text:
|Publication number
|Publication type
|Application number
|Publication date
|Sep 1, 1998
|Filing date
|Sep 17, 1996
|Priority date
|Sep 17, 1996
|Fee status
|Also published as
|Publication number
|Gerald Soff, Stephen T. Gately, Przemyslaw Twardowski
|Original Assignee
|Northwestern University
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (1), Non-Patent Citations (46), Referenced by (27), Classifications (34), Legal Events (8)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to methods and compositions for making angiostatin, an inhibitor of angiogenesis. The invention also relates to the treatment of diseases caused by, or involving, angiogenesis.
Angiostatin, a proteolytic fragment of plasminogen including kringles 1 though 4, is a potent inhibitor of angiogenesis and the growth of tumor cell metastases. O'Reilly et al., Cell, 79, 315-328 (1994). Angiostatin is found in vivo in tumor-bearing mice. O'Reilly et al., Cell, 79, 315-328 (1994); O'Reilly et al., Nature Med. 2, 689-692 (1996). The enzymatic mechanism by which angiostatin is generated in vivo remains unknown.
Angiostatin activity can be generated in vitro by limited elastase proteolysis of plasminogen. Sottrup-Jensen et al., in Progress in Chemical Fibrinolysis and Thrombolysis, 3, 191-209 (Davidson et al. eds. 1978). A recent abstract proposes that angiostatin is generated by macrophages infiltrating primary tumors and releasing elastase activity, which then cleaves plasminogen to form a protein having angiostatin activity. Dong et al., Proc. Am. Assoc. Cancer Res., 37 58 (1996). However, while limited elastase cleavage of plasminogen will yield a fragment or fragments having angiostatin activity, elastase will further digest the fragment(s) to inactive peptides, and therefore, is probably not the enzyme that generates angiostatin in vivo.
As noted above, angiostatin may be generated in vitro by limited elastase proteolysis of plasminogen. This method has several disadvantages. First, while elastase cleaves plasminogen to generate a fragment containing kringles 1-3, it is not known if this cleavage is at the normal sites where cleavage occurs to produce angiostatin in vivo. Therefore, the elastase-derived angiostatin may have altered in vivo processing with altered activity in humans. It may also be immunogenic if the sites of peptide cleavage are different from normal angiostatin.
A second means of producing angiostatin is by expressing the desired kringle 1-4 domains, or subdomains or other domains, of the plasminogen cDNA or gene in an expression vector in prokaryotic or eukaryotic cells. See PCT application WO 95/29242. This approach is also limited since the appropriate domains to express are not known. The product may also be immunogenic and may not be processed in humans as would be the product generated by cleavage of plasminogen by the normal in vivo enzymes.
Finally, angiostatin can be isolated from the body fluids of animals in which it is produced. See PCT application WO 95/29242. However, angiostatin cannot be produced in sufficient quantities for disease treatment in this manner, and the angiostatin may be contaminated with infectious agents when isolated from such sources.
Clearly a need exists for a method of producing native angiostatin in large quantities. "Native angiostatin" is defined herein to be the angiostatin produced in vivo or angiostatin, no matter how produced, which is the same as the angiostatin produced in vivo.
The present invention provides such a method. In particular, the invention provides a method of generating angiostatin in vitro comprising contacting plasminogen or plasmin with a plasminogen activator and a sulfhydryl donor. It has been found that a conditioned culture medium (CCM) produced by culturing cancer cells, primary endothelial cells, smooth muscle cells or fibroblasts produces angiostatin when contacted with plasminogen or plasmin. The active factors in the CCM have been identified to be a plasminogen activator and a sulfhydryl donor. Thus, the angiostatin produced by the use of a plasminogen activator and sulfhydryl donor is the same as angiostatin produced in vivo, i.e., it is native angiostatin.
The angiostatin produced by the method of the invention, along with the rest of the reaction medium, or angiostatin purified or partially purified from the reactants, may be administered to an animal, including a human, in need thereof. Animals in need of angiostatin are animals suffering from an angiogenic disease.
The invention further provides a composition for generating angiostatin. The composition comprises a sulfhydryl donor and a plasminogen activator. The composition may be CCM produced by culturing cells capable of producing plasminogen activator, or a lysate of such cells.
The invention also provides a method of treating an angiogenic disease comprising administering to an animal suffering from such a disease an amount of a sulfhydryl donor effective to cause the conversion of plasminogen or plasmin to angiostatin by plasminogen activator. The plasminogen, plasmin and plasminogen activator may be those found endogenously in the animal or, preferably, effective amounts of the plasminogen, plasmin and/or plasminogen activator are also administered to the animal.
The invention further provides a container holding a plasminogen activator, alone or in combination with sulfhydryl donor. The container has a label thereon instructing administration of the plasminogen activator or the combination of the plasminogen activator and sulfhydryl donor to an animal suffering from an angiogenic disease. The invention also provides a container holding a sulfhydryl donor with a label thereon instructing administration of the sulfhydryl donor in an amount effective to cause the conversion of plasminogen or plasmin to angiostatin by plasminogen activator.
FIG. 1A: Western blot showing conversion of plasminogen and plasmin to angiostatin by serum-free conditioned medium (SFCM) produced by PC-3 cells. Lane 1, molecular weight standard; lane 2, human plasminogen; lane 3, human plasminogen incubated overnight at 37� C. in non-conditioned RPMI; lane 4, human plasminogen incubated overnight at 37� C. in SFCM from PC-3 cells; lane 5, human plasmin incubated in non-conditioned RPMI; lane 6, human plasmin incubated in SFCM produced by PC-3 cells.
FIG. 1B: Western blot showing that the generation of angiostatin from plasminogen was time dependent. PC-3 SFCM was incubated with plasminogen and, at the time-points indicated, aliquots were removed and snap frozen prior to western blot analysis. Trace generation of angiostatin was first observed at 3 hours, and complete conversion was noted at 24 hours.
FIG. 1C: Western blots showing that the generation of angiostatin by PC-3 SFCM was concentration dependent. SFCM was diluted with various amounts of fresh RPMI as indicated and incubated with plasminogen for 24 hours.
FIG. 1D: Graph illustrating the relationship of angiostatin generation to the amount of SFCM. The relative angiostatin signal was quantitated by scanning densitometer with background subtraction. At 18 hours incubation, there was a linear relationship between the amount of angiostatin generated and the amount of PC-3 SFCM present in the reaction mixture.
FIG. 2: Western blots after affinity purification of angiostatin generated by incubation of plasminogen with SCFM produced by PC-3 cells. Lane 1, molecular weight standards; lane 2, human plasminogen incubated overnight at 37� C. in non-conditioned RPMI; lane 3, angiostatin produced by incubation of plasminogen with PC-3 SCFM and then affinity purified on lysine-sepharose and detected on western blot by staining with Coomassie blue; lane 4, angiostatin produced by incubation of plasminogen with PC-3 SCFM and then affinity purified on lysine-sepharose and detected on western blot using the monoclonal antibody K1-3 to kringles 1-3.
FIGS. 3A-B: Graphs showing that angiostatin produced by incubating plasminogen with PC-3 SCFM inhibits in vitro steps critical for angiogenesis. FIG. 3A: Endothelial cell proliferation. The data are mean � standard deviation. FIG. 3B: Basic fibroblast growth factor (bFGF)-induced migration. Background migration without the inducer and in the presence of stimulatory bFGF are indicated. Toxicity was measured in parallel by trypan blue exclusion and was <10% at all concentrations.
FIGS. 4A-B: Photographs showing that angiostatin produced by incubating plasminogen with PC-3 SCFM inhibits human endothelial cell tube formation in vitro. Human umbilical vein endothelial cells (HUVEC) were plated on gels of Matrigel in 24-well dishes and then were treated with 15 μg/ml of angiostatin produced using PC-3 SFCM in non-conditioned RPMI. FIG. 4A: Control HUVEC form branching, interconnecting networks. FIG. 4B: By contrast angiostatin produced using PC-3 SFCM caused a significant disruption of the tube network.
FIGS. 5A-B: Photographs showing the inhibition of angiogenesis in vivo by angiostatin produced using PC-3 SCFM. FIG. 5A: A hydron pellet (indicated by the arrow) containing bFGF induced a positive neovascular response 7 days after implantation. FIG. 5B: By contrast, no vessels are observed approaching a hydron pellet containing bFGF and 10 μg/ml angiostatin produced using PC-3 SFCM (indicated by the arrow).
FIG. 6: Western blot showing that the batch eluate from Reactive Red 120-Agarose generates angiostatin when combined with Reactive Red 120-Agarose flow-through, RPMI or RPMI amino acids. Lane 1 -- SFCM + plasminogen; Lane 2 -- Reactive Red 120-Agarose flow-through + plasminogen; Lane 3 -- Reactive Red 120-Agarose batch eluate after dialysis to TBS + plasminogen; Lane 4 -- dialyzed batch eluate + Reactive Red 120-Agarose flow-through + plasminogen; Lane 5 -- dialyzed batch eluate + RPMI + plasminogen; Lane 6 -- dialyzed batch eluate + RPMI vitamin mix + plasminogen; Lane 7 -- dialyzed batch eluate + RPMI amino acid mix + plasminogen; Lane 8 --dialyzed batch eluate + RPMI vitamin mix and amino acid mix + plasminogen; Lane 9 -- plasminogen + unconditioned RPMI.
FIG. 7: Graph showing that urokinase-type plasminogen activator (u-PA) activity and plasminogen-angiostatin converting activity (PACA) co-elute on a gradient elution from Hi-Q anion exchange column. Optical density readings at 280 nm demonstrated several protein peaks. u-PA activity was determined by measuring the cleavage of a chromogenic peptide substrate for plasmin (Val-Leu-Lys p-NA) at 405 nm. The peak fractions were assayed for PACA by western blot.
FIG. 8: Western blot showing that addition of u-PA and plasminogen to boiled Reactive Red 120-Agarose flow-through or fresh RPMI medium generated angiostatin. Lane 1 -- Reactive Red 120-Agarose flow-through + plasminogen; Lane 2 -- Reactive Red 120-Agarose flow-through + plasminogen + u-PA; Lane 3-- Reactive Red 120-Agarose boiled flow-through + plasminogen; Lane 4 -- Reactive Red 120-Agarose boiled flow-through + plasminogen + u-PA; Lane 5-- unconditioned RPMI + plasminogen; Lane 6-- unconditioned RPMI + plasminogen + u-PA.
FIG. 9: Western blot showing that the Reactive Red 120-Agarose flow-through produces angiostatin in the presence of plasminogen activators. Lane 1-- Reactive Red 120-Agarose flow-through + plasminogen; Lane 2 -- Reactive Red 120-Agarose flow-through + plasminogen + u-PA; Lane 3 -- Reactive Red 120-Agarose flow-through + plasminogen + t-PA.
FIG. 10: Western blot showing the production of angiostatin by u-PA and glutathione. Lane 1--plasminogen + u-PA; Lane 2 -- plasminogen + u-PA + 5 μM glutathione; Lane 3 -- plasminogen + u-PA + 50 μM glutathione; Lane 4 -- plasminogen + u-pA + 100 μM glutathione; Lane 5 -- plasminogen + u-PA + boiled 5 μM glutathione; Lane 6 -- plasminogen + u-PA + boiled 50 μM glutathione; Lane 7 -- plasminogen + u-PA + boiled 100 μM glutathione.
FIG. 11: Western blot showing that the combination of u-PA and D-penicillamine produces angiostatin. Lane 1 -- plasminogen + 100 μM D-penicillamine; Lane 2 -- plasminogen + u-PA +100 μM D-penicillamine; Lane 3 -- plasminogen + u-PA +1.0 mM D-penicillamine.
The invention provides an in vitro method of generating native angiostatin. The method comprises contacting plasminogen or plasmin with a plasminogen activator and a sulfhydryl donor.
The plasminogen may be from any animal species. Preferably, however, plasminogen from the species of animal to be treated with the angiostatin is used to avoid immune reactions upon administration of the angiostatin. Thus, if a human is to be treated with the angiostatin, human plasminogen is preferably used.
Methods of making plasminogen are well known in the art. Plasminogen may also be purchased commercially. Preferably the plasminogen is prepared by recombinant DNA or other techniques that avoid the inclusion of infectious agents in the plasminogen preparation.
Plasmin may also be used to prepare angiostatin. Plasmin may be prepared from plasminogen by methods known in the art. Plasmin may also be purchased commercially.
All types of plasminogen activators may be used to convert plasminogen and plasmin to angiostatin, including urokinase-type plasminogen activators, tissue-type plasminogen activators and streptokinase. The plasminogen activator may be from any animal species. Methods of making plasminogen activators are well known in the art and many plasminogen activators are available commercially. Preferably the plasminogen activator is prepared by recombinant DNA or other techniques that avoid the inclusion of infectious agents in the plasminogen activator preparation.
Any sulfhydryl donors may be used for converting plasminogen and plasmin to angiostatin. Sulfhydryl donors are well known and are available commercially. Suitable sulfhydryl donors include L-cysteine, D-cysteine, DL-cysteine, N-acetyl-L-cysteine, reduced glutathione, D-penicillamine and captopril. The sulfhydryl donor is believed to reduce or alter disulfide bond formation in one of the reactants (plasminogen, plasmin or plasminogen activator) and/or the product(s) (angiostatin or an intermediate).
The plasminogen or plasmin is contacted with the plasminogen activator and sulfhydryl donor in amounts and under conditions effective to cause the conversion of the plasminogen or plasmin to angiostatin. These amounts and conditions can be determined empirically as is known in the art. In particular, from about 1 ng/ml to about 1 μ/ml of urokinase plasminogen activator and from about 10 μM to about 1 mM of sulfhydryl donor for each microgram of plasminogen or plasmin in a 1 ml reaction have been found to give complete conversion to angiostatin after about 24 hours of incubation at 37� C.
The invention further provides a composition for generating angiostatin from plasminogen or plasmin. The composition comprises a plasminogen activator and a sulfhydryl donor as described above. The plasminogen activator and sulfhydryl donor may be contained in any physiologically-acceptable solution (e.g., saline, buffers, culture medium) or may be present in crystalline or lyophilized form. Compositions suitable for therapeutic use are described below.
The composition may be a conditioned culture medium (CCM) prepared by culturing cells capable of producing plasminogen activator. Malignant animal cells, human and non-human, which express a plasminogen activator can produce CCM capable of converting plasminogen and plasmin into angiostatin. Suitable malignant cells include human prostate carcinoma cell lines PC-3, DU-145, LN-CaP, human breast carcinoma cell lines MDA-MB-231 and MCF-7, human glioma cell lines U-373, U-118, A-172, and U-87, and mouse melanoma cell line B16F10. Many non-malignant animal cells are known to produce plasminogen activator. Suitable non-malignant cells include primary endothelial cells (e.g., bovine aortic endothelial cells), smooth muscle cells (e.g., bovine smooth muscle cells), and fibroblasts. In addition, bacterial cells are known which produce plasminogen activator (e.g., streptokinase), and cells of any type can be transformed by recombinant DNA techniques to produce plasminogen activator. Suitable cells and cell lines are well known in the art and may be obtained commercially, from cell depositories, and by methods well known in the art.
Suitable culture conditions for these cells are also well known in the art. The culture medium used must contain a sulfhydryl donor, or a sulfhydryl donor may be added to the CCM after it is produced. Suitable culture media include those available commercially, such as RPMI, DMEM, etc. The CCM may be produced by simply culturing the cells under normal culture conditions for a sufficient time to produce CCM capable of converting plasminogen or plasmin to angiostatin. This time can be determined empirically. In particular, it has been found that culturing the mammalian cells for 24-72 hours after a monolayer has formed at 37� C. is sufficient.
Alternatively, or in addition, the cells can be lysed after culturing for a time sufficient to allow synthesis of plasminogen activator. This time can be determined empirically, but culturing the cells until a monolayer has formed should be sufficient. The lysate can be used to convert plasminogen and plasmin to angiostatin.
The angiostatin produced by the method of the invention may be purified from the reaction mixture. Methods of protein purification are well known in the art. In particular, angiostatin may be purified by affinity chromatography using lysine-Sepharose. Residual plasmin activity should be removed with, e.g., soybean trypsin inhibitor-Sepharose, aprotinin-Sepharose, or other affinity chromatography procedures that remove serine proteases or the plasmin catalytic domain.
The angiostatin produced by the method of the invention has been found to have the N-terminal sequence of plasmin. For angiostatin produced from human plasminogen, the N-terminal sequence has been found to be Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly SEQ ID NO: 1!. Its C-terminal sequence has not yet been determined. The angiostatin reacts with a monoclonal antibody specific for kringles 1-3 of plasminogen and has a molecular weight of about 50 kD as determined by polyacrylamide gel electrophoresis under non-reducing conditions. It inhibits angiogenesis, as assessed by a variety of tests in vitro and in vivo.
The invention also provides methods of treating an angiogenic disease. An angiogenic disease is one caused by, involving or dependent on angiogenesis. Angiogenic diseases include neoplastic diseases (e.g., tumors and tumor metastasis), benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyrogenic granulomas), connective tissue disorders (e.g., rheumatoid arthritis and atherosclerosis), ocular angiogenic diseases (e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis), cardiovascular diseases, cerebral vascular diseases, diabetes-associated diseases and immune disorders (e.g., chronic inflammation and autoimmunity).
The angiogenic disease may be treated by administering an effective amount of the angiostatin produced by the method of the invention. The angiogenic disease may also be treated by administering an amount of a sulfhydryl donor sufficient to cause conversion of plasminogen or plasmin to angiostatin by plasminogen activator. The plasminogen, plasmin and plasminogen activator may be those found endogenously in the animal or, preferably, effective amounts of plasminogen, plasmin and/or plasminogen activator are also administered to the animal. Animals treatable according to the invention include mammals, such as dogs, cats, other domestic animals, and humans.
Effective dosage forms, modes of administration and dosage amounts for the various compounds for treating angiogenic diseases may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the activity of the particular compound employed, the severity of the angiogenic disease, the route of administration, the rate of excretion of the compound, the duration of the treatment, the identify of any other drugs being administered to the animal, the age, size and species of the animal, and like factors known in the medical and veterinary arts. In general, a suitable daily dose of a compound of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. However, the daily dosage will be determined by an attending physician or veterinarian within the scope of sound medical judgment. If desired, the effective daily dose may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
The compounds of the present invention may be administered to an animal patient for therapy by any suitable route of administration, including orally, nasally, rectally, vaginally, parenterally (e.g., intravenously, intraspinally, intraperitoneally, subcutaneously, or intramuscularly), intracisternally, transdermally, intracranially, intracerebrally, and topically (including buccally and sublingually). The preferred routes of administration are orally and intravenously. The use of biodegradable polymers similar to that described by Brem, et al., Lancet, 345, 1571 (1995) for the local sustained release of pharmacological agents following incorporation into the biodegradable polymers is also a preferred method of administration. Implantation of the drug-impregnated polymer at, e.g., a tumor site, allows prolonged local exposure with minimal systemic exposure.
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The pharmaceutical compositions of the invention comprise a compound of the invention as an active ingredient in admixture with one or more pharmaceutically-acceptable carriers and, optionally, with one or more other compounds, drugs or other materials. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
Pharmaceutical formulations of the present invention include those suitable for oral, nasal, ophthalmic, topical, rectal, vaginal and/or parenteral administration. Regardless of the route of administration selected, the compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
The amount of active ingredient which will be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration and all of the other factors described above. The amount of active ingredient which will be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which is the lowest dose effective to produce a therapeutic effect or the maximally-tolerated dose that yields a therapeutic increment for life-threatening illnesses, such as cancer.
Methods of preparing pharmaceutical formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar--agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form.
Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar--agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the invention to the body. Such dosage forms can be made by dissolving, dispersing or otherwise incorporating a compound of the invention in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.
Pharmaceutical compositions of this invention suitable for parenteral administrations comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monosterate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug from is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
Since the invention provides, for the first time, a method of producing large quantities of native angiostatin, the amino acid sequence of native angiostatin can finally be determined. Once this is accomplished, native angiostatin can be made by recombinant DNA techniques. DNA coding for native angiostatin can also be used for gene therapy, so that native angiostatin can be produced in vivo in animals suffering from angiogenic diseases. Recombinant DNA techniques and gene therapy techniques are well known in the art. See, e.g., PCT WO 95/29242, the complete disclosure of which is incorporated herein by reference.
This example demonstrates that a variety of cells express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin. Affinity-purified angiostatin generated by incubating plasminogen or plasmin with serum-free conditioned medium (SFCM) inhibited human endothelial cell proliferation, migration induced by angiogenic factor basic fibroblast growth factor (bFGF), endothelial cell tube formation, and bFGF-induced corneal angiogenesis. Serine proteinase inhibitors, but not inhibitors of metallo-, cysteine, or aspartic proteinases, blocked angiostatin generation. Elastatinal, a specific inhibitor of elastase, failed to block angiostatin generation, indicating that an elastase is not responsible for the conversion of plasminogen to angiostatin. Instead, the data show that serine proteinase activity is necessary for angiostatin generation.
A. Methods
1. Cell Culture. The human umbilical vein endothelial cells (HUVEC), were grown in RPMI supplemented with 20% bovine calf serum (Hyclone Laboratories Inc., Logan Utah #A-2151-L), 100 U/ml penicillin G, 100 mg/ml streptomycin, L-glutamine, (Gibco BRL), 2500 U Sodium heparin (Fisher Scientific, Itasca, Ill.), and 50 mg/ml endothelial cell growth supplement (Collaborative Biomedical Research, Bedford, Mass.). The other cells listed in Table 1 were grown in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, 100 mg/ml streptomycin (Gibco BRL, Gaithersburg, Md.). Cells were maintained at 37� C. in a humidified incubator in an atmosphere of 5% CO2. To generate SFCM, confluent cell monolayers were washed twice with phosphate buffered saline, then serum-free RPMI was added. The next day the SFCM was collected and centrifuged at 3000 rpm for 15 minutes to remove insoluble cellular debris.
2. Angiostatin Generation. Two micrograms of human plasminogen, obtained by lysine-sepharose affinity chromatography of human plasma (Castellino & Powell, Methods Enzymol, 80, 365-78 (1981)), or human plasmin (#527624, Calbiochem-Novabiochem Corp., La Jolla, Calif.) were added to 100 μl aliquots of SFCM and the mixture incubated at 37� C. overnight. Aliquots were analyzed for angiostatin generation by western blot (see below). Plasminogen cleavage by SFCM was also assessed in the presence of proteinase inhibitors (Boehringer Mannheim, Indianapolis, Ind.).
3. Western Blot. Samples were electrophoresed under non-reducing conditions on 12% polyacrylamide gels (NOVEX, San Diego, Calif.) in Tris-Glycine running buffer (Laemmli, Nature, 227, 680-685 (1970)), and electrotransferred to a 0.45 μM polyvinylene difluoride (PVDF) membrane (Immobilon, Millipore, Bedford, Mass.). The membrane was then blocked for 30 minutes in blocking buffer (1% bovine serum albumin in Tris-buffered saline) and probed with a 1:1000 dilution of a monoclonal antibody to the kringles 1-3(K1-3) fragment of human plasminogen (VAP 230L, Enzyme Research Laboratories, Inc., South Bend, Ind.). Following washing, the membrane was incubated for 30 minutes with an alkaline phosphatase conjugated goat anti-mouse IgG secondary antibody (Kirkegaard & Perry Laboratories (KPL), Gaithersburg, Md.) and developed using 5-bromo-4-chloro-3-indoyl-phosphate/nitroblue tetrazolium (KPL).
4. Zymographic Analysis. Zymograms to detect matrix metalloproteinase activity were performed as described previously. Heussen & Dowdle, Anal. Biochem., 102, 196-202 (1980).
5. Chromogenic Peptide Substrates. To determine if an elastase was present, 50 μl of SFCM were incubated with 0.3 mM of chromogenic peptide substrates specific for elastase (substrate I, MeOSuc-Ala-Ala-Pro-Val-pNA; substrate II, Boc-Ala-Ala-Pro-Ala-pNA); substrate III, pGlu-Pro-Val-pNA; substrate IV, Suc-Ala-Ala-Pro-Abu-pNA)(Calbiochem-Novabiochem Corp.), at 37� C. for 2-18 hours. Substrate cleavage was determined by monitoring the absorbance at 405 nm (Molecular Devices, Menlo Park, Calif.).
6. Lysine-Sepharose Purification of Angiostatin. To generate purified angiostatin for bioactivity analyses, human plasminogen was incubated with PC-3 SFCM at 20 μ/ml overnight at 37� C. The reaction product was applied to a lysine-sepharose column (Pharmacia Biotech), pre-equilibrated with TBS (50 mM Tris, pH 7.5, and 150 mM NaCl) Following washes with TBS to remove non-specifically bound protein, angiostatin was eluted in 0.2 M epsilon aminocaproic acid (EACA) in TBS. The eluted fraction was dialyzed (molecular weight cut off 12,000-14,000) to phosphate buffered saline. To remove residual plasmin, the angiostatin was applied to a soybean trypsin inhibitor agarose (Sigma Chemical Co., St. Louis, Mo.) column, and the flow-through collected, filter-sterilized and stored at -80� C. until used. Angiostatin was quantitated by measuring the absorbance at 280 nm, using an A1% /1cm of 8.0. Sottrup-Jensen et al., in Progress in Chemical Fibrinolysis and Thrombolysis, vol. 3, pages 191-209 (Davidson et al. eds. 1978). The purified angiostatin was also examined by Coomassie brilliant blue staining of polyacrylamide gels, and immunodetection by western blot. Elastase-generated angiostatin, purified from human plasma as described in O'Reilly, et al., Nature Med., 2, 689-692 (1996), was a generous gift from M.S. O'Reilly, Children's Hospital, Harvard University, Boston, Mass.
7. Microsequence Analysis of Angiostatin. To determine the NH2 -terminus of the angiostatin bands, 10 μg/ml of the affinity-purified angiostatin prepared by incubating plasminogen with PC-3 SFCM was electrophoresed on a 12% SDS-polyacrylamide gel, electroblotted to a PVDF membrane, and stained with Coomassie blue. The bands were excised, placed on Porton sample support discs, and sequenced using a pulse liquid-phase sequencer with phenylthiohydantoin analysis.
8. Endothelial Cell proliferation Assay. Cell proliferation was determined utilizing the CellTiter 96™ AQ Non-Radioactive Cell Proliferation Assay (Promega Corp., Madison, Wis.). The human endothelial cells were plated in a 96-well tissue culture plates (Becton Dickinson, Lincoln Park, N.J.) at a concentration of 5.0�103 cells/well. The following day, 1, 5, 8, or 10 μg/ml of angiostatin in fresh medium was added to triplicate wells. Wells without angiostatin served as control. The cells were incubated for 72 hours, and an absorbance read at 490 nm, reflecting the number of proliferating cells, was measured using an automated microplate reader (Molecular Devices). The results are reported as the percent of non-treated control cell number.
9. Endothelial Cell Migration Assay. To determine the ability of angiostatin prepared by incubation of plasminogen with PC-3 SFCM to block migration of endothelial cells towards an angiogenic factor, bFGF, migration assays were performed in a modified Boyden chamber using bovine capillary endothelial cells (a kind gift of Dr. Folkman, Harvard Medical School, Boston, Mass.) as described previously. Dameron et al., Science, 265, 1582-84 (1994). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% donor calf serum and 100 mg/ml endothelial cell mitogen and used at passage 15. To assess migration, the cells were serum starved overnight in DMEM supplemented with 0.1% bovine serum albumin (BSA), harvested, suspended in DMEM/BSA, plated at 106 /ml on the lower surface of a gelatinized membrane (Nucleopore Corp., Plesanton, Calif.) in an inverted Boyden chamber, and incubated for 1.5-2 hours to allow cell attachment. The chambers were reinverted, test material was added to the top well, and the chamber incubated for an additional 3-4 hours. Membranes were then fixed and stained and the number of cells that migrated to the top of the filter in 10 high-power fields was determined. DMEM with 0.1% BSA was used as a negative control, and bFGF (provided by Dr. Noel Bouck and prepared as described in Dameron et al., Science, 265, 1582-1584 (1994)) at 10 ng/ml was used as a positive control.
10. Endothelial Cell Tube Formation. HUVEC were plated on gels of Matrigel (kindly provided by Hynda Kleinman, National Institute of Dental Research) in 24-well tissue culture plates as described previously. Schnaper et al., J. Cell. Physiol, 156, 235-246 (1993). Angiostatin, prepared by incubation with PC-3 SFCM, in non-conditioned RPMI was added to the wells, followed by cells at a final concentration of 4.0�104 cells in 1 ml of 50% HUVEC culture medium, 50% RPMI. Each angiostatin or control condition was assayed in triplicate. The cultures were incubated for 16-18 hours at 37� C., in a 5% CO2 humidified atmosphere, then fixed with Diff-Quick Solution II (Baxter, McGraw Park, Ill.). A representative area of the tube network was photographed using a Polaroid MicroCam camera at a final magnification of 35�. The photographs were then quantitated by a blinded observer who measured the length of each tube, correcting for portions of tubes that were incomplete. The total length of the tubes was determined for each photograph and the mean tube length was determined. The results were expressed as the mean � standard error of the mean.
11. Corneal Angiogenesis Assay. The corneal assay was performed as described previously. Polverini et al., Methods Enzymol, 198, 440-450 (1991). Briefly, 5 μl hydron pellets (Hydron Laboratories, New Brunswick, N.J.) containing 10 μ/ml bFGF or bFGF plus 1 or 10 μg/ml angiostatin were implanted into the cornea of anesthetized rats. After 7 days, the animals were sacrificed and corneal vessels were stained with colloidal carbon and corneas were examined for angiogenic activity.
B. Results
1. Angiostatin Generation By Conditioned Culture Medium. Incubation of human plasminogen with the SFCM produced by PC-3 cells resulted in the generation of multiple immunoreactive bands at approximately 50 kD (FIG. 1A), similar to those observed by O'Reilly et al. Cell, 79, 315-328 (1994). Examination of SFCM from additional cell lines also revealed the generation of the multiple bands, similar to the PC-3 SFCM (data not shown). These cell lines are listed in Table 1 below.
The initial indication that the product was angiostatin was based on the immunoreactivity with the monoclonal antibody specific for kringles 1-3 (K1-3) of plasminogen and the size of the cleavage product that approximated the predicted mass of kringles 1-4 of human plasminogen. Subsequent confirmation that the plasminogen cleavage product was bioactive angiostatin is described below.
Angiostatin generation by PC-3 SFCM was time-dependent. There was a significant decrease in the plasminogen substrate and a corresponding increase in angiostatin beginning at 3 hours, with complete conversion to angiostatin by 24 hours (FIG. 1B). Dilution of the PC-3 SFCM resulted in a proportional decrease in angiostatin generation (FIGS. 1C and 1D).
To determine whether plasmin, the activated form of the zymogen plasminogen, could also be converted to angiostatin, plasmin was evaluated as a potential substrate. Incubation of plasmin with PC-3 SFCM yielded a product indistinguishable from the plasminogen-derived angiostatin (FIG. 1A). In kinetic studies, plasmin was converted to angiostatin at a comparable rate to the plasminogen; 50% conversion by 8 hours, with complete conversion by 24 hours (data not shown). These data suggest that in vitro both plasminogen and plasmin are substrates from which angiostatin can be generation.
TABLE 1______________________________________Cell Lines Tested for Angiostatin-Generating Activity Activity______________________________________Human Prostate CarcinomaPC-3 +++DU-145 ++Ln-CaP +Human Breast CarcinomaMDA-MB-231 ++MCF-7 +/-Human GliomaU-373 +U-118 +A-172 ++U-87 +Mouse MelanomaB16F10 ++Bovine Smooth MusclePrimary cell line ++Bovine Aortic Endothelial CellsBAEC ++______________________________________
2. Enzymatic Class Of Plasminogen-Angiostatin Converting Activity. To determine the proteolytic class of the angiostatin generating activity, PC-3 SFCM was incubated with plasminogen in the presence of various proteinase inhibitors. The proteinase inhibitors were added to the SFCM/plasminogen mix prior to the overnight incubation. Samples were analyzed by western blot for evidence of inhibition of angiostatin generation.
Only serine proteinase inhibitors blocked angiostatin generation (see Table 2 below). By contrast none of the other classes of proteinase inhibitors were effective.
Angiostatin can be generated in vitro by limited proteolysis of plasminogen by elastase. Sottrup-Jensen et al. in Progress in Chemical Fibrinolysis and Thrombolysis, 3, 191-209 (Davidson et al. eds. 1978); O'Reilly et al., Nature Med., 2, 689-692 (1996); Dong et al., Proc. Am. Assoc. Cancer Res., 37, 58 (1996). In the present study, angiostatin generation was not inhibited by elastatinal, a specific inhibitor of elastase (see Table 2 below). Additionally, no elastase activity was detected in PC-3 SFCM based on co-incubation of SFCM with 4 elastase-sensitive chromogenic substrates for 24 hours (data not shown). These data indicate that the human plasminogen-angiostatin converting activity is unlikely to depend on the action of an elastase. Furthermore, gelatin zymograms revealed no evidence of active or latent metalloproteinases in the PC-3 SFCM (not shown).
TABLE 2______________________________________Proteinase InhibitoryInhibitor Concentration Class Activity *______________________________________Pefabloc 4.0 mM Serine Proteinases CompleteAprotinin 0.3 μM Serine Proteinases CompleteSoybean Trypsin 2.0 mM Serine Proteinases CompleteInhibitorBenzamidine 1-10 mM Serine Proteinases WeakElastatinal 50-100 μM Elastase NoneAntipain 100 μM Limited Serine Nonedihydrochloride ProteinasesLeupeptin 100 μM Serine and Thiol None ProteinasesChymostatin 100 μM Chymotrypsin NoneBestatin 10 μM Aminopeptidases WeakE-64 10 μM Cysteine None ProteinasesPepstatin 1.0 μM Aspartic None ProteinasesEDTA 1-10 mM Metalloproteinases None1-10 10 μM Metalloproteinases NonePhenanthrolinePhosphoramidon 100 μM Metalloproteinases None______________________________________ *Complete inhibition is defined as no immunoreactive angiostatin bands; weak inhibition results in the developinent of faint angiostatin immunoreactive bands; and none refers to the full generation of angiostatin.
3. Purification Of Angiostatin. Angiostatin generated by PC-3 SFCM was affinity purified on lysine-sepharose (O'Reilly et al., Nature Med., 2, 689-692 (1996)), and the resulting product examined by western blot and Coomassie blue staining (FIG. 2). The amino-terminal sequence of all three bands was KVYLSECKTG SEQ. ID NO: 1! that corresponds to residues 78-87 of the plasminogen molecule, confirming that the product was an internal fragment of plasminogen.
4. Angiostatin Generated By PC-3 SFCM Inhibits Angiogenesis. Because angiogenesis represents a cascade of cellular processes that includes endothelial cell proliferation, migration, and tube formation, (Folkman & Shing, J. Biol. Chem, 267, 10931-10934 (1992)), multiple in vitro and in vivo assays related to angiogenesis were utilized to confirm that the product generated by incubating plasminogen with PC-3 SFCM was bioactive angiostatin.
Affinity purified angiostatin generated by PC-3 SFCM inhibited human endothelial cell proliferation in a concentration-dependent manner, with significant inhibition observed at 10 μg/ml (P<0.05) in comparison to the non-treated control cell proliferation (FIG. 3A).
Angiostatin generated by PC-3 SFCM also inhibited the bFGF-induced migration of bovine capillary endothelial cells (FIG. 3B) with an ED50 of 0.35 μg/ml. The dose/response curve of angiostatin generated by PC-3 SFCM was indistinguishable from that of elastase-generated angiostatin. Inhibition of migration occurred at a 10-fold lower concentration than required to inhibit proliferation, a finding that has been reported for other inhibitors of angiogenesis. Takano et al., Cancer Res., 54, 2654-2660 (1994). This may be due to the fact that the proliferation assay, in contrast to the migration assay, was conducted in RPMI supplemented with 20% calf serum and endothelial cell growth supplement, and therefore contained multiple stimulatory factors.
Endothelial cell tube formation on Matrigel was significantly inhibited at 15 μg/ml (FIGS. 4A and B); the mean length of tubes in non-treated control was 674.5�54 mm in comparison to angiostatin produced by PC-3 SFCM, 287.7�47 mm (P<0.005).
To determine the effect of angiostatin generated by PC-3 SFCM on corneal angiogenesis in vivo, its ability to block bFGF-induced angiogenesis in the corneal angiogenesis assay was tested. The bFGF pellet induced angiogenesis in 100% of implanted corneas (FIG. 5A). In contrast, angiostatin at 10 μg/ml completely inhibited the bFGF-induced angiogenic response in 3 of 3 animals (FIG. 5B). At a lower dose of 1.0 μg/ml, angiostatin completely blocked angiogenesis in 2 of 3 animals, with partial inhibition in the third animal.
Taken together, these data indicate that the angiostatin generated by the PC-3 SFCM is a potent inhibitor of both in vitro and in vivo angiogenesis.
The human prostate carcinoma cell line PC-3 was grown and PC-3 SFCM was prepared as described in Example 1. Angiostatin was generated by incubation with PC-3 SFCM or other materials identified below as described in Example 1. Western Blots were performed as described in Example 1.
PC-3 SFCM was applied to a Reactive Red 120-Agarose column (Sigma Chemical Co.). The flow-through had no residual plasminogen-angiostatin generating activity (PACA) as demonstrated by western blot analysis (FIG. 6). The bound material was eluted with 1 M KCl according to the manufacturer's protocol, then dialyzed to Tris-buffered saline (TBS, 20 mM Tris, pH 7.4, 100 mM NaCl), with a molecular cut-off of 6000-8000 Dalton. PACA was not detected in the dialyzed fraction (FIG. 6). The observation that PACA was not detected in either the flow-through or the eluate led to the hypothesis that two or more factors are necessary to generate angiostatin from plasminogen or plasmin, and that the factors were separated by the Reactive Red 120-Agarose chromatography, with one or more factors being present in the elaute and one more factors being contained in the flow-through.
To test this hypothesis, the dialyzed eluate was recombined with the flow-through. The recombined materials were able to convert plasminogen into angiostatin. Supplementation of the eluate with fresh RPMI culture medium, as well as the Reactive Red 120-Agarose flow-through, restored the capacity of the eluate to generate angiostatin, suggesting that the necessary factor was a component of RPMI, and not a protein or other factor unique to the SFCM.
To further define the putative cofactor, the individual components of RPMI were evaluated for the ability to complement the Reactive Red 120-Agarose eluate. The cofactor was present in the RPMI amino acid mix (FIG. 6).
To determine which amino acid was capable of restoring PACA to the Reactive Red 120-Agarose eluate, the 20 amino acids found in RPMI were tested individually. L-cysteine was the only amino acid capable of restoring PACA to the Reactive Red 120-Agarose eluate (data not shown).
Because the addition of L-cysteine to the Reactive Red 120-Agarose eluate restored angiostatin generating activity, it was hypothesized that the cofactor was a sulfhydryl donor. Pharmacological reducing agents, D-penicillamine and captopril were therefore examined for the ability to restore PACA to the Reactive Red 12-agarose eluate. Addition of 100 μM D-penicillamine to the Reactive Red 120-Agarose eluate restored angiostatin generating activity. Captopril also restored angiostatin generating activity to the Reactive Red 120-Agarose eluate.
PC-3 SFCM was diluted to 50 mM Tris, pH 10.0, 20 mM NaCl and applied to a Hi-Q Sepharose anion exchange resin (Bio Rad). No PACA was detected in the flow-through.
Preliminary experiments indicated that PACA eluted from the Hi-Q Sepharose column with 300 mM NaCl. Therefore, the bound material was eluted utilizing a linear gradient from 20 mM to 300 mM NaCl. PACA and urokinase-type plasminogen activator (u-PA) activity were measured in the fractions (after dilution to restore physiological NaCl concentrations). The u-PA activity and PACA co-purified (FIG. 7). Examination of the Reactive Red 120-Agarose eluate revealed it also contained u-PA.
As noted in Example 1, the NH2 -terminal cleavage of angiostatin is at Lys77, a site that results from cleavage of Glu-plasminogen by plasmin. This suggests that plasmin generation may be a necessary intermediate step in angiostatin generation from plasminogen.
To determine if the factor in the Reactive Red 120-Agarose eluate was u-PA, u-PA was tested as a substitute for the Reactive Red 120-Agarose eluate. As illustrated in FIG. 8, u-PA was capable of generating angiostatin in the presence of boiled Reactive Red 120-Agarose flow-through or RPMI, both sources of sulfhydryl donors. This indicates that the only protein necessary for conversion of plasminogen to angiostatin is u-PA.
Next, u-PA, tissue-type plasminogen activator (t-PA), and streptokinase were tested in combination with the Reactive Red 120-Agarose flow-through for PACA. The plasminogen activators alone failed to generate angiostatin from plasminogen but, in the presence of the flow-through, angiostatin was produced (FIG. 9). These data suggest that plasmin generation is an intermediate for angiostatin generation, and that angiostatin generation is not dependent on which plasminogen activator is present.
Having demonstrated that the only protein necessary for conversion of plasminogen to angiostatin is a plasminogen activator and that a sulfhydryl donor is a necessary cofactor, it was next determined if these components are sufficient for angiostatin generation. Incubation of u-PA with plasminogen and at least 5 μM reduced glutathione produced angiostatin (FIG. 10). Use of 100 μM or 1 MM D-penicillamine in combination with u-PA was also capable of generating angiostatin (FIG. 11). These data demonstrate that angiostatin is produced in the setting of plasminogen activation in the presence of physiological (L-cysteine, reduced glutathione) and pharmacological (captopril, D-penicillamine) reducing agents.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 1(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:LysValTyrLeuSerGluCysLysThrGly1510__________________________________________________________________________
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|Jan 8, 2007
|May 17, 2007
|Shirin Asina
|Composition of restricted cancer cells which produce cancer cell proliferation suppressive materials, and uses thereof
|Jun 23, 2010
|Dec 30, 2010
|Tyco Healthcare Group Lp
|Catheterization System
|May 7, 2010
|Jul 14, 2011
|Waisman David M
|Compositions and methods for inhibiting tumor growth and metastasis
|Nov 12, 2004
|Apr 10, 2013
|Excipients in drug delivery vehicles
|Nov 17, 1999
|Jun 2, 2000
|Bristol-Myers Squibb Company
|Compositions comprising angiostatin and kringle 5 region of plasminogen and methods of use thereof
|Nov 12, 2004
|Jun 2, 2005
|Alza Corporation
|Excipients in drug delivery vehicles
|U.S. Classification
|435/68.1, 435/212, 435/183, 435/219, 435/69.6
|International Classification
|Cooperative Classification
|European Classification
|Dec 12, 1996
Owner name: NORTHWESTERN UNIVERSITY, ILLINOIS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SOFF, GERALD;GATELY, STEPHEN T.;TWARDOWSKI, PRZEMYSLAW;REEL/FRAME:008302/0580
Effective date: 19961122
|Feb 20, 2002
|Fee payment
Year of fee payment: 4
|Mar 22, 2006
|Maintenance fee reminder mailed
|Jun 5, 2006
|Fee payment
Year of fee payment: 8
|Jun 5, 2006
|Surcharge for late payment
Year of fee payment: 7
|Apr 5, 2010
|Maintenance fee reminder mailed
|Sep 1, 2010
|Lapse for failure to pay maintenance fees
|Oct 19, 2010
|Expired due to failure to pay maintenance fee
Effective date: 20100901


text:
Fri, 21 July 2017
Jen, Kelly and Maria share their stories from Financial Gym clients.
Visit the https://financialgym.com for a free intro or what we call a "Warm-up Session"
Direct download: MAYM169_GC_Happy_Hour.mp3
Category:investing -- posted at: 2:00am EDT
Fri, 14 July 2017
Mariela Dabbah, founder and CEO of the Red Shoe Movement, talks about changes in the way businesses support upward mobility for women and how women can climb the corporate ladder more effectively. Her cause and mission are near and dear to my heart and I hope you're as inspired to make change as I am after listening to her.
Direct download: MAYM168_Mariela_Dabbah.mp3
Category:investing -- posted at: 2:00am EDT
Fri, 7 July 2017
We all are susceptible to the influences of the jealousy game whether we like them or not. What's important is to not avoid jealousy entirely but to manage it.
Despite the fact that many people may think the four of us have nothing to worry about, we all struggle with comparing ourselves to other people and in this episode we share the ups and downs of these emotions.
For more information and resources mentioned in this episode, visit http://financially-blonde.com/the-comparison-game-happy-hour-style
Direct download: MAYM167_REPLAY_Comparision_Game_Happy_Hour.mp3
Category:investing -- posted at: 2:00am EDT
Fri, 30 June 2017
Today's Happy Hour episode is our mid-year catch-up to let you know where we and the Happy Hour girls are with our goals.
Notes for this show as well as links to everyone's sites can be found on my blog, http://financially-blonde.com. Also, if you have any topics you would like for us to talk about during happy hour, please feel free to email me to email@example.com or tweet to me @Blonde_Finance or join the private Martinis and Your Money Facebook group and let us know.
Come with us to Lola Retreat August 18-20, 2017. Use the coupon code MAYM to get a discount on Melanie's big goal for this year, the Lola Retreat, at http://LolaRetreat.com
Direct download: MAYM166_Happy_Hour.mp3
Category:happy hour -- posted at: 2:00am EDT
Fri, 23 June 2017
What if our banking systems collapse or there is a successful attack on electronic data? Or money is really only numbers on a computer screen that we trust banks to maintain. What if that's all wiped out? It's an unlikely scenario, but I like to plan for all possibilities.
Direct download: MAYM165_Zombie_Apocolypse_REPLAY.mp3
Category:investing -- posted at: 2:00am EDT
Fri, 16 June 2017
Kate Gremillion, founder of Mavenly and Co, provides young women with resources to design a career and lifestyle with purpose about going from being an Employee to being an Intrapreneur. Kate is going to share her best practices and tips for how you can craft the right job for you without having to create it on your own.
Direct download: MAYM164_Kate_Gremillion.mp3
Category:investing -- posted at: 2:00am EDT
Fri, 9 June 2017
Harlan Landes, blogger, podcaster and founder of both the Plutus Awards and the Plutus Foundation, answers a listener's question about how you can run a charitable foundation. Harlan has great information for a number of ways you can give back before and after you achieve your personal financial goals and now here's Harlan.
Direct download: MAYM163_Harlan_Landes.mp3
Category:investing -- posted at: 2:00am EDT
Fri, 2 June 2017
Elle Martinez, host of the Couple Money Podcast has authored a new book – Jump Start Your Marriage and Your Money. It's about the various challenges we've seen couples face and how they can improve them to improve both their romantic and financial lives.
Whether you are in a relationship or not, there is plenty to learn from this episode. Cheers!
Notes for this show as well as links to everyone's sites can be found on my blog, Financially-blonde.com. Also, if you have any topics you would like for us to talk about during happy hour, please feel free to email me to email@example.com or tweet to me blonde_finance or join the private Facebook group and let us know. Until next time, take care!!
Direct download: MAYM162_Elle_Martinez.mp3
Category:Relationships -- posted at: 2:00am EDT
Fri, 26 May 2017
This month's Happy Hour topic comes from Tonya on the Happy Hour crew. She wanted to talk about the excuses we make as to why we aren't making financial changes in our lives.
Direct download: MAYM161_Happy_Hour_May_2017.mp3
Category:spending -- posted at: 2:00am EDT
Fri, 19 May 2017
Natalie Jean Baptiste is an attorney who found herself in the uncomfortable position of having to declare bankruptcy due to over $200,000 of consumer and student loan debt. Natalie shares her reasons for declaring bankruptcy, why it makes sense for some people and how the whole experience prepared her for this next chapter in her life where she's helping others conquer their student loan debt burdens.
Direct download: MAYM160_Natalie.mp3
Category:investing -- posted at: 2:00am EDT

subset: commoncrawl


text:
Morgan Mare, Black - 1988 Only
The Morgan Mare has been made in black since the mold was first licensed by Breyer in 1975. Early production saw many variations, but we can pinpoint this pretty girl to 1988. In that year only, she was made with a left hind sock over a buff hoof. Earlier models all have dark hooves. So, even though she's "young" on the age scale of this mold, she's harder to come by.
This girl is Near Mint with a tiny rub on her right hip. She features a beautiful satin finish, bordering on semi-gloss.
Size: Stablemate
Breyer Model #5039 - 1988 Only


text:
Snow White
Written by Josephine Poole
Illustrated by Angela Barratt
when in stock
As soon as this book is back in stock we will send you an email.
Snow White by Josephine PooleMirror, mirror on the wall, Who is the fairest of us all?' the mirror answered, 'Queen, you are full fair, 'tis true, But Snow White is fairer than you.' When the wicked queen orders a huntsman to take Snow White into the forest, kill her and bring back her heart as proof, he is so taken by her beauty that he spares her. Venturing into the depths of the forest, Snow White finds a little house belonging to a family of dwarves who take her in. However, when the queen discovers that Snow White is still alive she is furious and again takes up her mission to do away with fairest maiden in the land. This is a fresh and enchanting retelling of a classic children's story with breath-taking illustrations.
About the Author
Other Formats
Book Info
32 pages
Interest Age: from 5 to 7
Josephine Poole
More books by Josephine Poole
Red Fox an imprint of Random House Children's Publishers UK
Publication date
18th November 1993
It has introduced my children to books we hadn't come across before. Real children's reviews gives a great insight into what others think toLorraine Woods
We love Lovereading4kids because it promotes reading choices, new authors and a sense of community for children of all ages!Rachel Bridgeman
We love Lovereading as my 5 year old loves to read new books before anyone else has a chance, she says it makes reading exciting!Tracey Chorley
It's really exciting to read and review new books, thrilling to see my reviews online and I love finding the final published copies on sale.Sam Harper – age 10
You give me age appropriate ideas of books I can read and buy for the children and find out what other children their age think of them too.Katie Lonsdale
LoveReading4Kids is a modern and creative way of emphasising the value and importance of books in this digital age #booksforlifeAmrit Bunet – Teen
It is fantastic, you get to read lots of books and you always find something new and amazing in them.Erica Motoc, age 7
I love finding new books to read. My mummy and me look at the new ones coming out. I have written reviews of some of them!Jessica Cobbin – age 7


text:
AEG 6030M-BR Owner's Manual
Product Reviews
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Hubert Hubert - 2006-08-10
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Patrick De Coninck - 2006-08-16
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john cheadle - 2006-08-17
Superb service... product is exactly as described. Thank you!
luii hernandez - 2006-09-07
tenk^you for mi diagram soy latino y soy tecnico en electronica
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text: Content Monitor tool allows you to monitor changes on sites in provided URL list and configure monitoring rules (triggers). You can setup the tool to monitor the changes on the auto markets' sites (monitoring prices on interesting cars), tickets' sales (monitoring for discounts), different flea markets, stocks changes. The tool will work and inform you about the changes through the notifying messages in tray. Configuring new monitoring scenario is very easy and takes less than few minutes – you just choose trigger values by selecting them in embedded browser and setup how often to check the data.


text:
Fair Grounds trainer Neil Pessin was all smiles on
the local backstretch Saturday morning following Giant Oak's adjudged
victory in Friday's Grade I Clark Handicap at Churchill Downs, and why
shouldn't he be? Pessin trains the Virginia Tarra-owned Giant Oak during
the winter months at Fair Grounds.
"We had to get put up to win it," said Pessin of Giant Oak,
who finished second by a head under the wire, "but we'll take it. A win is a
"Giant Oak will be going to Ocala
for about 45 days," said Pessin. "Then he's scheduled to be sent down here
where he will be pointed toward the (Grade II) New Orleans Handicap."
The 4-year-old son of Giant's
Causeway finished fifth in last season's New Orleans
Handicap and was third in last season's Grade III Mineshaft Handicap.
Adele Dilschneider's Apart, who won three straight stakes
including the Grade II Super Derby at Louisiana Downs and the Grade III
Ack Ack Handicap at Churchill Downs before finishing seventh in the
roughly-run Clark Handicap, will also be coming to Fair Grounds and be pointed
toward the New Orleans Handicap, trainer Al Stall Jr. indicated Saturday
morning when interviewed by Churchill's Jill Byrne on her paddock show.
New Orleans native
Stall, the Fair Grounds' leading trainer three different seasons, is in the
midst of his own career year after saddling Claiborne Farm and Adele
Dilschneider's Blame to win the Grade I Breeders' Cup Classic and
possible Horse of the Year honors.
Stall also humorously recalled his own youth in New
Orleans for Byrne during the
"Whenever I got suspended, I was happy," said Stall, who
would make use of the time by going right to Fair Grounds to watch the
races. "Finally, they figured out that what they really had to do was
making me sit on a bench in the hallway for six hours."
Keeneland's November dispersal of the horses owned...
Brad Grady's Girvin and the Fair Grounds Racing Cl...
Brittlyn Stable's popular multiple graded stakes-p...
Georgia Farm's homebred Bam Bam Bryan served notic...
By the Moon, Paulassilverlining and Finley'sluckyc...
Grade 1 Kentucky Derby winner Always Dreaming turn...
Hence will go for back-to-back graded stakes wins ...
The unthinkable has happened. Coming into thi...
Horse Racing Nation frequently publishes excellen...
Copyright © 2010 -
Horse Racing Labs, LLC


text:
Explore Hair Loss Treatment, Hair Treatments and more!
Explore related topics
Side Effects of Hair Transplant Surgery. For anyone dealing with hair loss issues, hair transplant surgery can be the answer to gaining back that self confidence. While a hair transplant surgery is a fairly easy procedure, there are minor complications or side effects that can occur so it's important to prepare yourself in advance. Here are some side effects. #hairtransplant #Haircare #Turkey #Sideeffects
ginger for hair - Ginger for hair is highly recommended to use for hair growth, dandruff and hair loss treatment in Ayurveda. Check out ginger remedies for hair problems.
Tea tree oil for hair is considered as one of the best herb's oil to treat hair problems. It removes dandruff, promotes hair growth & has many more benefits.
15 Causes of Women's Hair loss and How To Treat it
Natural Ways to Control Hair Loss Massaging your scalp and hair with proper hair oil will help to increase the blood circulation to your hair follicles, condition them and thus strengthens your hair roots. It also helps to reduce the stress and promotes relaxation. #HairLoss
ginger for hair - Ginger for hair is highly recommended to use for hair growth, dandruff and hair loss treatment in Ayurveda. Check out ginger remedies for hair problems.
Green tea application will soothe the scalp and hair to reduce psoriasis, eczema, and other scalp irritations along with clearing dandruff, moisturizing the dry or itchy scalp and finally enhance your hair growth.
Hair loss, also known as alopecia or baldness, is a popular problem nowadays. It is estimated that a considerable number of people are suffering from this syndrome, which is sign of a worsening health, and hair loss leads to the decrease of self-esteem. However, after great efforts over a long period of time, the causes of this problem were figured out and so was the idea of how to stop hair loss.
Hair Grow
TOP 10 NATURAL REMEDIES FOR EVERY HAIR PROBLEM http://fancytemplestore.com


text:
Mumby Model Car Museu Read more [...]
Mumby Model Car Museum
Place Category: Activities
Mumby Model Car Museum houses a superb collection of model vehicles and motor racing memorabilia spanning the history of motoring around the world such as Formula 1, GT Racing, Rallying, Indycar and Supercars!
It's a definite day out for the boys. The collection is housed in a bespoke barn conversion and has bright and colourful displays where around 4000 models are presented every season and the displays are changed for each new season.
All the cabinets are themed to display items related to TV & Film, American Cars, Batman, James Bond,Ferrari, The Best of British … and many many more!
When you get tired of ogling the cars you can take a pitstop at the cafe where there's a wide range of drinks and snacks, tea/coffee and cakes.
Then on to try out your racing skills on the 45m Scalextric track with its four lanes! Apart from a day trip you may want to arrange a birthday party, corporate event or a car club night! It's all part of motor mania here at 'On your Marques'.
Opening times: Every day but Thursdays and kids are free up to age 16!
- Museum
- Pit Stop Cafe
- Race Track: four-lane slot-car circuit
- Over 1200 model cars display
- Motoring art and memorabilia on display and for sale
Mumby Model Car Museum Weather Forecast
Light rain today through Sunday, with temperatures rising to 22°C on Sunday.Light rain throughout the day and breezy starting in the afternoon, continuing until evening.Today's Forecast
14->1652% 69% 15 mph
5:04 am 9:08 pmMostly cloudy until evening.Tuesday, Jul 25th
14->193% 78% 10 mph
5:05 am 9:07 pmLight rain and breezy starting in the afternoon, continuing until evening.Wednesday, Jul 26th
14->2054% 68% 11 mph
5:07 am 9:05 pmDrizzle starting in the afternoon.Thursday, Jul 27th
12->1918% 45% 16 mph
5:08 am 9:04 pmBreezy starting in the afternoon, continuing until evening, and mostly cloudy starting in the evening.Friday, Jul 28th
11->210% 22% 18 mph
5:10 am 9:02 pmLight rain in the morning and evening and breezy in the morning.Saturday, Jul 29th
13->2119% 76% 17 mph
5:12 am 9:01 pmDrizzle in the evening.Sunday, Jul 30th
14->2220% 91% 12 mph
5:13 am 8:59 pmMostly cloudy starting in the evening.Monday, Jul 31st
13->213% 27% 14 mph
5:15 am 8:57 pm
Photos from around the surrounding area
Leave a Review


text:
Aberdeen is a neighbourhood in Kamloops with 4072 properties and 28 condo, townhouse, and apartment buildings on record at Snap Up Real Estate. Upper Sahali , Mount Dufferin , Knutsford and Southgate are nearby neighbourhoods.
The average (median) house price in Aberdeen is $445,900.00, the median condo price is $279,900.00, and the average rent is $1,125.00 per month. There are 62 houses and 5 condos for sale on Snap Up Real Estate while there are 2 rentals of all types combined.


text:
'The Vampire Diaries' Trailer for Season 6: Elena Is Still Grieving
The CW has finally released a trailer for "The Vampire Diaries" season 6 with actual footage. It teases the aftermath of the events in the season 5 finale which ended with Bonnie and Damon waiting for the oblivion to suck them.
While their fate remains unknown, it appears that they won't reunite with Elena and others in Mystic Falls soon. Everybody is still mourning the loss of their loved ones, but Elena insists that she needs to see Damon one last time.
Stefan tells her that it's time "to say goodbye" to Damon. In another scene, she lashes out and Caroline thinks Elena is hallucinating about Damon. At the end of the snippet, Damon does appear, telling teary-eyed Elena to "move on."
"The Vampire Diaries" season 6 premieres Thursday, October 2 at 8 P.M. on The CW. At the Comic-Con, Nina Dobrev said Elena's "definitely not in a happy place [with Damon gone]. Although she has an interesting and strange way of dealing with her grief."
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- 'Ant-Man and the Wasp': Evangeline Lilly Reacts to Michelle Pfeiffer's Casting as Her On-Screen Mom
- Celebrity Police Unit to Be Discontinued Following Big Bang's T.O.P's Marijuana Scandal
- Cara Delevingne Says She Wants to Be James Bond Amid Bond Girl Rumors
- Johnny Depp Demands Claim He Has 'Psychological Issues' Removed From Court Record
- Cheeky! Emily Ratajkowski Flashes Ample Cleavage in Polka Dot Outfit
- Stephen Colbert Mocks Anthony Scaramucci Hiring as New White House Press Secretary


text:
Division Of Actual Estate Contracts And Types
Our packages supply every thing you require to be sell your property and attain a great outcome. This may well be described as a home that is far more costly, gives much more elements to industry and generates much more intrigue — characteristics may well include a swimming pool, tennis court, grand public rooms, a ballroom or in depth grounds situated on an expansive house, which are all elements of a luxury house that give the photographer far more to photograph and an agent more to marketplace. By law, only an attorney can provide you legal guidance – not a genuine estate agent, loan officer, or closing agent.
Be cautious if you acquired the property in a tax-deferred Section 1031 like-kind exchange – where you swapped an additional home for the 1 you're considering about promoting. REIWA President Hayden Groves explains the risks related with agent referral web sites, and provides handy guidelines for picking the very best agent for your requirements. Not all licensed genuine estate sellers are members of NAR, but that organization has, nationally, 12.7 percent a lot more members nowadays than it had a year ago, a total of 1.2 million. Deciding on an Agent: The right agent will know the nearby real estate market place effectively and will have a established track record of good results.
It takes time to develop relationships, produce context, show houses, and go even though the approach of a transaction. My prime tip for real estate agents and those in the industry is to develop their email list. As the economy expands and a lot more individuals look to purchase homes, job competitors might enhance as more folks obtain their real estate license. Below this arrangement, the broker pays a charge to be affiliated with a broadly recognized genuine estate organization. The Provide/Counteroffer method might go back and forth till each the purchaser and seller are happy.
No wonder there are now some 2 million licensed genuine estate agents in the United States, according to estimates from the National Association of Realtors, and more signing up every day. Afterwards, purchasers will have a likelihood to negotiate with the sellers to cover the fees of specific repairs or to ask for concessions. An improving job industry and increasing customer spending also will drive demand for brokers and agents to manage commercial, retail, and industrial genuine estate transactions. The profitable bidder must sign the sale contract and pay you a deposit on the spot (usually 10%).
At closing, all the paperwork is signed by the buyer and seller, each parties pay any settlement fees and the documents are officially recorded. While you might have a page on the site of your broker, a individual seeking to do organization with you may well finish up with another agent right after visiting your broker's website. Staging costs can variety from a couple hundred dollars for a fundamental consultation to many thousand dollars, particularly if you rent contemporary, neutral furniture for displaying your property. If the commission on your home is decrease, fewer agents will show it. This does not imply you cannot negotiate a slightly reduce commission if a single agent ends up both listing and selling the home. A brief sale is a a lot more advantageous option to foreclosure and has grow to be commonplace in the United States because the 2007 actual estate recession.
Numerous of the processes described above apply in any genuine estate transaction, such as the sale or acquire of vacant land and holiday properties. With nearly four million house listings, is your location for genuine estate MLS listings. With a 1031 exchange, you defer paying the tax on a obtain from promoting one property by exchanging it for yet another home. About our credentialed tax experts: Reside tax tips service is accessible through phone for your toughest tax concerns fees may possibly apply. Try to put your self in the buyer's footwear and then decide what a fair price may be Have close friends, neighbors and genuine estate professionals tour the residence and weigh in as properly.
Despite the higher stakes, numerous buyers and sellers give little thought to selecting an agent, no matter whether they are purchasing or selling. To employ this approach is tantamount to quitting just ahead of the finish line and wastes a worthwhile opportunity for an agent to acquire added listings and attract possible purchasers and agents to the house. The agent will then present all delivers to the seller who will weigh up which, if any, they want to accept. About VREB – Founded in 1921, the Victoria Real Estate Board is a important player in the development of requirements and innovative programs to boost the professionalism of REALTORS®. Established in 1969, Frank Salt Actual Estate has become one particular of Malta's most recognisable and trusted genuine estate companies, supplying a 1-quit-shop solution to all your home requirements. Actual estate brokers and sales agents aid consumers purchase, sell, and rent properties.
No wonder there are now some 2 million licensed true estate agents in the United States, according to estimates from the National Association of Realtors, and far more signing up every single day. Afterwards, buyers will have a likelihood to negotiate with the sellers to cover the charges of certain repairs or to ask for concessions. An improving job marketplace and rising consumer spending also will drive demand for brokers and agents to deal with commercial, retail, and industrial real estate transactions. The successful bidder have to sign the sale contract and spend you a deposit on the spot (generally 10%).
Please take a minute and browse my regional neighborhood and college specifics, true estate mortgage info and interactive maps for the Central Texas marketplace. We clarify the procedure, offer suggestions on agent selection, how to price, and presenting your residence for sale. Auctions have become an increasingly popular way to sell or acquire residential property, but prior to you determine to go down that path, do your homework and familiarise your self with the process and what it includes. Ginger just did a webinar yesterday for true estate pros about advertising and marketing to Millennials with wonderful info and tips.
Some neighborhood colleges, colleges, and universities offer you courses in actual estate. Men and women are watching the quality of perform an agent produces, and our signature marketing is one particular of the crucial elements and the worth proposition we offer you our customers. 1st impressions are important when selling a residence, so just before the 'for sale' sign goes up, put in the time to make confident it really is in ship shape. This variety of pondering runs rampant amongst the personality types who get into real estate. In 2009 the government implemented the Creating Residence Reasonably priced Plan (MHA) two to address the genuine estate recession and the require to support homeowners deal with their real estate loans.
These courses cover a selection of topics, such as true estate fundamentals, real estate law, and mortgage financing. The most efficient strategies for actual estate agents are the ones that operate for their buyers and sellers. Promoting or Influencing Others — Convincing other people to get merchandise/goods or to otherwise alter their minds or actions. These sellers do not just want you to sell their home they want you to achieve the highest attainable sales value in the marketplace you happen to be promoting into. While increasing mortgage prices and a shortage of 1st-time purchasers may temper that outlook some, the coming year need to be another seller's market for actual estate. There will be two copies of the sale contract: one particular for you and one for the purchaser.
Interacting With Computer systems — Employing computer systems and personal computer systems (such as hardware and software program) to system, write software program, set up functions, enter information, or method data. The agent does not know the genuine estate landscape in your neighborhood Discovering a neighborhood specialist is specially critical in locations exactly where moving a block can raise or reduced the value of a house by $100,000. Genuine estate brokers and sales agents need to have at least a high school diploma or equivalent. I know it sounds ridiculous, but that's precisely what you do when you attempt to begin a company (and that's what selling properties is) and you do not have a program to follow.
Sellers' brokers and agents meet with consumers to help them choose how considerably to ask for and to convince them that the agent or broker can uncover them a certified buyer. In most areas, commissions are traditionally 5 to 7 %, split among the buying and promoting agent. Robust interpersonal expertise are vital for genuine estate brokers and sales agents, because they spend a lot of their time interacting with consumers and clients. Buyers are increasingly turning to on the internet photography as the initial step in the approach of getting house. Selling at an advertised price tag indicates that a property is marketed with an asking price tag – the amount the seller expects to be paid for it. This quantity is decided by the seller in discussion with the agent. If you uncover a property that you like, the buyers agent will then set up a showing for you.
With CENTURY 21 Genuine Estate, you will have the advantage of a professional local true estate sales associate who knows the ins and outs of residence sales in your neighborhood, plus the attain of a global brand presence. Other pre-occupancy fees ought to incorporate house insurance coverage, title insurance coverage and deed-recording fee, and possibly title insurance coverage, survey charges, credit report fees, flood insurance coverage and property owners association dues/insurance. Judgment and Choice Making — Thinking about the relative costs and rewards of prospective actions to decide on the most appropriate one particular. Sales Contract: As soon as the buyers are confident about the situation of the residence, they need to have to sign a sales contract. If you choose much more than 1 category for your listing, insertion and sophisticated listing upgrade fees apply for each and every category.
In addition, some of the larger real estate companies give formal classroom education for new agents as a way to obtain information and encounter, whilst other people supply coaching to employees studying for their true estate licensing exam. Real estate brokers and sales agents want to be capable to speedily (at times instantly) address concerns clientele or possible clients might have with a house. Normally, when you work with both of these agents, they are going to split the genuine estate commission that is due on the home. CENTURY 21® and the CENTURY 21 Logo are registered service marks owned by Century 21 Actual Estate LLC.


text: Now that you have either the MCV-1 or the MCV-1e Video input device, now is the time to install the driver. The driver comes on a mini CD with the MCV which you can use. If your computer cannot handle a mini CD then you can visit the Jack's Website and download the CD's contents.