2. Description of the Background Art 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). The microcirculation in most organs and tissues allows the free passage of these blood cells. When microthrombii (blood clots) of size greater than 10-15 microns are present in circulation, a risk of infarction or blockage of the capillaries results, leading to ischemia or oxygen deprivation and possible tissue death. Injection into the circulation of particles greater than 10-15 microns in diameter, therefore, must be avoided. 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 <BR> <BR> <BR> human body. For example, 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. Furthermore, 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.

U. S. Pat. No. 5,264,221, Tagawa, et al., issued Nov. 23,1993, describes drug- containing liposomes which have specifically defined targeting agents on their surface. 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.

The advantages have not been previously suggested by the art. The incorporation of fibrinogen into a hydrophobic structure could provide many advantages, for example, use of such hydrophobic structures could be virtually without risk of blood-borne infection to the recipient.

Conventional approaches of binding proteins to liposomes involve the use of a bifunctional reagent that cross-links a reactive group contributed by a liposomal component to a reactive group contributed by the protein. In the case of fibrinogen, such approaches do not yield liposomes coated densely with demonstrably functional protein. Acylation of the fibrinogen prior to incorporating it into liposomes is also problematic. In the absence of liposomes or other amphiphilic/hydrophobic surfaces, the acylation of fibrinogen yields an insoluble, amorphous material unsuitable for coating liposomes.

The poor aqueous solubility of many biologics 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.

Pharmaceuticals 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). The 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. These emulsions may be injected intravenously, provided the components of the emulsion are pharmacologically inert.

Additional biologics 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).

BREIF SUMMARY OF THE INVENTION 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

of inflammation, and/or clotting, 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. Examples of liquid hydrophobic phases include mineral oils, straight chain hydrocarbons, and various plant-and animal-derived oils. As a consequence, oil droplets coated with fibrinogen can participate in a host of biologically important adhesive processes in which the protein would be expected to participate.

BREIF DESCRIPTION OF THE FIGURES 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.

Fig. 3. Effect of lipid amphiphiles on the binding of fibrinogen to droplets of olive oil. 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.

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 51. 8 u. 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. 7. Effect of various agents on the adherence of droplets of olive oil: [1- l4C] dodecane, 95/5 (v/v), to a solution phase fibrin clot. Results are expressed as the mean SEM (n = 4) percentage of control radioactivity associated with clots in the absence of any treatment. See reference 12 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. A, 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. A, plasma fibrinogen concentration = 13.2 x 10-6 mol/L; B, plasma fibrinogen concentration = 7.1 x 10-6 mol/L; C, same as B but hirudin was added prior to the addition of thrombin; D, serum. The arrow indicates the addition of thrombin.

Fig. 12. Dose-dependent inhibition of thrombin-inducible aggregation of fibrinogen- coated droplets of mineral oil (Drakeol 32). (0), Heparin, Mr (ave) = 14,250; (§), heparin, Mr (ave) = 5,100; (O), pentosan polysulfate; (A), dextran sulfate, Mr (ave) = 8,000; (), heparin, Mr (ave) = (), suramin; and (+), dextran sulfate, Mr (ave) = 40,000. See reference 10 and text for details.

DETAILED DESCRIPTION OF THE INVENTION 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. The nomenclature of the chains derives from the fact that relatively small polypeptides, called 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 entire amino acid sequence of human fibrinogen, a total of 1,482 residues in each set of three polypeptide chains, has been determined by classic protein chemistry techniques. There are 610,461, and 411 amino acid residues in the

common forms of the A alpha, B beta, and gamma chains, respectively. The computed respective molecular weights of these chains are 66,500,52,000, and 46,500, for a total molecular weight of approximately 330,000 for two copies of each chain. Less common structural variants exist, with the most frequent one (less than 10%) being a variant of the gamma chain in which residues 408-411 have been replaced with a 20-residue sequence ending in leucine at position 427. In addition to their amino acid content, the B beta and gamma chains contain carbohydrate, each of molecular weight 2,500, attached covalently at residues 364 and 52, respectively.

Thus, 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.

The conversion of 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 XIIIa-catalyzed crosslinking.

Preferred blood fractions for producing the compositions of the invention are plasma, cryoprecipitate, and/or Factor VIII-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.

More specifically, the preferred method for producing the compositions of the present invention uses frozen human plasma from one or more donors as a starting material. Preferably, the plasma to be used will have been screened using conventional assay techniques for the presence of infectious viral contaminants, such

as hepatitis B and human immunodeficiency virus (HIV) to eliminate plasma for use as a starting material which contains detectable levels of such contaminants.

Whether or not the plasma has been previously screened, the 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. For purposes of this disclosure, the phrase"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"logl0 reduction factor". Generally, using the available techniques for reducing viral activity in a protein containing composition which are known in the art and/or described herein, it can be expected that the compositions of the inventions will have a logl0 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.

To process the frozen plasma to a cryoprecipitate, the plasma is thawed in a controlled environment. However, to limit the total protein content of the compositions of the invention to fibrinogen, residual amounts of fibronectin and any added protein components (such as albumin), 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. One of the other well known methods in the art may be used. Precipitation may be achieved in any conventional manner. Cryoprecipitation is preferred and may be carried out as follows: (a) freezing fresh frozen plasma at temperature in the range from about-70°C. to about-80°C. for a time in the range from about 1 hour to about 24 hours, preferably for at least about 12 hours; (b) raising the temperature of the frozen plasma to between about 0°C. and room temperature, preferably about 4°C., so as to form a supernatant and a cryoprecipitated suspension containing fibrinogen; and

(c) recovering the cryoprecipitated suspension.

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. Such 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.

Alternative sources of human fibrinogen are also envisioned. For example, 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.

To remove any prothrombin complex which may be present in the cryo-or cold-precipitate, the precipitate suspension is transferred to a buffer solution

containing a salt such as tri-calcium phosphate. Exposure to the salt-containing buffer will minimize the likelihood of prothrombin conversion to thrombin which, if such a reaction were to occur, could lead to the conversion of fibrinogen to fibrin. In this state, the resulting composition can be considered to be essentially free of prothrombin complex. The calcium phosphate, in turn, 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.

In a preferred embodiment, 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. For example, using the lysine-Sepharose material referred to above, the resulting final composition will contain no more than 10 ig plasminogen/milliliter.

In all embodiments, 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. 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.

To some extent, 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. Because 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

from the compositions of the invention. Such process steps will be known to, or can readily be ascertained by, one of ordinary skill in the art and include viral partitioning and adsorption/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"lgepon 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-dodecylaminoethanesulfonic acid, ethylene oxide-propylene oxide condensates ("Pluronic"copolymers), polyoxyethylated derivatives of esters (sold commercially under the tradenames "Tween 80"and"Polysorbate 80"), polyoxyethylene fatty alcohol ethers (sold commercially under the tradenames"Brij 35"), tetramethylsutylphenyl ethers of polyethylene glycols (sold commercially as"octoxynols"), as well as detergents sold commercially under the tradenames"nonidet P-40"and"Lubrox PX"..

In the preferred embodiment, 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. As described in Examples 1 and 5, 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- 0.3%) and octoxynol 9 (concentration 0.1-1.0%). In addition, chaotropic agents may also be utilized to inactivate viruses, providing the agent does not denature fibrinogen.

Alternatively, 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

1.0 mg/ml, preferably between about 0.1 mg/ml to about 10 mg/ml. The amount of detergent or wetting agent utilized is not crucial since its function is to improve the contact between the organic solvent and the virus. For most of the nonionic materials which are useful, 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.

Whatever viral activity reducing agent is used, it will be removed after it is contacted with the compositions of the invention for a period of time sufficient to reduce the viral activity in the composition to undetectable levels (at least one minute). In the preferred embodiment, 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. Other 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). When calculating the amount of fibrinogen to be added to the reaction, 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

surface. The precise amount this translates into depends upon the size and surface area of the hydrophobic structures utilized. However, utilizing hydrophobic structures of the size defined herein, it is preferred that from about 0.1 to about 500 u. g of fibrinogen, preferably from about 1 to about 100 gg of fibrinogen, more preferably from about 1 to about 10 pg of fibrinogen, and most preferably about 10 pg 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.

The fibrinogen-coated hydrophobic structures of the present invention may be effectively utilized as excellent hemostatic agents, without containing any encapsulated pharmaceutical compounds. However, the hydrophobic structures of the present invention may also contain pharmaceutically-active agents within them. In this configuration, 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 <BR> <BR> <BR> 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. The presence of such agents allows 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. Such 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. Various

reagents (such as dye, clotting factors, heparin or chromogenic polypeptide substrates) can also be incorporated within the hydrophobic structures in order to carry out clinical, clot-based coagulation assays. Finally, nucleic acids and/or their analogues, <BR> <BR> <BR> <BR> 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.

The pharmaceutical 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. For example, 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.

In such compositions, a safe and effective amount of the fibrinogen-coated hydrophobic structures component is utilized in order to achieve the desired pharmaceutical result. For intravenous uses, the 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.

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) could also be added to the wound site to catalyze the blood clot formation. 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.

Where the pharmaceutical 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.

As used herein, the term"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.

As used herein, the term"biologic"refers to pharmaceutically active 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.

As used herein, the term"micron"refers to a unit of measure of one one- thousandth of a millimeter.

As used herein, the term"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.

Examples of mineral oils are petroleum oil and the distilled fractions thereof such as kerosene, gasoline, naphtha and paraffin oil. Examples of silicone oils are siloxanes, polydimethylsiloxanes, fluorosilicone oil, and dimethylsiloxane ethylene oxide- propylene oxide copolymers (DMSiEPO). Examples of 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.

One skilled in the art will recognize that several variations are possible within the scope and spirit of this invention. For example, the applications are fairly wide ranging. Other than 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.

In accordance with one embodiment of the present invention, fibrinogen- coated hydrophobic structures prepared as described above are used for the in vivo delivery of biologics, such as pharmaceutically active agents, diagnostic agents or agents of nutritional value (i. e. nutriceuticals). Examples of 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,

and the like), 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), anti-hypertensive agents (e. g., propanolol, propafenone, oxyprenolol, nifedipine, reserpine, and the like), steroidal anti-inflammatory agents (e. g., cortisone, hydrocortisone, dexamethasone, prednisolone, prednisone, fluazacort, and the like), non-steroidal anti-inflammatory agents (e. g., indomethacin, ibuprofen, ramifenizone, piroxicam, and the like), anti-neoplastic agents (e. g., adriamycin, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU), cisplatin, etoposide, interferons, phenesterine, taxol (as used herein, the term"taxol"is intended to include taxol analogs and prodrugs, taxanes, and other taxol-like drugs, e. g., Taxotere, and the like), camptothecin and derivatives thereof (which compounds have great promise for the treatment of colon cancer), vinblastine, vincristine, as well as hormonal anti- neoplastic agents such as estrogens, progestogens, tamoxifen, and the like), anxiolytic agents (e. g., dantrolene, diazepam, and the like), enzymatically active agents (e. g., DNAse, ribozymes, and the like), nucleic acid constructs (e. g., IGF-1 encoding sequence, Factor VIII encoding sequence, Factor IX encoding sequence, antisense <BR> <BR> nucleotide sequences, and the like), immunostimulating agents (i. e., interleukins, interferons, vaccines, and the like), immunosuppressive agents (e. g., cyclosporine (CsA), azathioprine, mizorobine, FK506, prednisone, and the like), physiologically active gases (e. g., air, oxygen, 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), as well as other pharmacologically active agents, such as cimetidine, mitotane, visadine, halonitrosoureas, anthracyclines, ellipticine, benzocaine, barbiturates, and the like.

Examples of 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).

Key differences between the biologic-containing fibrinogen-coated hydrophobic structures of the invention and protein microspheres of the prior art are in the nature of formation and the final state of the protein after formation of the polymeric shell, and its ability to carry poorly aqueous-soluble or substantially aqueous-insoluble agents. In contrast to the invention process, the prior art methods utilize glutaraldehyde crosslinking is nonspecific and essentially reactive with any nucleophilic group present in the protein structure (e. g., amines, sulfhydryls and hydroxyls). Heat denaturation as taught by the prior art significantly and irreversibly alters protein structure.

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.

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.

As used herein, the term"nutriceutical"refers to an agent of nutritional value.

Examples of 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

use in the practice of the present invention, 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.

In accordance with another embodiment of the present invention, 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. As an example, 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.

Several useful 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,

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. First, an aqueous drug solution is emulsified with a biocompatible oil to obtain a water-in-oil (W/O) emulsion. The 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). 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.

Preferred routes for in vivo administration are the intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, oral, inhalational, topical, transdermal, suppository, pessary and the like.

In accordance with another embodiment of the present invention, there is provided an approach to the problem of administration of substantially water insoluble drugs such as taxol that has not been described in the literature. Thus, it has been discovered that delivery of such drugs can be accomplished as an aqueous suspension of micron size particles, or an aqueous suspension containing either particles of such drug or drug dissolved in a biocompatible non-aqueous liquid. This approach would facilitate the delivery of such drugs at relatively high concentrations, and thereby obviate the use of emulsifiers and their associated toxic side effects.

Methods Reagents and Chemicals 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-HCI, pH 7.40, or 0.01 mol/L Na2HP04 and NaH2PO4, pH 7.40, containing 0.145 mol/L NaCl (PBS) as eluent. Desalted fibrinogen was aliquoted and stored frozen at-20°C until use. 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. For some experiments, fibrinogen was uniformly labeled using NaI from Amersham, Arlington Heights, IL, and Iodo-Gen from Pierce, Rockford, IL. Egg yolk L-a-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. [1-14C] 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 (Mr (ave)-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. The sodium salt of unfractionated pharmaceutical grade porcine mucosa heparin, 5,000 U/mL, was from Elkins-Sinn, Cherry Hill, NJ. Sodium suramin was provided by the Drug Service of the Centers for Disease Control, Atlanta, GA. The sodium salt of dextran sulfate of Mr (ave)-8,000 was from Sigma, and the sodium salt of dextran sulfate of Mr (ave) -40,000 (range 37,000-43,000) was from Chemical Dynamics, South Plainfield, NJ.

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.

Fresh human plasma and fresh serum were prepared from the blood of healthy donors. For plasma, 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. For 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 1,500g for 15 min, and the corresponding plasma or serum was then aspirated and, unless specified otherwise, used immediately thereafter. As necessary, the fibrinogen concentration of citrated plasma samples was determined using the method of Clauss. ll Inactivation of proteinases in citrated plasma was accomplished by heating the plasma sample at 60°C for 30 min. With heating, a fibrin (ogen)-rich coagulum developed in the plasma. After cooling the sample to room temperature, this coagulum was removed.

Lyophilized fibrinogen was then added to the medium yielding a fibrinogen concentration of 2.9 x 10-6 mol/L.

Emulsification of Liquid Hydrophobic Phases High pressure extrusion was used to prepare emulsions. For this purpose, either 140 pL or 220 uL of a liquid hydrophobic phase was first added to a clean, 12 mm x 75 mm glass tube. To a tube containing 140 uL of oil was then added 3.5 mL of PBS ; to a tube containing 220 pL of oil was then added 5.0 mL of PBS. After agitating briefly, 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).

Isolation of Droplets of Liquid Hydrophobic Phases Using centrifugation, droplets were separated from the aqueous medium in which they were prepared. After aspirating the droplet-free medium, droplets were washed thrice using each time 2.0 mL of fresh buffer. For all droplets other than those containing lecithin, the relative centrifugal force and duration of the initial centrifugation were 1,500g and 20 min, respectively. Subsequently, washes of lecithin-free droplets were performed using centrifugation at 1,500g for 5.0 min. To isolate droplets that had been emulsified in the presence of lecithin, the entire

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-HCI, pH 7.40, containing 1.0 mg/mL BSA.' Visualization of Droplets of Liquid Hydrophobic Phases Light microscopy was used to visualize both monodisperse droplets and aggregates of droplets. For this purpose, a small volume of an emulsion was placed onto a microscope slide and then overlaid with a coverslip. Permanent records of microscopic views of droplets were obtained using photomicroscopy.

Sizing of Droplets of Liquid Hydrophobic Phases For one set of experiments, the size of droplets of various oils was determined using a laser diffraction particle size analyzer (LS 230, Coulter, Miami, FL).

Refractive indices of 1.47 (olive oil) and 1.33 (water) were used when fitting the light-scattering data to the instrument's pre-programmed sizing algorithm.

Fibrinogen Binding Studies We assessed the binding of l2sI-fibrinogen from buffer to emulsified droplets of several liquid hydrophobic phases. For this purpose, 1.0 mL of PBS containing 2.80 mg of 12sI-fibrinogen was added to 4.0 mL of PBS containing 176 IL of emulsified oil droplets. Following centrifugation of the dispersion, the radioactivity associated with the droplet-free reaction medium was measured. When droplets contained lecithin, the medium was cleared by centrifugation at 9,400g for 1.0-6.0 h.

When droplets did not contain lecithin, the medium was cleared by centrifugation at 1,500g for 20 min. The difference between the total radioactivity of a sample and that remaining in the medium after separation of the droplets yielded the radioactivity, hence fibrinogen, bound to the oil droplets.

The binding of fibrinogen from a citrated, plasma-like medium to olive oil droplets was also assessed. We used for these studies citrated plasma that had been

supplemented with various amounts of 12sI-fibrinogen. The fibrinogen concentration of the citrated, virgin plasma was 7.1 x 10-6 mol/L. Three mL of l2sI-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 l2sI-fibrinogen supplementing that medium, and the radioactivity associated with the oil droplets, the fibrinogen bound to the olive oil droplets was determined.

The time dependence of the association of l2sI-fibrinogen with oil droplets dispersed in either buffer or heat-treated, citrated plasma was assessed as follows.

Two mL of oil droplets that had been coated with 125I-fibrinogen was dispersed with continuous stirring in 20 mL of one of the two aqueous phases. After various lengths of time, 1.0 mL aliquots of the stirred dispersion were removed, and the oil and aqueous phases of these aliquots were separated using centrifugation. Subsequently, the radioactivities associated with the oil and aqueous phases were determined.

Aggregation of Fibrin-coated Oil Droplets and Dissociation of Aggregates of Fibrin-coated Oil Droplets When stirred in the presence of thrombin, fibrinogen-coated oil droplets aggregate, a consequence of interparticle fibrin formation. l Thus, 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 nL 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 u. L, using a wooden spatula. With stirring, droplets coated with a dense layer of fibrinogen aggregate within seconds after adding the enzyme, a process obvious to the naked eye. With the addition of certain substances, aggregates of fibrin-coated particles dissociate yielding monodisperse particles.'' This

dissociation was monitored visually. Permanent records of these phenomena were obtained using photographic means.

Another method used to monitor both the aggregation of droplets and the dissociation of aggregates of droplets involved a platelet aggregometer. typical aggregation assay was performed at room temperature as follows. A 0.5 mL dispersion of droplets in 0.02 mol/L Tris-HCI, 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 pm (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.

Once the baseline signal of the stirred (1,000 rpm) test sample was established, 20 uL 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. For the purpose of this report, 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).

Just as particle aggregation can be monitored turbidimetrically using a platelet aggregometer, so, too, can dissociation of aggregates of particles be monitored using an aggregometer. 1, 10 Dissociation of aggregates of fibrin-coated oil droplets was assessed using the same volumes, conditions and instrument parameters described for aggregation assays except that fibrinogen-coated droplets in the sample cuvette had first to be aggregated. For that purpose, 20 u. L of a buffered aqueous solution of thrombin, 0.5 NIH U, was added to stirred, monodisperse, fibrinogen-coated droplets.

Within 15.0 min after adding the enzyme, droplets had aggregated maximally and the resulting aggregates could be used to assess aggregate dissociation. Test reagents were added in 20 pL of water to the reaction cuvette, and the state of aggregation of the droplets was followed turbidimetrically as a function of time after adding the reagent.

Studies Involving Solution Phase Fibrin Clots Round bottom, 12 mm x 75 mm glass tubes were used as reaction vessels to explore interactions of variously coated droplets of olive oil: [1-14C] dodecane, 95/5 (v/v), with solution phase fibrin clots. Ten microliters of buffer containing 0.25 NIH U thrombin was added to 200 uL of PBS containing 1.8 x 10-5 mol/L fibrinogen.

Solution phase clots formed in this fashion'2 were then left undisturbed for 60 min.

For one experiment, 2.0 IU hirudin in 10 ut of buffer was placed on each of several clots. The exposed, uppermost surface of each clot, 1.13 cm2, was overlaid with 100 u. of buffer containing 10 uL of droplets of a particular olive oil: [1-14C] dodecane emulsion. The contents of a reaction vessel were then agitated for 6 s using a vortex apparatus. To remove unbound oil droplets, 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. In the case of clots that had been overlaid with fibrinogen-coated oil droplets, an attempt was made to dissociate from the clots any bound droplets using one of several solutions. These solutions included: 1) 100 uL of buffer containing 0.25 IU plasmin, 2) 100 u. L of buffer containing 5.0 x 10-3 mol/L GPRP, and 3) 100 u. L of buffer containing 100 USP U unfractionated pharmaceutical grade heparin. Both untreated clots and clots that had been treated with one of the test solutions were then washed another two times as described above using for each wash 1.0 mL of fresh buffer. After removing from the surface those oil droplets that could be liberated, the amount of radioactivity remaining associated with the clots was determined. Prior to measuring this radioactivity, clots were dissolved by the addition of 0.5 mL of 8.0 mol/L urea. The entire volume of the solubilized material, 7 mL, was then added to 5.0 mL of scintillation cocktail (Ultimo Gold XR, Puckered, Meridian, CT) for measurement. Each test sample was prepared in quadruplicate.

Analysis of Data 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. l3

Results Fibrinogen Binds to and Stabilizes Droplets of Emulsified Liquid Hydrophobic Phases.

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. In contrast, in the presence of fibrinogen the oil droplets remain monodisperse, eventually forming cream layers over a time course ranging in oil-dependent fashion from hours to days. 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.

The amounts of fibrinogen that bind to various oil droplets prepared by high pressure extrusion and then exposed to the protein according to our standard protocol are shown in Fig. 2.

Lecithin Prevents the Binding of Fibrinogen to Droplets of Liquid Hydrophobic Phases; Cholesteryl Oleate does not.

Earlier, we showed that lecithin preexisting at rather high packing density, i. e., -0.014 molecule/A2 ( 70 A2/molecule), on the surface of hydrophobic beads prevents the binding of fibrinogen to those beads. 8 In contrast, 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. We wondered whether lecithin or cholesteryl oleate, when included in the formulation of an oil emulsion, would affect the association of fibrinogen with oil droplets in the same way they affect the binding of the protein to hydrophobic beads.

To address this, we first prepared emulsions containing olive oil and various quantities of either lecithin or cholesteryl oleate. Next, we used laser diffraction to size droplets prepared from olive oil alone, olive oil and 1.0 mole % lecithin, and olive oil and 2.0 mole % cholesteryl oleate. We found that all of the measured droplets have virtually the same size distribution, mean diameter 5 1.8. m.1.8. m. We then quantitated the binding of 12sI-fibrinogen to the droplets. From the data of Fig. 2 and the diameter of the droplets, we determined that the fibrinogen on droplets of virgin olive oil likely exists as a monomolecular layer. 21819 Furthermore, the packing density of the fibrinogen, 5.8 x 10-5 molecules/A2 (i. e., ~ 17, 300 Å2/molecule), is consistent with the molecules of the protein being oriented with their long axis more normal than tangential to the interface. 219 We found next that no amount of cholesteryl oleate used for these experiments-up to 4 mole % of the lipid of an emulsion-reduces the quantity of fibrinogen that associates with droplets of otherwise virgin olive oil, Fig. 3. This is in keeping with both the oil solubility and the marginal amphiphilicity of the cholesterol ester. 8 4~l6 As shown in the same figure, however, as little as 1.0 mole % lecithin reduces to nearly zero the amount of fibrinogen that otherwise binds to olive oil droplets. Indeed, from the volume of oil used for the experiment (176 uL); the mean diameter of the spherical droplets; and the weight average molecular weights of lecithin (760) and triolein (885,'olive oil'), one calculates that the point of intersection of the two extrapolated linear regions of the figure corresponds to a nominal molecular area for each lecithin molecule of-43 ~ 43 Å2.

This nominal molecular area is in good agreement with that of lecithin maximally packed at the air-water interface, A. The difference between these two areas may be more apparent than real since, in the case of oil droplets, the phospholipid partitions between the bulk and surface phases of the oil. Taken together, these data are eminently consistent with the proposal that fibrinogen is excluded from the surface of the droplets when that surface is rendered hydrophilic as a consequence of occupancy by'tightly packed'phospholipid. 8

Fibrinogen Bound to Droplets of Liquid Hydrophobic Phases is Functional.

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. As expected, 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.

Droplet aggregation and related phenomena can be monitored conveniently using either a platelet aggregometer or another photometric device. As shown in Fig.

6, the apparent absorbance of a stirred dispersion of fibrinogen-coated oil droplets decreases rapidly following the addition of thrombin to the dispersion. This decrease in absorbance corresponds to the aggregation of droplets, a consequence of interparticle fibrin dimerization.'By inhibiting thrombin, hirudin prevents aggregation. Thus, just as fibrin (ogen) binds to, and remains operational on, solid microscopic hydrophobic beads, l so, too, does fibrinogen bind to, and remain operational on, droplets of liquid hydrophobic phases.

We assessed next whether various measures would liberate fibrin-coated oil droplets from the surface of a solution phase fibrin clot. l2 For this purpose, we first prepared olive oil droplets containing 0.5 mole % [1-14C] dodecane. These radiolabeled droplets were coated with fibrinogen, washed, and concentrated. An aliquot of the concentrated, fibrinogen-coated droplets was then overlaid onto the exposed surface of a uniform, thrombin-containing, fibrin clot, and the radioactivity remaining associated with the clot following one of several treatments was determined, Fig. 7. As expected, 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

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. l2 Heparinl° and GPRP, on the other hand, likely interfere with noncovalent interactions between fibrin on the droplets and the fibrin at the clot's surface.

We conclude from these experiments that the functionality of fibrin (ogen) bound to oil droplets is similar in all measured respects to that of fibrin (ogen) in solution.

Ristocetin Flocculates Fibrinogen-coated Oil Droplets.

Ristocetin dimers flocculate fibrinogen, a consequence of complexation of the bifunctional dimers with certain ß-turns of the protein. 22 As shown in Fig. 8, dimeric ristocetin also flocculates fibrinogen-coated oil droplets. Ristocetin dimers do not, however, flocculate droplets coated with an irrelevant protein, i. e., BSA. Thus, as assessed using ristocetin as a structural probe, 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 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. Such results are consistent with the notion that the binding of fibrinogen to hydrophobic surfaces is essentially irreversible, a phenomenon due, at least in part, to interfacial 'denaturation'of the protein. l81819 The protein is not so denatured as to lose function, however. As shown in Fig. 9, the thrombin-inducible aggregation of fibrinogen-coated olive oil droplets changes little, if any, following incubation for 24 h in citrated plasma.

Fibrinogen Adsorbs from Plasma to Droplets of Liquid Hydrophobic Phases and is Functional.

Fibrinogen adsorbs rapidly and in relatively large quantity from blood to solid hydrophobic surfaces in contact with that medium."*' 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. 10, olive oil droplets exposed to fresh citrated plasma-but not droplets exposed to fresh serum-aggregate in the presence of thrombin, indicating that fibrinogen adsorbs from plasma to the droplets and is functional. As shown in Fig. 11, the rate and extent of aggregation of plasma-exposed oil droplets depend, in turn, on the concentration of the fibrinogen in the plasma. Using 125I- fibrinogen as tracer, we found that the quantity of 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, and 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. Thus, while plasma proteins in addition to fibrinogen must contribute to the final layer of protein adsorbed from plasma to oil droplets, the fibrinogen that does adsorb is functional, and its interfacial concentration increases with increasing plasma concentration of the protein.

Heparins and Other Polyanions Prevent the Mutual Adhesion of Fibrin-coated Surfaces.

Earlier, we demonstrated that heparins and related polyanions-in the absence of any cofactor-bind with high affinity to fibrin (ogen) adsorbed to solid polymeric beads As consequences of this binding, 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

adsorbed fibrin (ogen) might serve some general, cofactor-independent,'anti- adhesive'function.

Believing, 10 as do others, 26-29 that the direct interaction of heparin with fibrin (ogen) contributes in vivo to the anticoagulant activity of the mucopolysaccharide, we tested whether heparins and other polyanions prevent thrombin-inducible aggregation of fibrinogen-coated droplets of mineral oil. For these studies, we used unfractionated heparin, low molecular weight heparins, pentosan polysulfate, suramin and dextran sulfates. As shown in Fig. 12, all of the materials tested reduce in dose-dependent fashion the maximal rate of aggregation of <BR> <BR> <BR> droplets. For all but dextran sulfate of Mr (ave) ~ 40, 000 the reduction in rate as a function of polyanion concentration obeys well the relationship Vobs = V, (l + P/Kd) where Vmax is the maximal rate of aggregation in the absence of polyanion, Vobs is the observed rate of aggregation in the presence of polyanion, P is the molarity of the polyanion, and Kd is the equilibrium dissociation constant of the complex. The <BR> <BR> <BR> polyanions fall into four distinct groups, Table 1. Unfractionated heparin (Mr (aV¢) ~ 14,250) is by far the most potent inhibitor of the series followed, in decreasing order of potency, by the group consisting of the sulfated polysaccharides of low molecular weight (i. e., Mur (aveus between 4,000 and 8,000), by suramin (Mr = 1429), and by high <BR> <BR> <BR> molecular weight dextran sulfate (Mr (aVe) ~ 40, 000). For the heparins and the dextran sulfates, these results are the same as those obtained using fibrin (ogen)-coated solid polymeric beads. l° The apparent dependence on the length of the dextran sulfate polymer as compared to that of heparin, and the relative impotency of suramin with respect to unfractionated heparin, probably indicate some fundamental structural requirement for complex formation.

Discussion We have demonstrated that 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.

As a consequence, oil droplets coated with fibrinogen can participate in a host of biologically important adhesive processes in which the protein would otherwise be expected to participate. Certain polyanions appear to bind to adsorbed fibrin (ogen) and prevent thrombin-dependent adhesion of fibrin-coated surfaces. What follows is a discussion of potential practical applications and theoretical implications of these findings.

Since so many pathologic lesions, e. g., tumors, granulomas, bacterial abscesses, atherosclerotic plaques-indeed, virtually all sites of inflammation- have a thrombotic component, 30~32 it would seem worthwhile generally to develop vehicles to deliver therapeutic drugs or imaging agents to sites of fibrin deposition.

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. 33 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. Earlier, we proposed that 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. 534 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. 35 Using the bead system, we demonstrated conclusively that fibrinogen binds avidly and with high affinity to beads coated with adjuvant lipids, and that the bound protein acts synergistically with the lipid microenvironment to produce the biologic effects traditionally associated with adjuvant oil emulsions. Since then, many adjuvant preparations that include oil and one or another amphiphilic lipid have been formulated for successful vaccine use. 36 We suggest that precoating adjuvant oil

droplets with fibrinogen might optimize the immune-enhancing potential of the droplets, perhaps by either facilitating or promoting the interactions of the particles with macrophages.

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. 423 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. Thus, 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. 37) Another aim of biomedical materials researchers is identification of the region (s) of fibrinogen that actually contacts hydrophobic polymeric surfaces. ' Attempts at this second aim have met with limited success both because the binding of fibrinogen to hydrophobic surfaces is essentially irreversible and because the protein and its remnants are difficult to elute from hydrophobic surfaces.' We propose that droplets of liquid hydrophobic phases may help determine the identify of the'surface recognition site (s)' of fibrinogen. Because droplets of the oils used here are readily soluble in organic phases, there is no need to elute the protein or an adherent remnant of the protein from the droplets. It is enough to simply dissolve away the underlying hydrophobic scaffold leaving as residual only proteinaceous material.

Perhaps one of the more important concepts to derive from this study is that 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,' begging further investigation of the phenomenon. As concerns practical application, we suspect that 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

For quite some time, biomedical scientists studying atherosclerosis have focused on the role (s) of lipids in that disorder. More recently, investigators have begun to consider seriously mechanistic roles for fibrinogen in atherogenesis, 4l-43 this, in part, because fibrin (ogen) is a ubiquitous and significant component of advanced lesions of atherosclerosis, i. e., plaques. 324254044~46 We, ourselves, have proposed that the affinity of fibrinogen for extracellular deposits of atheromatous lipids contributes to the morbid and mortal thrombotic consequences of atherosclerosis: 8 because clots are nucleated by adsorbed fibrinogen, fibrinogen-coated lipid surfaces should be predisposed to thrombosis. But fibrinogen is a significant component of even the earliest detectable precursor of the atherosclerotic plaque, the fatty streak. 42 What role, if any, could fibrinogen play in the earliest stages of plaque development? Blood, a fibrinogen-rich medium, is replete with lipid-laden lipoproteins that percolate through the walls of blood vessels and into the tissues. Popular theories hold that lipoproteins, particularly low density lipoproteins, are somehow retained within arterial walls, and this retention accounts for the accumulation there of the lipids that will eventually constitute the plaque. 47, 48 Given the results of our studies, those of others, 46, 49 and current opinion regarding plaque initiation, development and growth, it is reasonable to propose that the localization and accumulation in vivo of lipoproteins-or, for that matter, any lipid particle-will be mediated, at least in part, by fibrinogen adsorbed to those particles. This adsorption, in turn, will be dictated by the expression of hydrophobic domains on the surface of the particles.

Thus, localization and accumulation of 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 any site of inflammation. 32 Many approaches could be used to assess the validity of this proposal. One approach might involve measuring directly the binding of fibrinogen to various lipoproteins, and, if fibrinogen binds to them, its functionality once bound (GSR, manuscript in preparation). Another approach might involve correlating plasma fibrinogen concentration with some lipid-dependent aspect of plaque growth. Still

another approach might involve use of anticoagulants as a means to prevent or limit plaque development. All of these biological approaches have merit, and all would likely yield valuable mechanistic insight relevant to both atherosclerosis and its therapy. But physicochemical approaches of the sort presented here should also provide valuable information pertinent to mechanistic understanding of atherosclerosis since the adhesive potential conferred to a lipid particle by adsorbed fibrinogen makes the presence of that protein on the particle singularly important.

What factors should be expected to influence the'non-specific'binding of fibrinogen to hydrophobic lipid particles? We have demonstrated several, including the solution phase concentration of fibrinogen, the solution phase concentration of species that compete with fibrinogen for the surface of particles, and amphiphiles preexisting on the surface of particles. The concentration of particles-a measure of lipid'load'-will also influence the equilibrium distribution of the protein, as will measures that affect the affinity and/or capacity of individual particles for the protein.

Given all of these, it is reasonable to presume that the disposition of lipid particles that percolate through the walls of the vasculature will depend on the dynamic equilibrium that must normally exist between bound and free forms of fibrinogen.

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