WO1999055314A1 - Gouttelettes, enrobees de fibrinogene, de phases hydrophobes liquides - Google Patents

Gouttelettes, enrobees de fibrinogene, de phases hydrophobes liquides Download PDF

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Publication number
WO1999055314A1
WO1999055314A1 PCT/US1999/009940 US9909940W WO9955314A1 WO 1999055314 A1 WO1999055314 A1 WO 1999055314A1 US 9909940 W US9909940 W US 9909940W WO 9955314 A1 WO9955314 A1 WO 9955314A1
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fibrinogen
oil
coated
droplets
hydrophobic structure
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PCT/US1999/009940
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English (en)
Inventor
Gregory S. Retzinger
Ashley P. Deanglis
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University Of Cincinnati
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Priority to CA002327469A priority Critical patent/CA2327469A1/fr
Priority to EP99921733A priority patent/EP1073425A1/fr
Publication of WO1999055314A1 publication Critical patent/WO1999055314A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin

Definitions

  • the present invention relates generally to methods for treating or assessing diseases associated with fibrin clots, other f ⁇ brin(ogen)-coated surfaces and or f ⁇ brin(ogen)-associated processes.
  • the present invention is a new method for treating a mammal with fibrinogen bound to microscopic hydrophobic (oil) droplets which can then mediate adhesion of these droplets to other fibrin-coated surfaces or cells. This adhesion can be prevented by certain polyanions.
  • the present methods can be used for the formulation of vehicles for the targeted delivery of drugs and can also be used as an adjuvant for vaccines.
  • Microparticles and foreign bodies present in the blood are generally cleared from the circulation by the 'blood filtering organs', namely the spleen, lungs and liver.
  • the particulate matter contained in normal whole blood comprises red blood cells (typically 8 microns in diameter), white blood cells (typically 6-8 microns in diameter), and platelets (typically 1-3 microns in diameter).
  • red blood cells typically 8 microns in diameter
  • white blood cells typically 6-8 microns in diameter
  • platelets typically 1-3 microns in diameter.
  • the microcirculation in most organs and tissues allows the free passage of these blood cells.
  • microthrombii blood clots
  • a suspension of particles less than 7-8 microns is however, relatively safe and has been used for the delivery of pharmacologically active agents in the form of liposomes and emulsions, nutritional agents, and contrast media for imaging applications.
  • Liposomes are known for the delivery of pharmaceutical actives to various sites in the human body.
  • U.S. Pat. No. 4,394,448, Szoka, Jr., et al, issued Jul. 19, 1983 describes lipid vesicles which encapsulate DNA or DNA fragments and are used to insert those DNA materials into living cells.
  • U.S. Pat. No. 5,658,588, Retzinger and DeAnglis issued Aug. 19, 1997, describes a method for preparing fibrinogen-coated liposomes. In this process, fibrinogen and an acylating agent are reacted in the presence of a dispersion of liposomes under specifically defined reaction conditions. The liposomes formed are used to enhance blood clotting at wound sites and to deliver pharmaceutical agents and/or other chemicals to specific sites in vivo or in vitro.
  • These targeting agents are thiol-containing proteins and residues of thiol- containing compounds which include a polyalkylene glycol moiety bound to a maleimide residue on the surface of the liposome.
  • aqueous solubility of many biologies presents a problem for human administration. Indeed, the delivery of pharmacologically active agents that are inherently insoluble or poorly soluble in aqueous medium can be seriously impaired if oral delivery is not effective. Accordingly, currently used formulations for the delivery of pharmacologically active agents that are inherently insoluble or poorly soluble in aqueous medium require the addition of agents to solubilize the pharmacologically active agent. Frequently, however, severe allergic reactions are caused by the agents (e.g., emulsifiers) employed to solubilize pharmacologically active agents. Thus, a common regimen of administration involves treatment of the patient with antihistamines and steroids prior to injection of the pharmacologically active agent to reduce the allergic side effects of the agents used to aid in drug delivery.
  • agents e.g., emulsifiers
  • compositions that are water-insoluble or poorly water- soluble and sensitive to acid environments in the stomach cannot be conventionally administered (e.g., by intravenous injection or oral administration).
  • parenteral administration of such pharmaceuticals has been achieved by emulsification of oil-solubilized drug with an aqueous liquid (such as normal saline) in the presence of surfactants or emulsion stabilizers to produce stable microemulsions.
  • aqueous liquid such as normal saline
  • surfactants or emulsion stabilizers to produce stable microemulsions.
  • emulsions may be injected intravenously, provided the components of the emulsion are pharmacologically inert.
  • Additional biologies which are frequently inherently insoluble or poorly soluble in aqueous medium, and which are desirable to administer dissolved in an innocuous carrier such as normal saline, while promoting a minimum of undesired side-reactions and/or allergic reactions, are diagnostic agents such as contrast agents.
  • Contrast agents are desirable in radiological imaging because they enhance the visualization of organs (i.e., their location, size and conformation) and other cellular structures from the surrounding medium.
  • the soft tissues for example, have similar cell composition (i.e., they are primarily composed of water) even though they may have remarkably different biological functions (e.g., liver and pancreas).
  • the present invention relates to a method for effectively incorporating fibrinogen onto the surface of a hydrophobic structure (e.g., an oil droplet). It further encompasses the fibrinogen-coated hydrophobic structures, themselves, and pharmaceutical compositions containing those structures. Those compositions can be used as platelet-like biochemical hemostats, drug delivery systems which target sites - 4 -
  • reagents for the imaging of clot-containing lesions reagents for clinical, clot-based coagulation assays, a bioadhesive reagent to introduce molecules into cells or facilitate adhesion between chemical reactants, an adhesive reagent for facilitating chemical reactions between environmentally-incompatible reactants, a vaccine component or an adjuvant for vaccines, a reagent for modifying the lipid composition of biological membranes, and a reagent for transfection of genes into target cells.
  • Fibrinogen adsorbs spontaneously from aqueous media containing that protein to droplets of liquid hydrophobic phases dispersed in those same media.
  • liquid hydrophobic phases include mineral oils, straight chain hydrocarbons, and various plant- and animal-derived oils.
  • oil droplets coated with fibrinogen can participate in a host of biologically important adhesive processes in which the protein would be expected to participate.
  • Fig. 1 Fibrinogen stabilizes emulsified droplets of liquid hydrophobic phases. Left, Mineral oil (Drakeol 32) floats as a discrete phase on aqueous buffer; right, fibrinogen stabilizes microscopic droplets of mineral oil dispersed in an aqueous phase.
  • Fig. 2. Fibrinogen binds to microscopic droplets of various liquid hydrophobic phases. Oil droplets were dispersed in PBS containing 1.65 x 10 "6 mol/L fibrinogen. See text for details.
  • lecithin When included in the oil phase, lecithin (•) reduces the binding of fibrinogen to droplets of otherwise virgin olive oil; cholesteryl oleate (O) does not.
  • the straight line regions of the data that relate to lecithin represent an empiric fit of the data to a titration curve.
  • the arrow indicates the lecithin content at the intersection of the extrapolated straight line fits. All data points represent the mean ⁇ SEM of duplicate determinations. - 5 -
  • Fig. 4 Macroscopic assessment of fibrinogen-coated droplets of mineral oil (Drakeol 32).
  • A In the absence of thrombin, fibrinogen-coated droplets are monodisperse and form a confluent layer.
  • B With the addition of thrombin, fibrinogen-coated droplets aggregate.
  • C Hirudin inhibits the aggregation of fibrinogen-coated droplets that otherwise occurs in the presence of thrombin.
  • Fig. 5 Microscopic assessment of fibrinogen-coated droplets of mineral oil (Drakeol 32). The droplets are of diameter ⁇ 3.5 ⁇ 1.8 ⁇ m.
  • A Fibrinogen- coated droplets in the absence of thrombin;
  • B fibrinogen-coated droplets in the presence of thrombin.
  • Fig. 6. Aggregometry tracing of fibrinogen-coated droplets of mineral oil (Drakeol 32).
  • A In the presence of thrombin, the apparent absorbance of a stirred dispersion of fibrinogen-coated oil droplets decreases with time.
  • B Hirudin inhibits the aggregation of fibrinogen-coated droplets that otherwise occurs after the addition of thrombin. The arrow indicates the addition of thrombin. See reference 1 and text for details.
  • Fig. 8 Ristocetin dimers flocculate fibrinogen-coated droplets of olive oil. See text for details.
  • Fig. 9 Aggregometry tracing of fibrinogen-coated droplets of olive oil before, A, and after, B, incubating them for 24 h in heat-treated, fibrinogen- supplemented (2.9 x 10 "6 mol/L), citrated normal plasma. The arrow indicates the addition of thrombin.
  • Fig. 10 Macroscopic assessment of fibrinogen-coated droplets of olive oil.
  • Droplets of olive oil coated from an aqueous solution containing fibrinogen alone as protein; B, droplets of olive oil coated with fibrinogen alone and then exposed to thrombin; C, droplets of olive oil coated with fibrinogen alone and then exposed simultaneously to hirudin and thrombin; D, olive oil droplets isolated and washed after exposure to citrated plasma (fibrinogen 7.1 x 10 " mol/L); E, same as D but after addition of thrombin; F, same as D but after addition simultaneously of hirudin and thrombin; G, droplets of olive oil isolated and washed after exposure to normal serum; H, same as G but after the addition of thrombin.
  • Fig. 11 Aggregometry tracing of droplets of olive oil isolated and washed after exposure to either citrated plasma or serum.
  • C same as B but hirudin was added prior to the addition of thrombin;
  • D serum.
  • the arrow indicates the addition of thrombin.
  • Fibrinogen is a well known, naturally occurring material. It is described in Hantgan, et al, Fibrinogen Structure and Physiology, in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd Edition, edited by Robert W. Colman, et al, J. B. Lippincott Company, Philadelphia, 1994, incorporated herein by reference.
  • the fibrinogen molecule is a dimeric molecule consisting of three pairs of disulfide- bonded polypeptide chains, designated A alpha , B beta and gamma .
  • fibrinopeptides A and B which constitute about 2% of the total protein content, are released from A alpha and B beta chains when thrombin acts on fibrinogen. Molecules devoid of fibrinopeptides A and/or B are referred to as fibrin monomers.
  • the B beta and gamma chains contain carbohydrate, each of molecular weight 2,500, attached covalently at residues 364 and 52, respectively.
  • the computed molecular weight of fibrinogen is approximately 340,000.
  • the overall length of fibrinogen is 475 A (“Angstrom"), and the molecule is composed of two roughly spherical nodules 65 A in diameter connected by thin threads which are 8 A to 15 A in diameter to a central nodule about 50 A in diameter.
  • soluble fibrinogen into an insoluble polymer to form a blood clot at a wound site takes place in essentially three steps: (1) cleavage of fibrinopeptides by thrombin resulting in the formation of fibrin monomer; (2) a three step non-covalent assembly process wherein the fibrin fragments are oriented in a specific manner; and (3) covalent stabilization of fibrin by Factor XHIa-catalyzed crosslinking.
  • Preferred blood fractions for producing the compositions of the invention are plasma, cryoprecipitate, and/or Factor Vlll-depleted cold-precipitate. Because the preferred blood fraction for use as a starting material is human plasma, the starting material will hereafter be referred to as plasma, although it will be understood by those of skill in the art that the compositions of the invention can be produced by starting with any human blood-derived fraction which has not been significantly depleted of fibrinogen. Generally, the process involves the formation from plasma of a cryoprecipitate which is high in Factor XIII (F XIII) and fibrinogen. This step may be followed by cold-precipitation of proteins from the cryoprecipitate.
  • F XIII Factor XIII
  • the preferred method for producing the compositions of the present invention uses frozen human plasma from one or more donors as a starting material.
  • the plasma to be used will have been screened using conventional assay techniques for the presence of infectious viral contaminants, such - 8 -
  • hepatitis B hepatitis B and human immunodeficiency virus (HIV) to eliminate plasma for use as a starting material which contains detectable levels of such contaminants.
  • HIV human immunodeficiency virus
  • cryoprecipitate or cold-precipitate product of the plasma to be used in the compositions of the invention will be treated as further described below to reduce the viral activity therein to undetectable levels.
  • undetectable levels will refer to levels of viral activity which can be detected by viral assay protocols which are well known to those of ordinary skill in the art, such as detection of plaque forming units in infected tissue or cells.
  • a reduction in the known viral activity level in a given composition will be conventionally described herein as the "log 10 reduction factor".
  • compositions of the inventions will have a log 10 reduction factor for lipid enveloped viruses of at least 4 logs (preferably at least 6 logs) and a lesser reduction factor for other viral pathogens.
  • the plasma is thawed in a controlled environment.
  • the cryoprecipitate will preferably be further processed to a cold-precipitate.
  • Fibrinogen is separated from the plasma by precipitation to produce a fibrinogen -containing precipitate and a thrombin-containing supernatant.
  • Precipitation may be achieved in any conventional manner. Cryoprecipitation is preferred and may be carried out as follows:
  • the concentrated fibrinogen -containing cryoprecipitate may be prepared well in advance of its intended use, i.e., pre-formed, and stored as long as two months or more at about - 80°C. before use.
  • the buffer solution with which the cryoprecipitate is treated typically has a pH of about 6.0 to about 8.0.
  • the cold-soluble plasma protein is generally separated by centrifugation on maintaining a temperature of about 0° to about 4° C. at from about 4000 g to about 5000 g for a short time, typically about 5 min to about 10 min.
  • the supernatant is then decanted, leaving the precipitate which contains most of the fibrinogen.
  • the purified precipitate is then washed with the buffer solution.
  • the fibrinogen for use in the present invention may be in an intimate admixture with other proteins that are typically found uncoagulated whole blood, in platelet-rich plasma, in plasma, in cryoprecipitate, or in precipitates of plasma obtained by a method such as Cohn precipitation of plasma.
  • additional protein components may include fibronectin, immunoglobulin, particularly IgG, factor XIII, plasminogen, and albumin.
  • the fibrinogen preparations used in the present invention can be virally inactivated by one or more well known methods prior to their employment in the invention.
  • fibrinogen made by recombinant techniques could also be employed in the present invention. It is expected that future developments will lead to the ability to produce usable amounts of fibrinogen by such techniques in other types of cells.
  • Normal or mutant recombinant fibrinogens may be employed in the compositions formulated with the types of oil droplets as described herein. Where cold-precipitation is used, the addition of a calcium ion source during the process will enhance the precipitation of fibrinogen, as well as fibronectin, thereby increasing the concentration of these substances in the cold-precipitate.
  • the fibrinogen can also be further concentrated by the addition of polyethylene glycol (PEG) to the cold-precipitate.
  • PEG polyethylene glycol
  • the resulting composition can be considered to be essentially free of prothrombin complex.
  • the calcium phosphate is removed from the process by centrifugation and/or filtration. Additional techniques for removal of prothrombin are well known in the art. Thus, by removing prothrombin and inhibiting thrombin in the compositions of the invention, the final compositions will be essentially free of fibrin molecules.
  • the dissolved cryo- or cold-precipitate is warmed from about 23°to about 27°C. and contacted with a lysine affinity column, such as the matrix column product sold commercially under the tradename lysine-Sepharose 4B. Residual plasminogen present in the cryo- or cold-precipitate will be adsorbed by the matrix while fibrinogen will not, thus rendering the resulting solution essentially plasminogen-free.
  • a lysine affinity column such as the matrix column product sold commercially under the tradename lysine-Sepharose 4B.
  • Residual plasminogen present in the cryo- or cold-precipitate will be adsorbed by the matrix while fibrinogen will not, thus rendering the resulting solution essentially plasminogen-free.
  • the resulting final composition will contain no more than 10 ⁇ g plasminogen/milliliter.
  • the compositions (preferably the concentrated solution) will be treated with an effective amount of a viral activity reducing agent, such as a detergent, which, typically, acts by disrupting the lipid envelope of such viruses as Hepatitis B, HIV, and HTLV.
  • a viral activity reducing agent such as a detergent
  • the term "effective amount of viral activity reducing" agent means that the concentration of viral activity reducing agent added to the composition is sufficient to reduce the viral activity in the compositions of the invention to undetectable levels. Of course, the concentration of viral activity reducing agent should not significantly effect the patient.
  • viral contaminants may be removed from the compositions of the invention by virtue of process steps which do not involve the addition of a viral activity reducing agent to the composition.
  • viral activity reducing agents typically affect only lipid enveloped viruses (by disrupting the integrity of the lipid envelope)
  • the process steps will likely be the principal means given the current state of the art by which any viruses present which lack a lipid envelope will be removed - 11 -
  • compositions of the invention are prepared from the compositions of the invention.
  • process steps will be known to, or can readily be ascertained by, one of ordinary skill in the art and include viral partitioning and adso tion/filtration with calcium phosphate.
  • Nondenaturing detergents which are useful as such agents can be selected by one of ordinary skill in the art from such recognized groups as anionic, cationic, and non-ionic detergents.
  • examples include sulfated alcohols and sodium acid salts, such as sulfated oxyethylated alkylphenol (sold commercially under the tradenames "Triton W-30” and “Triton X-100"), sodium dodecylbenzensulfonate (sold commercially under the tradename “Nacconol NR”), sodium 2-sulfoethyl oleate (sold commercially under the tradename “Igepon A”), sodium cholate, sodium deoxycholate, sodium dodecylsulfonate, dodecyldimethylbenzylammonium chloride (sold commercially under the tradename "Triton K-60”), oxyethylated amines (sold commercially under the tradename "Ethomeen”), N-do
  • the viral activity-reducing agent will consist of an organic solvent, preferably tri-n-butyl phosphate (TNBP) mixed with nonionic detergents, preferably polysorbate 80 and octoxynol 9.
  • TNBP tri-n-butyl phosphate
  • nonionic detergents preferably polysorbate 80 and octoxynol 9.
  • undenatured compositions of the invention in which viral activity is at undetectable levels can be produced using a preferred viral activity-reducing agent comprised of TNBP (concentration 0.03-0.3%), polysorbate 80 (concentration 0.03-
  • chaotropic agents may also be utilized to inactivate viruses, providing the agent does not denature fibrinogen.
  • the concentration of organic solvent and detergent used in the practice of the preferred embodiments of the invention can vary, depending upon the composition to be treated, and upon the solvent or detergent selected.
  • the alkyl phosphates can be used in concentrations from about 0.10 mg/ml of mixture treated to - 12 -
  • the wetting agent can vary from about 0.001% to 10%, preferably from about 0.01% to about 2% of the aqueous mixture, depending upon the amount of fatty material in the treated aqueous mixture.
  • DEAE diethylaminoethyl cellulose (sold commercially under the tradename "DE 52") is the matrix utilized for the removal of the solvent detergent from the fibrinogen composition.
  • the fibrinogen binds to the diethylaminoethyl cellulose and, after thorough washing to remove unbound material and detergent, is eluted with, for example, 0.3M NaCl.
  • ion exchange materials which can be utilized for removal of the solvent/detergent include virtually any of the commercially available anion exchange matrices including, but not limited to, cellulose and agarose matrices.
  • the specific parameters for binding and eluting from these various ion exchange materials are known to those of skill in the art, or can be readily ascertained without undue experimentation.
  • the precise amount of fibrinogen incorporated onto the hydrophobic structures is difficult to define in absolute terms since it will depend upon the size of the hydrophobic structures utilized and the particular characteristics which the finished hydrophobic structures are desired to have. However, the amount to be used in a given situation may be determined by one skilled in the art based on the information provided herein.
  • the amount of fibrinogen which is incorporated onto the hydrophobic structures is a hemostatically/adhesively-effective amount (i.e., that amount which will be sufficient to direct that oil droplet to the site of interest (i.e., site of fibrin(ogen) concentration) and provide hemostatic or adhesive properties).
  • a hemostatically/adhesively-effective amount i.e., that amount which will be sufficient to direct that oil droplet to the site of interest (i.e., site of fibrin(ogen) concentration) and provide hemostatic or adhesive properties.
  • that amount should be sufficient such that the hydrophobic structures formed contain either a saturated or subsaturated film (preferably a saturated monolayer coating) of fibrinogen on their - 13 -
  • hydrophobic structures of the size defined herein, it is preferred that from about 0.1 to about 500 ⁇ g of fibrinogen, preferably from about 1 to about 100 ⁇ g of fibrinogen, more preferably from about 1 to about 10 ⁇ g of fibrinogen, and most preferably about 10 ⁇ g of fibrinogen, per milligram of hydrophobic structures be incorporated. It is generally preferred that the fibrinogen molecules be packed "end on" at the oil droplet surface (i.e., with respect to its long axis, each fibrinogen molecule is approximately perpendicular to the plane of the liposome surface). This will allow for the formation of a monolayer containing the greatest concentration of fibrinogen. The minimum surface area that the fibrinogen can take up at the oil droplet surface in this case is about 10,000 A 2 .
  • the fibrinogen-coated hydrophobic structures of the present invention may be effectively utilized as excellent hemostatic agents, without containing any encapsulated pharmaceutical compounds.
  • the hydrophobic structures of the present invention may also contain pharmaceutically-active agents within them.
  • the hydrophobic structures may be used to target those specific pharmaceutically-active agents to, for example, a site of inflammation or blood clots (i.e., any site at which fibrin would be present).
  • Examples of pharmaceutical agents which may be encapsulated within the hydrophobic structures of the present invention include anti-inflammatory agents (e.g., aspirin, ibuprofen, indocin, prostacyclin, a phospholipase A2 inhibitor, interleukin or lymphokine), clot dissolving agents (e.g., plasmin, plasminogen, tissue plasminogen activator, urokinase, streptokinase or atroxin), and mixtures thereof.
  • Radiopaque materials may also be encapsulated within the hydrophobic structures in order to provide reagents for imaging fibrin(ogen) containing lesions.
  • agents allow the physician to monitor the progression of wound healing occurring internally, such as at the liver, gall bladder, heart, lungs, brain, bone, gastrointestinal tract, or blood vessels.
  • agents include barium sulfate, as well as various organic compounds containing iodine. Examples of such compounds include iocetamic acid, iodipamide, iodoxamate meglumine, iopanoic acid, as well as diatrizoate derivatives such as diatrizoate sodium.
  • reagents such as dye, clotting factors, heparin or chromogenic polypeptide substrates
  • reagents can also be incorporated within the hydrophobic structures in order to carry out clinical, clot-based coagulation assays.
  • nucleic acids and/or their analogues such as those described in U.S. Pat. No. 4,394,448, Szoka, Jr., et al, issued Jul. 19, 1983, may be incorporated into the fibrinogen-coated hydrophobic structures as a means for inserting that material into cells, for example, in gene therapy.
  • compositions of the present invention may be formulated for a variety of uses, such as control of clotting at wound sites, the targeted delivery of pharmaceutical agents to sites where fibrin is present, use as an imaging agent at fibrin(ogen)-containing lesion sites, vaccine adjuvant, targeting of cells types with fibrinogen receptors such as platelets, megakaryocytes, hepatocytes, macrophages, other white blood cells, tumor cells and viruses, and the insertion of nucleic acids and/or their analogues into specific cell sites.
  • Specific components included in the pharmaceutical compositions will vary depending on the use envisioned for those compositions. Where control of clotting or drug delivery is to be desired, the composition may be formulated either for topical or parenteral use.
  • the pharmaceutically-acceptable carriers utilized in the present invention are those conventionally known and used. Any pharmaceutically-acceptable carrier which is compatible with the fibrinogen-coated hydrophobic structures of the present invention and with the blood may be used in formulating the compositions of the present invention.
  • suitable carriers are aqueous solutions including electrolyte solutions, sugar solutions, or saline solutions.
  • the aqueous component of these solutions should be pyrogen-free water, buffered as appropriate.
  • Such solutions can be used whether administered topically or parentally.
  • a safe and effective amount of the fibrinogen-coated hydrophobic structures component is utilized in order to achieve the desired pharmaceutical result.
  • compositions will preferably contain from about 0.001% to about 98%, more preferably from about 5% to about 50%, and most preferably from about 5% to about 20% of the fibrinogen-coated hydrophobic structures.
  • the balance of the compositions is the pharmaceutically acceptable carrier. - 15 -
  • Topical treatment regimens of the present invention comprise applying the compositions herein directly to the skin at the site of the wound to be treated.
  • the rate of application and duration of treatment will depend upon the severity of the condition, the response of the particular patient, and related factors within the sound medical judgment of the attending physician or patient. In general, from about 0.1 to about 100.0 milligrams of oil droplets per square centimeter of afflicted sites are used.
  • An effective amount of thrombin i.e., from about 0.01 NIH unit to about 5.0 NIH units per square centimeter of skin
  • Application can be made once or several times to control the bleeding from a wound or may be made over the course of a more extended period of time where drug delivery to the sites or promotion of wound healing is desired.
  • compositions of the present invention are to be administered parenterally, a safe and effective amount of the composition (preferably from about 0.5 to about 50 ml/minute) is given to the patient either subcutaneously, intramuscularly or intravenously.
  • in vivo delivery refers to delivery of a biologic by such routes of administration as oral, intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, intracranial, inhalational, topical, transdermal, suppository (rectal), pessary (vaginal), and the like.
  • biological refers to pharmaceutically active agents
  • analgesic agents such as analgesic agents, anesthetic agents, anti-asthmatic agents, antibiotics, anti- depressant agents, anti-diabetic agents, anti-fungal agents, anti-hypertensive agents, anti-inflammatory agents, anti-neoplastic agents, anxiolytic agents, enzymatically active agents, nucleic acid constructs, immunostimulating agents, immunosuppressive agents, physiologically active gases, vaccines, and the like), diagnostic agents (such as ultrasound contrast agents, radiocontrast agents, or magnetic contrast agents), agents of nutritional value, and the like.
  • diagnostic agents such as ultrasound contrast agents, radiocontrast agents, or magnetic contrast agents
  • micron refers to a unit of measure of one one- thousandth of a millimeter. - 16 -
  • biocompatible describes a substance that does not appreciably alter or affect in any adverse way, the biological system into which it is introduced.
  • Materials which can be used to form the hydrophobic portion of the fibrinogen-coated hydrophobic structures are those that occur naturally, such as mineral oils, silicone oils, animal oils, vegetable oils, and the like, and synthetic oils.
  • mineral oils are petroleum oil and the distilled fractions thereof such as kerosene, gasoline, naphtha and paraffin oil.
  • silicone oils are siloxanes, polydimethylsiloxanes, fluorosilicone oil, and dimethylsiloxane ethylene oxide- propylene oxide copolymers (DMSiEPO).
  • animal oils are fish oil and lard oil.
  • Examples of vegetable oils are peanut oil, linseed oil, soybean oil, castor oil, corn oil, grapeseed oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.
  • Examples of synthetic oils are biphenyl derivatives such as dichlorobiphenyl and trichlorobiphenyl, phosphoric acid derivatives such a triphenyl phosphate, naphthalene derivatives such as alkyl naphthalenes (e.g., isopropyl naphthalene) phthalate derivatives such as diethylphthalate and dibutyl phthalate and dioctylphthalate, salicylate derivatives such as ethylsalicylate, and the like.
  • the applications are fairly wide ranging.
  • biomedical applications such as the delivery of drugs, diagnostic agents (in imaging applications), artificial blood (crosslinked hemoglobin) and parenteral nutritional agents
  • the fibrinogen-coated hydrophobic structures of the invention may be incorporated into cosmetic applications such as skin creams or hair care products, in perfumery applications, in pressure sensitive inks, pesticides, and the like.
  • fibrinogen- coated hydrophobic structures prepared as described above are used for the in vivo delivery of biologies, such as pharmaceutically active agents, diagnostic agents or agents of nutritional value (i.e. nutriceuticals).
  • pharmacologically active agents contemplated for use in the practice of the present invention include analgesic agents (e.g., acetominophen, aspirin, ibuprofen, morphine and derivatives thereof, - 17 -
  • anesthetic gases e.g., cyclopropane, enfluorane, halothane, isofluorane, methoxyfluorane, nitrous oxide, and the like
  • anti-asthmatic agents e.g., azelastine, ketotifen, traxanox, and the like
  • antibiotics e.g., neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline, and the like
  • anti-depressant agents e.g., nefopam, oxypertine, imipramine, trazadone, and the like
  • anti-diabetic agents e.g., biguanidines, hormones, sulfonylurea derivatives, and the like
  • anti-fungal agents e.g., amphotericin B, nystatin, candicidin, and the like
  • diagnostic agents contemplated for use in the practice of the present invention include ultrasound contrast agents, radiocontrast agents (e.g., iodo- octanes, halocarbons, renografin, and the like), magnetic contrast agents (e.g., fluorocarbons, lipid soluble paramagnetic compounds, GdDTPA, aqueous paramagnetic compounds, and the like), as well as other agents (e.g., gases such as argon, nitrogen, carbon monoxide, carbon dioxide, helium, xenon, nitrous oxide, nitric oxide, nitrogen dioxide, and the like, as well as combinations of any two or more thereof).
  • radiocontrast agents e.g., iodo- octanes, halocarbons, renografin, and the like
  • magnetic contrast agents e.g., fluorocarbons, lipid soluble paramagnetic compounds, GdDTPA, aqueous paramagnetic compounds, and the
  • the fibrinogen-coated hydrophobic structures containing biologic allows for the delivery of high doses of biologic in relatively small volumes. This minimizes patient discomfort at receiving large volumes of fluid and minimizes hospital stay.
  • nutriceuticals in accordance with another embodiment of the present invention, there are provided methods for preparing articles for in vivo delivery of nutriceuticals, said method comprising coating a hydrophobic medium with a fibrinogen material wherein the hydrophobic material contains biocompatible material.
  • the term "nutriceutical” refers to an agent of nutritional value.
  • nutriceuticals contemplated for use in the practice of the present invention include amino acids, sugars, proteins, Carbohydrates, fat-soluble vitamins (e.g., vitamins A, D, E, K, and the like) or fat, or combinations of any two or more thereof.
  • Articles prepared in accordance with the above methods are useful vehicles for the administration of nutriceuticals to subjects in need thereof. As contemplated for - 19 -
  • the articles are suitable for "in vivo delivery" administration in a variety of forms and formulations as discussed, including pediatric dosage formulations and formulations for inhalation therapy.
  • fibrinogen- coated hydrophobic structures as described herein, when prepared containing hemoglobin are useful as blood substitutes.
  • Hemoglobin is a 64,500 MW protein that consists of a tetramer (two alpha and two beta chains) and can be made soluble in the hydrophobic material of the present invention.
  • Hemoglobin, the protein for oxygen transport and delivery can be separated from the red blood cell wall membranes or stroma (stroma contain the specific antigens that determine blood type) and from other cell and plasma components. If such separation and isolation is effected, the resulting stroma-free hemoglobin contains no antigenic materials; thus, blood typing and matching are no longer necessary.
  • the invention lends itself to the use of other oxygen binding proteins as RBC substitutes.
  • the protein myoglobin which possesses a single oxygen binding heme group (but no crosslinkable cysteine residues) may be expected to behave in the same way.
  • a genetically engineered myoglobin with at least two crosslinkable cysteine residues may be utilized to generate an insoluble myoglobin construct.
  • a combination of oxygen binding proteins with proteins that have no affinity for oxygen may be utilized in formation of the insoluble constructs of the present invention, e.g., hemoglobin and albumin may be used.
  • cytotoxic drugs are oil-soluble. These drugs may be dissolved in a fluorocarbon or other biocompatible oil such as soybean oil, safflower oil, coconut oil, olive oil, cottonseed oil, and the like.
  • the oil/drug solution is used to produce a fibrinogen-coated hydrophobic structure.
  • the suspension may be oxygenated prior to intravascular administration.
  • Oil-soluble cytotoxic drugs include cyclophosphamide, BCNU, melphalan, mitomycins, taxol and derivatives, taxotere and derivatives, camptothecin, adriamycin, etoposide, tamoxifen, vinblastine, vincristine and the like; nonsteroidal antiinflammatories such as ibuprofen, aspirin, piroxicam, cimetidine, and the like; steroids such as estrogen, prednisolone, cortisone, hydrocortisone, diflorasone, and the like, drugs such as phenesterine, mitotane, - 20 - visadine, halonitrosoureas, anthrocyclines, ellipticine, diazepam, and the like; immunosuppressive agents such as cyclosporine, azathioprine, FK506, and the like.
  • Water- soluble drugs may also be encapsulated within the fibrinogen-coated hydrophobic structures by a method of double emulsion.
  • an aqueous drug solution is emulsified with a biocompatible oil to obtain a water-in- oil (W/O) emulsion.
  • W/O emulsion is treated as an oil phase and subjected to ultrasonic irradiation with an aqueous solution to produce fibrinogen-coated hydrophobic structures containing within their hydrophobic region, a microemulsion of the desired water- soluble drug.
  • Emulsifiers contemplated for use in this embodiment of the present invention include the Pluronics (block copolymers of polyethylene oxide and polypropylene oxide), phospholipids of egg yolk origin (e.g., egg phosphatides, egg yolk lecithin, and the like); fatty acid esters (e.g., glycerol mono- and di-stearate, glycerol mono- and di- palmitate, and the like).
  • Pluronics block copolymers of polyethylene oxide and polypropylene oxide
  • phospholipids of egg yolk origin e.g., egg phosphatides, egg yolk lecithin, and the like
  • fatty acid esters e.g., glycerol mono- and di-stearate, glycerol mono- and di- palmitate, and the like.
  • Water- soluble drugs contemplated for use in this embodiment of the present invention include antineoplastic drugs such as actinomycin, bleomycin, cyclophosphamide, duanorubicin, doxorubicin, epirubicin, fluorouracil, carboplatin, cisplatin, interferons, interleukins, methotrexate, mitomycins, tamoxifen, estrogens, progestogens, and the like.
  • antineoplastic drugs such as actinomycin, bleomycin, cyclophosphamide, duanorubicin, doxorubicin, epirubicin, fluorouracil, carboplatin, cisplatin, interferons, interleukins, methotrexate, mitomycins, tamoxifen, estrogens, progestogens, and the like.
  • Preferred routes for in vivo administration are the intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, oral, inhalational, topical, transdermal, suppository, pessary and the like.
  • fibrinogen Human fibrinogen, FIB3, was from Enzyme Research Laboratories, South Bend, IN. Prior to use, fibrinogen was desalted by gel permeation chromatography using Sephadex G-25 (Pharmacia, Piscataway, NJ) as matrix material and either 0.02 mol/L Tris-HCl, pH 7.40, or 0.01 mol/L Na 2 HPO 4 and NaH 2 PO 4 , pH 7.40, containing 0.145 mol/L NaCl (PBS) as eluent. Desalted fibrinogen was aliquoted and stored frozen at -20°C until use.
  • fibrinogen Prior to use, a frozen aliquot of fibrinogen solution was thawed to room temperature, diluted in buffer as appropriate, and then heated to 37°C to dissolve any residual cryoprecipitate.
  • Fatty acid-free bovine serum albumin (BSA) was from ICN, Aurora, OH.
  • fibrinogen was uniformly labeled using Na I from Amersham, Arlington Heights, IL, and Iodo-Gen from Pierce, Rockford, IL.
  • Egg yolk L- ⁇ -lecithin was from Avanti Polar Lipids, Alabaster, AL. Drakeol 6-VR and Drakeol 32, white mineral oils containing paraffin and naphthene hydrocarbons ranging from approximately 18 to 36 carbon atoms, were from Penreco, Butler, PA.
  • [l- 14 C]Dodecane of specific activity 151.7 MBq/mmol was from Sigma, St. Louis, MO.
  • Dodecane, olive oil, safflower oil, soybean oil, triolein, cholesteryl oleate, squalene, squalane, ristocetin sulfate, human thrombin (> 3,000 NIH U/mg), hirudin (3,000 U/mg), the sodium salt of pentosan polysulfate (M r ( aV e) ⁇ 3,800), and the acetate salt of the tetrapeptide Gly-Pro-Arg-Pro (GPRP) were also from Sigma.
  • the sodium salts of porcine mucosa heparins of average molecular weights 14,250, 3,700 and 5,100 were from Calbiochem, San Diego, CA.
  • Sodium suramin was provided by the Drug Service of the Centers for Disease Control, Atlanta, GA.
  • the sodium salt of dextran sulfate of M r(ave) -8,000 was from Sigma, and the sodium salt of dextran sulfate of M r ( ave ) -40,000 (range 37,000-43,000) was from Chemical Dynamics, South Plainfield, NJ.
  • water Prior to its use, water was first deionized, and then distilled using an all-glass apparatus. All organic solvents were of a grade suitable for high-performance liquid chromatography. All other chemicals were of the highest quality available commercially. - 22 -
  • Fresh human plasma and fresh serum were prepared from the blood of healthy donors.
  • blood was drawn directly into evacuated siliconized glass tubes (Becton Dickinson, Franklin Lakes, NJ) containing sodium citrate, yielding a final concentration of the anticoagulant of 0.013 mol/L.
  • serum blood was drawn directly into evacuated glass tubes (Becton Dickinson) where it was left undisturbed for 2 h while it clotted at room temperature. Cells were removed from anticoagulated blood and from clotted blood by centrifugation at l,500g for 15 min, and the corresponding plasma or serum was then aspirated and, unless specified otherwise, used immediately thereafter.
  • the fibrinogen concentration of citrated plasma samples was determined using the method of Clauss.
  • High pressure extrusion was used to prepare emulsions.
  • either 140 ⁇ L or 220 ⁇ L of a liquid hydrophobic phase was first added to a clean, 12 mm x 75 mm glass tube.
  • To a tube containing 140 ⁇ L of oil was then added 3.5 mL of PBS; to a tube containing 220 ⁇ L of oil was then added 5.0 mL of PBS.
  • an oil - water mixture was passed five times under high pressure, 15,000 psi, through the aperture of an automated homogenizer (EmulsiFlexTM-20,000- B3, Avestin, Ottawa).
  • emulsion was first transferred to a clean, round bottom, glass tube. The emulsion was then centrifuged at 9,400g for 60 min. After removing the droplet-free medium, droplets were washed thrice using each time 2.0 mL of fresh buffer. Each of these washes involved centrifugation at 9,400g for 20 min. Following their isolation and wash, droplets were redispersed as necessary to an apparent absorbance of 1.0 at 500 nm using a cuvette of 1.0 cm path length. The medium for this purpose was 0.02 mol/L Tris-HCl, pH 7.40, containing 1.0 mg/mL BSA.
  • the fibrinogen concentration of the citrated, virgin plasma was 7.1 x 10 "6 mol/L.
  • Three mL of 125 I-fibrinogen- supplemented plasma was added to an equivalent volume of PBS containing 132 ⁇ L of freshly prepared olive oil droplets. After 30 min, this dispersion was mixed with an equivalent volume of aqueous sucrose, 78% (w/v), and the droplets were then floated by centrifugation for 3 h at 7,000g.
  • the resulting cream layer was washed twice using each time 10 ml of aqueous sucrose and centrifugation for 1.0 h at 7,000g. The radioactivity associated with the washed cream layer was then measured. Using the known concentration of endogenous fibrinogen in the plasma, the specific activity of the 125 I-fibrinogen supplementing that medium, and the radioactivity associated with the oil droplets, the fibrinogen bound to the olive oil droplets was determined.
  • thrombin-inducible aggregation of such droplets is a convenient measure of the functionality of bound fibrinogen.
  • Several methods were used to monitor both aggregation of fibrin-coated oil droplets and dissociation of aggregates of fibrin-coated droplets.
  • One method involved simply applying 200 ⁇ L of an emulsion containing fibrinogen-coated oil droplets to a smooth surface and then mixing into this emulsion an amount of thrombin, 0.5 NIH U in 20 ⁇ L, using a wooden spatula.
  • a typical aggregation assay was performed at room temperature as follows. A 0.5 mL dispersion of droplets in 0.02 mol/L Tris-HCl, pH 7.40, containing 1.0 mg/mL BSA was added to a cylindrical, glass, sample cuvette (internal diameter, 6 mm). The apparent absorbance of test dispersions was 1.0 at 500 nm when using a cuvette of 1.0 cm path length. As reference material, a dispersion of polystyrene beads of diameter 0.945 ⁇ 0.0064 ⁇ m (Seradyn, Indianapolis, IN) was used.
  • the apparent absorbance of this reference material was 0.5 at 500 nm when using a cuvette of 1.0 cm path length.
  • 20 ⁇ L of an aqueous solution containing 0.5 NIH U thrombin was added to the reaction cuvette.
  • the relative absorbance of the sample as a function of time after the addition of the enzyme was then recorded.
  • a value of 1.0 was assigned arbitrarily to the maximal change of the aggregometry signal that occurred after the addition of 0.5 NIH U thrombin to a dispersion of fibrinogen-coated droplets of mineral oil (Drakeol 32).
  • each clot 1.13 cm 2
  • 100 ⁇ L of buffer containing 10 ⁇ L of droplets of a particular olive oil:[l- 14 C]dodecane emulsion was overlaid with 100 ⁇ L of buffer containing 10 ⁇ L of droplets of a particular olive oil:[l- 14 C]dodecane emulsion.
  • the contents of a reaction vessel were then agitated for 6 s using a vortex apparatus.
  • a clot was 'washed' twice using each time 1.0 mL of fresh buffer and gentle agitation. Following each wash, the unbound oil droplets were decanted.
  • fibrinogen-coated oil droplets an attempt was made to dissociate from the clots any bound droplets using one of several solutions.
  • Concentration-dependent data were paired with the corresponding concentrations and then fit to an appropriate equation described in the text. The best values for the parameters of an equation were determined using the paired data and a nonlinear least squares regression method. - 27 -
  • Surfactants can be used to stabilize emulsions consisting of two partially miscible or immiscible phases such as mineral oil and water, Fig. 1. By occupying the interface between the continuous and discontinuous phases, the surfactant lowers the interfacial tension of the system reducing the drive toward self-coalescence of the individual phases. As shown on the right in Fig. 1, fibrinogen — an amphiphilic protein and, as such, a surfactant — effectively stabilizes microscopic droplets of emulsified mineral oil. In the absence of fibrinogen or other added surfactant, the water and oil of the emulsion separate back into discrete, continuous phases within 20 min.
  • Liquid hydrophobic phases that we have found can be emulsified in the presence of fibrinogen with the resulting droplets remaining monodisperse for various extended periods include, in addition to mineral oils, olive oil, olive oil/cholesteryl oleate mixtures, triolein, triolein/cholesteryl oleate mixtures, soybean oil, safflower oil, squalene, squalane and dodecane.
  • Lecithin Prevents the Binding of Fibrinogen to Droplets of Liquid Hydrophobic Phases; Cholesteryl Oleate does not.
  • lecithin preexisting at rather high packing density, i.e., - 0.014 molecule/ A 2 ( ⁇ 70 A 2 /molecule), on the surface of hydrophobic beads prevents the binding of fibrinogen to those beads.
  • cholesteryl oleate coated to a similar nominal packing density does not influence the rather significant amount of fibrinogen that otherwise binds to the surface of the beads.
  • Fibrinogen-coated droplets of liquid hydrophobic phases can be isolated and then redispersed in fresh aqueous medium containing either no protein or proteins other than fibrinogen, e.g., BSA.
  • the fibrinogen bound to oil droplets is functional as demonstrated by the macroscopic aggregation of the droplets when they are stirred in the presence of thrombin, Fig. 4B.
  • thrombin-induced aggregation of fibrinogen-coated droplets is inhibited by hirudin, a rapid and potent inhibitor of the enzyme, Fig 4C. Photomicrographs of monodisperse, fibrinogen-coated mineral oil droplets and aggregates of fibrin-coated mineral oil droplets are shown in Fig. 5.
  • hirudin co-administered with fibrinogen-coated oil droplets reduces significantly the association of droplets with the clot, indicating that adherence of the droplets to the clot is likely a consequence of noncovalent interactions between the fibrin of the clot and the fibrin generated on the droplets.
  • Plasmin, GPRP, and unfractionated pharmaceutical grade heparin each effectively dislodge droplets bound to the surface of clots.
  • the mechanism by which plasmin - 30 - liberates droplets undoubtedly involves digestion of fibrin existing between the droplets and the clot surface since plasminogen does not free fibrin-coated particles from the surface of a solution phase clot.
  • Heparin and GPRP ' on the other hand, likely interfere with noncovalent interactions between fibrin on the droplets and the fibrin at the clot's surface.
  • dimeric ristocetin also flocculates fibrinogen-coated oil droplets.
  • Ristocetin dimers do not, however, flocculate droplets coated with an irrelevant protein, i.e., BSA.
  • fibrinogen retains important solution phase features when it is bound to oil droplets.
  • Fibrinogen is Stable on Droplets of Liquid Hydrophobic Phases.
  • Fibrinogen dissociates rather slowly from oil droplets dispersed in buffer: only - 4% of the radioactivity bound initially to saturation on droplets of either mineral oil (Drakeol 32) or olive oil is liberated from the droplets when they are incubated in buffer for 48 h. In the case of olive oil, this same holds true even if the aqueous phase is heat-treated, fibrinogen-supplemented plasma: 95% of the radioactivity remains associated with the droplets after 48 h.
  • Fibrinogen adsorbs rapidly and in relatively large quantity from blood to solid hydrophobic surfaces in contact with that medium. 4 ' 23 For this reason, we assessed next whether fibrinogen would adsorb from citrated plasma to oil droplets. Such an assessment seemed apropos since biologically relevant lipid particles in vivo, i.e., lipoproteins, are bathed continuously in a fibrinogen-rich medium, and fibrinogen appears to contribute to the initiation, development and growth of atherosclerotic plaques. 3 ' 24 ' 25 As shown in Fig.
  • fibrinogen adsorbed to droplets incubated in plasma containing 7.1 x 10 "6 mol/L (241 mg%) fibrinogen was 0.40 mg per mL oil
  • the quantity of fibrinogen bound to droplets from the plasma containing 13.2 x 10 "6 mol/L (450 mg%) fibrinogen was 0.87 mg per mL oil.
  • Heparins and Other Polyanions Prevent the Mutual Adhesion of Fibrin-coated Surfaces.
  • heparins and related polyanions in the absence of any cofactor — bind with high affinity to fibrin(ogen) adsorbed to solid polymeric beads. 10
  • the mutual adhesion of fibrinogen-coated beads that otherwise occurs in the presence of thrombin can be prevented, and preexisting aggregates of fibrin-coated beads can be dissociated. Discovery of these phenomena led us to suggest that the binding of heparin to - 32 -
  • adsorbed fibrin(ogen) might serve some general, cofactor-independent, 'anti- adhesive' function.
  • M r(ave ) low molecular weight
  • M r(aVe) - 40,000 high molecular weight dextran sulfate
  • fibrinogen adsorbs spontaneously from aqueous media containing that protein to droplets of liquid hydrophobic phases dispersed in those same media.
  • Lecithin preexisting on the surface of oil droplets reduces significantly the amount of fibrinogen that can otherwise bind to the droplets. When bound, fibrinogen is stable and remains active in the classic sense of fibrin gelation. - 33 -
  • Fibrinogen-coated oil droplets might provide such a vehicle, especially for the delivery of water-insoluble molecules. While the use of oil droplets for the delivery of hydrophobic molecules is not new, the site-specific targeting of such droplets has been problematic. Functional fibrinogen adsorbed 'irreversibly' to drug-loaded oil droplets might serve to focus the droplets to pathologic sites that express thrombin activity, thereby increasing locally drug concentration while limiting globally nonspecific effects of therapy.
  • Fibrinogen-coated oil droplets might also be formulated as adjuvants for vaccines.
  • fibrinogen adsorbs from plasma to the surface of mineral oil droplets of Freund's and related adjuvants, and acts synergistically with amphiphilic lipids expressed on the surface of those droplets to elicit acute inflammation, granuloma formation, immune adjuvancy and hemorrhage. 5 ' 34 Since at that time it was our intention to expose the surface of oil droplets as the site of biological activity of adjuvant lipids, we substituted solid, microscopic, hydrophobic beads for the oil droplets of conventional adjuvant emulsions.
  • Biomedical materials scientists have long appreciated that fibrinogen, of all the plasma proteins, adsorbs rapidly and preferentially to hydrophobic, polymeric materials in contact with blood. 4 ' 23 As a consequence of this adsorption, blood clots are nucleated on the surface of the material often leading, in the case of circulatory prosthetics, to the development of an occlusive thrombus.
  • one aim of biomedical materials researchers is formulation of polymers that do not bind fibrinogen. (Importantly, a successful means by which to prevent the adsorption of fibrinogen and other proteins to a surface involves coating the surface with phospholipids.
  • heparin and related polyanions prevent adhesion between fibrin-coated surfaces, modeled here by droplets of liquid hydrophobic phases.
  • the sensitivity and specificity of the phenomenon to unfractionated heparin supports the notion that this anti-adhesive property exists by design, 10 begging further investigation of the phenomenon.
  • these polyanions might be used advantageously to prevent or even reverse a host of deleterious, fibrin(ogen)-mediated, adhesive events, particularly those occurring at sites of inflammation. 6 ' 7 ' 30 - 32 ' 39"40 - 35 -
  • lipid particles within the vasculature need not involve any specific interaction between some particle-resident amphiphile, e.g., apolipoprotein (a), 50 and fibrin(ogen): for fibrinogen-coated particles to accumulate, there needs only to be the focal production of thrombin such as that which occurs at
  • Baier RE Initial events in interactions of blood with a foreign surface. J Biomed Mater Res. 1969;3:191-206.
  • Laudano AP Doolittle RF. Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences. Biochemistry. 1980;19:1013-1019.
  • Bini A Fenoglio JJ, Mesa-Tejada R, Kudryk B, Kaplan KL. Identification and distribution of fibrinogen, fibrin, and fibrin(ogen) degradation products in atherosclerosis. Use of monoclonal antibodies. Arteriosclerosis. 1989;9:109-121. - 41 -

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Abstract

Le fibrinogène s'adsorbe spontanément, à partir de milieux aqueux contenant cette protéine, sur les gouttelettes de phases hydrophobes liquides dispersées dans lesdits milieux. Parmi ces phases, on trouve les huiles minérales, les hydrocarbures à chaîne droite, ainsi que diverses huiles d'origine végétale ou animale. La lécithine préexistant à la surface des gouttelettes d'huile diminue de manière significative la quantité de fibrinogène susceptible de se fixer sur lesdites gouttelettes. Une fois fixé, le fibrinogène reste actif dans le sens classique de gélification fibrineuse. Les huiles enrobées de fibrinogène peuvent donc participer chez un hôte à d'importants processus biologiques d'adhésion auxquels la protéine est censée participer. Certains polyanions, tels que l'héparine, le polysulfate de pentosane, le sulfate de dextrane et la suramine, se fixent sur la fibrine/le fibrinogène adsorbé et empêche l'adhésion thrombine-dépendante sur les surfaces revêtues de fibrinogène. Ces polyanions peuvent donc être utilisés pour prévenir l'adhésion entre les gouttelettes d'huile enrobées de fibrine/fibrinogène et d'autres surfaces revêtues de fibrine/fibrinogène. D'éventuelles applications pratiques et implications biologiques de ces phénomènes sont présentées et discutées.
PCT/US1999/009940 1998-04-30 1999-04-28 Gouttelettes, enrobees de fibrinogene, de phases hydrophobes liquides WO1999055314A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10255106A1 (de) * 2002-11-24 2004-06-09 Novosom Ag Liposomale Glucocorticoide
WO2005039535A1 (fr) * 2003-10-24 2005-05-06 Alza Corporation Preparation de particules lipidiques

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WO1996029990A1 (fr) * 1995-03-31 1996-10-03 University Of Cincinnati Liposomes enduits de fibrinogene

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029990A1 (fr) * 1995-03-31 1996-10-03 University Of Cincinnati Liposomes enduits de fibrinogene

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10255106A1 (de) * 2002-11-24 2004-06-09 Novosom Ag Liposomale Glucocorticoide
WO2005039535A1 (fr) * 2003-10-24 2005-05-06 Alza Corporation Preparation de particules lipidiques

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