Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to conjugates of RGD-containing peptides and endogenous carriers, particularly to RGD-containing peptides and various mobile blood components, particularly mobile endogenous proteins.
• RGD tripeptide Arg-Gly-Asp
• disintegrins small synthetic peptides that contain the tripeptide Arg-Gly-Asp (RGD) sequence or natural RGD- containing small proteins derived from snake venoms, collectively termed disintegrins, inhibit the binding of fibrinogen to the GPMb/llla receptor and abolish aggregation.
• RGD peptides are generally not suitable for in vivo use because of their short half life in the body. It would be desirable to be able to develop therapeutic agents which retain the positive properties of RGD-containing peptides for longer periods of time than is normally currently the case.
• This invention relates to novel chemically reactive derivatives of RGD-containing peptides which can react with available functionalities on mobile blood proteins to form covalent linkages, and in which the resulting covalently bound conjugates have RGD peptide activity.
• the conjugated molecules As compared with RGD peptide drugs the conjugated molecules have extended lifetimes in the bloodstream and are, therefore, capable of maintaining RGD peptide activity for extended periods of time as compared to the unconjugated parent drug, and provide such activity with reduced centrally mediated side effects.
• the invention also includes the selective labeling of mobile endogenous proteins such as serum albumin and IgG with RGD- maleimide peptides.
• the invention includes the nonspecific labeling of mobile blood proteins with NHS-RGD peptides and sulfo-NHS peptides.
• NHS is N-hydroxysuccinimide.
• Sulfo-NHS is N-hydroxysulfosuccinimide.
• the invention further includes the conjugates of the RGD peptide derivatives with mobile blood components and methods for providing RGD peptide activity in vivo comprising administering to a mammalian host the novel RGD peptide derivatives or their conjugates.
• This invention also relates to use of antibodies to locate and bind to such conjugates, for instance, to remove undesirable excesses of them from the host's blood stream.
• the invention also relates to the use of antibodies to detect levels of RGD peptides in blood.
• SEQ ID NO: 1 is an acetylated RGD peptide.
• SEQ ID NO:2 is an acetylated maleimide RGD peptide.
• SEQ ID NO:3 is an acetylated NHS RGD peptide.
• RGD Peptides are peptides comprised of amino acids that include the sequence R-G-D where R is arginine, G is glycine and D is aspartic acid.
• Reactive entities are entities capable of forming a covalent bond. Such reactive agents are coupled or bonded to an RGD peptide of interest. Reactive entities will generally be stable in an aqueous environment and will usually be carboxy, phosphoryl, or convenient acyl group, either as an ester or a mixed anhydride, or an imidate, thereby capable of forming a covalent bond with functionalities such as an amino group, a hydroxy or a thiol at the target site on mobile blood components. For the most part, the esters will involve phenolic compounds, or be thiol esters, alkyl esters, phosphate esters, or the like.
• Functionalities are groups on blood components including mobile proteins or RGD peptide derivatives to which reactive entities react to form covalent bonds. Functionalities include hydroxyl groups for bonding to ester reactive entities; thiol groups for bonding to imidates and thioester groups; amino groups for bonding to carboxy, phosphoryl or acyl groups on reactive entities and carboxyl groups for bonding to amino groups.
• Mobile blood proteins are soluble blood proteins. These blood proteins are not membrane-associated and are present in the blood for extended periods of time and are present in a minimum concentration of at least 0.1 ⁇ g/ml. Mobile blood proteins include serum albumin, transferrin, ferritin and immunoglobulins such as IgM and IgG. The half-life of mobile blood proteins is at least about 1 2 hours.
• Protective groups are chemical moieties utilized to protect RGD peptide derivatives from reacting with themselves.
• Various protective groups are disclosed in U.S. 5,493,007 which is hereby incorporated by reference.
• Such protective groups include acetyl, fluorenylmethyloxycarbonyl (FMOC), BOC, CBZ, and the like.
• Linking groups are chemical moieties that link or connect reactive entities to RGD peptides.
• Linking groups may comprise one or more alkyl groups, alkoxy group, alkenyl group, alkynyl group or amino group substituted by alkyl groups, cycloalkyl group, polycyclic group, aryl groups, polyaryl groups, substituted aryl groups, heterocyclic groups, and substituted heterocyclic groups.
• this invention relates to compositions which are derivatives of RGD peptides which can react with the available reactive functionalities on blood components via covalent linkages.
• the invention also relates to such derivatives, such combinations with blood components, and methods for their use.
• the RGD peptide derivative is of a type designated as a DAC (drug affinity complex) which comprises the RGD peptide molecule and a linking group together with a chemically reactive group capable of reaction with a reactive functionality of a mobile blood protein.
• DAC drug affinity complex
• the RGD peptide derivative or DAC may be delivered via the blood to appropriate sites or receptors of the patient such as platelet glycoprotein llb/llla or other integrin receptors such as v ⁇ 3 .
• N-hydroxysuccinimide N-hydroxy-sulfosuccinimide
• MBS maleimide-benzoyl- succinimide
• GMBS gamma-maleimido-butyryloxy succinimide ester
• MPA maleimidopropionic acid
• the functional group on this protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as (GMBA or MPA).
• GMBA stands for gamma-maleimide-butrylamide.
• the RGD peptide for conjugation to cells, erythrocytes or mobile endogenous proteins in accordance with the present invention includes a sequence of amino acids, preferably naturally occurring L-amino acids and glycine, having the following formula: R r Arg-Gly-Asp-R 2
• R, and R 2 represent an amino acid or a sequence of more than one amino acid or a derivatized or chemically modified amino acid or more than one derivatized or chemically modified amino acids.
• R. represents XY(Z) friendship, in which X, Y and Z independently represent an amino acid; and n represents 0 or 1 ; R 2 represents OH or NH 2 ; or any amino acid; or a sequence of more than one amino acid or a derivatized or chemically modified amino acid.
• R 2 represents an amino acid other than serine, threonine or cysteine or the amide thereof wherein the amino acid is rendered a carboxyamide.
• R 2 is more than one amino acid, the first amino acid in the sequence, which is attached to aspartic acid, being other than serine, threonine or cysteine, or the amide of any free carboxyl groups wherein R 2 includes a derivatized or chemically modified amino acid.
• R 2 includes a linking group having a chemically reactive group which covalently bonds to reactive functionalities or proteins and R, includes a protective group to prevent the chemically reactive group of R 2 from reacting with R
• R includes a linking group having a chemically reactive group which covalently bonds to reactive functionalities on proteins and R 2 includes a protective group to prevent the chemically reactive group of R ⁇ from reacting with R 2 .
• both R and R 2 include a linking group having a chemically reactive entity which covalently bonds to functionalities on mobile proteins.
• the linking groups may be similar or different.
• I, R, and R 2 may include any amino acid or sequence thereof.
• the amino acids are preferably naturally occurring. The most common naturally-occurring amino acids are shown in Table I:
• R. and R 2 in the RGD peptide of this invention are not limited to the 20 natural-amino acids.
• R and R 2 can be D-amino acids, non-classical amino acids or cyclic peptides or peptidomimetics (chemical peptide analogs).
• Non- classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
• Arg and/or Asp in the RGD sequence can be the D (dextrarotary) or L (levorotary) amino acid.
• R, and/or R 2 are a sequence of amino acids
• the polypeptide for conjugation to mobile blood proteins can be any size, and encompasses what might otherwise be called an oligopeptide, a protein, an organic molecule or a polymer such as polyethylene glycol.
• the polypeptide will have no more than about 1 ,000 amino acids.
• the polypeptide may be prepared by methods that are known in the art.
• solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
• the protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
• the coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Pat. No. 4,244,946.
• Such solid phase syntheses have been described, for example, by Merrifield, 1 964, J. Am. Chem. Soc.
• RGD peptides and their analogs which can conjugate with proteins and other mobile blood proteins are prepared as is known in the art by the use of linking groups having chemically reactive groups which covalently bond to reactive functionalities on proteins.
• the RGD peptides of this invention are designed to specifically react with thiol groups on mobile blood proteins.
• Such reaction is preferably established by covalent bonding of a maleimide link (e.g. prepared from GMBS, MPA or other maleimides) to a thiol group on a mobile blood protein such as albumin or IgG.
• a maleimide link e.g. prepared from GMBS, MPA or other maleimides
• maleimide RGD peptide derivatives of this invention will covalently bond to fewer proteins.
• albumin the most abundant blood protein
• RGD-maleimide- albumin conjugates will tend to comprise approximately a 1 : 1 ratio of RGD peptide derivatives to albumin.
• IgG molecules class II also have free thiols. Since IgG molecules and serum albumin make up the majority of the soluble protein in blood they also make up the majority of the free thiol groups in blood that are available (to covalently bond to) maleimide-RGD peptide derivatives.
• maleimide-RGD peptide Through controlled administration of maleimide-RGD peptide in vivo, one can control the specific labeling of albumin and IgG in vivo.
• the administered maleimide- RGD peptide will label albumin and 1 0-20% will label IgG. Trace labeling of free thiols such as glutathione will also occur. Such specific labeling is preferred for in vivo use as it permits an accurate calculation of the estimated half-life of the administered agent.
• maleimide-RGD peptides can provide specific labeling of serum albumin and IgG ex vivo.
• ex vivo labeling involves the addition of maleimide-RGD to blood, serum or saline solution containing serum albumin. Once modified ex vivo with RGD-Mal, the blood, serum or saline solution can be readministered to the blood for in vivo treatment.
• maleimide-RGD peptides are generally quite stable in the presence of aqueous solutions and in the presence of free amines. Since maleimide RGD derivatives will only react with free thiols, protective groups are generally not necessary to prevent the maleimide-RGD peptide derivative from reacting with itself. In addition, the increased stability of the peptide permits the use of further purification steps such as HPLC to prepare highly purified products suitable for in vivo use. Lastly, the increased chemical stability provides a product with a longer shelf life. Non-Specific Labeling.
• Bonds to amino groups will also be employed, particularly with the formation of amide bonds for non-specific labeling.
• the various sites with which the chemically reactive group of the subject non-specific RGD peptide derivatives may react in vivo include cells, particularly red blood cells (erythrocytes) and platelets, and proteins, such as immunoglobulins, including IgG and IgM, serum albumin, ferritin, steroid binding proteins, transferrin, thyroxin binding protein, ⁇ -2-macroglobulin, and the like.
• erythrocytes red blood cells
• platelets proteins, such as immunoglobulins, including IgG and IgM, serum albumin, ferritin, steroid binding proteins, transferrin, thyroxin binding protein, ⁇ -2-macroglobulin, and the like.
• proteins indicated above including the proteins of the cells
• reaction will be with mobile components in the blood, particularly blood proteins and cells, more particularly blood proteins and erythrocytes.
• mobile is intended that the component does not have a fixed situs for any extended period of time, generally not exceeding 5, more usually one minute, although some of the blood component may be relatively stationary for extended periods of time.
• functionalized proteins and cells there will be a relatively heterogeneous population of functionalized proteins and cells.
• the population within a few days will vary substantially from the initial population, depending upon the half-life of the functionalized proteins in the blood stream. Therefore, usually within about three days or more, IgG will become the predominant functionalized protein in the blood stream.
• IgG, serum albumin and erythrocytes will be at least about 60 mole %, usually at least about 75 mole %, of the conjugated components in blood, with IgG, IgM (to a substantially lesser extent) and serum albumin being at least about 50 mole %, usually at least about 75 mole %, more usually at least about 80 mole %, of the non-cellular conjugated components.
• the RGD peptide derivative is conjugated to albumin.
• the desired conjugates of non-specific RGD peptides to blood components may be prepared in vivo by administration of the RGD peptide derivatives to the patient, which may be a human or other mammal.
• the administration may be done in the form of a bolus or introduced slowly over time by infusion using metered flow or the like.
• the subject conjugates may also be prepared ex vivo by combining blood with derivatized RGD peptides of the present invention, allowing covalent bonding of the derivatized RGD peptides to reactive functionalities on blood components and then returning or administering the conjugated blood to the host.
• the above may also be accomplished by first purifying an individual blood component or limited number of components, such as red blood cells, immunoglobulins, serum albumin, or the like, and combining the component or components ex vivo with the chemically reactive RGD peptide derivatives.
• the functionalized blood or blood component may then be returned to the host to provide in vivo the subject therapeutically effective conjugates.
• the blood also may be treated to prevent coagulation during handling ex vivo.
• the RGD peptide derivatives will be administered in a physiologically acceptable medium, e.g. deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose, alcohol, vegetable oil, or the like.
• a physiologically acceptable medium e.g. deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose, alcohol, vegetable oil, or the like.
• Other additives which may be included include buffers, where the media are generally buffered at a pH in the range of about 5 to 1 0, where the buffer will generally range in concentration from about 50 to 250 mM, salt, where the concentration of salt will generally range from about 5 to 500 mM, physiologically acceptable stabilizers, and the like.
• the compositions may be lyophil
• the subject RGD peptide derivatives will for the most part be administered parenterally, such as intravascularly (IV), intraarterially (IA), intramuscularly (IM), subcutaneously (SC), or the like. Administration may in appropriate situations be by transfusion. In some instances, where reaction of the functional group is relatively slow, administration may be oral, nasal, rectal, transdermal or aerosol, where the nature of the conjugate allows for transfer to the vascular system. Usually a single injection will be employed although more than one injection may be used, if desired.
• the RGD peptide derivatives may be administered by any convenient means, including syringe, trocar, catheter, or the like.
• the administration will be intravascularly, where the site of introduction is not critical to this invention, preferably at a site where there is rapid blood flow, e.g., intravenously, peripheral or central vein. Other routes may find use where the administration is coupled with slow release techniques or a protective matrix.
• the intent is that the RGD peptide, analog or derivative be effectively distributed in the blood, so as to be able to react with the blood components.
• the concentration of the conjugate will vary widely, generally ranging from about 1 pg/ml to 50 mg/ml.
• the total administered intravascularly will generally be in the range of about 0.1 mg/ml to about 10 mg/ml, more usually about 1 mg/ml to about 5 mg/ml.
• RGD peptide compound By bonding to long-lived components of the blood, such as immunoglobulin, serum albumin, red blood cells and platelets, a number of advantages ensue.
• the activity of the RGD peptide compound is extended for days to weeks. Only one administration need be given during this period of time. Greater specificity can be achieved, since the active compound will be primarily bound to large molecules, where it is less likely to be taken up intracellularly to interfere with other physiological processes.
• the blood of the mammalian host may be monitored for the presence of the RGD peptide compound one or more times. By taking a portion or sample of the blood of the host, one may determine whether the RGD peptide has become bound to the long- lived blood components in sufficient amount to be therapeutically active and, thereafter, the level of RGD peptide compound in the blood. If desired, one may also determine to which of the blood components the RGD peptide or RGD peptide derivative molecule is bound. This is particularly important when using non-specific RGD peptides. For specific maleimide-RGD peptides, it is much simpler to calculate the half life of serum albumin and IgG.
• this invention relates to such conjugates of RGD peptides, RGD peptide analogs and their derivatives with blood components, particularly blood proteins such as albumin, as well as methods of administrating them to human and other mammal patients.
• Another aspect of this invention relates to methods for determining the concentration of the RGD peptides and/or analogs, or their derivatives and conjugates in biological samples (such as blood) using antibodies specific to the RGD peptides or RGD peptide analogs or their derivatives and conjugates, and to the use of such antibodies as a treatment for toxicity potentially associated with such RGD peptides, analogs, and/or their derivatives or conjugates.
• anti-therapeutic agent antibodies either monoclonal or polyclonal, having specificity for a particular RGD peptide, RGD peptide analog or derivative thereof, can assist in mediating any such problem.
• the antibody may be generated or derived from a host immunized with the particular RGD peptide, analog or derivative thereof, or with an immunogenic fragment of the agent, or a synthesized immunogen corresponding to an antigenic determinant of the agent.
• Preferred antibodies will have high specificity and affinity for native, derivatized and conjugated forms of the RGD peptide or RGD peptide analog.
• Such antibodies can also be labeled with enzymes, fluorochromes, or radiolables.
• Antibodies specific for derivatized RGD peptides may be produced by using purified RGD peptides for the induction of derivatized RGD peptide-specific antibodies. By induction of antibodies, it is intended not only the stimulation of an immune response by injection into animals, but analogous steps in the production of synthetic antibodies or other specific binding molecules such as screening of recombinant immunoglobulin libraries. Both monoclonal and polyclonal antibodies can be produced by procedures well known in the art.
• the anti-therapeutic agent antibodies may be used to treat toxicity induced by administration of the RGD peptide, analog or derivative thereof, and may be used ex vivo or in vivo. Ex vivo methods would include immuno-dialysis treatment for toxicity employing anti-therapeutic agent antibodies fixed to solid supports. In vivo methods include administration of anti-therapeutic agent antibodies in amounts effective to induce clearance of antibody- agent complexes.
• the antibodies may be used to remove the RGD peptides, analogs or derivatives thereof, and conjugates thereof, from a patient's blood ex vivo by contacting the blood with the antibodies under sterile conditions.
• the antibodies can be fixed or otherwise immobilized on a column matrix and the patient's blood can be removed from the patient and passed over the matrix.
• the RGD peptides or RGD peptide analogs, derivatives or conjugates will bind to the antibodies and the blood containing a low concentration of the RGD peptide, analog, derivative or conjugate, then may be returned to the patient's circulatory system.
• the amount of RGD peptide compound removed can be controlled by adjusting the pressure and flow rate.
• Preferential removal of the RGD peptides, analogs, derivatives and conjugates from the plasma component of a patient's blood can be effected, for example, by the use of a semipermeable membrane, or by otherwise first separating the plasma component from the cellular component by ways known in the art prior to passing the plasma component over a matrix containing the anti-therapeutic antibodies.
• the preferential removal of RGD peptide-conjugated blood cells, including red blood cells can be effected by collecting and concentrating the blood cells in the patient's blood and contacting those cells with fixed anti-therapeutic antibodies to the exclusion of the serum component of the patient's blood.
• the anti-therapeutic antibodies can be administered in vivo, parenterally, to a patient that has received the RGD peptide, analogs, derivatives or conjugates for treatment.
• the antibodies will bind the RGD peptide compounds and conjugates. Once bound the RGD peptide activity will be hindered if not completely blocked thereby reducing the biologically effective concentration of RGD peptide compound in the patient's bloodstream and minimizing harmful side effects.
• the bound antibody-RGD peptide complex will facilitate clearance of the RGD peptide compounds and conjugates from the patient's blood stream.
• RGD peptides and their analogs may be used in several different ways and to achieve several different ends. As mentioned above, these materials may be used in place of typical RGD peptide drugs as an anti-platelet or anti- adhesive agent. As compared with RGD peptide drugs currently available, the materials of this invention can reduce clot formation with less side effects and are available for reducing clot formation for a substantially longer time than conventionally administered RGD peptide drugs. In addition, the derivatized RGD peptides of this invention may be utilized (in accordance with U.S.
• Such therapies include the use of aspirin, hirudin, argatroban and argatroban derivatives, streptokinase, tissue plasminogen activator and the like.
• Analytical HPLC were performed using a Varian (Rainin) binary HPLC system: gradient elution of 5-60% B (0.045% TFA in H 2 O (A) and 0.045% TFA in CH 3 CN (B)) at 0.5 mL/min using a Dynamax C 18 , 60A, 8 ⁇ m, 4.6 mm x 25 cm column equipped with a Dynamax C 18 , 60A, 8 ⁇ m guard module and an UV detector (Varian Dynamax UVD II) detecting at ⁇ 21 4 and 254 nm. Mass spectrometry was performed on a PE Sciex API III electro-spray Biomolecular Mass Analyzer.
• Fmoc protected amino acids 4 eq. of a 0.45 M O-benzotriazol-1 -yl- N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and 1 - hydroxybenzotriazole (HOBt) in N,N-dimethylformamide solution and activation with 4 eq. of 2 M N,N,-diisopropylethylamine (DIEA) in 1 - methyl-2-pyrrolidinone (NMP), and piperidine deprotection of Fmoc groups.
• DIEA N,N,-diisopropylethylamine
• NMP 1 - methyl-2-pyrrolidinone
• the peptide was removed from the resin by shaking 609 mg of Ac-RIARGDFPDDRK-MBHA-Resin with two-5 mL portions of a cleavage cocktail (comprised of: 10 mL of trifluoroacetic acid (TFA);
• reaction was quenched by the addition of 1 36 ⁇ L of TFA (1 .68 mmol), vortexed for 30 sec and product oiled out by the addition of 30 mL of dry-ice cold Et 2 O.
• the Et 2 O was removed and oil washed with 30 mL more of dry-ice cold Et 2 O, then dissolved in 2 mL of 0.045% TFA in CH 3 CN and 8 mL 0.045% TFA in water, purified by preparative HPLC and desired fractions collected and lyophilized to afford 83.1 mg of product as a pale yellow solid (0.042 mmol, 50%).
• Example 2 in 1 00 ⁇ L of DMF and following addition the reaction was allowed to stand at RT for 1 .5 h. To this was added 9.88 ⁇ L of TFA (0.1 22 mmol), vortexed and product precipitated out by the addition of 1 5-mL of dry-ice cold Et 2 O. The precipitate was collected by centrifugation and solid taken up in 1 mL of 0.045% TFA in
• RGD peptides were anchored to human plasma proteins by the following procedures.
• Triflavin-like peptide acetylated RIARGDFPDDRK (Example 2)
• Example 4 For each peptide, a 40 mM stock-solution was prepared in DMSO.
• PPP Platelet Poor Plasma
• Anchoring to Human Plasma Proteins Venous blood was withdrawn from normal donor and placed in citrated tubes. Blood samples were centrifuged for 1 0 minutes at 2500 g in order to obtain platelet-poor-plasma (PPP).
• PPP was mixed with RGD peptides at 1 00 ⁇ M final concentration. After 1 hour-incubation at room temperature with gentle stirring, plasma samples were extensively dialyzed against
• Venous blood was withdrawn on 0.1 29 M sodium citrate from normal donors who had not ingested any drugs (i.e. aspirin) for at least 1 0 days prior to blood donation.
• Blood was centrifuged at 250 g for 10 min. and the PRP (supernatant) was removed with plastic pipette.
• Platelet-poor-plasma (PPP) was obtained by spinning the red cell sediment at 2500 g for 1 0 min. Platelet concentration of PRP was determined using Coulter T40 and adjusted to 280,000 to
• the IC50 calculations indicate a loss of potency about of 3.8 to 4.8 times after derivatization and conjugation to plasma proteins. However, the derivatized RGD peptide remains active when covalently attached to proteins.
• the peptides are the same as those described in example 6.
• Peptides were solubilized at 40 mM in DMSO and incubated with 300 ⁇ l RSA (rabbit serum albumin, Sigma A9638) at 100 ⁇ M (6.6 mg/ml) in PBS at peptide/RSA ratio of 5/1 (final concentration of peptide is 500 ⁇ M). After a 30-minute incubation at room temperature, conjugates were filtrated on PD1 0 column (5 ml desalting column, Pharmacia) equilibrated in PBS, and recovered in a 2 ml fraction. Conjugates were concentrated by ultrafiltration on Microsep (cut-off 10kDa) and adjusted to a concentration of 5 mg/ml as RSA after protein dosage (BCA, Pierce).
• Platelet-rich plasma was isolated as described in Example 6.
• lysis buffer 10 mM Tris-HCI, 1 50 mM NaCI, 1 % Triton X-1 00, 1 mM CaCI2, 10 ⁇ 5 M Leupeptin, pH 7.4, cold
• lysis buffer 10 mM Tris-HCI, 1 50 mM NaCI, 1 % Triton X-1 00, 1 mM CaCI2, 10 ⁇ 5 M Leupeptin, pH 7.4, cold
• Glycoprotein llb-llla complexes were purified by three successive affinity chromatographies.
• the first purification step was performed on Concanavalin A-Sepharose 4B (Sigma, C-901 7, lot 1 25H1071 ) which retains glycoprotein (gp) lib, gpllla, thrombospondin and fibrinogen. Platelet lysates were thawed by immersion into a 37 °C bath and centrifuged 30 min at 30,000 g at 4°C to remove insoluble material.
• the second purification step consisted in removing thrombospondin component by chromatography on Heparin Agarose (Sigma, H-6508, lot 25H9508).
• the column was equilibrated with buffer A and gp llb-llla containing fractions from the Con A column were applied at a flow rate of 1 ml/min. Flow through fractions containing gpllb-llla and fibrinogen were stored at + 4°C. Thrombospondin and degradation products binding to heparin- agarose were eluted with 0.5 M NaCI and column was used again for a new run.
• Wells of a flat-bottom 96-well plate (Maxisorp, Nunc) were coated with 50 ⁇ l of purified llb-llla (2 ⁇ g/ml in 20 mM Tris-HCI, 1 50 mM NaCI, 1 mM CaCI 2 , 0.02% NaN 3 pH7.4) overnight at 4 C. Plate was flicked free of solution and saturated with 3% BSA in buffer described above, for 3 hours at room temperature (RT). After three washes with TBS, the plate was incubated with 50 ⁇ l/well of biotinylated fibrinogen diluted in TBS-1 mM CaCI 2 for 1 .5 hours at RT.

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