rate determination for the high affinity interaction between the minimal DNA ligands and PDGF-AB.
- the fraction of 5' 32 P end-labeled ligands 20t (o), 36t ( ⁇ ), .and 41t (D), all at 0.17 nM, bound to PDGF-AB (1 nM) was measured by nitrocellulose filter binding at the indicated time points following the addition of a 500-fold excess of the unlabeled competitor.
- the dissociation rate constant (k off ) values were determined by fitting the data points to eq 3 in Example 1. The experiments were performed at 37°C in binding buffer.
- Figure 5 shows the thermal denaturation profiles for the minimal high affinity DNA ligands to PDGF-AB.
- the change in absorbance at 260 nm was measured in PBS containing 1 mM MgCl 2 as a function of temperature for ligands 20t (o), 36t ( ⁇ ), and 411 (D).
- Figure 6 shows the effect of DNA ligands on the binding of 125 I-PDGF-BB to PDGF ⁇ -receptors expressed in PAE cells.
- Figure 7 shows the effect of DNA ligands on the mitogenic effect of PDGF-BB on
- PAE cells expressing the PDGF ⁇ -receptors PAE cells expressing the PDGF ⁇ -receptors.
- Figures 8A-8B show the substitution pattern compatible with high affinity binding to PDGF-AB.
- the underlined symbols indicate 2'-O-methyl-2'-deoxynucleotides; italicized symbols indicate 2'-fTuoro-2'-deoxynucleotides; normal font indicates 2'-deoxyribonucleotides; [3'T] indicates inverted orientation (3'3') thymidine nucleotide (Glen Research, Sterling, VA); PEG in the loops of helices II and III of Figure 8B indicates pentaethylene glycol spacer phosphoramidite (Glen Research,
- Figure 8C shows the predicted secondary structure of a scrambled Nucleic Acid Ligand sequence that was used as a control in Examples 8 and 9. The scrambled region is boxed to accent the overall similarity of the scrambled Nucleic Acid Ligand to the Nucleic Acid Ligand shown in Figure 8B.
- FIGS. 9A-9E show the molecular descriptions NX31975-40K PEG (SEQ ID NO:
- FIG 10 shows the stabilities of DNA (36ta) and modified DNA (NX21568)
- Nucleic Acid Ligands in rat serum over time at 37°C. 36ta is shown by the symbol •; and NX21568 is shown by the symbol A.
- Figure 11 shows that NX31975-40K PEG significantly inhibited (p ⁇ 0.05) about
- Figure 12 shows the effects of NX31975-40K PEG on mitogen-stimulated proliferation of mesangial cells in culture (all mitogens were added at 100 ng/ml final concentration). Scrambled Nucleic Acid Ligand NX31976 and 40K PEG were also tested.
- Data are optical densities measured in the XTT assay and are expressed as percentages of baseline, i.e., cells stimulated with medium plus 200 ⁇ g/ml 40K PEG (i.e., the amount equivalent to the PEG attached to 50 ⁇ g/ml Nucleic Acid Ligand).
- Figures 13A-13E show effects of NX31975-40K PEG on glomerular cell proliferation (Figure 13 A), expression of glomerular PDGF B-chain (Figure 13B), proteinuria in rats with anti-Thy 1.1 nephritis (Figure 13C), mesangial cell activation (as assessed by glomerular de novo expression of ⁇ -smooth muscle actin) (Figure 13D), and monocyte/macrophage influx (Figure 13E).
- NX31975-40K PEG is shown as black
- NX31976-40K PEG is shown as cross-hatched
- 40K PEG is shown as white
- PBS is shown as hatched
- the normal range is shown as stippled.
- Figures 14A-C show the effects of NX31975-40K PEG on glomerular matrix accumulation. Glomerular immunostaining scores for fibronectin and type IV collagen as well as glomerular scores for type IV collagen mRNA expression (in situ hydridization) are shown. NX31975-40K PEG is shown as black, NX31976-40K PEG is shown as cross- hatched, 40K PEG is shown as white, PBS is shown as hatched, and the normal range is shown as stippled.
- Lipophilic Compounds are compounds which have the propensity to associate with or partition into lipid and/or other materials or phases with low dielectric constants, including structures that are comprised substantially of lipophilic components. Lipophilic Compounds include lipids as well as non-lipid containing compounds that have the propensity to associate with lipid (and/or other materials or phases with low dielectric const.ants). Cholesterol, phospholipids, .and glycerohpids, such as dialkylglycerol, and diacylglycerol, and glycerol amide lipids are further examples of Lipophilic Compounds.
- Complex as used herein describes the molecular entity formed by the covalent linking of a PDGF Nucleic Acid Ligand to a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.
- the Complex is depicted as A-B-Y, wherein A is a Lipophilic Compound or Non-Immunogenic, High Molecular Weight Compound as described herein; B is optional, and comprises a Spacer which may comprise one or more linkers Z; and Y is a PDGF Nucleic Acid Ligand.
- Lipid Constructs for purposes of this invention, .are structures containing lipids, phospholipids, or derivatives thereof comprising a variety of different structural arrangements which lipids are known to adopt in aqueous suspension. These structures include, but are not limited to, Lipid Bilayer Vesicles, micelles, Liposomes, emulsions, lipid ribbons or sheets, and may be complexed with a variety of drugs .and components which .are known to be pharmaceutically acceptable. In the preferred embodiment, the Lipid Construct is a Liposome.
- the preferred Liposome is unilamellar and has a relative size less than 200 nm.
- Common additional components in Lipid Constructs include cholesterol and alpha-tocopherol, .among others.
- the Lipid Constructs may be used alone or in any combination which one skilled in the art would appreciate to provide the characteristics desired for a particular application.
- the technical aspects of Lipid Constructs and Liposome formation are well known in the art and any of the methods commonly practiced in the field may be used for the present invention.
- Nucleic Acid Ligand as used herein is a non-naturally occurring Nucleic Acid having a desirable action on a Target.
- the Target of the present invention is PDGF, hence the term PDGF Nucleic Acid Ligand.
- a desirable action includes, but is not limited to, binding of the Target, catalytically changing the Target, reacting with the Target in a way which modifies/alters the Target or the functional activity of the Target, covalently attaching to the Target as in a suicide inhibitor, or facilitating the reaction between the Target and another molecule.
- the action is specific binding affinity for PDGF, wherein the Nucleic Acid Ligand is not a Nucleic Acid having the known physiological function of being bound by PDGF.
- the PDGF Nucleic Acid Ligand of the Complexes and Lipid Constructs of the invention are identified by the SELEX methodology.
- PDGF Nucleic Acid Ligands are identified from a Candidate Mixture of Nucleic Acids, said Nucleic Acid being a ligand of PDGF, by the method comprising a) contacting the Candidate Mixture with PDGF, wherein Nucleic Acids having an increased affinity to PDGF relative to the Candidate Mixture may be partitioned from the remainder of the Candidate Mixture; b) partitioning the increased affinity Nucleic Acids from the remainder of the Candidate Mixture; .and c) .amplifying the increased affinity Nucleic Acids to yield a ligand-enriched mixture of Nucleic Acids (see United States Patent Application Serial No. 08/479,725, filed June 7, 1995, entitled "High Affinity PDGF Nucleic Acid Ligands," now United States Patent No. 5,674,685, United States Patent Application
- portions of the PDGF Nucleic Acid Ligand (Y) may not be necessary to maintain binding and certain portions of the contiguous PDGF Nucleic Acid
- Ligand can be replaced with a Spacer or Linker.
- Y can be represented as Y-B'-Y'-B"-Y", wherein Y, Y' and Y" are parts of a PDGF Nucleic Acid Ligand or segments of different PDGF Nucleic Acid Ligands and B' and/or B" are Spacers or Linker molecules that replace certain nucleic acid features of the original PDGF Nucleic Acid Ligand.
- B' and B" are present and Y, Y', and Y" are parts of one PDGF Nucleic Acid Ligand, a tertiary structure is formed that binds to PDGF.
- B' and B" are not present, Y, Y', and Y" represent one contiguous PDGF Nucleic Acid Ligand.
- PDGF Nucleic Acid Ligands modified in such a manner are included in this definition.
- Candidate Mixture is a mixture of Nucleic Acids of differing sequence from which to select a desired ligand.
- the source of a Candidate Mixture can be from naturally- occurring Nucleic Acids or fragments thereof, chemically synthesized Nucleic Acids, enzymatically synthesized Nucleic Acids or Nucleic Acids made by a combination of the foregoing techniques.
- each Nucleic Acid has fixed sequences surrounding a randomized region to facilitate the amplification process.
- Nucleic Acid means either DNA, RNA, single-standed or double-str.anded and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the Nucleic Acid Lig.and bases or to the Nucleic Acid Ligand as a whole.
- modifications include, but are not limited to, 2'- position sug.ar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications such as internucleoside phosphorothioate linkages, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
- Non-Immunogenic, High Molecular Weight Compound is a compound between approximately 1000 Da to 1,000,000 Da, more preferably approximately 1000 Da to 500,000 Da, and most preferably approximately 1000 Da to 200,000 Da, that typically does not generate an immunogenic response.
- a - n immunogenic response is one that causes the organism to make antibody proteins.
- Examples of Non- Immunogenic, High Molecular Weight Compounds include Polyalkylene Glycol and polyethylene glycol.
- the molecular weight is about between 10-80 kDa.
- the molecular weight of the polyalkylene glycol is about between 20-45 kDa.
- the Non-Immunogenic, High Molecular Weight Compound can also be a Nucleic Acid Ligand.
- Lipid Bilayer Vesicles are closed, fluid-filled microscopic spheres which are formed principally from individual molecules having polar (hydrophilic) and non-polar
- hydrophilic portions may comprise phosphato, glycerylphosphato, carboxy, sulfato, amino, hydroxy, choline and other polar groups.
- non-polar groups are saturated or unsaturated hydrocarbons such as alkyl, alkenyl or other lipid groups.
- Sterols e.g., cholesterol
- other pharmaceutically acceptable components including anti- oxidants like alpha-tocopherol may also be included to improve vesicle stability or confer other desirable characteristics.
- Liposomes are a subset of Lipid Bilayer Vesicles and are comprised principally of phospholipid molecules which contain two hydrophobic tails consisting of long fatty acid chains. Upon exposure to water, these molecules spontaneously align to form a bilayer membrane with the lipophilic ends of the molecules in each layer associated in the center of the membrane and the opposing polar ends forming the respective inner and outer surface of the bilayer membrane. Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium.
- These membranes when formed are generally arranged in a system of concentric closed membranes separated by interlamellar aqueous phases, in a manner not dissimilar to the layers of an onion, around an internal aqueous space.
- MLV multilamellar vesicles
- UV unilamellar vesicles
- “Cationic Liposome” is a Liposome that contains lipid components that have an overall positive charge at physiological pH.
- SELEX methodology involves the combination of selection of Nucleic Acid Ligands which interact with a Target in a desirable manner, for example binding to a protein, with amplification of those selected Nucleic Acids. Iterative cycling of the selection/amplification steps allows selection of one or a small number of Nucleic Acids which interact most strongly with the Target from a pool which contains a very large number of Nucleic Acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved.
- the SELEX methodology is described in the SELEX Patent Applications.
- Target means any compound or molecule of interest for which a ligand is desired.
- a Target can be a protein (such as PDGF, thrombin, .and selectin), peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation.
- the principal Target of the subject invention is PDGF.
- “Improved Pharmacokinetic Properties” means that the PDGF Nucleic Acid Ligand covalently linked to a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound or in association with a Lipid Construct shows a longer circulation half- life in vivo relative to the same PDGF Nucleic Acid Ligand not in association with a Non- Immunogenic, High Molecular Weight Compound or Lipophilic Compound or in association with a Lipid Construct.
- Linker is a molecular entity that connects two or more molecular entities through Covalent Bond or Non-Covalent Interactions, and can allow spatial separation of the molecular entities in a manner that preserves the functional properties of one or more of the molecular entities.
- a linker can also be known as a Spacer. Examples of Linkers, include but are not limited to, the structures shown in Figures 9C-9E and the PEG spacer shown in Figure 9A. In the preferred embodiment, the linker B' and B" are pentaethylene glycols.
- “Therapeutic” as used herein, includes treatment and/or prophylaxis. When used,
- Therapeutic refers to humans and other animals.
- This invention includes ssDNA and RNA lig-ands to PDGF. This invention further includes the specific ssDNA and RNA ligands to PDGF shown in Tables 2-3, 6-7, and 9 and
- Figures 1-2A, 2B and 2C, 8A and 8B (SEQ ID NOS:4-35, 39-87, 97-144, 148-149). More specifically, this invention includes nucleic acid sequences that are substantially homologous to and that have substantially the same ability to bind PDGF as the specific nucleic acid ligands shown in Tables 2-3, 6-7, and 9 and Figures 1-2 A, 2B and 2C, 8 A and 8B (SEQ ID NOS:4-35, 39-87, 97-144, 148-149).
- substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%, and even more preferably in excess of 90%, 95%, or 99%.
- the percentage of homology as described herein is calculated as the percentage of nucleotides found in the smaller of the two sequences which align with identical nucleotide residues in the sequence being compared when 1 gap in a length of 10 nucleotides may be introduced to assist in that alignment.
- Substantially the same ability to bind PDGF means that the affinity is within one or two orders of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence - substantially homologous to those specifically described herein - has the same ability to bind PDGF.
- Tables 2-3, 6-7, and 9 and Figures 1-2A, 2 B and 2C, 8A and 8B shows that sequences with little or no primary homology may have substantially the same ability to bind PDGF.
- this invention also includes Nucleic Acid Ligands that have substantially the same postulated structure or structural motifs and ability to bind PDGF as the nucleic acid ligands shown in Tables 2-3, 6-7, and 9 and Figures 1-2A, 2B and 2C, 8A and 8B (SEQ ID NOS:4-35, 39-87, 97-144, 148-149).
- Substantially the same structure or structural motifs can be postulated by sequence alignment using the Zukerfold program (see Zuker (1989) Science 244:48-52). As would be known in the art, other computer programs can be used for predicting secondary structure .and structural motifs. Substantially the same structure or structural motif of Nucleic Acid Ligands in solution or as a bound structure can also be postulated using NMR or other techniques as would be known in the art. Further included in this invention is a method for preparing a Complex comprised of a
- Lipophilic Compound by the method comprising identifying a Nucleic Acid Ligand from a
- Candidate Mixture of Nucleic Acids where the Nucleic Acid is a ligand of PDGF by the method of (a) contacting the Candidate Mixture of Nucleic Acids with PDGF, (b) partitioning between members of said Candidate Mixture on the basis of affinity to PDGF, and c) amplifying the selected molecules to yield a mixture of Nucleic Acids enriched for Nucleic
- Acid sequences with a relatively higher affinity for binding to PDGF and covalently linking said identified PDGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular Weight Compound or a Lipophilic Compound.
- Such Complexes have one or more of the following advantages over a PDGF Nucleic Acid Ligand not in association with a Non- Immunogenic, High Molecular Weight Compound or Lipophilic Compound: 1) Improved Pharmacokinetic Properties, and 2) improved capacity for intracellular delivery, or 3) improved capacity for t.argeting.
- Complexes further associated with a Lipid Construct have the same advantages.
- a Lipid Construct of the present invention may be comprised of a) a Liposome, b) a drug that is encapsulated within the interior of the Liposome, .and c) a Complex comprised of a PDGF Nucleic Acid Ligand and Lipophilic Compound, wherein the PDGF Nucleic Acid Ligand component of the Complex is associated with and projecting from the exterior of the Lipid Construct.
- the Lipid Construct comprising a Complex will 1) have Improved Pharmacokinetic Properties, 2) have enhanced capacity for intracellular delivery of the encapsulated drug, and 3) be specifically targeted to the preselected location in vivo that is expressing PDGF by the exteriorly associated PDGF Nucleic Acid Ligand.
- this invention provides a method for improving the pharmacokinetic properties of a PDGF Nucleic Acid Ligand by covalently linking the PDGF Nucleic Acid Lig.and with a Non-Immunogenic, High Molecule Weight Compound or Lipophilic Compound to form a Complex and administering the Complex to a patient.
- the invention further relates to a method for improving the pharmacokinetic properties of a PDGF
- the Complex of the present invention is comprised of a
- PDGF Nucleic Acid Ligand covalently attached to a Lipophilic Compound, such as a glycerolipid, or a Non-Immunogenic, High Molecular Weight Compound, such as polyalkylene glycol or polyethylene glycol (PEG).
- a Lipophilic Compound such as a glycerolipid
- a Non-Immunogenic, High Molecular Weight Compound such as polyalkylene glycol or polyethylene glycol (PEG).
- PEG polyethylene glycol
- Ligand is enhanced relative to the PDGF Nucleic Acid Ligand alone when the PDGF Nucleic Acid Ligand is covalently attached to a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound and is further associated with a Lipid Construct or the PDGF Nucleic Acid Ligand is encapsulated within a Lipid Construct.
- the Complex is comprised of multiple PDGF Nucleic Acid Ligands
- the pharmacokinetic properties of the Complex will be improved relative to one PDGF Nucleic Acid Ligand alone.
- the Pharmacokinetic Properties of the PDGF Nucleic Acid Ligand may be improved relative to Lipid Constructs in which there is only one Nucleic Acid Ligand or Complex.
- the Complex of the present invention is comprised of a PDGF Nucleic Acid Ligand attached to one (dimeric) or more (multimeric) other Nucleic Acid Ligands.
- the Nucleic Acid Ligand can be to PDGF or a different Target.
- there are multiple PDGF Nucleic Acid Ligands there is an increase in avidity due to multiple binding interactions with PDGF.
- the Complex is comprised of a PDGF Nucleic Acid Ligand attached to one or more other PDGF Nucleic Acid Ligands, the pharmacokinetic properties of the Complex will be improved relative to one PDGF Nucleic Acid Ligand alone.
- the Non-Immunogenic, High Molecular Weight compound or Lipophilic Compound may be covalently bound to a variety of positions on the PDGF Nucleic Acid Ligand, such as to an exocyclic amino group on the base, the 5-position of a pyrimidine nucleotide, the 8- position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5' or 3' terminus of the PDGF Nucleic Acid Ligand.
- the Non-Immunogenic, High Molecul-ar Weight Compound is polyalkylene glycol or polyethylene glycol, preferably it is bonded to the 5' or 3' hydroxyl of the phosphate group thereof.
- the Non-Immunogenic, High Molecular Weight Compound is polyalkylene glycol or polyethylene glycol, preferably it is bonded to the 5' or 3' hydroxyl of the phosphate group thereof.
- the Non-Immunogenic, High Molecular Weight is
- Compound is bonded to the 5' hydroxyl of the phosphate group of the Nucleic Acid Ligand.
- Lipid Construct comprises a Complex
- PDGF Nucleic Acid Ligands are encapsulated within the Liposome
- oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest.
- Certain chemical modifications of the PDGF Nucleic Acid Ligand can be made to increase the in vivo stability of the PDGF Nucleic Acid Ligand or to enhance or to mediate the delivery of the PDGF Nucleic Acid Ligand.
- Modifications of the PDGF Nucleic Acid Ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the PDGF Nucleic Acid Ligand bases or to the PDGF Nucleic Acid Ligand as a whole.
- modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base- pairing combinations such as the isobases isocytidine .and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
- the modifications can be pre- or post- SELEX modifications.
- Pre-SELEX modifications yield PDGF Nucleic Acid Ligands with both specificity for PDGF .and improved in vivo stability.
- Post-SELEX modifications made to 2'-OH Nucleic Acid Ligands can result in improved in vivo stability without adversely affecting the binding capacity of the Nucleic Acid Ligands.
- the preferred modifications of the PDGF Nucleic Acid Ligands of the subject invention are 5' and 3' phosphorothioate capping and 3 '3' inverted phosphodiester linkage at the 3' end.
- the preferred modification of the PDGF Nucleic Acid Ligand is 3 '3' inverted phosphodiester linkage at the 3'end. Additional 2' fluoro (2'-F), 2' amino (2'-NH 2 ) .and
- 2' OMethyl (2'-OMe) modification of all or some of the nucleotides is preferred.
- the preferred modification is 2'-OMe and
- the PDGF Nucleic Acid Ligand can be post-SELEX modified to substitute Linkers or Spacers such as hexaethylene glycol Spacers for certain portions.
- the covalent linking of the PDGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound results in Improved Pharmacokinetic Properties (i.e., slower clearance rate) relative to the PDGF Nucleic Acid Ligand not in association with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.
- the Complex comprising a PDGF Nucleic Acid Ligand and Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound can be further associated with a Lipid Construct. This association may result in Improved Pharmacokinetic Properties relative to the PDGF Nucleic Acid Ligand or Complex not in association with a Lipid Construct.
- the PDGF Nucleic Acid Ligand or Complex can be associated with the Lipid Construct through covalent or Non-Covalent Interactions.
- the PDGF Nucleic Acid Ligand can be associated with the Lipid Construct through Covalent or Non-Covalent Interactions. In a preferred embodiment, the association is through Non-Covalent Interactions.
- the Lipid Construct is a Lipid Bilayer Vesicle.
- the Lipid Construct is a Liposome.
- Liposomes for use in the present invention can be prepared by any of the various techniques presently known in the art or subsequently developed. Typically, they are prepared from a phospholipid, for example, distearoyl phosphatidylcholine, and may include other materials such as neutral lipids, for example, cholesterol, and also surface modifiers such as positively charged (e.g., sterylamine or aminomannose or aminomannitol derivatives of cholesterol) or negatively charged (e.g., diacetyl phosphate, phosphatidyl glycerol) compounds.
- positively charged e.g., sterylamine or aminomannose or aminomannitol derivatives of cholesterol
- negatively charged e.g., diacetyl phosphate, phosphatidyl glycerol
- Multilamellar Liposomes can be formed by conventional techniques, that is, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase is then added to the vessel with a swirling or vortexing motion which results in the formation of MLVs. UVs can then be formed by homogenization, sonication or extrusion (through filters) of MLVs. In addition,
- UVs can be formed by detergent removal techniques.
- the Lipid Construct comprises a targeting
- the Lipid Construct is a Liposome.
- Preformed Liposomes can be modified to associate with the PDGF Nucleic Acid Ligands.
- a Cationic Liposome associates through electrostatic interactions with the PDGF Nucleic Acid Ligand.
- a PDGF Nucleic Acid Ligand covalently linked to a Lipophilic Compound, such as a glycerolipid can be added to preformed Liposomes whereby the glycerolipid, phospholipid, or glycerol amide lipid becomes associated with the liposomal membrane.
- the PDGF Nucleic Acid Ligand can be associated with the Liposome during the formulation of the Liposome.
- Liposomes are advantageous for encapsulating or incorporating a wide variety of therapeutic and diagnostic agents. Any variety of compounds can be enclosed in the internal aqueous compartment of the Liposomes.
- Illustrative therapeutic agents include antibiotics, antiviral nucleosides, antifungal nucleosides, metabolic regulators, immune modulators, chemotherapeutic drugs, toxin antidotes, DNA, RNA, antisense oligonucleotides, etc.
- the Lipid Bilayer Vesicles may be loaded with a diagnostic radionuclide (e.g., indium 111, iodine 131, yttrium 90, phosphorous 32, or gadolinium) and fluorescent materials or other materials that are detectable in in vitro .and in vivo applications.
- a diagnostic radionuclide e.g., indium 111, iodine 131, yttrium 90, phosphorous 32, or gadolinium
- fluorescent materials or other materials that are detectable in in vitro .and in vivo applications.
- the therapeutic or diagnostic agent can be encapsulated by the Liposome walls in the aqueous interior.
- the carried agent can be a part of, that is, dispersed or dissolved in the vesicle wall-forming materials.
- water soluble carrier agents may be encapsulated in the aqueous interior by including them in the hydrating solution, and lipophilic molecules incorporated into the lipid bilayer by inclusion in the lipid formulation.
- lipophilic molecules e.g., cationic or anionic lipophilic drugs
- loading of the drug into preformed Liposomes may be accomplished, for example, by the methods described in U.S.
- Patent No. 4,946,683 the disclosure of which is incorporated herein by reference.
- the Liposomes are processed to remove unencapsulated drug through processes such as gel chromatography or ultrafiltration.
- the Liposomes are then typically sterile filtered to remove any microorganisms which may be present in the suspension. Microorganisms may also be removed through aseptic processing.
- larger unilamellar vesicles can be formed by methods such as the reverse-phase evaporation (REV) or solvent infusion methods.
- REV reverse-phase evaporation
- Other standard methods for the formation of Liposomes are known in the art, for example, methods for the commercial production of Liposomes include the homogenization procedure described in U.S. Patent No. 4,753,788 and the thin- film evaporation method described in U.S. Patent No. 4,935,171, which are incorporated herein by reference.
- the therapeutic or diagnostic agent can also be associated with the surface of the Lipid Bilayer Vesicle.
- a drug can be attached to a phospholipid or glyceride (a prodrug).
- the phospholipid or glyceride portion of the prodrug can be incorporated into the lipid bilayer of the Liposome by inclusion in the lipid formulation or loading into preformed Liposomes (see U.S. Patent Nos 5,194,654 and 5,223,263, which are incorporated by reference herein). It is readily apparent to one skilled in the art that the particular Liposome preparation method will depend on the intended use and the type of lipids used to form the bilayer membrane.
- oligo- PEG-lipid conjugates can be prepared and then formulated into pre-formed liposomes via spontaneous incorporation ("anchoring") of the lipid tail into the existing lipid bilayer.
- the lipid group undergoes this insertion in order to reach a lower free energy state via the removal of its hydrophobic lipid anchor from aqueous solution and its subsequent positioning in the hydrophobic lipid bilayer.
- the key advantage to such a system is that the oligo-lipid is anchored exclusively to the exterior of the lipid bilayer. Thus, no oligo-lipids are wasted by being unavailable for interactions with their biological targets by being in an inward-facing orientation.
- the efficiency of delivery of a PDGF Nucleic Acid Ligand to cells may be optimized by using lipid formulations and conditions known to enhance fusion of
- Liposomes with cellular membranes For example, certain negatively charged lipids such as phosphatidylglycerol and phosphatidylserine promote fusion, especially in the presence of other fusogens (e.g., multivalent cations like Ca 2+ , free fatty acids, viral fusion proteins, short chain PEG, lysolecithin, detergents and surfactants). Phosphatidylethanolamine may also be included in the Liposome formulation to increase membrane fusion and, concomitantly, enhance cellular delivery.
- fusogens e.g., multivalent cations like Ca 2+ , free fatty acids, viral fusion proteins, short chain PEG, lysolecithin, detergents and surfactants.
- Phosphatidylethanolamine may also be included in the Liposome formulation to increase membrane fusion and, concomitantly, enhance cellular delivery.
- free fatty acids and derivatives thereof may be used to prepare pH-sensitive Liposomes which are negatively charged at higher pH and neutral or protonated at lower pH.
- pH-sensitive Liposomes are known to possess a greater tendency to fuse.
- the PDGF Nucleic Acid Ligands of the present invention are derived from the SELEX methodology.
- SELEX is described in United States Patent Application Serial No. 07/536,428, entitled “Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, United States Patent Application Serial No. 07/714,131, filed June 10, 1991 , entitled “Nucleic Acid Ligands," now United States Patent No. 5,475,096, United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled “Methods of Identifying Nucleic Acid Ligands," now United States Patent No. 5,270,163 (see also WO 91/19813).
- the SELEX process provides a class of products which are Nucleic Acid molecules, each having a unique sequence, and each of which has the property of binding specifically to a desired Target compound or molecule.
- Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules.
- SELEX methodology can also be used to target biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure. In its most basic form, the SELEX process may be defined by the following series of steps:
- a Candidate Mixture of Nucleic Acids of differing sequence is prepared.
- the Candidate Mixture generally includes regions of fixed sequences (i.e., each of the members of the Candidate Mixture contains the same sequences in the same location) and regions of randomized sequences.
- the fixed sequence regions are selected either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the Target, or (c) to enhance the concentration of a given structural arrangement of the Nucleic Acids in the Candidate Mixture.
- the randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
- the newly formed Candidate Mixture contains fewer and fewer unique sequences, .and the average degree of affinity of the Nucleic Acids to the target will generally increase.
- the SELEX process will yield a Candidate Mixture containing one or a small number of unique Nucleic Acids representing those Nucleic Acids from the original Candidate Mixture having the highest affinity to the target molecule.
- the basic SELEX method has been modified to achieve a number of specific objectives. For example, United States Patent Application Serial No. 07/960,093, filed
- Nucleic Acid Ligands describes a SELEX based method for selecting Nucleic Acid Ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule.
- the SELEX method encompasses the identification of high-affinity Nucleic Acid Ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified Nucleic Acid Ligands containing modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Lig-ands Containing Modified Nucleotides," abandoned in favor of United States Patent Application Serial No. 08/430,709, now United States Patent
- Nucleophilic Displacement describes oligonucleotides containing various 2'-modified pyrimidines.
- the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” now United States Patent No. 5,637,459, and United States Patent Application Serial No. 08/234,997, filed April 28, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,” now United States Patent No. 5,683,867, respectively.
- the SELEX method further encompasses combining selected Nucleic Acid Ligands with Lipophilic Compounds or Non-Immunogenic, High Molecul-ar Weight Compounds in a diagnostic or therapeutic Complex as described in United States Patent Application Serial No. 08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes.”
- the SELEX method further encompasses combining selected VEGF Nucleic Acid Ligands with lipophilic compounds, such as diacyl glycerol or dialkyl glycerol, as described in United States Patent Application Serial No.
- VEGF Vascular Endothelial Growth Factor Nucleic Acid Ligand Complexes.
- VEGF Nucleic Acid Ligands that are associated with a High Molecular Weight, Non-Immunogenic Compound, such as polyethylene glycol, or a Lipophilic Compound, such as glycerolipid, phospholipid, or glycerol amide lipid, in a diagnostic or therapeutic complex are described in United States Patent Application Serial No.
- SELEX identifies Nucleic Acid Lig-ands that are able to bind targets with high affinity and with outstanding specificity, which represents a singular achievement that is unprecedented in the field of Nucleic Acids research. These characteristics are, of course, the desired properties one skilled in the art would seek in a therapeutic or diagnostic ligand.
- the Nucleic Acid Ligand (1) binds to the target in a manner capable of achieving the desired effect on the target; (2) be as small as possible to obtain the desired effect; (3) be as stable as possible; and (4) be a specific ligand to the chosen target. In most situations, it is preferred that the Nucleic Acid Ligand has the highest possible affinity to the target.
- Nucleic Acid Ligands can have facilitating properties.
- the SELEX process has been used to identify a group of high affinity RNA Ligands to
- the PDGF Nucleic Acid Ligand(s) can serve in a targeting capacity
- the PDGF Nucleic Acid Lig.ands adopt a three dimensional structure that must be retained in order for the PDGF Nucleic Acid Ligand to be able to bind its target.
- the Lipid Construct comprises a Complex and the PDGF Nucleic Acid
- Ligand of the Complex is projecting from the surface of the Lipid Construct, the PDGF
- Nucleic Acid Ligand must be properly oriented with respect to the surface of the Lipid Construct so that its target binding capacity is not compromised. This can be accomplished by attaching the PDGF Nucleic Acid Ligand at a position that is dist-ant from the binding portion of the PDGF Nucleic Acid Ligand. The three dimensional structure and proper orientation can also be preserved by use of a Linker or Spacer as described supra. Any variety of therapeutic or diagnostic agents can be attached to the Complex for targeted delivery by the Complex. In addition, any variety of therapeutic or diagnostic agents can be attached encapsulated, or incorporated into the Lipid Construct as discussed supra for targeted delivery by the Lipid Construct.
- the Complex is comprised of a Lipophilic Compound and a PDGF Nucleic Acid Ligand in association with a Liposome
- the PDGF Nucleic Acid Ligand could target rumor cells expressing PDGF (e.g., in Kaposi's sarcoma) for delivery of an antitumor drug (e.g., daunorubicin) or imaging agent (e.g., radiolabels).
- an antitumor drug e.g., daunorubicin
- imaging agent e.g., radiolabels
- cells and tissues surrounding the tumor may also express PDGF, and targeted delivery of an antitumor drug to these cells would also be effective.
- the therapeutic or diagnostic agent to be delivered to the Target cell could be another Nucleic Acid Ligand.
- the agent to be delivered can be incorporated into the Complex in such a way as to be associated with the outside surface of the Liposome (e.g., a prodrug, receptor antagonist, or radioactive substance for treatment or imaging).
- the agent can be associated through covalent or Non-Covalent Interactions.
- the Liposome would provide targeted delivery of the agent extracellularly, with the Liposome serving as a Linker.
- a Non-Immunogenic, High Molecular Weight Compound (e.g., PEG) can be attached to the Liposome to provide improved pharmacokinetic properties for the Complex.
- PDGF Nucleic Acid Ligands may be attached to the Liposome membrane or may be attached to a Non-Immunogenic, High Molecular Weight Compound which in turn is attached to the membrane.
- the Complex may be shielded from blood proteins and thus be made to circulate for extended periods of time while the PDGF Nucleic Acid Ligand is still sufficiently exposed to make contact with and bind to its Target.
- more than one PDGF Nucleic Acid is still sufficiently exposed to make contact with and bind to its Target.
- Ligand is attached to the surface of the same Liposome. This provides the possibility of bringing the same PDGF molecules in close proximity to each other and can be used to generate specific interactions between the PDGF molecules.
- PDGF Nucleic Acid Ligands and a Nucleic Acid Ligand to a different Target can be attached to the surface of the same Liposome. This provides the possibility of bringing PDGF in close proximity to a different
- Target and can be used to generate specific interactions between PDGF and the other Target.
- agents could be encapsulated in the Liposome to increase the intensity of the interaction.
- the Lipid Construct comprising a Complex allows for the possibility of multiple binding interactions to PDGF. This, of course, depends on the number of PDGF Nucleic Acid Ligands per Complex, and the number of Complexes per Lipid Construct, and mobility of the PDGF Nucleic Acid Ligands and receptors in their respective membranes. Since the effective binding constant may increase as the product of the binding constant for each site, there is a substantial advantage to having multiple binding interactions. In other words, by having many PDGF Nucleic Acid Ligands attached to the Lipid Construct, and therefore creating multivalency, the effective affinity (i.e., the avidity) of the multimeric Complex for its Target may become as good as the product of the binding constant for each site.
- the effective affinity i.e., the avidity
- the Complex of the present invention is comprised of a PDGF Nucleic Acid Ligand attached to a Lipophilic Compound.
- the pharmacokinetic properties of the Complex will be improved relative to the PDGF Nucleic Acid Ligand alone.
- the Lipophilic Compound may be covalently bound to the PDGF Nucleic Acid Ligand at numerous positions on the PDGF Nucleic Acid Ligand.
- the Lipid Construct comprises a PDGF
- the glycerolipid can assist in the incorporation of the PDGF Nucleic Acid Ligand into the Liposome due to the propensity for a glycerolipid to associate with other Lipophilic Compounds.
- the glycerolipid in association with a PDGF Nucleic Acid Ligand can be incorporated into the lipid bilayer of the Liposome by inclusion in the formulation or by loading into preformed Liposomes.
- the glycerolipid can associate with the membrane of the Liposome in such a way so as the PDGF Nucleic Acid Ligand is projecting into or out of the Liposome.
- the PDGF Nucleic Acid Ligand is projecting out of the Complex, the PDGF Nucleic
- Acid Ligand can serve in a targeting capacity. It is to be understood that additional compounds can be associated with the Lipid Construct to further improve the pharmacokinetic properties of the Lipid Construct.
- a PEG may be attached to the exterior-facing part of the membrane of the Lipid Construct.
- the Complex of the present invention is comprised of a PDGF
- the pharmacokinetic properties of the Complex are improved relative to the PDGF Nucleic Acid Ligand alone.
- the polyalkylene glycol or PEG may be covalently bound to a variety of positions on the PDGF Nucleic Acid Ligand. In embodiments where polyalkylene glycol or PEG are used, it is preferred that the PDGF Nucleic Acid Ligand is bonded through the 5' hydroxyl group via a phosphodiester linkage.
- a plurality of Nucleic Acid Ligands can be associated with a single Non-Immunogenic, High Molecular Weight Compound, such as polyalkylene glycol or PEG, or a Lipophilic Compound, such as a glycerolipid.
- the Nucleic Acid Ligands can all be to PDGF or PDGF and a different Target. In embodiments where there are multiple PDGF Nucleic Acid Ligands, there is an increase in avidity due to multiple binding interactions with PDGF.
- a plurality of polyalkylene glycol, PEG, glycerol lipid molecules can be attached to each other.
- one or more PDGF Nucleic Acid Ligands or Nucleic Acid Ligands to PDGF and other Targets can be associated with each polyalkylene glycol, PEG, or glycerol lipid. This also results in an increase in avidity of each Nucleic Acid Ligand to its Target. In embodiments where multiple PDGF Nucleic Acid Ligands are attached to polyalkylene glycol, PEG, or glycerol lipid, there is the possibility of bringing PDGF molecules in close proximity to each other in order to generate specific interactions between PDGF.
- Nucleic Acid Ligands specific for PDGF and different Targets are attached to polyalkylene glycol, PEG, or glycerol lipid
- a drug can also be associated with polyalkylene glycol, PEG, or glycerol lipid.
- the Complex would provide targeted delivery of the drug, with polyalkylene glycol, PEG, or glycerol lipid serving as a Linker.
- PDGF Nucleic Acid Ligands selectively bind PDGF.
- a Complex comprising a
- PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound or a Lipid Construct comprising a PDGF Nucleic Acid Ligand or a
- Ligand-containing Complexes and Lipid Constructs can be used to treat, inhibit, prevent or diagnose any disease state that involves inappropriate PDGF production, for example, cancer, angiogenesis, restenosis, and fibrosis.
- PDGF is produced and secreted in varying amounts by many tumor cells.
- the present invention includes methods of treating, inhibiting, preventing, or diagnosing cancer by administration of a Complex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound, a Lipid Construct comprising a Complex, or a PDGF Nucleic Acid Ligand in association with a Lipid Construct without being part of the Complex.
- Angiogenesis rarely occurs in healthy adults, except during the menstrual cycle and wound healing.
- Angiogenesis is a central feature, however, of various disease states, including, but not limited to cancer, diabetic retinopathy, macular degeneration, psoriasis and rheumatoid arthritis.
- the present invention includes methods of treating, inhibiting, preventing, or diagnosing angiogenesis by administration of a Complex comprising PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound, a Lipid Construct comprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.
- PDGF is also produced in fibrosis in organs, such as lung, bone marrow .and kidney. Fibrosis can also be associated with radiation treatments.
- the present invention includes methods of treating, inhibiting, preventing or diagnosing lung, bone marrow, kidney and radiation treatment-associated fibrosis by administration of a Complex comprising PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound, a Lipid Construct comprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.
- PDGF is a prominent growth factor involved in restenosis. Restenosis, the reocclusion of a diseased blood vessel after treatment to eliminate stenosis, is a common occurrence that develops following coronary interventions and some peripheral vessel interventions.
- stents have been used in the treatment of or in conjunction with treatment of coronary and non-coronary vessels; however, restenosis is also associated with use of stents
- in-stent restenosis occurs in about 15-30% of coronary interventions and frequently in some peripheral vessel interventions.
- in-stent restenosis is a significant problem in small vessels, with frequencies ranging from 15% to 40% in stented femoral or popliteal arteries.
- Intermediate-sized vessels, such as renal arteries, have an in-stent restenosis rate of 10-20%.
- the present invention includes methods of treating, inhibiting, preventing or diagnosing restenosis by administration of a Complex comprising PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound, a Lipid Construct comprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.
- the present invention also includes methods of treating, inhibiting, preventing or diagnosing restenosis in coronary and non-coronary vessels.
- the present invention also includes methods of treating, inhibiting, preventing or diagnosing in-stent restenosis.
- a Complex comprising PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound a Lipid Construct comprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound can be used in conjunction with Complexes comprising Nucleic Acid Ligands to other growth factors (such as bFGF, TGF ⁇ , hKGF, etc.) and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound, a Lipid Construct comprising PDGF Nucleic Acid Ligand or a Complex comprising a PDGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.
- the Lipid Construct comprises a Complex comprised of a PDGF Nucleic Acid Ligand and a Lipophilic Compound with an additional diagnostic or therapeutic agent encapsulated in the Lipid Construct or associated with the interior of the Lipid Construct.
- the Lipid Construct is a Lipid
- Bilayer Vesicle and more preferably a Liposome.
- the therapeutic use of Liposomes includes the delivery of drugs which are normally toxic in the free form. In the liposomal form, the toxic drug is occluded, and may be directed away from the tissues sensitive to the drug and targeted to selected areas. Liposomes can also be used therapeutically to release drugs over a prolonged period of time, reducing the frequency of administration. In addition, liposomes can provide a method for forming aqueous dispersions of hydrophobic or amphiphilic drugs, which are normally unsuitable for intravenous delivery.
- liposomes In order for many drugs and imaging agents to have therapeutic or diagnostic potential, it is necessary for them to be delivered to the proper location in the body, and the liposome can thus be readily injected and form the basis for sustained release and drug delivery to specific cell types, or parts of the body.
- Several techniques can be employed to use liposomes to target encapsulated drugs to selected host tissues, and away from sensitive tissues. These techniques include manipulating the size of the liposomes, their net surface charge, and their route of administration.
- MLVs primarily because they are relatively large, are usually rapidly taken up by the reticuloendothelial system (principally the liver and spleen). UVs, on the other hand, have been found to exhibit increased circulation times, decreased clearance rates and greater biodistribution relative to MLVs.
- PDGF Nucleic Acid Ligand is associated with the outside surface of the liposome, and serves in a targeting capacity. Additional targeting components, such as antibodies or specific receptor ligands can be included on the liposome surface, as would be known to one of skill in the art.
- Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound, and a PDGF Nucleic Acid Ligand in association with a Lipid Construct without being part of a Complex may be administered parenterally by injection, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iotophoresis or suppositories, are also envisioned. They may also be applied locally by direct injection, can be released from devices, such as implanted stents or catheters, or delivered directly to the site by an infusion pump.
- One preferred carrier is physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers may also be used.
- the carrier and the PDGF Nucleic Acid Ligand Complex constitute a physiologically-compatible, slow release formulation.
- the primary solvent in such a carrier may be either aqueous or non-aqueous in nature.
- the carrier may contain other pharmacologically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
- the carrier may contain still other pharmacologically-acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the PDGF Nucleic Acid Ligand.
- excipients are those substances usually and customarily employed to formulate dosages for parental administration in either unit dose or multi-dose form.
- the therapeutic or diagnostic composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in ready to use form or requiring reconstitution immediately prior to administration.
- the manner of administering formulations containing PDGF Nucleic Acid Ligand for systemic delivery may be via subcutaneous, intramuscular, intravenous, intr.an.asal or vaginal or rectal suppository.
- the advantages of the Complexes and Lipid Constructs of the invention include: i) improving the plasma pharmacokinetics of the Nucleic Acid Lig.and; ii) presenting Nucleic Acid Lig.ands in a multivalent array with the aim of increasing the avidity of interaction with their targets; iii) combining two or more presenting Nucleic Acid Lig-ands with different specificities in the same liposome p.article; iv) enh.ancing the delivery of presenting Nucleic Acid Ligands to tumors by taking advantage of the intrinsic tumor targeting properties of liposomes; and v) using the high affinity and specificity of presenting Nucleic Acid Ligands, which is comparable to that of antibodies, to guide liposomal contents to specific targets.
- Nucleic Acid Ligands are well suited for the kinds of preparations described here since, unlike most proteins, the denaturation of presenting Nucleic Acid Ligands by heat, various molecular denatur.ants .and org.anic solvents is readily reversible.
- Example 1 describes the various materials and experimental procedures used in Ex.amples 2-4 for the generation of ssDNA ligands to PDGF and tests associated therewith.
- Example 2 describes the ssDNA ligands to PDGF and the predicted secondary structure of selected nucleic acid ligands and a shared secondary structure motif.
- Example 3 describes the minimum sequence necessary for high affinity binding, the sites on the nucleic acid ligands and PDGF that are in contact, inhibition by DNA ligands of PDGF isoforms on cultured cells, and inhibition of mitogenic effects of PDGF in cells by DNA ligands.
- Example 4 describes substitutions of SELEX-derived ligands with modified nucleotides.
- Example 5 describes synthesis of PEG-modified PDGF Nucleic Acid Ligands.
- Example 6 describes stability of modified ligands in serum.
- Example 7 describes efficacy of a modified ligand (NX31975-40K PEG) in restenosis.
- Example 8 describes the various materials and method used in Example 9 for testing the inhibition of PDGF in glomerulonephritis.
- Example 9 describes inhibition of PDGF in glomerulonephritis.
- Example 10 describes the experimental procedures for evolving 2'-fiuoro-2'-deoxypyrimidine RNA ligands to PDGF and the RNA sequences obtained.
- EXAMPLE 1 EXPERIMENTAL PROCEDURES This example provides the general procedures followed and incorporated in
- DNA ligands were synthesized by NeXstar Pharmaceuticals, Inc. (Boulder, CO) or by
- the ssDNA library was purified by electrophoresis on an 8% polyacrylamide/7 M urea gel.
- the band that corresponds to the full-length DNA was visualized under UV light, excised from the gel, eluted by the crush and soak method, ethanol precipitated and pelleted by centrifugation.
- the ssDNA Prior to incubation with the protein, the ssDNA was heated at 90°C for 2 minutes in PBSM and cooled on ice. The first selection was initiated by incubating approximately 500 pmol (3 x 10 14 molecules) of 5' 32 P end-labeled random ssDNA with PDGF-AB in binding buffer (PBSM containing 0.01% human serum albumin (HSA)). The mixture was incubated at 4°C overnight, followed by a brief (15 min) incubation at 37°C.
- PBSM binding buffer
- HSA human serum albumin
- the DNA bound to PDGF-AB was separated from unbound DNA by electrophoresis on an 8% polyacrylamide gel (1 :30 bis-acrylamide:acrylamide) at 4°C and at 5 V/cm with 89 mM Tris-borate (pH 8.3) containing 2 mM EDTA as the running buffer.
- the ssDNA was incubated with PDGF-AB for 15 minutes at 37°C in binding buffer and the PDGF-bound ssDNA was separated from the unbound DNA by nitrocellulose filtration, as previously described (Green et al. (1995) Chemistry and Biology 2:683-695). All affinity-selected ssDNA pools were amplified by PCR in which the DNA was subjected to
- the 5' PCR primer was 5' end-labeled with polynucleotide kinase and
- [o.- 32 P]ATP and the 3' PCR primer was biotinylated at the 5' end using biotin phosphoramidite (Glen Research, Sterling, VA).
- streptavidin Piereptavidin (Pierce, Rockford, IL) was added to the unpurified PCR reaction mixture at a 10-fold molar excess over the biotinylated primer and incubated for 15 min at room temperature.
- the dsDNA was denatured by adding an equal volume of stop solution (90% formamide, 1% sodium dodecyl sulfate, 0.025% bromophenol blue and xylene cyanol) and incubating for 20 min at room temperature.
- the radiolabeled strand was separated from the streptavidin-bound biotinylated strand by electrophoresis on 12% polyacrylamide/7 M urea gels.
- the faster migrating radiolabeled (non-biotinylated) ssDNA strand was cut out of the gel and recovered as described above.
- the amount of ssDNA was estimated from the absorbance at 260 nm using the extinction coefficient of 33 ⁇ g/ml/absorbance unit (Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual, 2nd Ed. 3 vols., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
- the amplified affinity-enriched pool from SELEX round 12 was purified on a 12% polyacrylamide gel and cloned between HindlU .and P>ytl sites in JM109 strain of E. coli (Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual, 2nd Ed. 3 vols., Cold Spring Harbor Laboratory Press, Cold Spring Harbor). Individual clones were used to prepare plasmids by alkaline lysis. Plasmids were sequenced at the insert region using the forward sequencing primer and Sequenase 2.0 (Amersham, Arlington Heights, IL) according to the manufacturer's protocol.
- ssDNA for all binding experiments were purified by electrophoresis on 8% (>80 nucleotides) or 12% ( ⁇ 40 nucleotides) polyacrylamide/7 M urea gels. All ssDNA ligands were heated at 90°C in binding buffer at high dilution ( ⁇ 1 nM) for
- binding mixtures were typically incubated for 15 min at 37°C before partitioning on nitrocellulose filters:
- ⁇ , and ⁇ 2 are the mole fractions of L, and L 2 .
- the K d values for the binding of DNA ligands to PDGF were calculated by fitting the data points to equation 1 (for PDGF-AA) or equation 2 (for PDGF-AB and PDGF-BB) using the non-linear least squares method.
- the dissociation rate constants were determined by measuring the amount of 3 P 5'-end labeled minimal ligands (0.17 nM) bound to PDGF-AB (1 nM) as a function of time following the addition of 500-fold excess of unlabeled ligands, using nitrocellulose filter binding as the partitioning method.
- the k off values were determined by fitting the data points to the first-order rate equation 3 :
- a primer complementary to the 3' invariant sequence region of a DNA ligand template (truncated primer 5N2, Table 1) (SEQ ID NO:3) was radiolabeled at the 5' end with [ ⁇ - 32 P]-ATP and T4 polynucleotide kinase, annealed to the template and extended with Sequenase (Amersham, Arlington Heights, IL) and a mixture of all four dNTPs and ddNTPs.
- DNA ligands were radiolabeled at the 3' end with [ ⁇ - 32 P]-cordycepin-5'-triphosphate (New England Nuclear, Boston, MA) and T4 RNA ligase (Promega, Madison, WI), phosphorylated at the 5' end with ATP and T4 polynucleotide kinase, and partially digested with lambda exonuclease
- T-_ measurements Melting temperature (T-_ measurements. Melting profiles for the minimal DNA ligands were obtained on a Cary Model IE spectrophotometer. Oligonucleotides (320-400 nM) were heated to 95°C in PBS, PBSM or PBS with 1 mM EDTA and cooled to room temperature prior to the melting profile determination. Melting profiles were generated by heating the samples at the rate of l°C/min from 15-95°C and recording the absorbance every 0.1 °C. The first derivative of the data points was calculated using the plotting program KaleidaGraph (Synergy Software, Reading, PA). The first derivative values were smoothed using a 55 point smoothing function by averaging each point with 27 data points on each side. The peak of the smoothed first derivative curves was used to estimate the T m values.
- KaleidaGraph Synergy Software, Reading, PA
- the binding mixture was transferred to a 1 cm path length cuvette thermostated at 37°C and irradiated at 308 nm for 25-400 s at 20 Hz using a XeCl charged Lumonics Model EX748 excimer laser.
- the cuvette was positioned 24 cm beyond the focal point of a convergent lens, with the energy at the focal point measuring 175 mjoules/pulse.
- aliquots were mixed with an equal volume of formamide loading buffer containing 0.1 % SDS and incubated at 95°C for 5 min prior to resolution of the crosslinked PDGF/ligand complex from the free ligand on 8% polyacrylamide/7 M urea gels.
- PDGF-AB and 5' 32 P end-labeled ligand were incubated and irradiated (300 s) as described above in two 1 ml reaction vessels.
- the reaction mixtures were combined, ethanol precipitated and resuspended in 0.3 ml of Tris-HCl buffer (100 mM, pH 8.5).
- the PDGF-AB/ligand crosslinked complex was digested with 0.17 ⁇ g/ ⁇ l of modified trypsin (Boehringer
- EXAMPLE 2 ssDNA LIGANDS OF PDGF High affinity DNA ligands to PDGF-AB were identified by the SELEX process from a library of ⁇ 3 x 10 H molecules (500 pmol) of single str-anded DNA randomized at forty contiguous positions (Table 1) (SEQ ID NO:l). The PDGF-bound DNA was separated from unbound DNA by polyacrylamide gel electrophoresis in the first round and by nitrocellulose filter binding in the subsequent rounds. After 12 rounds of SELEX, the affinity-enriched pool bound to PDGF-AB with an apparent dissociation constant (K d ) of
- the relative affinities for PDGF-AB were determined by measuring the fraction of 5' 32 P end-labeled ligands bound to PDGF-AB over a range of protein concentrations.
- the affinity toward PDGF-BB and PDGF-AA was also examined. In all cases, the affinity of ligands for PDGF-AB and PDGF-BB was comparable while the affinity for PDGF-AA was considerably lower (data not shown).
- Twenty-one of the thirty-two unique ligands can be grouped into a sequence family shown in Table 3 (SEQ ID NOS:4, 5, 7-9, 14-24, 26, 31, 32, 34 and 35).
- the sequences of the initially randomized region (uppercase letters) are aligned according to the consensus three-way helix junction motif. Nucleotides in the sequence-invariant region (lowercase letters) are only shown where they participate in the predicted secondary structure.
- Several ligands were "disconnected" (equality symbol) in order to show their relatedness to the consensus motif through circular permutation.
- the nucleotides predicted to participate in base pairing are indicated with underline inverted arrows, with the arrow heads pointing toward the helix junction.
- sequences are divided into two groups, A and B, based on the first single stranded nucleotide (from the 5' end) at the helix junction (A or G, between helices II and III). Mismatches in the helical regions are shown with dots under the corresponding letters (G-T and T-G base pairs were allowed). In places where single nucleotide bulges occur, the mismatched nucleotide is shown above the rest of the sequence between its neighbors.
- This classification is based in part on sequence homology among these ligands, but in greater part on the basis of a shared secondary structure motif: a three-way helix junction with a three nucleotide loop at the branch point ( Figure 1) (SEQ ID NO:82).
- Figure 1 SEQ ID NO:82.
- These ligands were subdivided into two groups; for ligands in group A, the loop at the branch point has an invariant sequence AGC and in group B, that sequence is G(T/G)(C/T).
- the proposed consensus secondary structure motif is supported by base-pairing covariation at non-conserved nucleotides in the helices (Table 4).
- PDGF-AB The highly conserved nucleotides are indeed found near the helix junction
- the minimal sequence necessary for high affinity binding was determined for six of the best ligands to PDGF-AB.
- the information about the 3' and 5' minimal sequence boundaries can be obtained by partially fragmenting the nucleic acid ligand and then selecting for the fragments that retain high affinity for the target.
- the fragments can be conveniently generated by mild alkaline hydrolysis (Tuerk et al. (1990) J. Mol. Biol. 211:749-761; Jellinek et al (1994) Biochemistry 33:10450-10456; Jellinek et al (1995) Biochemistry 34:11363-11372; Green et al (1995) J. Mol. Biol. 247:60-68).
- the 5' boundary was determined in an analogous manner except that a population of 3' end-labeled ligand fragments serially truncated at the 5' end was generated by limited digestion with lambda exonuclease.
- the minimal ligand is then defined as the sequence between the two boundaries. It is important to keep in mind that, while the information derived from these experiments is useful, the suggested boundaries are by no means absolute since the boundaries are examined one terminus at the time. The untruncated (radiolabeled) termini can augment, reduce or have no effect on binding (Jellinek et al (1994) Biochemistry 33:10450-10456).
- the six minimal ligands for which the boundaries were determined experimentally two (20t (SEQ ID NO:83) ( Figure 2A) and 41t (SEQ ID NO:85) ( Figure
- the higher affinity binding component is the most populated ligand species in all cases ( Figures 3A-3C).
- a 39-mer DNA ligand that binds to human thrombin with a K d of 0.5 nM (ligand T39 (SEQ ID NO:88)): 5'-CAGTCCGTGGTAGGGCAGGTTGGGGTGACTTCGTGGAA[3 * T], where [3'T] represents a 3 '-3' linked thymidine nucleotide added to reduce 3 '-exonuclease degradation) and has a predicted stem-loop structure, binds to PDGF-AB with a K d of 0.23 ⁇ M (data not shown).
- the dissociation rates at 37°C for the complexes of minimal ligands 20t (SEQ ID NO:83), 36t (SEQ ID NO:84) and 41t (SEQ ID NO:85) with PDGF-AB were determined by measuring the amount of radiolabeled ligands (0.17 nM) bound to PDGF-AB (1 nM) as a function of time following the addition of a large excess of unlabeled ligands ( Figure 4). At these protein and DNA ligand concentrations, only the high affinity fraction of the DNA ligands binds to PDGF-AB.
- T m 's Melting temperatures
- T m 's Melting temperatures
- oligonucleotide concentrations used in these experiments 320-440 nM
- the T m values were obtained from the first derivative replots of the melting profiles.
- Ligands 20t and 411 exhibited monophasic melting with T m values of 44°C and 49°C.
- the excited triplet state species then reacts with electron rich amino acid residues (such as Trp, Tyr and His) that are in its close proximity to yield a covalent crosslink.
- electron rich amino acid residues such as Trp, Tyr and His
- This method has been used extensively in studies of nucleic acid-protein interactions since it allows irradiation with >300 nm light which minimizes photodamage (Willis et al. (1994) Nucleic Acids Res. 22:4947-4952;
- ligands 20t-Il through 20t-I7 S'-TGGGAGGGCGCGT'T'CTVCGT'GGT ⁇ ACT'TVTAGT'CCCG ⁇ ' (SEQ ID NOS:89-95) where the numbers indicate IdU substitutions at indicated thymidine nucleotides for the seven ligands).
- efficient crosslinking to PDGF-AB was observed only with ligand 20t-I4 (SEQ ID NO:92).
- the photo-reactive IdU position corresponds to the 3' proximal thymidine in the loop at the helix junction ( Figure 2).
- DNA ligand T39 (SEQ ID NO:88) (described supra), directed against thrombin and included as a control, showed no effect. None of the ligands was able to inhibit the binding of 125 I-PDGF-AA to the PDGF ⁇ -receptor (data not shown), consistent with the observed specificity of ligands 20t, 36t and 41t for PDGF-BB and PDGF-AB.
- nucleic acids to nucleases are an important consideration in efforts to develop nucleic acid-based therapeutics.
- many, and in some cases most of the nucleotides in SELEX-derived ligands can be substituted with modified nucleotides that resist nuclease digestion, without compromising high affinity binding
- FIGS 8A and 8B summarize the substitutions examined and their effect on the affinity of the modified ligands for PDGF-AB or PDGF-BB.
- 2-Fluoropyrimidine nucleoside phosphoramidites were obtained from JBL Scientific (San Louis Obispo, CA).
- 2'-O- Methylpurine phosphoramidites were obtained from PerSeptive Biosystems (Boston, MA). All other nucleoside phosphoramidites were from PerSeptive Biosystems (Boston, MA). Not all substitution combinations were examined. Nevertheless, these experiments have been used to identify the pattern of 2'-O-Me and 2'-F substitutions that are compatible with high affinity binding to PDGF-AB or PDGF-BB.
- trinucleotide loops on helices II and III in ligand 36t can be replaced with pentaethylene glycol (18-atom) spacers (Spacer Phosphoramidite 18, Glen Research, Sterling, VA) (see Example 5 for description of synthesis of pentaethylene gly col-substituted ligand) without compromising high affinity binding to PDGF-AB or -BB.
- pentaethylene glycol (18-atom) spacers Spacer Phosphoramidite 18, Glen Research, Sterling, VA
- the syntheses were carried out using 4,5-dicyanoimidazole as the activator on controlled pore glass (CPG) support of 600A pore size, 80-120 mesh, and 60 -70 ⁇ mol/g loading with 5'-succinyl thymidine. After the synthesis, the oligos were deprotected with 40% NH 4 OH, at 55°C for 16 hours. The support was filtered, and washed with water and 1 : 1 acetonitrile/water and the combined washings were evaporated to dryness.
- CPG controlled pore glass
- the ammonium counterion on the backbone was exchanged for triethylammonium ion by reverse phase salt exchange and the solvent was evaporated to afford the crude oligo as the triethylammonium salt.
- Hexaethylene glycol spacers on the loops are attached to the nucleotides through phosphate linkages.
- the structures of the 2 loops are shown in Figures 9A and 9B.
- the 5' phosphate group shown is from the hexaethylene glycol phosphoramidite.
- the modified ligand (NX21568) exhibited a substantially greater stability in rat serum compared with the DNA ligand (36ta), which was degraded with a half-life of about 35 minutes ( Figure 10).
- the increase in stability in serum results from the 2'-substitutions.
- EXAMPLE 7 EFFICACY OF NX31975-40K PEG (SEQ ID NO:146) IN RESTENOSIS Rat Restenosis Model and Efficacy Results.
- the plasma residence time of Nucleic Acid Ligands is dramatically improved by the addition of large, inert functional groups such as polyethylene glycol (see for example PCT/US 97/18944).
- 40K PEG was conjugated to NX31975 to create NX31975-40K PEG as described in Example 5B (see Figure 9A for molecular description).
- the addition of 40 kDa PEG group at the 5'-end of the ligand does not affect its binding affinity for PDGF-BB.
- a total of 30 rats were randomly allocated to one of two treatment groups: 15 rats in group one received 10 mg/kg body weight of NX31975-40K PEG in phosphate buffered saline (PBS) twice daily delivered by intraperitoneal (i.p.) injections and 15 rats in group two (the control group) received an equal volume of PBS (about 1 ml). The duration of treatment was 14 days. The first injections in both groups were given one hour before arterial injury.
- PBS phosphate buffered saline
- the left common carotid artery was traumatized by intraluminal passage of 2F Fogarty embolectomy catheter introduced through the external carotid artery.
- the catheter was passed three times with the balloon expanded sufficiently with 0.06 ml distilled water to achieve a distension of the carotid itself.
- the external carotid was ligated after removal of the catheter and the wound was closed. All surgical procedures were performed by a surgeon blinded to the treatment groups. Fourteen days after the catheter injury, the animals were anesthetized as above.
- the carotid arteries were perfused with ice-chilled PBS in situ at 100 mm Hg, via a large cannula placed retrograde in the abdominal aorta until the effluent ran clear via inferior caval vein vent. A distal half of the right and left common carotid arteries, up to the level of the bifurcation, were removed and frozen in liquid nitrogen.
- the remaining proximal segment was perfusion-fixed through the same aortic cannula at 100 mm Hg pressure with 2.5% glutaraldehyde in phosphate buffer, pH 7.3.
- the animals were killed by an overdose of phenobarbital.
- the remaining proximal right and left common carotid arteries were retrieved for further preparation, including the aortic arch and innominate artery.
- Example 9 provides the general procedures followed and incorporated in Example 9.
- NX31975-40K PEG (SEQ ID NO:146) ( Figure 9A).
- NX31975-40K PEG was created by conjugating NX31975 (SEQ ID NO: 148) (Table 7) to 40K PEG as described in Example 5.
- eight nucleotides in the helix junction region of NX31975 were interchanged without formally changing the consensus secondary structure (see Figure 8C).
- the binding affinity of the sequence-scrambled control Nucleic Acid Ligand for PDGF-BB is ⁇ l ⁇ M, which is 10,000 fold lower compared to NX21617 (SEQ ID NO:143).
- the sequence-scrambled control Nucleic Acid Ligand was then conjugated to PEG and named NX31976-40K PEG (SEQ ID NO: 147) (see Figure 9B for molecular description).
- the covalent coupling of PEG to the Nucleic Acid Ligand (or to the sequence-scrambled control) was accomplished as described in Example 5.
- Rat PDGF-BB for cross-reactivity binding experiments was derived from E. coli transfected with sCR-Script Amp SK(+) plasmid containing the rat PDGF-BB sequence.
- Rat PDGF-BB sequence was derived rat lung poly A+ RNA (Clonetech, San Diego, CA) through RT-PCR using primers that amplify sequence encoding the mature form of PDGF- BB.
- Rat PDGF-BB protein expression and purification was performed at R&D Systems.
- Anti-Thy 1.1 mesangial proliferative glomerulonephritis was induced in 33 male Wistar rats (Charles River, Sulzfeld, Germany) weighing 150-160 g by injection of 1 mg/kg monoclonal anti-Thy 1.1 antibody (clone OX-7; European Collection of Animal Cell Cultures, Salisbury, England). Rats were treated with Nucleic Acid Ligands or PEG (see below) from day 3 to 8 after disease induction. Treatment consisted of twice daily i.v. bolus injections of the substances dissolved in 400 ⁇ l PBS, pH 7.4. The treatment duration was chosen to treat rats from about one day after the onset to the peak of mesangial cell proliferation (Floege et al.
- Renal biopsies for histological evaluation were obtained on days 6 and 9 after disease induction. Twenty-four hour urine collections were performed from days 5 to 6 and 8 to 9 after disease induction.
- the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU; Sigma, Deisenhofen, Germany; 100 mg/kg body weight) was injected intraperitoneally at 4 hours prior to sacrifice on day 9. Normal ranges of proteinuria and renal histological parameter (see below) were established in 10 non-manipulated Wistar rats of similar age.
- a murine monoclonal antibody (clone 1 A4) to ⁇ -smooth muscle actin; a murine monoclonal antibody (clone PGF-007) to PDGF B-chain; a murine monoclonal IgG antibody (clone EDI) to a cytoplasmic antigen present in monocytes, macrophages and dendritic cells; affinity purified polyclonal goat anti-human/bovine type IV collagen IgG preabsorbed with rat erythrocytes; an affinity purified IgG fraction of a polyclonal rabbit anti-rat fibronectin antibody; plus appropriate negative controls as described previously (Burg et al (1997) Lab. Invest. 76:505-516; Yoshimura et al (1991) Kidney Int. -40:470- 476). Evaluation of all slides was performed by an observer, who was unaware of the origin of the slides.
- Double immunostaining for the identification of the type of proliferating cells was performed as reported previously. (Kliem et al. (1996) Kidney Int. 49:666-678; Hugo et al. (1996) J. Clin. Invest. 97:2499-2508) by first staining the sections for proliferating cells with a murine monoclonal antibody (clone BU-1) against bromo-deoxyuridine containing nuclease in Tris buffered saline (Amersham, Braunschweig, Germany) using an indirect immunoperoxidase procedure.
- clone BU-1 murine monoclonal antibody against bromo-deoxyuridine containing nuclease in Tris buffered saline
- Sections were then incubated with the IgG] monoclonal antibodies 1 A4 against ⁇ -smooth muscle actin and EDI against monocytes/macrophages.
- Cells were identified as proliferating mesangial cells or monocytes/macrophages if they showed positive nuclear staining for BrdU and if the nucleus was completely surrounded by cytoplasm positive for ⁇ -smooth muscle actin.
- Negative controls included omission of either of the primary antibodies in which case no double-staining was noted.
- RNA probe for type IV collagen a digoxigenin-labelled anti-sense RNA probe for type IV collagen (Eitner et al. (1997) Kidney Int. 51:69-78) as described (Yoshimura et al. (1991) Kidney Int. 40:470-476). Detection of the RNA probe was performed with an alkaline phosphatase coupled anti-digoxigenin antibody (Genius Nonradioactive Nucleic Acid Detection Kit, Boehringer-Mannheim, Mannheim, Germany) with subsequent color development.
- Controls consisted of hybridization with a sense probe to matched serial sections, by hybridization of the anti-sense probe to tissue sections which had been incubated with RNAse A before hybridization, or by deletion of the probe, antibody or color solution described (Yoshimura et al (1991) Kidney Int. 40:470-476). Glomerular mRNA expression was semiquantitatively assessed using the scoring system described above.
- Urinary protein was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, Munchen, Germany) and bovine serum albumin (Sigma) as a standard. Statistical Analysis
- the modified DNA Nucleic Acid Ligand was conjugated to 40K PEG as described in Examples 5 and 8 and shown in Figures 9A and 9B. Since most Nucleic Acid Lig-ands have molecular weights ranging between 8 to 12 kDa (the modified PDGF Nucleic Acid Ligand has MW of 10 kDa), the addition of a large inert molecular entity such as PEG dramatically improves the residence times of Nucleic Acid Ligands in vivo (see for example PCT/US 97/18944). Importantly, the addition of the PEG moiety to the 5' end of the Nucleic Acid Ligand has no effect on the binding affinity of the Nucleic Acid Ligand for PDGF-BB (Kef lxl O' 10 M).
- the sequence of PDGF is highly conserved among species, and human and rat PDGF B-chain sequences are 89% identical (Herren et al. (1993) Biochim. Biophys. Acta 1173:294; Lindner et al. (1995) Circ. Res. 76:951). Nevertheless, in view of the high specificity of Nucleic Acid Ligands (Gold et al. (1995) Ann. Rev. Biochem. 64:763-797), the correct interpretation of the in vivo experiments requires understanding of the binding properties of the Nucleic Acid Ligands to rat PDGF B-chain. We have therefore cloned and expressed the mature form of rat PDGF-BB in E. coli. The PDGF Nucleic Acid Ligands bound to rat and human recombinant PDGF-BB with the same high affinity (data not shown).
- PDGF B-Chain DNA-Ligand Specifically Inhibits Mesangial Cell Proliferation in vitro In growth arrested mesangial cells, the effects of NX31975-40K PEG (SEQ ID NO:
- PBS treated animals developed the typical course of the nephritis, which is characterized by early mesangiolysis and followed by a phase of mesangial cell proliferation and matrix accumulation on days 6 and 9 (Floege et al. (1993) Kidney Int. Suppl. 39:S47-54). No obvious adverse effects were noted following the repeated injection of Nucleic Acid Ligands or PEG alone, and all rats survived and appeared normal until the end of the study.
- PDGF-B aptamer treated rates 2.8 ⁇ 1.1 BrdU+/ED-l+ cells per 100 glomerular cross sections; scrambled aptamer treated rats: 2.7 ⁇ 1.8).
- EXAMPLE 10 EXPERIMENTAL PROCEDURE FOR EVOLVING 2'-FLUORO-2'-DEOXYPYRIMIDINE RNA LIGANDS TO PDGF AND RNA SEQUENCES OBTAINED.
- *B biotin phosphoramidite (e. g., Glen Research, Sterling, VA)
- III-l 21 0 0 0 0 0 0 0 0 0 0 0 0
- ⁇ elices are numbered with Roman numerals as shown in Figure 1. Individual base pairs are numbered with Arabic numerals starting with position 1 at the helix junction and increasing with increased distance from the junction. The TG and GT base pairs to the Watson-Crick base pairs for this analysis were included. There is a total of 21 sequences in the set.
- **Kd value of 0.178 ⁇ 88 pM used for the calculation is average, with standard deviation, of four independent measurements (94 ⁇ 1 1, 161 ⁇ 24, 155 ⁇ 30 and 302 ⁇ 32 pM).
- NX21568 CAGGC-i/ACG-S-CGTAGAGCAC/CA-S-TGATCCC/G[ 3 'T] 0.63 142
- **Kd a 0.159 ⁇ 13 pM.
- ATCCGCCTGA TTAGCGATAC TATGGGAGGG CGCGTTCTTC GTGGTTACTT 50
Abstract
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JP2000539042A JP4413428B2 (en) | 1997-12-16 | 1998-04-29 | Platelet-derived growth factor (PDGF) nucleic acid ligand complex |
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SI9830941T SI1042348T1 (en) | 1997-12-16 | 1998-04-29 | Platelet derived growth factor (pdgf) nucleic acid ligand complexes |
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ES98920194T ES2426160T3 (en) | 1997-12-16 | 1998-04-29 | Platelet-derived growth factor nucleic acid ligand complexes (PDGF) |
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Also Published As
Publication number | Publication date |
---|---|
EP1042348A4 (en) | 2004-07-07 |
EP1042348A1 (en) | 2000-10-11 |
CA2315271C (en) | 2010-11-23 |
US20030036642A1 (en) | 2003-02-20 |
EP1042348B1 (en) | 2013-07-03 |
US6582918B2 (en) | 2003-06-24 |
ES2426160T3 (en) | 2013-10-21 |
CA2315271A1 (en) | 1999-06-24 |
JP2002508387A (en) | 2002-03-19 |
DK1042348T3 (en) | 2013-09-08 |
AU749273B2 (en) | 2002-06-20 |
US20050042273A1 (en) | 2005-02-24 |
US20080207883A1 (en) | 2008-08-28 |
US20100029922A1 (en) | 2010-02-04 |
JP4413428B2 (en) | 2010-02-10 |
CY1114352T1 (en) | 2016-08-31 |
US7939654B2 (en) | 2011-05-10 |
PT1042348E (en) | 2013-10-09 |
US6229002B1 (en) | 2001-05-08 |
US7879993B2 (en) | 2011-02-01 |
AU7282398A (en) | 1999-07-05 |
SI1042348T1 (en) | 2013-10-30 |
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